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Sample records for dna adduct formation

  1. Temporal and spatial features of the formation of DNA adducts in sulfur mustard-exposed skin

    Energy Technology Data Exchange (ETDEWEB)

    Batal, Mohamed [Laboratoire «Lésions des Acides Nucléiques», Université Joseph Fourier – Grenoble 1, CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France); Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche (France); Boudry, Isabelle; Mouret, Stéphane; Wartelle, Julien; Emorine, Sandy; Bertoni, Marine [Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche (France); Bérard, Izabel [Laboratoire «Lésions des Acides Nucléiques», Université Joseph Fourier – Grenoble 1, CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France); Cléry-Barraud, Cécile [Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche (France); and others

    2013-12-15

    Sulfur mustard (SM) is a chemical warfare agent that targets skin where it induces large blisters. DNA alkylation is a critical step to explain SM-induced cutaneous symptoms. We determined the kinetics of formation of main SM–DNA adducts and compare it with the development of the SM-induced pathogenesis in skin. SKH-1 mice were exposed to 2, 6 and 60 mg/kg of SM and treated skin was biopsied between 6 h and 21 days. Formation of SM DNA adducts was dose-dependent with a maximum immediately after exposure. However, adducts were persistent and still detectable 21 days post-exposure. The time-dependent formation of DNA adducts was also found to be correlated with the appearance of apoptotic cells. This temporal correlation suggests that these two early events are responsible for the severity of the damage to the skin. Besides, SM–DNA adducts were also detected in areas located next to contaminated zone, thus suggesting that SM diffuses in skin. Altogether, this work provides for the first time a clear picture of SM-induced genotoxicity using DNA adducts as a marker. - Highlights: • Sulfur mustard adducts are formed in DNA after skin exposure. • DNA damage formation is an early event in the pathological process of skin burn. • The amount of SM–DNA adducts is maximal at the earliest time point investigated. • Adducts are still detected 3 weeks after exposure. • Sulfur mustard diffuses in skin especially when large doses are applied.

  2. DNA adducts as molecular dosimeters

    International Nuclear Information System (INIS)

    Lucier, G.W.

    1990-01-01

    There is compelling evidence that DNA adducts play an important role in the actions of many pulmonary carcinogens. During the last ten years sensitive methods (antibodies and 32 P-postlabeling) have been developed that permit detection of DNA adducts in tissues of animals or humans exposed to low levels of some genotoxic carcinogens. This capability has led to approaches designed to more reliably estimate the shape of the dose-response curve in the low dose region for a few carcinogens. Moreover, dosimetry comparisions can, in some cases, be made between animals and humans which help in judging the adequacy of animal models for human risk assessments. There are several points that need to be considered in the evaluation of DNA adducts as a molecular dosimeter. For example, DNA adduct formation is only one of many events that are needed for tumor development and some potent carcinogens do not form DNA adducts; i.e., TCDD. Other issues that need to be considered are DNA adduct heterogeneity, DNA repair, relationship of DNA adducts to somatic mutation and cell specificity in DNA adduct formation and persistence. Molecular epidemiology studies often require quantitation of adducts in cells such as lymphocytes which may or may not be reliable surrogates for adduct concentrations in target issues. In summary, accurate quantitation of low levels of DNA adducts may provide data useful in species to species extrapolation of risk including the development of more meaningful human monitoring programs

  3. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  4. DNA adduct formation among workers in a Thai industrial estate and nearby residents.

    Science.gov (United States)

    Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Meunier, Aurelie; Sangrajrang, Suleeporn; Piro, Sara; Ceppi, Marcello; Boffetta, Paolo

    2008-01-25

    The genotoxic effects of air pollutant exposures have been studied in people living and working in Map Ta Phut, Rayong province, Thailand, a site where is located the Map Ta Phut Industrial Estate (MIE) one of the largest steel, refinery and petrochemical complex in the South-Eastern Asia. This was done by the conduction of a transversal study aimed to compare the prevalence of bulky DNA adducts in groups of subjects experiencing various degree of air pollution. DNA adduct analysis was performed in the leukocytes of 201 volunteers by the (32)P-postlabelling assay: 79 were workers in the MIE complex, including 24 refinery workers, 40 steel workers and 15 tinplate workers, 72 were people residing downwind in the MIE area and 50 were residents in a control district of the same Rayong province but without industrial exposures. The groups of workers were analyzed separately to evaluate if DNA adduct formation differs by the type of industry. The levels of bulky DNA adducts were 1.17+/-0.17 (SE) adducts/10(8) nucleotides in refinery workers, 1.19+/-0.19 (SE) in steel workers, 0.87+/-0.17 (SE) in tinplate workers, 0.85+/-0.07 (SE) in MIE residents and 0.53+/-0.05 (SE) in district controls. No effects of smoking habits on DNA adducts was found. The multivariate regression analysis shows that the levels of DNA adducts were significantly increased among the individuals living near the MIE industrial complex in respect to those resident in a control district (pindustrial air pollution can experiment an excess of DNA adduct formation. The emissions from the MIE complex are the main source of air pollution in this area and can be the cause of such increment in the levels of DNA damage.

  5. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidende in rodent bioassays

    NARCIS (Netherlands)

    Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O'Brien, J.; Bladeren, van P.J.; Rietjens, I.

    2011-01-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate

  6. Theoretical investigations on the formation of nitrobenzanthrone-DNA adducts.

    Science.gov (United States)

    Arlt, Volker M; Phillips, David H; Reynisson, Jóhannes

    2011-09-07

    3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. The thermochemical formation cascades were calculated for six 3-NBA-derived DNA adducts employing its arylnitrenium ion as precursor using density functional theory (DFT). Clear exothermic pathways were found for four adducts, i.e., 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone, 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone. All four have been observed to be formed in cell-free experimental systems. The formation of N-(2'-deoxyadenosin-8-yl)-3-aminobenzanthrone is predicted to be not thermochemically viable explaining its absence in either in vitro or in vivo model systems. However, 2-(2'-deoxyadenosin-8-yl)-3-aminobenzanthrone, can be formed, albeit not as a major product, and is a viable candidate for an unknown adenine adduct observed experimentally. 2-nitrobenzanthrone (2-NBA), an isomer of 3-NBA, was also included in the calculations; it has a higher abundance in ambient air than 3-NBA, but a much lower genotoxic potency. Similar thermochemical profiles were obtained for the calculated 2-NBA-derived DNA adducts. This leads to the conclusion that enzymatic activation as well as the stability of its arylnitrenium ion are important determinants of 2-NBA genotoxicity.

  7. Formation of monofunctional cisplatin-DNA adducts in carbonate buffer.

    Science.gov (United States)

    Binter, Alexandra; Goodisman, Jerry; Dabrowiak, James C

    2006-07-01

    Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768-12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8mM HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 5mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8mM carbonate buffer, 5mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

  8. DNA adduct formation among workers in a Thai industrial estate and nearby residents

    International Nuclear Information System (INIS)

    Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Meunier, Aurelie; Sangrajrang, Suleeporn; Piro, Sara; Ceppi, Marcello; Boffetta, Paolo

    2008-01-01

    The genotoxic effects of air pollutant exposures have been studied in people living and working in Map Ta Phut, Rayong province, Thailand, a site where is located the Map Ta Phut Industrial Estate (MIE) one of the largest steel, refinery and petrochemical complex in the South-Eastern Asia. This was done by the conduction of a transversal study aimed to compare the prevalence of bulky DNA adducts in groups of subjects experiencing various degree of air pollution. DNA adduct analysis was performed in the leukocytes of 201 volunteers by the 32 P-postlabelling assay: 79 were workers in the MIE complex, including 24 refinery workers, 40 steel workers and 15 tinplate workers, 72 were people residing downwind in the MIE area and 50 were residents in a control district of the same Rayong province but without industrial exposures. The groups of workers were analyzed separately to evaluate if DNA adduct formation differs by the type of industry. The levels of bulky DNA adducts were 1.17 ± 0.17 (SE) adducts/10 8 nucleotides in refinery workers, 1.19 ± 0.19 (SE) in steel workers, 0.87 ± 0.17 (SE) in tinplate workers, 0.85 ± 0.07 (SE) in MIE residents and 0.53 ± 0.05 (SE) in district controls. No effects of smoking habits on DNA adducts was found. The multivariate regression analysis shows that the levels of DNA adducts were significantly increased among the individuals living near the MIE industrial complex in respect to those resident in a control district (p < 0.05). In the groups of occupationally exposed workers, the highest levels of DNA adducts were found among the workers experiencing an occupational exposure to polycyclic aromatic hydrocarbons, e.g. the steel factory and refinery workers. When we have evaluated if the levels of DNA adducts of the PAH exposed workers were different from those of the MIE residents, a statistical significantly difference was found (p < 0.05). Our present study indicates that people living near point sources of industrial air

  9. Inhibition of nicotine-DNA adduct formation by polyphenolic compounds in vitro

    International Nuclear Information System (INIS)

    Cheng Yan; Wang Haifang; Sun Hongfang; Li Hongli

    2004-01-01

    Nicotine[3-(1-methyl-2-pyrrolidinyl)-pyridine], a major alkaloid in tobacco products, has proven to be a potential genotoxic compound. Some polyphenolic compounds can suppress the DNA adduction, and hence act as the potential inhibitors of carcinogenesis. In this study, the inhibitory effects of three polyphenolic compounds, curcumin (diferuloylmethane), resveratrol (trans-3, 5, 4-trihydroxystilbene) and tea polyphenols, on the nicotine-DNA adduction have been investigated in vitro using radiolabelled nicotine and liquid scintillation counting (LSC) technique. Also, the inhibition mechanism of these chemopreventive agents in regard to the activity of the biotransformation enzymes, including cytochrome P450 (CYP450), cytochrome b 5 (CYb 5 ) and glutathione S-transferase (GST), has been studied. The results demonstrated that these three polyphenols induced marked dose-dependent decrease in nicotine-DNA adducts as compared with the controls. The elimination rate of adducts reached above 46% at the highest dose for all the three agents with 51.6% for resveratrol. Correspondingly, three polyphenols all suppressed CYP450 and CYb 5 , whereas curcumin and resveratrol induced GST. The authors may arrive at a point that the three polyphenols are beneficial to prevent the nicotine adduct formation, and thus may be used to block the potential carcinogenesis induced by nicotine. (authors)

  10. DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

    International Nuclear Information System (INIS)

    Mei, Nan; Arlt, Volker M.; Phillips, David H.; Heflich, Robert H.; Chen, Tao

    2006-01-01

    Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32 P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N 6 -yl]-aristolactam I, 7-[deoxyadenosin-N 6 -yl]-aristolactam II and 7-[deoxyguanosin-N 2 -yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10 8 nucleotides in liver and 95-4598 adducts/10 8 nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10 -6 in liver compared with the MFs of 78-1319 x 10 -6 that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually

  11. DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

    Energy Technology Data Exchange (ETDEWEB)

    Mei, Nan [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)]. E-mail: nan.mei@fda.hhs.gov; Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Heflich, Robert H. [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States); Chen, Tao [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)

    2006-12-01

    Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by {sup 32}P-postlabeling and mutant frequency (MF) was determined using the {lambda} Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N {sup 6}-yl]-aristolactam I, 7-[deoxyadenosin-N {sup 6}-yl]-aristolactam II and 7-[deoxyguanosin-N {sup 2}-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10{sup 8} nucleotides in liver and 95-4598 adducts/10{sup 8} nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10{sup -6} in liver compared with the MFs of 78-1319 x 10{sup -6} that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T {sup {yields}} T:A transversion was the predominant mutation in AA-treated rats; whereas G:C {sup {yields}} A:T transition was the main type of mutation in control

  12. 32P-postlabeling DNA adduct assay: cigarette smoke-induced dna adducts in the respiratory and nonrespiratory rat tissues. Book chapter

    International Nuclear Information System (INIS)

    Gupta, R.C.; Gairola, C.G.

    1990-01-01

    An analysis of the tissue DNA adducts in rats by the sensitive (32)p-postlabeling assay showed one to eight detectable DNA adducts in lung, trachea, larynx, heart and bladder of the sham controls. Chronic exposure of animals to mainstream cigarette smoke showed a remarkable enhancement of most adducts in the lung and heart DNA. Since cigarette smoke contains several thousand chemicals and a few dozen of them are known or potential carcinogens, the difference between the DNA adducts of nasal and the other tissues may reflect the diversity of reactive constituents and their differential absorption in different tissues. In comparison to the lung DNA adducts, the adducts in nasal DNA were less hydrophobic. Identity of the predominant adducts was further investigated by comparison with several reference DNA adducts from 10 PAH and aromatic amines. Since some of these chemicals are present in cigarette smoke, the results suggest that these constituents of cigarette smoke may not be directly responsible for formation of DNA adducts in the lung and heart of the smoke-exposed animals

  13. Formation of DNA adducts in mouse tissues after 1-nitropyrene administration

    International Nuclear Information System (INIS)

    Mitchell, C.E.

    1986-01-01

    DNA adducts were isolated and characterized in mouse lung, liver and kidney after intratracheal instillation of [ 3 H]-1-nitropyrene (1-NP). HPLC analysis of the enzymatically digested DNA indicated the presence of multiple DNA adducts in mouse lung, liver and kidney. These results indicate that DNA adducts of 1-NP are formed in mouse lung, liver and kidney after intratracheal instillation of 1-NP; the HPLC profiles of the multiple adducts suggests that adducts may be formed via metabolic pathways that involve both nitroreduction and ring-oxidation. 6 references, 1 figure

  14. DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1,2,3-trichloropropane.

    Science.gov (United States)

    La, D K; Lilly, P D; Anderegg, R J; Swenberg, J A

    1995-06-01

    1,2,3-Trichloropropane (TCP) is a multispecies, multisite carcinogen which has been found to be an environmental contaminant. In this study, we have characterized and measured DNA adducts formed in vivo following exposure to TCP. [14C]TCP was administered to male B6C3F1 mice and Fischer-344 rats by gavage at doses used in the NTP carcinogenesis bioassay. Both target and nontarget organs were examined for the formation of DNA adducts. Adducts were hydrolyzed from DNA by neutral thermal or mild acid hydrolysis, isolated by HPLC, and detected and quantitated by measurement of radioactivity. The HPLC elution profile of radioactivity suggested that one major DNA adduct was formed. To characterize this adduct, larger yields were induced in rats by intraperitoneal administration of TCP (300 mg/kg). The DNA adduct was isolated by HPLC based on coelution with the radiolabeled adduct, and compared to previously identified adducts. The isolated adduct coeluted with S-[1-(hydroxymethyl)-2-(N7-guanyl)-ethyl]glutathione, an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray mass spectrometry suggested that the TCP-induced adduct and the DBCP-derived adduct were identical. The 14C-labeled DNA adduct was distributed widely among the organs examined. Adduct levels varied depending on species, organ, and dose. In rat organs, adduct concentrations for the low dose ranged from 0.8 to 6.6 mumol per mol guanine and from 7.1 to 47.6 mumol per mol guanine for the high dose. In the mouse, adduct yields ranged from 0.32 to 28.1 mumol per mol guanine for the low dose and from 12.2 to 208.1 mumol per mol guanine for the high dose. The relationship between DNA adduct formation and organ-specific tumorigenesis was unclear. Although relatively high concentrations of DNA adducts were detected in target organs, several nontarget sites also contained high adduct levels. Our data suggest that factors in addition to adduct formation

  15. Genotoxic Pyrrolizidine Alkaloids — Mechanisms Leading to DNA Adduct Formation and Tumorigenicity

    Directory of Open Access Journals (Sweden)

    Ming W. Chou

    2002-09-01

    Full Text Available Abstract: Plants that contain pyrrolizidine alkaloids are widely distributed in the world. Although pyrrolizidine alkaloids have been shown to be genotoxic and tumorigenic in experimental animals, the mechanisms of actions have not been fully understood. The results of our recent mechanistic studies suggest that pyrrolizidine alkaloids induce tumors via a genotoxic mechanism mediated by 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5Hpyrrolizine (DHP-derived DNA adduct formation. This mechanism may be general to most carcinogenic pyrrolizidine alkaloids, including the retronecine-, heliotridine-, and otonecinetype pyrrolizidine alkaloids. It is hypothesized that these DHP-derived DNA adducts are potential biomarkers of pyrrolizidine alkaloid tumorigenicity. The mechanisms that involve the formation of DNA cross-linking and endogenous DNA adducts are also discussed.

  16. Photochemical Reaction of 7,12-Dimethylbenz[a]anthracene (DMBA and Formation of DNA Covalent Adducts

    Directory of Open Access Journals (Sweden)

    Peter P. Fu

    2005-04-01

    Full Text Available DMBA, 7,12-dimethylbenz[a]anthracene, is a widely studied polycyclic aromatic hydrocarbon that has long been recognized as a probable human carcinogen. It has been found that DMBA is phototoxic in bacteria as well as in animal or human cells and photomutagenic in Salmonella typhimurium strain TA102. This article tempts to explain the photochemistry and photomutagenicity mechanism. Light irradiation converts DMBA into several photoproducts including benz[a]anthracene-7,12-dione, 7-hydroxy-12-keto-7-methylbenz[a]anthracene, 7,12-epidioxy-7,12-dihydro-DMBA, 7-hydroxymethyl-12-methylbenz[a]anthracene and 12-hydroxymethyl-7-methylbenz[a]anthracene. Structures of these photoproducts have been identified by either comparison with authentic samples or by NMR/MS. At least four other photoproducts need to be assigned. Photo-irradiation of DMBA in the presence of calf thymus DNA was similarly conducted and light-induced DMBA-DNA adducts were analyzed by 32P-postlabeling/TLC, which indicates that multiple DNA adducts were formed. This indicates that formation of DNA adducts might be the source of photomutagenicity of DMBA. Metabolites obtained from the metabolism of DMBA by rat liver microsomes were reacted with calf thymus DNA and the resulting DNA adducts were analyzed by 32P-postlabeling/TLC under identical conditions. Comparison of the DNA adduct profiles indicates that the DNA adducts formed from photo-irradiation are different from the DNA adducts formed due to the reaction of DMBA metabolites with DNA. These results suggest that photo-irradiation of DMBA can lead to genotoxicity through activation pathways different from those by microsomal metabolism of DMBA.

  17. Detection of Riddelliine-Derived DNA Adducts in Blood of Rats Fed Riddelliine

    Directory of Open Access Journals (Sweden)

    Ming W. Chou

    2002-09-01

    Full Text Available Abstract: We have previously shown that riddelliine, a naturally occurring genotoxic pyrrolizidine alkaloid, induces liver tumors in rats and mice through a genotoxic mechanism mediated by the formation of a set of eight 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5Hpyrrolizine ( DHP-derived DNA adducts. In this study we report the formation of these DHP-derived DNA adducts in blood DNA of rats fed riddelliine. In an adduct formation and removal experiment, male and female F344 rats (8 weeks of age were administered riddelliine by gavage at a single dose of 10.0 mg/kg body weight in 0.1 M phosphate buffer. At 8, 24, 48, and 168 hrs after dosing, the levels of DHP-derived DNA adduct in blood and liver were determined by 32P-postlabeling/HPLC. Maximum DNA adduct formation occurred at 48 hr after treatment. From 48 to 168 hours, the adduct levels in female rat blood were 4-fold greater than those in male rats. In a dose response experiment, female rats were gavaged 0.1 and 1.0 mg/kg doses of riddelliine for three consecutive days and the DHPderived DNA adducts in blood DNA were assayed. The levels of the DHP-derived DNA adducts in blood of rats receiving 0.1 and 1.0 mg/kg doses were 12.9 and 51.8 adducts/107 nucleotides. These results suggest that: (i leucocyte DNA can bind with DHP to form a set of DHP-derived DNA adducts generated in liver; (ii DHP-derived DNA adducts in blood can serve as a potential non-invasive biomarkers for assessing the exposure to riddelliine.

  18. DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats

    International Nuclear Information System (INIS)

    Arlt, Volker M.; Sorg, Bernd L.; Osborne, Martin; Hewer, Alan; Seidel, Albrecht; Schmeiser, Heinz H.; Phillips, David H.

    2003-01-01

    Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2 mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32 P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation

  19. Genetic polymorphisms in catalase and CYP1B1 determine DNA adduct formation by bento(a)pyrene ex vivo

    NARCIS (Netherlands)

    Schults, Marten A.; Chiu, Roland K.; Nagle, Peter; Kleinjans, J C; van Schooten, Frederik Jan; Godschalk, Roger W.

    Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification

  20. The effects of exposure route on DNA adduct formation and cellular proliferation by 1,2,3-trichloropropane.

    Science.gov (United States)

    La, D K; Schoonhoven, R; Ito, N; Swenberg, J A

    1996-09-01

    1,2,3-Trichloropropane (TCP) induces high incidences of tumors at multiple sites in mice and rats when administered chronically by gavage. The animal tumor data are being used to predict human risk from potential exposure to TCP in drinking water. Risk assessment may be affected by differences in the route of exposure. Gavage administration, which results in high bolus concentrations compared to drinking water exposure, may quantitatively affect toxicokinetics, cytotoxicity, and genotoxicity. We have examined the effects of TCP exposure by the two routes on the formation of DNA adducts and the induction of cellular proliferation. Male B6C3F1 mice were administered [14C]TCP for 1 week by gavage or in drinking water at the low dose (6 mg/kg) used in the NTP carcinogenesis bioassay. Two target organs (forestomach and liver) and two nontarget organs (glandular stomach and kidney) were examined for DNA adduct formation. Adducts were hydrolyzed from DNA, isolated by HPLC, and quantitated by measuring HPLC fractions for radioactivity. In the forestomach, liver, and kidney, gavage administration of TCP resulted in 1.4-to 2.4-fold greater yields of the major DNA adduct, previously identified as S-[1-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione. Significant differences in cell proliferation, as determined by incorporation of bromodeoxyuridine into DNA, were also observed for the two routes. Gavage administration of TCP for 2 weeks resulted in up to a threefold greater cell proliferation rate relative to administration in drinking water. Our findings of exposure-related differences in TCP-induced DNA adduct formation and cell proliferation suggest that a risk assessment based on the existing gavage study may overestimate human risk.

  1. Formation of 7-hydroxymethyl-12-methylbenz(a)anthracene-DNA adducts from 7,12-dimethylbenz(a)anthracene in mouse epidermis

    International Nuclear Information System (INIS)

    DiGiovanni, J.; Nebzydoski, A.P.; Decina, P.C.

    1983-01-01

    The formation of DNA adducts from [ 3 H]-7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA) and [ 3 H]-7,12-dimethylbenz(a)anthracene (DMBA) in the epidermis of Sencar mice was analyzed. Comparison of Sephadex LH-20 chromatographic profiles of DNA samples isolated from mice treated with DMBA or 7-OHM-12-MBA suggested that the DMBA-treated animals contained DNA adduct(s) derived from the further metabolism of 7-OHM-12-MBA. Further analysis of DNA samples from DMBA-treated mice by high-pressure liquid chromatography demonstrated the presence of 5 DNA adducts which were chromatographically indistinguishable from the DNA adducts formed in 7-OHM-12-MBA-treated mice. Epidermal homogenates were utilized to catalyze the covalent binding of [ 3 H]DMBA and [ 3 H]-7-OHM-12-MBA to calf thymus DNA in vitro. Under conditions of limiting concentrations of [ 3 H]DMBA, the majority of the DNA adducts formed chromatographed in regions where 7-OHM-12-MBA-DNA adducts eluted. A major DMBA-DNA adduct formed in this in vitro system eluted with the same retention time as did the major 7-OHM-12-MBA-DNA adduct formed in mouse skin in vivo. These results when coupled with the in vivo data suggest that 7-OHM-12-MBA is an intermediate for at least some of the binding of DMBA to epidermal DNA in Sencar mice

  2. Polycyclic aromatic hydrocarbons and PAH-related DNA adducts.

    Science.gov (United States)

    Ewa, Błaszczyk; Danuta, Mielżyńska-Švach

    2017-08-01

    Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.

  3. Activation of dihaloalkanes by glutathione conjugation and formation of DNA adducts

    International Nuclear Information System (INIS)

    Guengerich, F.P.; Peterson, L.A.; Cmarik, J.L.; Koga, N.; Inskeep, P.B.

    1987-01-01

    Ethylene dibromide (1,2-dibromoethane, EDB) can be activated to electrophilic species by either oxidative metabolism or conjugation with glutathione. Although conjugation is generally a route of detoxication, in this case it leads to genetic damage. The major DNA adduct has been identified as S-[2-(N 7 -guanyl)ethyl]glutathione, which is believed to arise via half-mustard and episulfonium ion intermediates. The adduct has a half-life of about 70 to 100 hr and does not appear to migrate to other DNA sites. Glutathione-dependent DNA damage by EDB was also demonstrated in human hepatocyte preparations. The possible relevance of this DNA adduct to genetic damage is discussed

  4. Metabolic Activation of the Tumorigenic Pyrrolizidine Alkaloid, Retrorsine, Leading to DNA Adduct Formation In Vivo

    Directory of Open Access Journals (Sweden)

    Ming W. Chou

    2005-04-01

    Full Text Available Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. The high toxicity of many pyrrolizidine alkaloids has caused considerable loss of free-ranging livestock due to liver and pulmonary lesions. Chronic exposure of toxic pyrrolizidine alkaloids to laboratory animals induces cancer. This investigation studies the metabolic activation of retrorsine, a representative naturally occurring tumorigenic pyrrolizidine alkaloid, and shows that a genotoxic mechanism is correlated to the tumorigenicity of retrorsine. Metabolism of retrorsine by liver microsomes of F344 female rats produced two metabolites, 6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP, at a rate of 4.8 ± 0.1 nmol/mg/min, and retrorsine-N-oxide, at a rate of 17.6±0.5 nmol/mg/min. Metabolism was enhanced 1.7-fold by using liver microsomes prepared from dexamethasone-treated rats. DHP formation was inhibited 77% and retrorsine N-oxide formation was inhibited 29% by troleandomycin, a P450 3A enzyme inhibitor. Metabolism of retrorsine with lung, kidney, and spleen microsomes from dexamethasone-treated rats also generated DHP and the N-oxide derivative. When rat liver microsomal metabolism of retrorsine occurred in the presence of calf thymus DNA, a set of DHP-derived DNA adducts was formed; these adducts were detected and quantified by using a previously developed 32P-postlabeling/HPLC method. These same DNA adducts were also found in liver DNA of rats gavaged with retrorsine. Since DHP-derived DNA adducts are suggested to be potential biomarkers of riddelliine-induced tumorigenicity, our results indicate that (i similar to the metabolic activation of riddelliine, the mechanism of retrorsine-induced carcinogenicity in rats is also through a genotoxic mechanism involving DHP; and (ii the set of DHP-derived DNA adducts found in liver DNA of rats gavaged with retrorsine or riddelliine can serve as biomarkers for the

  5. Metabolic Activation of the Tumorigenic Pyrrolizidine Alkaloid, Retrorsine, Leading to DNA Adduct Formation In Vivo

    Science.gov (United States)

    Wang, Yu-Ping; Fu, Peter P.; Chou, Ming W.

    2005-01-01

    Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. The high toxicity of many pyrrolizidine alkaloids has caused considerable loss of free-ranging livestock due to liver and pulmonary lesions. Chronic exposure of toxic pyrrolizidine alkaloids to laboratory animals induces cancer. This investigation studies the metabolic activation of retrorsine, a representative naturally occurring tumorigenic pyrrolizidine alkaloid, and shows that a genotoxic mechanism is correlated to the tumorigenicity of retrorsine. Metabolism of retrorsine by liver microsomes of F344 female rats produced two metabolites, 6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), at a rate of 4.8 ± 0.1 nmol/mg/min, and retrorsine-N-oxide, at a rate of 17.6±0.5 nmol/mg/min. Metabolism was enhanced 1.7-fold by using liver microsomes prepared from dexamethasone-treated rats. DHP formation was inhibited 77% and retrorsine N-oxide formation was inhibited 29% by troleandomycin, a P450 3A enzyme inhibitor. Metabolism of retrorsine with lung, kidney, and spleen microsomes from dexamethasone-treated rats also generated DHP and the N-oxide derivative. When rat liver microsomal metabolism of retrorsine occurred in the presence of calf thymus DNA, a set of DHP-derived DNA adducts was formed; these adducts were detected and quantified by using a previously developed 32P-postlabeling/HPLC method. These same DNA adducts were also found in liver DNA of rats gavaged with retrorsine. Since DHP-derived DNA adducts are suggested to be potential biomarkers of riddelliine-induced tumorigenicity, our results indicate that (i) similar to the metabolic activation of riddelliine, the mechanism of retrorsine-induced carcinogenicity in rats is also through a genotoxic mechanism involving DHP; and (ii) the set of DHP-derived DNA adducts found in liver DNA of rats gavaged with retrorsine or riddelliine can serve as biomarkers for the tumorigenicity induced by

  6. DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas

    International Nuclear Information System (INIS)

    Le Goff, J.; Gallois, J.; Pelhuet, L.; Devier, M.H.; Budzinski, H.; Pottier, D.; Andre, V.; Cachot, J.

    2006-01-01

    The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32 P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32 P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 μg g -1 dry weight) in comparison to individuals from the reference site (0.053 μg g -1 dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10 8 nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 μg g -1 dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 μg g -1 dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10 8 nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32 P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable biomarker to monitor

  7. DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas

    Energy Technology Data Exchange (ETDEWEB)

    Le Goff, J. [GRECAN, UPRES EA-1772, University of Caen, Caen (France); Gallois, J. [Laboratory F. Duncombe, Conseil General du Calvados, Caen (France); Pelhuet, L. [LPTC, UMR-5472 CNRS, University Bordeaux I, Bordeaux (France); Devier, M.H. [LPTC, UMR-5472 CNRS, University Bordeaux I, Bordeaux (France); Budzinski, H. [LPTC, UMR-5472 CNRS, University Bordeaux I, Bordeaux (France); Pottier, D. [GRECAN, UPRES EA-1772, University of Caen, Caen (France); Andre, V. [GRECAN, UPRES EA-1772, University of Caen, Caen (France); Cachot, J. [LEMA, UPRES EA-3222, IFRMP 23, University of Le Havre, 25 rue Philippe Lebon, B.P. 540, 76058 Le Havre Cedex (France)]. E-mail: jerome.cachot@univ-lehavre.fr

    2006-08-12

    The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a {sup 32}P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of {sup 32}P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 {mu}g g{sup -1} dry weight) in comparison to individuals from the reference site (0.053 {mu}g g{sup -1} dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10{sup 8} nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 {mu}g g{sup -1} dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 {mu}g g{sup -1} dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10{sup 8} nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced {sup 32}P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in

  8. An integrated QSAR-PBK/D modelling approach for predicting detoxification and DNA adduct formation of 18 acyclic food-borne α,β-unsaturated aldehydes

    Energy Technology Data Exchange (ETDEWEB)

    Kiwamoto, R., E-mail: reiko.kiwamoto@wur.nl; Spenkelink, A.; Rietjens, I.M.C.M.; Punt, A.

    2015-01-01

    Acyclic α,β-unsaturated aldehydes present in food raise a concern because the α,β-unsaturated aldehyde moiety is considered a structural alert for genotoxicity. However, controversy remains on whether in vivo at realistic dietary exposure DNA adduct formation is significant. The aim of the present study was to develop physiologically based kinetic/dynamic (PBK/D) models to examine dose-dependent detoxification and DNA adduct formation of a group of 18 food-borne acyclic α,β-unsaturated aldehydes without 2- or 3-alkylation, and with no more than one conjugated double bond. Parameters for the PBK/D models were obtained using quantitative structure–activity relationships (QSARs) defined with a training set of six selected aldehydes. Using the QSARs, PBK/D models for the other 12 aldehydes were defined. Results revealed that DNA adduct formation in the liver increases with decreasing bulkiness of the molecule especially due to less efficient detoxification. 2-Propenal (acrolein) was identified to induce the highest DNA adduct levels. At realistic dietary intake, the predicted DNA adduct levels for all aldehydes were two orders of magnitude lower than endogenous background levels observed in disease free human liver, suggesting that for all 18 aldehydes DNA adduct formation is negligible at the relevant levels of dietary intake. The present study provides a proof of principle for the use of QSAR-based PBK/D modelling to facilitate group evaluations and read-across in risk assessment. - Highlights: • Physiologically based in silico models were made for 18 α,β-unsaturated aldehydes. • Kinetic parameters were determined by in vitro incubations and a QSAR approach. • DNA adduct formation was negligible at levels relevant for dietary intake. • The use of QSAR-based PBK/D modelling facilitates group evaluations and read-across.

  9. O⁶-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat.

    Science.gov (United States)

    Vanden Bussche, Julie; Hemeryck, Lieselot Y; Van Hecke, Thomas; Kuhnle, Gunter G C; Pasmans, Frank; Moore, Sharon A; Van de Wiele, Tom; De Smet, Stefaan; Vanhaecke, Lynn

    2014-09-01

    Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO). In this study, the NOC-derived DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O(6)-CMG formation (p meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose-response association with O(6)-CMG (p = 0.004) and MDA (p = 0.008) formation. The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Yun-bo

    1988-03-01

    This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs.

  11. Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

    International Nuclear Information System (INIS)

    Shi, Yun-bo.

    1988-03-01

    This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs

  12. Etheno-DNA adduct formation in rats gavaged with linoleic acid, oleic acid and coconut oil is organ- and gender specific

    International Nuclear Information System (INIS)

    Fang Qingming; Nair, Jagadeesan; Sun Xin; Hadjiolov, Dimiter; Bartsch, Helmut

    2007-01-01

    Intake of linoleic acid (LA) increased etheno-DNA adducts induced by lipid peroxidation (LPO) in white blood cells (WBC) of female but not of male volunteers [J. Nair, C.E. Vaca, I. Velic, M. Mutanen, L.M. Valsta, H. Bartsch, High dietary ω-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA adducts in white blood cells of female subjects, Cancer Epidemiol. Biomarkers Prev. 6 (1997) 597-601]. Etheno-adducts were measured in rats gavaged with LA, oleic acid (OA) and saturated fatty acid rich coconut oil for 30 days. DNA from organs and total WBC was analyzed for 1, N 6 -ethenodeoxyadenosine (εdA) and 3, N 4 -ethenodeoxycytidine (εdC) by immunoaffinity/ 32 P-postlabeling. Colon was the most affected target with LA-treatment, where etheno-adducts were significantly elevated in both sexes. In WBC both adducts were elevated only in LA-treated females. Unexpectedly, OA treatment enhanced etheno-adduct levels in prostate 3-9 fold. Our results in rodents confirm the gender-specific increase of etheno-adducts in WBC-DNA, likely due to LPO induced by redox-cycling of 4-hydroxyestradiol. Colon was a target for LPO-derived DNA-adducts in both LA-treated male and female rats, supporting their role in ω-6 PUFA induced colon carcinogenesis

  13. Benzo(a)pyrene-DNA adduct formation in cells: time-dependent differences in the benzo(a)pyrene-DNA adducts present

    International Nuclear Information System (INIS)

    Baird, W.M.; Dumaswala, R.U.

    1980-01-01

    Procedures involving isolation of the DNA from tritium labelled hydrocarbon-treated cells are discussed. Enzymatic degradation of the DNA to deoxyribonucleosides, and chromatography of the adducts on columns of water gradients were covered as well

  14. Role of CYP1B1 in PAH-DNA adduct formation and breast cancer risk

    Energy Technology Data Exchange (ETDEWEB)

    Goth-Goldstein, Regine; Russell, Marion L.; Muller, A.P.; Caleffi, M.; Eschiletti, J.; Graudenz, M.; Sohn, Michael D.

    2010-04-01

    This study investigated the hypothesis that increased exposure to polycyclic aromatic hydrocarbons (PAHs) increases breast cancer risk. PAHs are products of incomplete burning of organic matter and are present in cigarette smoke, ambient air, drinking water, and diet. PAHs require metabolic transformation to bind to DNA, causing DNA adducts, which can lead to mutations and are thought to be an important pre-cancer marker. In breast tissue, PAHs appear to be metabolized to their cancer-causing form primarily by the cytochrome P450 enzyme CYP1B1. Because the genotoxic impact of PAH depends on their metabolism, we hypothesized that high CYP1B1 enzyme levels result in increased formation of PAH-DNA adducts in breast tissue, leading to increased development of breast cancer. We have investigated molecular mechanisms of the relationship between PAH exposure, CYP1B1 expression and breast cancer risk in a clinic-based case-control study. We collected histologically normal breast tissue from 56 women (43 cases and 13 controls) undergoing breast surgery and analyzed these specimens for CYP1B1 genotype, PAH-DNA adducts and CYP1B1 gene expression. We did not detect any difference in aromatic DNA adduct levels of cases and controls, only between smokers and non-smokers. CYP1B1 transcript levels were slightly lower in controls than cases, but the difference was not statistically significant. We found no correlation between the levels of CYP1B1 expression and DNA adducts. If CYP1B1 has any role in breast cancer etiology it might be through its metabolism of estrogen rather than its metabolism of PAHs. However, due to the lack of statistical power these results should be interpreted with caution.

  15. Protection by quercetin and quercetin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes

    NARCIS (Netherlands)

    Wilms, L.C.; Hollman, P.C.H.; Boots, A.W.; Kleinjans, J.C.S.

    2005-01-01

    Flavonoids are claimed to protect against cardiovascular disease, certain forms of cancer and ageing, possibly by preventing initial DNA damage. Therefore, we investigated the protective effects of the flavonoid quercetin against the formation of oxidative DNA damage and bulky DNA adducts in human

  16. DNA adducts in senescent cells

    International Nuclear Information System (INIS)

    Gaubatz, J.W.

    1987-01-01

    Perturbations in DNA repair and other metabolic processes during development and aging might affect the steady-state level of genomic damage. The persistence or accumulation of DNA lesions in postmitotic cells could have a significant impact on proper cellular function, interfering with gene regulation for example. To test the notion that DNA damage increases as a function of age in non-dividing cells, DNA was purified from heart tissue of C57BL/6Nia mice at different ages and analyzed by post labeling techniques to detect DNA adducts. In the present experiments, four-dimensional, thin-layer chromatography was used to isolate aromatic adducts that were labeled with carrier-free (γ- 32 P) ATP under DNA-P excess conditions. The complexity and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all preparations and were clearly more abundant in nucleotide maps of mature and old heart DNA. However, a direct correlation with age was not observed. In contrast, experiments in which aromatic adducts were first isolated by phase-transfer to 1-butanol, then labeled with excess (γ- 32 P)ATP indicated that there was an age-related increase in these adducts. The results are consistent with their earlier studies that showed alkyl adducts increased during aging of mouse myocardium and suggest that a common repair pathway might be involved

  17. Etheno-DNA adduct formation in rats gavaged with linoleic acid, oleic acid and coconut oil is organ- and gender specific

    Energy Technology Data Exchange (ETDEWEB)

    Fang Qingming [Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280 69120 Heidelberg (Germany); Nair, Jagadeesan [Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280 69120 Heidelberg (Germany)], E-mail: j.nair@dkfz.de; Sun Xin [Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280 69120 Heidelberg (Germany); Hadjiolov, Dimiter [National Oncological Centre, Sofia (Bulgaria); Bartsch, Helmut [Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280 69120 Heidelberg (Germany)

    2007-11-01

    Intake of linoleic acid (LA) increased etheno-DNA adducts induced by lipid peroxidation (LPO) in white blood cells (WBC) of female but not of male volunteers [J. Nair, C.E. Vaca, I. Velic, M. Mutanen, L.M. Valsta, H. Bartsch, High dietary {omega}-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA adducts in white blood cells of female subjects, Cancer Epidemiol. Biomarkers Prev. 6 (1997) 597-601]. Etheno-adducts were measured in rats gavaged with LA, oleic acid (OA) and saturated fatty acid rich coconut oil for 30 days. DNA from organs and total WBC was analyzed for 1, N{sup 6}-ethenodeoxyadenosine ({epsilon}dA) and 3, N{sup 4}-ethenodeoxycytidine ({epsilon}dC) by immunoaffinity/{sup 32}P-postlabeling. Colon was the most affected target with LA-treatment, where etheno-adducts were significantly elevated in both sexes. In WBC both adducts were elevated only in LA-treated females. Unexpectedly, OA treatment enhanced etheno-adduct levels in prostate 3-9 fold. Our results in rodents confirm the gender-specific increase of etheno-adducts in WBC-DNA, likely due to LPO induced by redox-cycling of 4-hydroxyestradiol. Colon was a target for LPO-derived DNA-adducts in both LA-treated male and female rats, supporting their role in {omega}-6 PUFA induced colon carcinogenesis.

  18. Quantitation of DNA adducts by stable isotope dilution mass spectrometry

    Science.gov (United States)

    Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

    2012-01-01

    Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

  19. Polycyclic Aromatic Hydrocarbon (PAH Exposure and DNA Adduct Semi-Quantitation in Archived Human Tissues

    Directory of Open Access Journals (Sweden)

    M. Margaret Pratt

    2011-06-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAHs are combustion products of organic materials, mixtures of which contain multiple known and probable human carcinogens. PAHs occur in indoor and outdoor air, as well as in char-broiled meats and fish. Human exposure to PAHs occurs by inhalation, ingestion and topical absorption, and subsequently formed metabolites are either rendered hydrophilic and excreted, or bioactivated and bound to cellular macromolecules. The formation of PAH-DNA adducts (DNA binding products, considered a necessary step in PAH-initiated carcinogenesis, has been widely studied in experimental models and has been documented in human tissues. This review describes immunohistochemistry (IHC studies, which reveal localization of PAH-DNA adducts in human tissues, and semi-quantify PAH-DNA adduct levels using the Automated Cellular Imaging System (ACIS. These studies have shown that PAH-DNA adducts concentrate in: basal and supra-basal epithelium of the esophagus, cervix and vulva; glandular epithelium of the prostate; and cytotrophoblast cells and syncitiotrophoblast knots of the placenta. The IHC photomicrographs reveal the ubiquitous nature of PAH-DNA adduct formation in human tissues as well as PAH-DNA adduct accumulation in specific, vulnerable, cell types. This semi-quantative method for PAH-DNA adduct measurement could potentially see widespread use in molecular epidemiology studies.

  20. Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells

    Directory of Open Access Journals (Sweden)

    Schins Roel PF

    2009-06-01

    Full Text Available Abstract Titanium dioxide (TiO2, also known as titanium (IV oxide or anatase, is the naturally occurring oxide of titanium. It is also one of the most commercially used form. To date, no parameter has been set for the average ambient air concentration of TiO2 nanoparticles (NP by any regulatory agency. Previously conducted studies had established these nanoparticles to be mainly non-cyto- and -genotoxic, although they had been found to generate free radicals both acellularly (specially through photocatalytic activity and intracellularly. The present study determines the role of TiO2-NP (anatase, ∅ in vitro. For comparison, iron containing nanoparticles (hematite, Fe2O3, ∅ 2-NP did not induce DNA-breakage measured by the Comet-assay in both cell types. Generation of reactive oxygen species (ROS was measured acellularly (without any photocatalytic activity as well as intracellularly for both types of particles, however, the iron-containing NP needed special reducing conditions before pronounced radical generation. A high level of DNA adduct formation (8-OHdG was observed in IMR-90 cells exposed to TiO2-NP, but not in cells exposed to hematite NP. Our study demonstrates different modes of action for TiO2- and Fe2O3-NP. Whereas TiO2-NP were able to generate elevated amounts of free radicals, which induced indirect genotoxicity mainly by DNA-adduct formation, Fe2O3-NP were clastogenic (induction of DNA-breakage and required reducing conditions for radical formation.

  1. Decay kinetics of nicotine/NNK-DNA adducts in vivo studied by accelerator mass spectrometry

    International Nuclear Information System (INIS)

    Sun, H.F.; He, L.; Liu, Y.F.; Liu, K.X.; Lu, X.Y.; Wang, J.J.; Ma, H.J.; Li, K.

    2000-01-01

    The decay kinetics of nicotine-DNA adducts and NNK-DNA adducts in mice liver after single dosing was studied by accelerator mass spectrometry (AMS). The decay is characterized by a two-stage process. The half-lives of nicotine-DNA adducts are 1.3 d (4-24 h) and 7.0 d (1-21 d), while for NNK-DNA adducts are 0.7 d (4-24 h) and 18.0 d (1-21 d). The relatively faster decay of nicotine-DNA adducts suggests that the genotoxicity of nicotine is weaker than that of NNK. The in vitro study shows that the metabolization of nicotine is necessary for the final formation of nicotine-DNA adducts, and nicotine Δ1'(5') iminium ion is a probable metabolite species that binds to DNA molecule covalently

  2. DNA adducts: Mass spectrometry methods and future prospects

    International Nuclear Information System (INIS)

    Farmer, P.B.; Brown, K.; Tompkins, E.; Emms, V.L.; Jones, D.J.L.; Singh, R.; Phillips, D.H.

    2005-01-01

    Detection of DNA adducts is widely used for the monitoring of exposure to genotoxic carcinogens. Knowledge of the nature and amounts of DNA adducts formed in vivo also gives valuable information regarding the mutational effects that may result from particular exposures. The power of mass spectrometry (MS) to achieve qualitative and quantitative analyses of human DNA adducts has increased greatly in recent years with the development of improved chromatographic interfaces and ionisation sources. Adducts have been detected on nucleic acid bases, 2'-deoxynucleosides or 2'-deoxynucleotides, with LC-MS/MS being the favoured technique for many of these analyses. Our current applications of this technique include the determination of N7-(2-carbamoyl-2-hydroxyethyl)-guanine, which was postulated to be found as a DNA repair product in urine following exposure to acrylamide, and of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyadenosine, as markers of oxidative damage in human lymphocyte DNA. Higher sensitivity (with a detection limit of 1-10 adducts/10 12 nucleotides) may be achieved by the use of accelerator mass spectrometry (AMS), although this requires the presence of certain isotopes, such as [ 14 C], in the material being analysed. In order to make this technique more amenable for studies of human exposure to environmental carcinogens, new postlabelling techniques, incorporating [ 14 C] into specific DNA adducts after formation, are being developed. It is expected that combining the use of advanced MS techniques with existing 32 P-postlabelling and immunochemical methodologies will contribute greatly to the understanding of the burden of human exposure to environmental carcinogens

  3. DNA adduct profiling of in vitro colonic meat digests to map red vs. white meat genotoxicity.

    Science.gov (United States)

    Hemeryck, Lieselot Y; Rombouts, Caroline; De Paepe, Ellen; Vanhaecke, Lynn

    2018-05-01

    The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O 6 -carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N 4 -etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes

    International Nuclear Information System (INIS)

    Suh, Myungkoo.

    1995-01-01

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7β, 8α-dihydoxy-9α, l0α-epoxy-7,8,9, 10-tetrahydrobenzo[α]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( ∼ 25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant π-π stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G 2 or G 3 (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N 2 -dG in DNA isolated from the skin of mice treated topically with benzo[α]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N 2 -dG

  5. DNA-adducts in fish exposed to alkylating carcinogens

    International Nuclear Information System (INIS)

    Giam, C.S.; Holliday, T.L.; Williams, J.L.; Bahnson, A.; Weller, R.; Hinton, D.E.

    1988-01-01

    There are limited studies on DNA-adduct formation following exposure of fish or fish cells to carcinogens. It will be essential to determine if procarcinogens and carcinogens form the same DNA-adducts in different liver cells and how these compare to those reported in mammalian livers. They are also interested in the influence of different alkylating agents on the type and quantity of DNA-adduct formation and repair in fish. While eggs or small fish are ideal for routine screening, large fish such as trout (Salmo gairdneri) is needed initially for the development of analytical procedures for the isolation, quantitation and identification of various adducts. Trout (Salmo gairdneri) weighing approximately 250 grams were acclimatized at 13 degree C before being given i.p. injection of diethylnitrosoamine (DEN). The exposure period varied, though most animals were sacrificed after 24 hours. Their livers were excised and DNA was isolated mainly according the procedure of Croy et al. The neutral thermal hydrolysate and the acid hydrolysate were analyzed by HPLC-Fluorescent detector for 7-ethylguanine and O 6 -ethylguanine, respectively. O 6 -ethylguanine was detected, 7-ethylguanine was not detected. Attempts are being made to improve the detection of the latter compound. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used to establish nanogram quantities of the ethylated bases. Laser desorption FT-IC-MS is particularly useful for characterizing thermally-labile and involatile nucleosides or nucleotides. Excretion of DEN was rapid and high. Exposure of trout (and other fish) to various ethylating agents will be discussed

  6. DNA adducts-chemical addons

    Directory of Open Access Journals (Sweden)

    T R Rajalakshmi

    2015-01-01

    Full Text Available DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde. This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers.

  7. Detection of Adriamycin-DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations.

    Science.gov (United States)

    Coldwell, Kate E; Cutts, Suzanne M; Ognibene, Ted J; Henderson, Paul T; Phillips, Don R

    2008-09-01

    Limited sensitivity of existing assays has prevented investigation of whether Adriamycin-DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin-DNA adducts/10(4) bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin-DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [(14)C]Adriamycin-DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin-DNA adducts at clinically-relevant Adriamycin concentrations. [(14)C]Adriamycin treatment (25 nM) resulted in 4.4 +/- 1.0 adducts/10(7) bp ( approximately 1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin-DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.

  8. Polycyclic aromatic hydrocarbon-DNA adducts in cervix of women infected with carcinogenic human papillomavirus types: An immunohistochemistry study

    International Nuclear Information System (INIS)

    Pratt, M. Margaret; Sirajuddin, Paul; Poirier, Miriam C.; Schiffman, Mark; Glass, Andrew G.; Scott, David R.; Rush, Brenda B.; Olivero, Ofelia A.; Castle, Philip E.

    2007-01-01

    Among women infected with carcinogenic human papillomavirus (HPV), there is a two- to five-fold increased risk of cervical precancer and cancer in women who smoke compared to those who do not smoke. Because tobacco smoke contains carcinogenic polycyclic aromatic hydrocarbons (PAHs), it was of interest to examine human cervical tissue for PAH-DNA adduct formation. Here, we measured PAH-DNA adduct formation in cervical biopsies collected in follow-up among women who tested positive for carcinogenic HPV at baseline. A semi-quantitative immunohistochemistry (IHC) method using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was used to measure nuclear PAH-DNA adduct formation. Cultured human cervical keratinocytes exposed to 0, 0.153, or 0.331 μM BPDE showed dose-dependent increases in r7,t8,t9-trihydroxy-c-10-(N 2 deoxyguanosyl)-7,8,9, 10-tetrahydro-benzo[a]pyrene (BPdG) adducts. For BPdG adduct analysis, paraffin-embedded keratinocytes were stained by IHC with analysis of nuclear color intensity by Automated Cellular Imaging System (ACIS) and, in parallel cultures, extracted DNA was assayed by quantitative BPDE-DNA chemiluminescence immunoassay (CIA). For paraffin-embedded samples from carcinogenic HPV-infected women, normal-appearing cervical squamous epithelium suitable for scoring was found in samples from 75 of the 114 individuals, including 29 cases of cervical precancer or cancer and 46 controls. With a lower limit of detection of 20 adducts/10 8 nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/10 8 nucleotides, with a median of 75/10 8 nucleotides. PAH-DNA adduct values above 150/10 8 nucleotides were found in eight samples, and in three samples adducts were non-detectable. There was no correlation between PAH-DNA adduct formation and either smoking or case status. Therefore, PAH-DNA adduct formation as measured by this methodology did not appear related to the increased risk

  9. Polycyclic aromatic hydrocarbon-DNA adducts in cervix of women infected with carcinogenic human papillomavirus types: An immunohistochemistry study

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, M. Margaret [Carcinogen-DNA Interactions Section, LCBG, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD (United States)], E-mail: prattm@mail.nih.gov; Sirajuddin, Paul; Poirier, Miriam C. [Carcinogen-DNA Interactions Section, LCBG, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD (United States); Schiffman, Mark [Hormonal and Reproductive Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD (United States); Glass, Andrew G.; Scott, David R.; Rush, Brenda B. [Northwest Kaiser Permanente, Portland, OR (United States); Olivero, Ofelia A. [Carcinogen-DNA Interactions Section, LCBG, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD (United States); Castle, Philip E. [Hormonal and Reproductive Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD (United States)

    2007-11-01

    Among women infected with carcinogenic human papillomavirus (HPV), there is a two- to five-fold increased risk of cervical precancer and cancer in women who smoke compared to those who do not smoke. Because tobacco smoke contains carcinogenic polycyclic aromatic hydrocarbons (PAHs), it was of interest to examine human cervical tissue for PAH-DNA adduct formation. Here, we measured PAH-DNA adduct formation in cervical biopsies collected in follow-up among women who tested positive for carcinogenic HPV at baseline. A semi-quantitative immunohistochemistry (IHC) method using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was used to measure nuclear PAH-DNA adduct formation. Cultured human cervical keratinocytes exposed to 0, 0.153, or 0.331 {mu}M BPDE showed dose-dependent increases in r7,t8,t9-trihydroxy-c-10-(N{sup 2}deoxyguanosyl)-7,8,9, 10-tetrahydro-benzo[a]pyrene (BPdG) adducts. For BPdG adduct analysis, paraffin-embedded keratinocytes were stained by IHC with analysis of nuclear color intensity by Automated Cellular Imaging System (ACIS) and, in parallel cultures, extracted DNA was assayed by quantitative BPDE-DNA chemiluminescence immunoassay (CIA). For paraffin-embedded samples from carcinogenic HPV-infected women, normal-appearing cervical squamous epithelium suitable for scoring was found in samples from 75 of the 114 individuals, including 29 cases of cervical precancer or cancer and 46 controls. With a lower limit of detection of 20 adducts/10{sup 8} nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/10{sup 8} nucleotides, with a median of 75/10{sup 8} nucleotides. PAH-DNA adduct values above 150/10{sup 8} nucleotides were found in eight samples, and in three samples adducts were non-detectable. There was no correlation between PAH-DNA adduct formation and either smoking or case status. Therefore, PAH-DNA adduct formation as measured by this methodology did not appear

  10. Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Myungkoo [Iowa State Univ., Ames, IA (United States)

    1995-12-06

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7β, 8α-dihydoxy-9α, l0α-epoxy-7,8,9, 10-tetrahydrobenzo[α]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( ~25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant π-π stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G2 or G3 (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N2-dG in DNA isolated from the skin of mice treated topically with benzo[α]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG.

  11. Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

    Science.gov (United States)

    Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.

    2012-01-01

    Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account. PMID:22157354

  12. Sequence distribution of acetaldehyde-derived N2-ethyl-dG adducts along duplex DNA.

    Science.gov (United States)

    Matter, Brock; Guza, Rebecca; Zhao, Jianwei; Li, Zhong-ze; Jones, Roger; Tretyakova, Natalia

    2007-10-01

    Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence

  13. DNA-nicotine adduction of lung and liver of mice exposed to passive smoking studied by AMS

    International Nuclear Information System (INIS)

    Hou Qin; Sun Hongfang; Shi Jingyuan; Liu Yuanfang; Wang Jianjun; Lu Xiangyang; Li Kun; Zhao Qiang

    1997-01-01

    The author presents the measurement of adduction of mice lung or liver DNA with nicotine by accelerator mass spectrometry (AMS). Mice were exposed in a toxicity infecting chamber filled up with cigarette smoke for a period of time of simulate the exposure of mice to passive smoking. The dose of nicotine inhaled by mice was determined. The results of AMS showed, when the dose of inhaled nicotine ranged from 33 μg/kg to 330 μg/kg, the adducts number of lung DNA was 10 3 -10 4 adducts/10 12 nucleotides, and the adducts increased linearly with increasing dose of nicotine; the adducts number of liver DNA reached to 10 4 -10 5 adducts/10 12 nucleotides, when the dose of nicotine ranged from 99 μg/kg to 330 μg/kg, and the adducts increased vigorously as dose of nicotine increased. Comparing the DNA adducts levels of the same nicotine dose, liver DNA adducts were more than lung DNA adducts. This study also suggested that the other components of cigarette smoke have synergic effect on the formation of nicotine derived DNA adducts

  14. Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

    Science.gov (United States)

    Hellman, Lance M.; Spear, Tyler J.; Koontz, Colton J.; Melikishvili, Manana; Fried, Michael G.

    2014-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs. PMID:25080506

  15. Pyrrolizidine alkaloid-derived DNA adducts are common toxicological biomarkers of pyrrolizidine alkaloid N-oxides.

    Science.gov (United States)

    He, Xiaobo; Xia, Qingsu; Woodling, Kellie; Lin, Ge; Fu, Peter P

    2017-10-01

    There are 660 pyrrolizidine alkaloids (PAs) and PA N-oxides present in the plants, with approximately half being possible carcinogens. We previously reported that a set of four PA-derived DNA adducts is formed in the liver of rats administered a series of hepatocarcinogenic PAs and a PA N-oxide. Based on our findings, we hypothesized that this set of DNA adducts is a common biological biomarker of PA-induced liver tumor formation. In this study, we determined that rat liver microsomal metabolism of five hepatocarcinogenic PAs (lasiocarpine, retrorsine, riddelliine, monocrotaline, and heliotrine) and their corresponding PA N-oxides produced the same set of DNA adducts. Among these compounds, lasiocarpine N-oxide, retrorsine N-oxide, monocrotaline N-oxide, and heliotrine N-oxide are for first time shown to be able to produce these DNA adducts. These results further support the role of these DNA adducts as potential common biomarkers of PA-induced liver tumor initiation. Copyright © 2017. Published by Elsevier B.V.

  16. Pyrrolizidine alkaloid-derived DNA adducts are common toxicological biomarkers of pyrrolizidine alkaloid N-oxides

    Directory of Open Access Journals (Sweden)

    Xiaobo He

    2017-10-01

    Full Text Available There are 660 pyrrolizidine alkaloids (PAs and PA N-oxides present in the plants, with approximately half being possible carcinogens. We previously reported that a set of four PA-derived DNA adducts is formed in the liver of rats administered a series of hepatocarcinogenic PAs and a PA N-oxide. Based on our findings, we hypothesized that this set of DNA adducts is a common biological biomarker of PA-induced liver tumor formation. In this study, we determined that rat liver microsomal metabolism of five hepatocarcinogenic PAs (lasiocarpine, retrorsine, riddelliine, monocrotaline, and heliotrine and their corresponding PA N-oxides produced the same set of DNA adducts. Among these compounds, lasiocarpine N-oxide, retrorsine N-oxide, monocrotaline N-oxide, and heliotrine N-oxide are for first time shown to be able to produce these DNA adducts. These results further support the role of these DNA adducts as potential common biomarkers of PA-induced liver tumor initiation.

  17. DNA adduct quantification in Eisenia fetida after subchronic exposures to creosote contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Charrois, J.W.A.; McGill, W.B. [Alberta Univ., Dept. of Renewable Resources, Edmonton, AB (Canada)

    1999-07-01

    Within soil ecosystems contaminant toxicity can vary from acute and chronic, depending on the time of exposure. Due to the long times involved chronic toxicity is difficult to determine. DNA adducts fall into the category of biochemical markers that act as an early warning system in environmental monitoring. It has been proposed that they could be used as a sensitive method to determine environmental exposures to compounds such as polycyclic aromatic hydrocarbons (PAHs), which can occur, although not exclusively, in creosote. In this connection, Benzo[a]pyrene (BaP) is a PAH that can be transformed into an electrophilic metabolite, which ultimately results in DNA adduct formation. Use was made of a 32P postlabeling method to quantify the number of DNA adducts occurring in the earthworm Eisenia fetida after exposure to weathered creosote contaminated- and biotreated-soils with and without additions of extra BaP. DNA adducts can be measured in earthworms exposed to creosote contaminated- and biotreated-soils. E. fetida exposed to weathered creosote contaminated soils had significantly more DNA adducts than those exposed to a pristine control soil. Exposures to creosote contaminated soils with additional BaP (1000 mg/kg) or biotreatment did not yield statistically significant increases in DNA adducts compared to the pristine control. (Abstract only)

  18. Direct reduction of N-acetoxy-PhIP by tea polyphenols: a possible mechanism for chemoprevention against PhIP-DNA adduct formation

    International Nuclear Information System (INIS)

    Lin Dongxin; Thompson, Patricia A.; Teitel, Candee; Chen Junshi; Kadlubar, Fred F.

    2003-01-01

    The chemopreventive effect of tea against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adduct formation and its mechanism were studied. Rats were exposed to freshly prepared aqueous extracts of green tea (3% (w/v)) as the sole source of drinking water for 10 days prior to administration with a single dose of PhIP (10 mg/kg body weight) by oral gavage. PhIP-DNA adducts in the liver, colon, heart, and lung were measured using the 32 P-postlabelling technique. Rats pre-treated with tea and given PhIP 20 h before sacrifice had significantly reduced levels of PhIP-DNA adducts as compared with controls given PhIP alone. The possible mechanism of protective effect of tea on PhIP-DNA adduct formation was then examined in vitro. It was found that an aqueous extract of green and black tea, mixtures of green and black tea polyphenols, as well as purified polyphenols could strongly inhibit the DNA binding of N-acetoxy-PhIP, a putative ultimate carcinogen of PhIP formed in vivo via metabolic activation. Among these, epigallocatechin gallate was exceptionally potent. HPLC analyses of these incubation mixtures containing N-acetoxy-PhIP and the tea polyphenols each revealed the production of the parent amine, PhIP, indicating the involvement of a redox mechanism. In view of the presence of relatively high levels of tea polyphenols in rat and human plasma after ingestion of tea, this study suggests that direct reduction of the ultimate carcinogen N-acetoxy-PhIP by tea polyphenols is likely to be involved in the mechanism of chemoprotection of tea against this carcinogen

  19. Low-dose DNA adduct dosimetry by accelerator mass spectrometry (AMS)

    International Nuclear Information System (INIS)

    Turteltaub, K.W.; Felton, J.S.; Vogel, J.S.; Gledhill, B.L.; Davis, J.C.; Snyderwine, E.G.; Thorgeirsson, S.S.; Adamson, R.H.

    1991-01-01

    DNA adduction was measured following exposure to low doses of [2- 14 C]-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) [2- 14 C]-2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and [U- 14 C]-2,3,7,8-tetrachlorodibenzo-p-dioxin by AMS, a technique used in the earth sciences but not previously in toxicological research. The ability to measure low concentrations of rare isotopes suggested that biomedical research was a potentially powerful application for this technology. Sensitivity of the method was found to be one adduct per 10 11 nucleotides. No DNA adduct formation could be detected in TCDD treated rodents. DNA adducts in cynomolgus monkey lymphocytes following exposure to 500 μg/kg IQ peaked between 6 and 18 hrs following exposure. Sensitivity was limited mainly by the abundance of 14 C in contemporary carbon. Hosts depleted in radiocarbon are being developed, potentially increasing sensitivity another 3 orders of magnitude. These results demonstrate the high sensitivity of AMS for tracing molecules following administration of low levels of isotopically-labeled xenobiotics. In addition to 14 C measurement, AMS offers potential to conduct studies with other isotopes, particularly 3 H and 41 Ca

  20. Formation of cyclic 1,N2-propanodeoxyguanosine and thymidine adducts in the reaction of the mutagen 2-bromoacrolein with calf thymus DNA

    International Nuclear Information System (INIS)

    Meerman, J.H.; Smith, T.R.; Pearson, P.G.; Meier, G.P.; Nelson, S.D.

    1989-01-01

    The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-3H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N2-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N2-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules. Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz 1H and 75.43 MHz 13C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2-bromo-3-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [3H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct

  1. Determination of adducts of polycyclic aromatic hydrocarbons to DNA

    International Nuclear Information System (INIS)

    Bean, R.M.; Chess, E.K.; Thomas, B.L.; Mann, D.B.; Dankovic, D.A.; Franz, J.A.; Springer, D.L.

    1987-01-01

    Adducts to deoxyribonucleic acid (DNA), formed from metabolites of polynuclear aromatic compounds, are relatively persistent and correlate with bioresponse (carcinogenicity). Therefore, qualitative and quantitative analysis of adducts in the DNA of individuals may provide valuable information as to recent exposure to carcinogenic hydrocarbons. Further, the ability to detect adducts in a large segment of a population may have significant epidemiological significance. The current thrust of the analytical development at PNL is to isolate the DNA, liberate the adducted hydrocarbon residue from the DNA with acid hydrolysis, and prepare derivatives of the hydrolyzed species that will enhance its detection, quantitation, and characterization using gas chromatography/mass spectrometry (GC/MS). They have initiated the development of the necessary techniques using benzo[a]pyrene (B[a]P). Samples of DNA adducts of radiolabeled B[a]P have been prepared for study by reacting DNA isolated from calf thymus with benzo[a]pyrene-7,8-diol-9,10-epoxide (the ultimate carcinogenic form of B[a]P). Other DNA/B[a]P samples have been prepared by painting the skin of mice with radiolabeled B[a]P. The ability to prepare research quantities of adducts using the hepatocyte preparation method reported by Dankovic et al is a significant development to their DNA adduct analysis program

  2. The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells : comparison of immunocytochemical determination with detection in isolated DNA

    NARCIS (Netherlands)

    Blommaert, F.A.; Floot, B.G.J.; Dijk-Knijnenburg, H.C.M. van; Berends, F.; Baan, R.A.; Schornagel, J.H.; Engelse, L. den; Fichtinger-Schepman, A.M.J.

    1998-01-01

    We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively.

  3. In vitro study of DNA Adduct 8-OHdG Formation by using Bisphenol A in Calf Thymus DNA and 2’-Deoxyguanosine

    Science.gov (United States)

    Budiawan; Cahaya Dani, Intan; Bakri, Ridla; Handayani, Sri; Ratna Dewi, Evi

    2018-01-01

    The in vitro study of DNA Adduct 8-OHdG Formation due to BisphenolA (BPA) as xenobiotics has been conducted by using calf thymus DNA and 2’deoxyguanosine. The method of study was conducted by incubating calf thymus DNA and 2’dG with compounds trigger to radicals in the variation of pH (7.4 and 8.4), temperature (37°C and 60°C), and BPA concentrations (2 ppm and 10 ppm). To represent the work of CYP 450 enzyme in metabolic process of xenobiotics in the body and the effect of metal presence to the formation of radicals that can lead to 8-OHdG formation, we used iron(II) solution and also fenton reagent (Fe(II) and H2O2). The DNA used has 1.8 purity ratio (checked at λ260/λ280 by using Spectrophotometry UV-Vis). The results by using HPLC method showed that BPA could interact with DNA and DNA base (represent as calf thymus and 2’dG) and potentially induced 8-OHdG formation. The presence of iron(II) metal and Fenton reagent also induced the higher 8-OHdG formation. The higher of pH, temperature and concentrations also lead to 8-OHdG formation (ranger between 4 - 70 ppb).

  4. Genotoxic Pyrrolizidine Alkaloids — Mechanisms Leading to DNA Adduct Formation and Tumorigenicity

    OpenAIRE

    Ming W. Chou; Ge Lin; Qingsu Xia; Peter P. Fu

    2002-01-01

    Abstract: Plants that contain pyrrolizidine alkaloids are widely distributed in the world. Although pyrrolizidine alkaloids have been shown to be genotoxic and tumorigenic in experimental animals, the mechanisms of actions have not been fully understood. The results of our recent mechanistic studies suggest that pyrrolizidine alkaloids induce tumors via a genotoxic mechanism mediated by 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5Hpyrrolizine (DHP)-derived DNA adduct formation. This mechanism may ...

  5. Adduction of DNA with MTBE and TBA in mice studied by accelerator mass spectrometry.

    Science.gov (United States)

    Yuan, Y; Wang, H F; Sun, H F; Du, H F; Xu, L H; Liu, Y F; Ding, X F; Fu, D P; Liu, K X

    2007-12-01

    Methyl tert-butyl ether (MTBE) is a currently worldwide used octane enhancer substituting for lead alkyls and gasoline oxygenate. Our previous study using doubly (14)C-labeled MTBE [(CH(3))(3) (14)CO(14)CH(3)] has shown that MTBE binds DNA to form DNA adducts at low dose levels in mice. To elucidate the mechanism of the binding reaction, in this study, the DNA adducts with singly (14)C-labeled MTBE, which was synthesized from (14)C-methanol and tert-butyl alcohol (TBA), or (14)C-labeled TBA in mice have been measured by ultra sensitive accelerator mass spectrometry. The results show that the methyl group of MTBE and tert-butyl alcohol definitely form adducts with DNA in mouse liver, lung, and kidney. The methyl group of MTBE is the predominant binding part in liver, while the methyl group and the tert-butyl group give comparable contributions to the adduct formation in lung and kidney.

  6. A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes

    NARCIS (Netherlands)

    Paini, A.; Punt, A.; Viton, F.; Scholz, G.; Delatour, T.; Marin-Kuan, M.; Schilter, B.; Bladeren, van P.J.; Rietjens, I.

    2010-01-01

    Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed

  7. Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks.

    Science.gov (United States)

    Borm, Paul J A; Cakmak, Gonca; Jermann, Erich; Weishaupt, Christel; Kempers, Pascal; van Schooten, Frederik Jan; Oberdörster, Günter; Schins, Roel P F

    2005-06-01

    The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m(3)). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 microM) and a mixture of 16 PAHs (0.1 microM) were used. No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 microg/cm(2)) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely.

  8. Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks

    International Nuclear Information System (INIS)

    Borm, Paul J.A.; Cakmak, Gonca; Jermann, Erich; Weishaupt, Christel; Kempers, Pascal; Schooten, Frederik Jan van; Oberdoerster, Guenter; Schins, Roel P.F.

    2005-01-01

    Objective: The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. Methods: In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m 3 ). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 μM) and a mixture of 16 PAHs (0.1 μM) were used. Results: No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 μg/cm 2 ) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. Conclusion: The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely

  9. Effects of thiourea and ammonium bicarbonate on the formation and stability of bifunctional cisplatin-DNA adducts : consequences for the accurate quantification of adducts in (cellular) DNA

    NARCIS (Netherlands)

    Fichtinger-Schepman, A.M.J.; Dijk-Knijnenburg, H.C.M. van; Dijt, F.J.; Velde-Visser, S.D. van der; Berends, F.; Baan, R.A.

    1995-01-01

    Cisplatin reacts with DNA by forming mainly bifunctional adducts via reactive monofunctional intermediates. When freshly platinated DNA was postincubated with thiourea (10 mM, at 23 or 37°C) for periods of up to 24 h, followed by determination of mono- and diadducts, a rapid initial decrease was

  10. DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas. Potential use for genotoxicant biomonitoring of fresh water ecosystems.

    Science.gov (United States)

    Le Goff, J; Gallois, J; Pelhuet, L; Devier, M H; Budzinski, H; Pottier, D; André, V; Cachot, J

    2006-08-12

    The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 microg g(-1) dry weight) in comparison to individuals from the reference site (0.053 microg g(-1) dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10(8) nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 microg g(-1) dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 microg g(-1) dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10(8) nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable

  11. K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts.

    Science.gov (United States)

    Ziegel, Rebecca; Shallop, Anthony; Jones, Roger; Tretyakova, Natalia

    2003-04-01

    The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O(6)-methylguanine, N7-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O(6)-methylguanine and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O(6)-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras gene-derived double-stranded DNA sequence (5'-G(1)G(2)AG(3)CTG(4)G(5)TG(6)G(7)CG(8)TA G(9)G(10)C-3') was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for G-->A and G-->T base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.

  12. Evaluation of Interindividual Human Variation in Bioactivation and DNA Adduct Formation of Estragole in Liver Predicted by Physiologically Based Kinetic/Dynamic and Monte Carlo Modeling

    NARCIS (Netherlands)

    Punt, Ans; Paini, Alicia; Spenkelink, Bert; Scholz, Gabriele; Schilter, Benoit; Bladeren, Van Peter J.; Rietjens, Ivonne M.C.M.

    2016-01-01

    Estragole is a known hepatocarcinogen in rodents at high doses following metabolic conversion to the DNA-reactive metabolite 1′-sulfooxyestragole. The aim of the present study was to model possible levels of DNA adduct formation in (individual) humans upon exposure to estragole. This was done by

  13. Oxidative Stress Induced Lipid Peroxidation And DNA Adduct Formation In The Pathogenesis Of Multiple Myeloma And Lymphoma

    Directory of Open Access Journals (Sweden)

    Tandon, Ravi

    2013-02-01

    Full Text Available Objective: To access the oxidative stress status by quantification of byproducts generated during lipid peroxidation and DNA breakdown products generated during DNA damage in the blood serum of multiple myeloma and lymphoma patients.Material & Methods: Case control study comprised of 40 patients of multiple myeloma and 20 patients of lymphoma along with 20 age and sex-matched healthy subjects as controls. Levels of Malondialdehyde and 8-hydroxy-2-deoxy-Guanosine were measured to study the oxidative stress status in the study subjects.Results: The level of markers of DNA damage and lipid peroxidation were found to be raised significantly in the study subjects in comparison to healthy controls. The results indicate oxidative stress and DNA damage activity increase progressively with the progression of disease.Conclusion: Oxidative stress causes DNA damage and Lipid peroxidation which results in the formation of DNA adducts leading to mutations thereby indicate the role of oxidative stress in the pathogenesis of multiple myeloma and lymphoma.

  14. Formation of adduct of cerium (4) thenoyltrifluoroacetonate

    International Nuclear Information System (INIS)

    Anyfrieva, S.I.; Polyakova, G.V.; Snezhko, N.I.; Pechurova, N.I.; Martynenko, L.I.; Spitsyn, V.I.

    1981-01-01

    Adduct formation of thenoyltrifluoroacetonate of Ce(4) [Ce(TTFA) 4 ] with seven nitrogen- and oxygen-containing donor additional ligands is studied using the methods of IR-spectroscopy, derivatography, X-ray phase analysis. The presence of formation of Ce(TTFA) 4 adducts with phosphorus-containing additional ligands tributyl phosphate (TBP), trioctylphosphine oxide (TOPO), triphenylphosphine oxide (TPPO); α, α'-dipyridyl (Dipy) and o-phenanthroline (Phen) is established. The adduct Ce(TTFA) 4 stable to reduction is formed with Dipy, and in the case of Phen, TBP, TOPO, TPPO in the process of adduct formation the reduction of Ce(4) to Ce(3) takes place [ru

  15. Inhibition of nitrobenzene-induced DNA and hemoglobin adductions by dietary constituents

    Energy Technology Data Exchange (ETDEWEB)

    Li Hongli; Cheng Yan; Wang Haifang; Sun Hongfang; Liu Yuanfang E-mail: yliu@pku.edu.cn; Liu Kexin; Peng Shixiang

    2003-03-01

    Nitrobenzene (NB), a widely used industrial chemical, is a likely human carcinogen. Many dietary constituents can suppress the DNA-adduction, acting as the inhibitors of cancer. In this study, we investigated the inhibitory effects of vitamin C (VC), vitamin E (VE), tea polyphenols (TP), garlic squeeze, curcumin, and grapestone extract on NB-DNA and NB-hemoglobin (Hb) adductions in mice using an ultrasensitive method of accelerator mass spectrometry (AMS) with {sup 14}C-labelled nitrobenzene. All of these dietary constituents showed their inhibitory effects on DNA or Hb adduction. VC, VE, TP and grapestone extract could efficaciously inhibit the adductions by 33-50%, and all of these six agents could inhibit Hb adduction by 30-64%. We also investigated resveratrol, curcumin, VC and VE as inhibitors of NB-DNA adduction in vitro using liquid scintillation counting technique. These agents in the presence of NADPH and S9 components also pronouncedly blocked DNA adduction in a dose-dependent profile. Our study suggests that these seven constituents may interrupt the process of NB-induced chemical carcinogenesis.

  16. Pyrrolizidine alkaloid-derived DNA adducts as a common biological biomarker of pyrrolizidine alkaloid-induced tumorigenicity.

    Science.gov (United States)

    Xia, Qingsu; Zhao, Yuewei; Von Tungeln, Linda S; Doerge, Daniel R; Lin, Ge; Cai, Lining; Fu, Peter P

    2013-09-16

    pyrrolizidine alkaloid-induced liver tumor formation. To date, this is the first finding that a set of exogenous DNA adducts are commonly formed from a series of tumorigenic xenobiotics.

  17. Formation of cigarette smoke-induced DNA adducts in the rat lung and nasal mucosa

    International Nuclear Information System (INIS)

    Gupta, R.C.; Sopori, M.L.; Gairola, C.G.

    1989-01-01

    The formation of DNA adducts in the nasal, lung, and liver tissues of rats exposed daily to fresh smoke from a University of Kentucky reference cigarette (2R1) for up to 40 weeks was examined. The amount of smoke total particulate matter (TPM) inhaled and the blood carboxyhemoglobin (COHb) values averaged 5-5.5 mg smoke TPM/day/rat and 5.5%, respectively. The pulmonary AHH activity measured at the termination of each experiment showed an average increase of about two- to threefold in smoke-exposed groups. These observations suggested that animals effectively inhaled both gaseous and particulate phase constituents of cigarette smoke. DNAs from nasal, lung, and liver tissue were extracted and analyzed by an improved 32 P-postlabeling procedure. The data demonstrate the DNA-damaging potential of long term fresh cigarette smoke exposure and suggest the ability of the tissue to partially recover from such damage following cessation of the exposure

  18. Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system

    International Nuclear Information System (INIS)

    Brown, W.C.; Romano, L.J.

    1991-01-01

    The authors have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. They show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, they present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, they observe an increase in synthesis but to a lesser extent than observed on oligonucleotide-primed synthesis. Finally, they have examined DNA synthesis on duplex templates and show the B[a]P adducts inhibit synthesis by the T7 DNA polymerase and gene 4 protein to the same extent regardless of whether the adducts are positioned in the leading or lagging strand, while synthesis by the polymerase alone is inhibited only when the adducts are in the template strand

  19. The long persistence of pyrrolizidine alkaloid-derived DNA adducts in vivo: kinetic study following single and multiple exposures in male ICR mice.

    Science.gov (United States)

    Zhu, Lin; Xue, Junyi; Xia, Qingsu; Fu, Peter P; Lin, Ge

    2017-02-01

    Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and the most common poisonous plants affecting livestock, wildlife, and humans. Our previous studies demonstrated that PA-derived DNA adducts can potentially be a common biological biomarker of PA-induced liver tumor formation. In order to validate the use of these PA-derived DNA adducts as a biomarker, it is necessary to understand the basic kinetics of the PA-derived DNA adducts formed in vivo. In this study, we studied the dose-dependent response and kinetics of PA-derived DNA adduct formation and removal in male ICR mice orally administered with a single dose (40 mg/kg) or multiple doses (10 mg/kg/day) of retrorsine, a representative carcinogenic PA. In the single-dose exposure, the PA-derived DNA adducts exhibited dose-dependent linearity and persisted for up to 4 weeks. The removal of the adducts following a single-dose exposure to retrorsine was biphasic with half-lives of 9 h (t 1/2α ) and 301 h (~12.5 days, t 1/2β ). In the 8-week multiple exposure study, a marked accumulation of PA-derived DNA adducts without attaining a steady state was observed. The removal of adducts after the multiple exposure also demonstrated a biphasic pattern but with much extended half-lives of 176 h (~7.33 days, t 1/2α ) and 1736 h (~72.3 days, t 1/2β ). The lifetime of PA-derived DNA adducts was more than 8 weeks following the multiple-dose treatment. The significant persistence of PA-derived DNA adducts in vivo supports their role in serving as a biomarker of PA exposure.

  20. Alcohol, Aldehydes, Adducts and Airways

    Directory of Open Access Journals (Sweden)

    Muna Sapkota

    2015-11-01

    Full Text Available Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA adduct and hybrid malondialdehyde-acetaldehyde (MAA protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease.

  1. Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

    Science.gov (United States)

    Bist, Itti; Bhakta, Snehasis; Jiang, Di; Keyes, Tia E; Martin, Aaron; Forster, Robert J; Rusling, James F

    2017-11-21

    Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S N 2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu 2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10 6 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD 50 and Comet assay results.

  2. Ochratoxin A: In Utero Exposure in Mice Induces Adducts in Testicular DNA

    Directory of Open Access Journals (Sweden)

    Jamie E. Jennings-Gee

    2010-06-01

    Full Text Available Ochratoxin A (OTA is a nephrotoxin and carcinogen that is associated with Balkan endemic nephropathy and urinary tract tumors. OTA crosses the placenta and causes adducts in the liver and kidney DNA of newborns. Because the testis and kidney develop from the same embryonic tissue, we reasoned that OTA also may cause adducts transplacentally in the testis. We tested the hypothesis that acute exposure to OTA, via food and via exposure in utero, causes adducts in testicular DNA and that these lesions are identical to those that can be produced in the kidney and testis by the consumption of OTA. Adult mice received a single dose of OTA (from 0–1,056 µg/kg by gavage. Pregnant mice received a single i.p. injection of OTA (2.5 mg/kg at gestation day 17. DNA adducts were determined by 32P-postlabeling. Gavage-fed animals sacrificed after 48 hours accumulated OTA in kidney and testis and showed DNA adducts in kidney and testis. Some OTA metabolites isolated from the tissues were similar in both organs (kidney and testis. The litters of mice exposed prenatally to OTA showed no signs of overt toxicity. However, newborn and 1-month old males had DNA adducts in kidney and testis that were chromatographically similar to DNA adducts observed in the kidney and testis of gavage-fed adults. One adduct was identified previously as C8-dG-OTA adduct by LC MS/MS. No adducts were observed in males from dams not exposed to OTA. Our findings that in utero exposure to OTA causes adducts in the testicular DNA of male offspring support a possible role for OTA in testicular cancer.

  3. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Binkova, Blanka [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Chvatalova, Irena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Lnenickova, Zdena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Milcova, Alena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Tulupova, Elena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Farmer, Peter B. [Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Sram, Radim J. [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic)]. E-mail: sram@biomed.cas.cz

    2007-07-01

    Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by {sup 32}P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 {mu}g/m{sup 3}, PM2.5 27-38 {mu}g/m{sup 3}, c-PAHs 18-22 ng/m{sup 3}; personal exposure to c-PAHs: 9.7 ng/m{sup 3} versus 5.8 ng/m{sup 3} (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 {+-} 0.28 adducts/10{sup 8} nucleotides versus 0.82 {+-} 0.23 adducts/10{sup 8} nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 {+-} 0.036 adducts/10{sup 8} nucleotides versus 0.099 {+-} 0.035 adducts/10{sup 8} nucleotides, P = 0

  4. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

    International Nuclear Information System (INIS)

    Binkova, Blanka; Chvatalova, Irena; Lnenickova, Zdena; Milcova, Alena; Tulupova, Elena; Farmer, Peter B.; Sram, Radim J.

    2007-01-01

    Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32 P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 μg/m 3 , PM2.5 27-38 μg/m 3 , c-PAHs 18-22 ng/m 3 ; personal exposure to c-PAHs: 9.7 ng/m 3 versus 5.8 ng/m 3 (P 8 nucleotides versus 0.82 ± 0.23 adducts/10 8 nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10 8 nucleotides versus 0.099 ± 0.035 adducts/10 8 nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-'like' DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine

  5. Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo

    International Nuclear Information System (INIS)

    Dračínská, Helena; Bárta, František; Levová, Kateřina; Hudecová, Alena; Moserová, Michaela; Schmeiser, Heinz H.; Kopka, Klaus; Frei, Eva; Arlt, Volker M.; Stiborová, Marie

    2016-01-01

    Highlights: • Oxidation and reduction of aristolochic acid I (AAI) dictate its (geno)toxicity in vivo. • Cytochrome P450 (CYP) 1A1 and 1A2 are induced in rats treated with Sudan I and AAI. • Induced CYP1A enzyme activity resulted in decreased AAI-DNA adduct levels in vivo. • CYP1A1 and 1A2 mainly detoxify AAI and attenuate its genotoxicity in vivo. - Abstract: Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H:quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the 32 P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.

  6. Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines

    International Nuclear Information System (INIS)

    Bedford, P.; Fichtinger-Schepman, A.M.; Shellard, S.A.; Walker, M.C.; Masters, J.R.; Hill, B.T.

    1988-01-01

    The formation and removal of four platinum-DNA adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (RT112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 microM (5 micrograms/ml) cisplatin. RT112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinated DNA may account for their hypersensitivity to cisplatin

  7. Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences.

    Science.gov (United States)

    Matter, Brock; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2004-06-01

    G --> T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligation-mediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (+/-)-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2'-deoxynucleosides. Capillary HPLC-ESI(+)-MS/MS was used to establish the amounts of (-)-trans-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N(2)-BPDE-dG, 5.6 and 16.7% for

  8. Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo

    International Nuclear Information System (INIS)

    Allaben, W.T.; Weis, C.C.; Fullerton, N.F.; Beland, F.A.

    1983-01-01

    The rat mammary carcinogen, N-hydroxy-2-acetylaminofluorene (N-hydroxy-2-AAF), has been proposed to be metabolically activated by mammary cytosolic N,O-acetyltransferase to a DNA binding species. To test this hypothesis, adult female Sprague-Dawley derived CD rats were treated, i.p., with 4.0 mg/kg [ring- 3 H]N-hydroxy-2-AAF. After 4 h, 1, 3, 14, and 28 days, the animals were killed, the mammary epithelium DNA was isolated and the carcinogen-deoxyribonucleoside adducts present were analyzed by high pressure liquid chromatography. At each time, only one adduct was detected and it was chromatographically identical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The level of the adduct was maximal at 4 h (1.5 adducts/10(6) nucleotides) and then decreased, following first order kinetics with a t1/2 of 14.2 days. The detection of a single non-acetylated aminofluorene adduct is consistent with N,O-acyltransferase being involved in the metabolic activation of N-hydroxy-2-AAF in the rat mammary gland

  9. Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

    Science.gov (United States)

    Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

    2007-02-01

    We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

  10. Potential use of DNA adducts to detect mutagenic compounds in soil

    International Nuclear Information System (INIS)

    Hua Guoxiong; Lyons, Brett; Killham, Ken; Singleton, Ian

    2009-01-01

    In this study, three different soils with contrasting features, spiked with 300 mg benzo[a]pyrene (BaP)/kg dry soil, were incubated at 20 deg. C and 60% water holding capacity for 540 days. At different time points, BaP and DNA were extracted and quantified, and DNA adducts were quantified by 32 P-postlabelling. After 540 days incubation, 69.3, 81.6 and 83.2% of initial BaP added remained in Cruden Bay, Boyndie and Insch soils, respectively. Meanwhile, a significantly different amount of DNA-BaP adducts were found in the three soils exposed to BaP over time. The work demonstrates the concept that DNA adducts can be detected on DNA extracted from soil. Results suggest the technique is not able to directly reflect bioavailability of BaP transformation products. However, this new method provides a potential way to detect mutagenic compounds in contaminated soil and to assess the outcomes of soil remediation. - A novel DNA adduct assay may provide a potential technique to detect mutagenic compounds in contaminated soil

  11. Protein Recognition in Drug-Induced DNA Alkylation: When the Moonlight Protein GAPDH Meets S23906-1/DNA Minor Groove Adducts.

    Science.gov (United States)

    Savreux-Lenglet, Gaëlle; Depauw, Sabine; David-Cordonnier, Marie-Hélène

    2015-11-05

    DNA alkylating drugs have been used in clinics for more than seventy years. The diversity of their mechanism of action (major/minor groove; mono-/bis-alkylation; intra-/inter-strand crosslinks; DNA stabilization/destabilization, etc.) has undoubtedly major consequences on the cellular response to treatment. The aim of this review is to highlight the variety of established protein recognition of DNA adducts to then particularly focus on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) function in DNA adduct interaction with illustration using original experiments performed with S23906-1/DNA adduct. The introduction of this review is a state of the art of protein/DNA adducts recognition, depending on the major or minor groove orientation of the DNA bonding as well as on the molecular consequences in terms of double-stranded DNA maintenance. It reviews the implication of proteins from both DNA repair, transcription, replication and chromatin maintenance in selective DNA adduct recognition. The main section of the manuscript is focusing on the implication of the moonlighting protein GAPDH in DNA adduct recognition with the model of the peculiar DNA minor groove alkylating and destabilizing drug S23906-1. The mechanism of action of S23906-1 alkylating drug and the large variety of GAPDH cellular functions are presented prior to focus on GAPDH direct binding to S23906-1 adducts.

  12. Fast repair of oxidizing OH adducts of DNA by hydroxycinnamic acid derivatives. A pulse radiolytic study

    International Nuclear Information System (INIS)

    Yue Jiang; Lin Weizhen; Yao Side; Lin Nianyun; Zhu Dayuan

    1999-01-01

    Using pulse radiolytic techniques, it has been demonstrated that the interactions of oxidizing OH adducts of DNA (ssDNA and dsDNA), polyA and polyG with hydroxycinnamic acid derivatives proceed via an electron transfer process (k=5-30x10 8 dm 3 mol -1 s -1 ). In addition, the rates for fast repair of OH adducts of dAMP, polyA and DNA (ssDNA and dsDNA) are slower than the corresponding rates for the rest OH adducts of DNA constituents. The slower rates for repair of oxidizing OH adducts of dAMP may be the rate determining step during the interaction of hydroxycinnamic acid derivatives with OH adducts of DNA containing the varieties of OH adducts of DNA constituents

  13. Environmental air pollution and DNA adducts in Copenhagen bus drivers - effect of GSTM1 and NAT2 genotypes on adduct level

    DEFF Research Database (Denmark)

    Nielsen, Per Sabro; de Pater, Nettie; Okkels, Henrik

    1996-01-01

    The lymphocyte bulky PAH-DNA adduct levels have been studied in persons occupationally exposed to ambient air pollution. The exposure group consisted of 90 healthy, nonsmoking bus drivers from the Copenhagen area, divided into three exposure groups according to driving area, and 60 rural controls...... (smokers and non-smokers). PAH-DNA adducts were determined by 32P-postlabelling with the butanol enrichment procedure. The bus drivers answered a comprehensive questionnaire on passive smoking, residential area, diet and other potential confounding variables. A significantly higher adduct level...... was observed in bus drivers working in central Copenhagen (1.214 fmol/microg DNA, n = 49) compared with both those driving in the dormitory (median: 0.507 fmol/microg DNA, P = 0.046, n = 16) and suburban (median: 0.585 fmol/microg DNA, P = 0.041, n = 25) areas. All three groups had higher adduct levels than...

  14. Detection of carcinogen-DNA adducts by radioimmunoassay

    International Nuclear Information System (INIS)

    Poirier, M.C.; Yuspa, S.H.; Weinstein, I.B.; Blobstein, S.

    1977-01-01

    Covalent binding of carcinogen to nucleic acids is believed to be an essential component of the carcinogenic process, so it is desirable to have highly sensitive and specific methods for detecting such adducts in cells and tissues exposed to known and suspected carcinogens. A radioimmunoassay is here described capable of detecting nanogram amounts of DNA adducts resulting from the covalent binding of the carcinogen N-2-acetylaminofluorene and its activated N-acetoxy derivative. (author)

  15. DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles

    Science.gov (United States)

    AbstractContext: Biodiesel and biodiesel-blend fuels offer a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. Objectives: We compared the formation of covalent DNA adducts by the in vitro metabol...

  16. Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA

    Energy Technology Data Exchange (ETDEWEB)

    Hang, Bo; Rodriguez, Ben; Yang, Yanu; Guliaev, Anton B.; Chenna, Ahmed

    2010-06-14

    Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N(2)-(4-Hydroxyphenyl)-2'-deoxyguanosine (N(2)-4-HOPh-dG) is the principal adduct identified in vivo by (32)P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N(2)-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N(2)-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson-Crick hydrogen bonds are present within the N(2)-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N(2)-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.

  17. DNA Adducts aand Human Atherosclerotis Lesions

    Czech Academy of Sciences Publication Activity Database

    Strejc, Přemysl; Boubelík, O.; Stávková, Zdena; Chvátalová, Irena; Šrám, Radim

    2001-01-01

    Roč. 42, - (2001), s. 662 ISSN 0008-5472. [Annual Meeting of Proceedings /92./. 24.03.2001-28.03.2001, New Orleans] R&D Projects: GA MZd NM10 Keywords : DNA adducts * LDL cholesterol Subject RIV: DN - Health Impact of the Environment Quality

  18. The study of DNA adduct 8-hydroxy-2‧deoxyguanosine (8-OHdG) formation of butylated hydroxyanisole (BHA) and its metabolite ter-butyl hydroquinone (TBHQ) through in vitro reaction with Calf Thymus DNA and 2‧deoxyguanosine

    Science.gov (United States)

    Budiawan; Purwaningsih, S. S.; Cahaya, D. I.

    2017-04-01

    Butylated Hydroxyanisole (BHA) and its metabolite Tert-Butyl Hydroquinone (TBHQ) are synthetic antioxidants, commonly used as food and beverage preservatives. Although WHO declared their safety, the use of these preservatives are still controversial because some studies showed that BHA induced proliferative effects in animal testing and TBHQ is considered as carcinogenic and causes DNA cleavage. This study is aimed to analyze the interaction between Calf Thymus DNA with BHA and TBHQ which are mediated with Copper (II) Chloride. The result of the study in spectrophotometric showed there was bathochromic shift as much as 2-3 nm in DNA treated with TBHQ. The next analysis used HPLC method in stationary phase of ODS, mobile phase of 10mM Natrium Hydrogen Phosphate Buffer and Methanol (85 : 15) for DNA adduct formation, 8-Hydroxy-2-Deoxyguanosine (8-OHDG) as biomarker of risk cancer. The resultof the study showed the formation of DNA adduct 8-OHDG in the interaction between DNA and 20-500 ppm of TBHQ. The 8-OHdG formation was greatly increased by the higher concentration of TBHQ. The relative amount of 8 OHDG which formed was reached 946/105 deoxyguanosine in DNA bases. Confirmation test by LCMS/MS was characterized with the detection of mother ion peak (m/z 284); fragment ion peaks at m/z 167.9, and 139.9; at retention time 3.52 min. Meanwhile the interaction between DNA and 50-250 ppm BHA did not induce 8-OHDG.

  19. Linking the generation of DNA adducts to lung cancer.

    Science.gov (United States)

    Ceppi, Marcello; Munnia, Armelle; Cellai, Filippo; Bruzzone, Marco; Peluso, Marco E M

    2017-09-01

    Worldwide, lung cancer is the leading cause of cancer death. DNA adducts are considered a reliable biomarker that reflects carcinogen exposure to tobacco smoke, but the central question is what is the relationship of DNA adducts and cancer? Therefore, we investigated this relationship by a meta-analysis of twenty-two studies with bronchial adducts for a total of 1091 subjects, 887 lung cancer cases and 204 apparently healthy individuals with no evidence of lung cancer. Our study shows that these adducts are significantly associated to increase lung cancer risk. The value of Mean Ratio lung-cancer (MR) of bronchial adducts resulting from the random effects model was 2.64, 95% C.I. 2.00-3.50, in overall lung cancer cases as compared to controls. The significant difference, with lung cancer patients having significant higher levels of bronchial adducts than controls, persisted after stratification for smoking habits. The MR lung-cancer value between lung cancer patients and controls for smokers was 2.03, 95% C.I. 1.42-2.91, for ex-smokers 3.27, 95% C.I. 1.49-7.18, and for non-smokers was 3.81, 95% C.I. 1.85-7.85. Next, we found that the generation of bronchial adducts is significantly related to inhalation exposure to tobacco smoke carcinogens confirming its association with volatile carcinogens. The MR smoking estimate of bronchial adducts resulting from meta-regression was 2.28, 95% Confidence Interval (C.I.) 1.10-4.73, in overall smokers in respect to non-smokers. The present work provides strengthening of the hypothesis that bronchial adducts are not simply relate to exposure, but are a cause of chemical-induced lung cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

    Science.gov (United States)

    Zimmermann, Maike; Wang, Si-Si; Zhang, Hongyong; Lin, Tzu-Yin; Malfatti, Michael; Haack, Kurt; Ognibene, Ted; Yang, Hongyuan; Airhart, Susan; Turteltaub, Kenneth W; Cimino, George D; Tepper, Clifford G; Drakaki, Alexandra; Chamie, Karim; de Vere White, Ralph; Pan, Chong-Xian; Henderson, Paul T

    2017-02-01

    We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [ 14 C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [ 14 C]carboplatin or [ 14 C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Exposure to meat-derived carcinogens and bulky DNA adduct levels in normal-appearing colon mucosa.

    Science.gov (United States)

    Ho, Vikki; Brunetti, Vanessa; Peacock, Sarah; Massey, Thomas E; Godschalk, Roger W L; van Schooten, Frederik J; Ashbury, Janet E; Vanner, Stephen J; King, Will D

    2017-09-01

    Meat consumption is a risk factor for colorectal cancer. This research investigated the relationship between meat-derived carcinogen exposure and bulky DNA adduct levels, a biomarker of DNA damage, in colon mucosa. Least squares regression was used to examine the relationship between meat-derived carcinogen exposure (PhIP and meat mutagenicity) and bulky DNA adduct levels in normal-appearing colon tissue measured using 32 P-postlabelling among 202 patients undergoing a screening colonoscopy. Gene-diet interactions between carcinogen exposure and genetic factors relevant to biotransformation and DNA repair were also examined. Genotyping was conducting using the MassARRAY ® iPLEX ® Gold SNP Genotyping assay. PhIP and higher meat mutagenicity exposures were not associated with levels of bulky DNA adducts in colon mucosa. The XPC polymorphism (rs2228001) was found to associate with bulky DNA adduct levels, whereby genotypes conferring lower DNA repair activity were associated with higher DNA adduct levels than the normal activity genotype. Among individuals with genotypes associated with lower DNA repair (XPD, rs13181 and rs1799179) or detoxification activity (GSTP1, rs1695), higher PhIP or meat mutagenicity exposures were associated with higher DNA adduct levels. Significant interactions between the XPC polymorphism (rs2228000) and both dietary PhIP and meat mutagenicity on DNA adduct levels was observed, but associations were inconsistent with the a priori hypothesized direction of effect. Exposure to meat-derived carcinogens may be associated with increased DNA damage occurring directly in the colon among genetically susceptible individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Dose-dependent reduction of 3,2'-dimethyl-4-aminobiphenyl-derived DNA adducts in colon and liver of rats administered celecoxib

    International Nuclear Information System (INIS)

    Ravoori, Srivani; Feng Yi; Neale, Jason R.; Jeyabalan, Jeyaprakash; Srinivasan, Cidambi; Hein, David W.; Gupta, Ramesh C.

    2008-01-01

    Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100 mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated 32 P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N 2 -DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer

  3. Exposure of bus and taxi drivers to urban air pollutants as measured by DNA and protein adducts

    DEFF Research Database (Denmark)

    Hemminki, K.; Zhang, L.F.; Krüger, J.

    1994-01-01

    Urinary 1-hydroxypyrene, lymphocyte DNA adducts, serum protein-bound PAH and hemoglobin-bound alkene adducts were analysed from 4 groups of non-smoking men: urban and suburban bus drivers, taxi drivers and suburban controls. The only differences between the groups were in DNA adducts between...... suburban bus drivers and controls, and in DNA adduct and plasma protein PAH-adducts between taxi drivers and controls....

  4. Molecular dosimetry of DNA adducts in rainbow trout (Oncorhynchus mykiss) exposed to benzo(a)pyrene by different routes

    International Nuclear Information System (INIS)

    Potter, D.; Clarius, T.M.; Wright, A.S.; Watson, W.P.

    1994-01-01

    Farm raised rainbow trout (Oncorhynchus mykiss) were exposed by various routes to benzo(a)pyrene (BP) as a representative carcinogenic polycyclic aromatic hydrocarbon (PAH). Following exposure of fish to the chemical by intraperitoneal (i.p.) injection, 32 P-postlabelling studies indicated that non-feral trout were relatively resistant to the formation of BP-DNA adducts in liver. No adducts were detected in fish exposed to single doses (20 mg/kg) of BP. Multiple exposures (e.g. 2 x 25 mg/kg) were necessary in order for adducts to be detected, indicating that induction of the metabolising enzymes required for the bioactivation of BP is necessary. These studies provided reference information on DNA adducts for comparison with data from subsequent experiments at environmentally realistic low level exposures. Two types of low level aquatic exposure were carried out. The first procedure exposed fish for 30 days to a nominally constant low level (1.2 and 0.4 μg/l) of a homogeneous dispersion of BP in water, to simulate low level aquatic environmental exposures. Following 32 P-postlabelling analysis of the liver DNA of exposed fish, BP-DNA adducts were not detected. In the second procedure, fish were exposed to a constant low level of BP (ca. 0.5 μg/l) for 15 days then to a pulse (60 μg/l) which was allowed to naturally decline (to ca. 2 μg/l) during a further 15 days. Following this exposure, significant levels of BP-DNA adducts were detected in livers of trout. The effect of dietary exposures was investigated by feeding trout a diet containing either 58 μg or 288 μg BP per day for 6 days, equivalent to total doses of 43 mg/kg and 216 mg/kg. In both cases BP-DNA adducts were detected in livers of exposed fish. The results provide useful information on the types of exposures to PAHs which may pose a genotoxic risk to fish in the environment. (orig.)

  5. /sup 32/P-postlabelling analysis of aromatic DNA adducts in human oral mucosal cells

    Energy Technology Data Exchange (ETDEWEB)

    Dunn, B.P.; Stich, H.F.

    1986-07-01

    Exfoliated mucosal cells were collected from the oral cavity of three groups at high risk for oral cancer: Indian betel nut chewers, Filipino inverted smokers (burning end of cigar in mouth) and Indian Khaini tobacco chewers. DNA was extracted from these samples, as well as from samples of exfoliated cells of Canadian non-smoking controls. DNA was analyzed for the presence of aromatic DNA adducts using /sup 32/P-postlabelling analysis. Five chromatographically distinct adducts were found in samples from both the high risk groups and the nonsmoking controls. Individual adducts were detectable in approximately 30-95% of samples, depending on the adduct and population group. Estimated levels of specific adducts ranged from non-detectable (prevalence relative to normal nucleotides less than 1 X 10(-9)) to occasionally greater than 1 X 10(-7). No adducts were found in high risk groups which did not also appear in control subjects.

  6. Bulky DNA adducts in white blood cells: a pooled analysis of 3,600 subjects

    DEFF Research Database (Denmark)

    Ricceri, Fulvio; Godschalk, Roger W; Peluso, Marco

    2010-01-01

    Bulky DNA adducts are markers of exposure to genotoxic aromatic compounds, which reflect the ability of an individual to metabolically activate carcinogens and to repair DNA damage. Polycyclic aromatic hydrocarbons (PAHs) represent a major class of carcinogens that are capable of forming such add...... such adducts. Factors that have been reported to be related to DNA adduct levels include smoking, diet, body mass index (BMI), genetic polymorphisms, the season of collection of biologic material, and air pollutants....

  7. Comparative DNA adduct formation and induction of colonic aberrant crypt foci in mice exposed to 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and azoxymethane

    Science.gov (United States)

    Kim, Sangyub; Guo, Jingshu; O’Sullivan, M. Gerald; Gallaher, Daniel D.; Turesky, Robert J.

    2015-01-01

    Considerable evidence suggests that environmental factors, including diet and cigarette smoke, are involved in the pathogenesis of colon cancer. Carcinogenic nitroso compounds (NOC), such as N-nitrosodimethylamine (NDMA), are present in tobacco and processed red meat, and NOC have been implicated in colon cancer. Azoxymethane (AOM), commonly used for experimental colon carcinogenesis, is an isomer of NDMA, and it produces the same DNA adducts as does NDMA. Heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and high-temperature cooking of meats are also associated with an elevated risk of colon cancer. The most abundant carcinogenic HAA formed in tobacco smoke is 2-amino-9H-pyrido[2,3-b]indole (AαC), whereas 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is the most potent carcinogenic HAA formed during the cooking of meat and fish. However, the comparative tumor-initiating potential of AαC, MeIQ, and AOM is unknown. In this report, we evaluate the formation of DNA adducts as a measure of genotoxicity, and the induction of colonic aberrant crypt foci (ACF) and dysplastic ACF, as an early measure of carcinogenic potency of these compounds in the colon of male A/J mice. Both AαC and AOM induced a greater number of DNA adducts than MeIQ in the liver and colon. AOM induced a greater number of ACF and dysplastic ACF than either AαC or MeIQ. Conversely, based on adduct levels, MeIQ-DNA adducts were more potent than AαC- and AOM-DNA adducts at inducing ACF. Long-term feeding studies are required to relate levels of DNA adducts, induction of ACF, and colon cancer by these colon genotoxicants. PMID:26734915

  8. Dose-dependent reduction of 3,2'-dimethyl-4-aminobiphenyl-derived DNA adducts in colon and liver of rats administered celecoxib

    Energy Technology Data Exchange (ETDEWEB)

    Ravoori, Srivani [James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Feng Yi; Neale, Jason R. [James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Jeyabalan, Jeyaprakash [James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Srinivasan, Cidambi [Department of Statistics, University of Kentucky, Lexington, KY 40502 (United States); Hein, David W. [James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Gupta, Ramesh C. [James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202 (United States)], E-mail: rcgupta@louisville.edu

    2008-02-01

    Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100 mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated {sup 32}P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N{sup 2}-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.

  9. Recognition and repair of the CC-1065-(N3-Adenine)-DNA adduct by the UVRABC nuclease

    International Nuclear Information System (INIS)

    Tang, M.; Lee, C.S.; Doisy, R.; Ross, L.; Needham-VanDevanter, D.R.; Hurley, L.H.

    1988-01-01

    The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, the authors have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, they have found that UVRABC nuclease incises at the eight phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure

  10. The role of reactive oxygen species (ROS and cytochrome P-450 2E1 in the generation of carcinogenic etheno-DNA adducts

    Directory of Open Access Journals (Sweden)

    Kirsten Linhart

    2014-01-01

    Full Text Available Exocyclic etheno-DNA adducts are mutagenic and carcinogenic and are formed by the reaction of lipidperoxidation (LPO products such as 4-hydoxynonenal or malondialdehyde with DNA bases. LPO products are generated either via inflammation driven oxidative stress or via the induction of cytochrome P-450 2E1 (CYP2E1. In the liver CYP2E1 is induced by various compounds including free fatty acids, acetone and ethanol. Increased levels of CYP2E1 and thus, oxidative stress are observed in the liver of patients with non-alcoholic steatohepatitis (NASH as well as in the chronic alcoholic. In addition, chronic ethanol ingestion also increases CYP2E1 in the mucosa of the oesophagus and colon. In all these tissues CYP2E1 correlates significantly with the levels of carcinogenic etheno-DNA adducts. In contrast, in patients with non-alcoholic steatohepatitis (NASH hepatic etheno-DNA adducts do not correlate with CYP2E1 indicating that in NASH etheno-DNA adducts formation is predominately driven by inflammation rather than by CYP2E1 induction. Since etheno-DNA adducts are strong mutagens producing various types of base pair substitution mutations as well as other types of genetic damage, it is strongly believed that they are involved in ethanol mediated carcinogenesis primarily driven by the induction of CYP2E1.

  11. Effect of green tea catechins and hydrolyzable tannins on benzo[a]pyrene-induced DNA adducts and structure-activity relationship.

    Science.gov (United States)

    Cao, Pengxiao; Cai, Jian; Gupta, Ramesh C

    2010-04-19

    Green tea catechins and hydrolyzable tannins are gaining increasing attention as chemopreventive agents. However, their mechanism of action is poorly understood. We investigated the effects of four green tea catechins and two hydrolyzable tannins on microsome-induced benzo[a]pyrene (BP)-DNA adducts and the possible structure-activity relationship. BP (1 microM) was incubated with rat liver microsomes and DNA in the presence of the test compound (1-200 microM) or vehicle. The purified DNA was analyzed by (32)P-postlabeling. The inhibitory activity of the catechins was in the following descending order: epigallocatechin gallate (IC(50) = 16 microM) > epicatechin gallate (24 microM) > epigallocatechin (146 microM) > epicatechin (462 microM), suggesting a correlation between the number of adjacent aromatic hydroxyl groups in the molecular structure and their potencies. Tannic acid (IC(50) = 4 microM) and pentagalloglucose (IC(50) = 26 microM) elicited as much DNA adduct inhibitory activity as the catechins or higher presumably due to the presence of more functional hydroxyl groups. To determine if the activity of these compounds was due to direct interaction of phenolic groups with electrophilic metabolite(s) of BP, DNA was incubated with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) (0.5 microM) in the presence of test compounds (200 microM) or vehicle. Significant inhibition of DNA adduct formation was found (tannic acid > pentagalloglucose > epigallocatechin gallate > epicatechin gallate). This notion was confirmed by analysis of the reaction products of anti-BPDE with the catechins and pentagalloglucose by electrospray ionization mass spectrometry and liquid chromatography-mass spectrometry. In conclusion, our data demonstrate that green tea catechins and the hydrolyzable tannins are highly effective in inhibiting BP-DNA adduct formation at least, in part, due to direct interaction of adjacent hydroxyl groups in their structures and that the activity is

  12. Effect of Green Tea Catechins and Hydrolyzable Tannins on Benzo[a]pyrene-Induced DNA Adducts and Structure–Activity Relationship

    Science.gov (United States)

    Cao, Pengxiao; Cai, Jian; Gupta, Ramesh C.

    2016-01-01

    Green tea catechins and hydrolyzable tannins are gaining increasing attention as chemopreventive agents. However, their mechanism of action is poorly understood. We investigated the effects of four green tea catechins and two hydrolyzable tannins on microsome-induced benzo[a]pyrene (BP)–DNA adducts and the possible structure–activity relationship. BP (1 μM) was incubated with rat liver microsomes and DNA in the presence of the test compound (1–200 μM) or vehicle. The purified DNA was analyzed by 32P-postlabeling. The inhibitory activity of the catechins was in the following descending order: epigallocatechin gallate (IC50 = 16 μM) > epicatechin gallate (24 μM) > epigallocatechin (146 μM) > epicatechin (462 μM), suggesting a correlation between the number of adjacent aromatic hydroxyl groups in the molecular structure and their potencies. Tannic acid (IC50 = 4 μM) and pentagalloglucose (IC50 = 26 μM) elicited as much DNA adduct inhibitory activity as the catechins or higher presumably due to the presence of more functional hydroxyl groups. To determine if the activity of these compounds was due to direct interaction of phenolic groups with electrophilic metabolite(s) of BP, DNA was incubated with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) (0.5 μM) in the presence of test compounds (200 μM) or vehicle. Significant inhibition of DNA adduct formation was found (tannic acid > pentagalloglucose > epigallocatechin gallate > epicatechin gallate). This notion was confirmed by analysis of the reaction products of anti-BPDE with the catechins and pentagalloglucose by electrospray ionization mass spectrometry and liquid chromatography–mass spectrometry. In conclusion, our data demonstrate that green tea catechins and the hydrolyzable tannins are highly effective in inhibiting BP–DNA adduct formation at least, in part, due to direct interaction of adjacent hydroxyl groups in their structures and that the activity is higher with an increasing

  13. Effect of turmeric and curcumin on BP-DNA adducts.

    Science.gov (United States)

    Mukundan, M A; Chacko, M C; Annapurna, V V; Krishnaswamy, K

    1993-03-01

    Many human cancers that are widely prevalent today can be prevented through modifications in life-styles, of which diet appears to be an important agent. Several dietary constituents modulate the process of carcinogenesis and prevent genotoxicity. Many plant constituents including turmeric appear to be potent antimutagens and antioxidants. Therefore the modulatory effects of turmeric and curcumin on the levels of benzo[a]pyrene induced DNA adducts in the livers of rats were studied by the newly developed 32P-postlabelling assay method. Turmeric when fed at 0.1, 0.5 and 3% and the active principle of turmeric (curcumin) when fed at a level of 0.03% in the diet for 4 weeks significantly reduced the level of BP-DNA adducts including the major adduct dG-N2-BP, formed within 24 h in response to a single i.p. injection of benzo[a]pyrene. The significance of these effects in terms of the potential anticarcinogenic effects of turmeric is discussed. Further, these results strengthen the various other biological effects of turmeric which have direct relevance to anticarcinogenesis and chemoprevention.

  14. 32P-postlabeling analysis of dibenz[a,j]acridine DNA adducts in mice: preliminary determination of initial genotoxic metabolites and their effect on biomarker levels.

    Science.gov (United States)

    Roh, J; Schamer, M; Reilman, R; Xue, W; Warshawsky, D; Talaska, G

    1993-01-01

    N-Heterocyclic aromatics (NHA) are widely occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. NHA are found in significant amounts in tobacco condensates, synthetic fuels, gasoline engine exhaust, and effluents from the heating of coal. Dibenz[a,j]acridine (DBA) is an example of NHA. The potency of many carcinogenic compounds is related, at least in part, to the efficiency of their biological activation. We undertook studies to determine which initial metabolites of DBA lead to the formation of high levels of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD), trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD), and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were applied to the skin of mice. DNA was isolated using enzyme-solvent extraction method. DNA was 32P-postlabeled under conditions of limiting [32P]ATP. In skin, DBA produced two distinct adducts. The same two adducts were seen when DBA-3,4-DHD was applied. In addition the total adduct level elicited by DBA-3,4-DHD was higher than that of parent compound. Two adducts were seen when DBA-5,6DHD was applied, but these were very different from adducts seen with DBA. These results suggested that activation of DBA to DNA-binding compounds in skin includes initial formation of DBA-3,4-DHD. The data support development of biomarkers for the exposure and effect of this compound, and also suggest that specific metabolic susceptibility markers might be able to predict populations at increased risk.

  15. DNA bulky adducts in a Mediterranean population correlate with environmental ozone concentration, an indicator of photochemical smog.

    Science.gov (United States)

    Palli, Domenico; Saieva, Calogero; Grechi, Daniele; Masala, Giovanna; Zanna, Ines; Barbaro, Antongiulio; Decarli, Adriano; Munnia, Armelle; Peluso, Marco

    2004-03-01

    Ozone (O(3)), the major oxidant component in photochemical smog, mostly derives from photolysis of nitrogen dioxide. O(3) may have biologic effects directly and/or via free radicals reacting with other primary pollutants and has been reported to influence daily mortality and to increase lung cancer risk. Although DNA damage may be caused by ozone itself, only other photochemical reaction products (as oxidised polycyclic aromatic hydrocarbons) may form bulky DNA adducts, a reliable biomarker of genotoxic damage and cancer risk, showing a seasonal trend. In a large series consisting of 320 residents in the metropolitan area of Florence, Italy, enrolled in a prospective study for the period 1993-1998 (206 randomly sampled volunteers, 114 traffic-exposed workers), we investigated the correlation between individual levels of DNA bulky adducts and a cumulative O(3) exposure score. The average O(3) concentrations were calculated for different time windows (0-5 to 0-90 days) prior to blood drawing for each participant, based on daily measurements provided by the local monitoring system. Significant correlations between DNA adduct levels and O3 cumulative exposure scores in the last 2-8 weeks before enrollment emerged in never smokers. Correlations were highest in the subgroup of never smokers residing in the urban area and not occupationally exposed to vehicle traffic pollution, with peak values for average concentrations 4-6 weeks before enrollment (r = 0.34). Our current findings indicate that DNA adduct formation may be modulated by individual characteristics and by the cumulative exposure to environmental levels of ozone in the last 4-6 weeks, possibly through ozone-associated reactive pollutants. Copyright 2003 Wiley-Liss, Inc.

  16. Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts

    International Nuclear Information System (INIS)

    McCormick, J.J.; Maher, V.M.

    1985-01-01

    A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B 1 dichloride, a model for the reactive 2,3-epoxide of aflatoxin B 1 . Exponentially growing cells were exposed to agents and assayed for mutations and cell killing. Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation of normal and repair-deficient cells showed this also to be true for transformation

  17. Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.

    Science.gov (United States)

    Gamboa da Costa, Gonçalo; Singh, Rajinder; Arlt, Volker M; Mirza, Amin; Richards, Meirion; Takamura-Enya, Takeji; Schmeiser, Heinz H; Farmer, Peter B; Phillips, David H

    2009-11-01

    The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more

  18. Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

    2008-01-01

    The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1¿-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

  19. Full structure assignments of pyrrolizidine alkaloid DNA adducts and mechanism of tumor initiation.

    Science.gov (United States)

    Zhao, Yuewei; Xia, Qingsu; Gamboa da Costa, Gonçalo; Yu, Hongtao; Cai, Lining; Fu, Peter P

    2012-09-17

    Pyrrolizidine alkaloid-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids are among the first chemical carcinogens identified in plants. Previously, we determined that metabolism of pyrrolizidine alkaloids in vivo and in vitro generated a common set of DNA adducts that are responsible for tumor induction. Using LC-ESI/MS/MS analysis, we previously determined that four DNA adducts (DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4) were formed in rats dosed with riddelliine, a tumorigenic pyrrolizidine alkaloid. Because of the lack of an adequate amount of authentic standards, the structures of DHP-dA-3 and DHP-dA-4 were not elucidated, and the structural assignment for DHP-dG-4 warranted further validation. In this study, we developed an improved synthetic methodology for these DNA adducts, enabling their full structural elucidation by mass spectrometry and NMR spectroscopy. We determined that DHP-dA-3 and DHP-dA-4 are a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N(6)-yl) dehydrosupinidine, while DHP-dG-4 is 7-hydroxy-9-(deoxyguanosin-N(2)-yl)dehydrosupinidine, an epimer of DHP-dG-3. With the structures of these DNA adducts unequivocally elucidated, we conclude that cellular DNA preferentially binds dehydropyrrolizidine alkaloid, for example, dehydroriddelliine, at the C9 position of the necine base, rather than at the C7 position. We also determined that DHP-dA-3 and DHP-dA-4, as well as DHP-dG-3 and DHP-dG-4, are interconvertible. This study represents the first report with detailed structural assignments of the DNA adducts that are responsible for pyrrolizidine alkaloid tumor induction on the molecular level. A mechanism of tumor initiation by pyrrolizidine alkaloids is consequently fully determined.

  20. Adduct formation in Ce(IV) thenolytrifluoroacetonate

    International Nuclear Information System (INIS)

    Anufrieva, S.I.; Polyakova, G.V.; Snezhko, N.I.; Pechurova, N.I.; Martynenko, L.I.; Spitsyn, V.I.

    1982-01-01

    The literature contains no information on adduct formation in Ce(IV) β-diketonates with additional ligands. Since tetrakis-β-diketonates of Ce(IV) have four six-membered chelate rings, we can suppose that the introduction of an additional monodentate or bidentate ligand into the coordination sphere of Ce(IV) β-diketonates would lead to an increase in the coordination number (CN) of the Ce(IV) to nine or ten. The possibility of realization of such a high CN for Ce(IV) has not been proved; a study of adduct formation by Ce(IV) tetrakis-β-diketonates is thus of theoretical interest. Such an investigation might also be of practical interest, because the introduction of an additional ligand into the coordination sphere of a rare-earth β-diketonate usually increases the solubility of the β-diketonate in nonpolar solvents and increases the volatility of the compound; such a modification of the properties is important for various practical purposes. The aim of our work was to study the possibility of separating solid adducts of Ce(IV) tetrakis-thenoyltrifluoroacetonate with certain oxygen-containing and nitrogen-containing donor monodentate and bidentate ligands, and also to investigate their properties. As the β-diketone we used thenoyltrifluoroacetone (HTTFA), since in a parallel investigation it was found that Ce(TTFA) 4 has a high oxidation-reduction stability

  1. Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1′-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

    2008-01-01

    The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

  2. Complex relationships between occupation, environment, DNA adducts, genetic polymorphisms and bladder cancer in a case-control study using a structural equation modeling.

    Directory of Open Access Journals (Sweden)

    Stefano Porru

    Full Text Available DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs and polycyclic aromatic hydrocarbons (PAHs. No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028, whereas XRCC1 Arg 399 (p<0.006 was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001, coffee (p<0.001, cumulative AAs exposure (p = 0.041 and MnSOD (p = 0.009 and a decreased risk by MPO (p<0.008 were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different

  3. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome

    Science.gov (United States)

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo. PMID:26559182

  4. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.

    Science.gov (United States)

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.

  5. Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light

    International Nuclear Information System (INIS)

    Strniste, G.F.; Rall, S.C.

    1976-01-01

    Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; a Sepharose 4B column assay in which protein eluted coincident with DNA; and a CsCl density gradient equilibrium assay which showed both protein and DNA banding at densities other than their respective native densities. Treatment of the irradiated chromatin with DNase provided further evidence that protein--DNA and not protein-protein adducts were being observed in the column assay. There is a fluence-dependent response of protein-DNA adduct formation when the chromatin is irradiated at low ionic strength and is linear for protein over the range studied. When the chromatin is exposed to differing conditions of pH, ionic strength, or divalent metal ion concentration, the quantity of adduct formed upon uv irradiation varies. Susceptibility to adduct formation can be partially explained in terms of the condensation state of the chromatin and other factors such as rearrangement, denaturation, and dissociation of the chromatin components. Besides providing information on the biological significance of these types of uv-induced lesions, this technique may be useful as a probe of chromatin structure

  6. γIrradiation induced formation of PCB-solvent adducts in aliphatic solvents

    International Nuclear Information System (INIS)

    Lepine, F.; Milot, S.; Gagne, N.

    1990-01-01

    γIrradiation induced formation of PCB-solvent adducts was investigated as a model for PCB residues in irradiated food. Formation of cyclohexyl adducts of PCBs was found to be significant when pure PCB congeners and Aroclor mixture were irradiated in cyclohexane and cyclohexene. Reaction pathways were investigated, and the effects of oxygen and electron scavenger were studied

  7. Nuclear magnetic resonance at the picomole level of a DNA adduct.

    Science.gov (United States)

    Kautz, Roger; Wang, Poguang; Giese, Roger W

    2013-10-21

    We investigate the limit of detection for obtaining NMR data of a DNA adduct using modern microscale NMR instrumentation, once the adduct has been isolated at the picomole level. Eighty nanograms (130 pmol) of a DNA adduct standard, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene 5'-monophosphate (AAF-dGMP), in 1.5 μL of D₂O with 10% methanol-d₄, in a vial, was completely picked up as a droplet suspended in a fluorocarbon liquid and loaded efficiently into a microcoil probe. This work demonstrates a practical manual method of droplet microfluidic sample loading, previously demonstrated using automated equipment, which provides a severalfold advantage over conventional flow injection. Eliminating dilution during injection and confining the sample to the observed volume produce the full theoretical mass sensitivity of a microcoil, comparable to that of a microcryo probe. With 80 ng, an NMR spectrum acquired over 40 h showed all of the resonances seen in a standard spectrum of AAF-dGMP, with a signal-to-noise ratio of at least 10, despite broadening due to previously noted effects of conformational exchange. Even with this broadening to 5 Hz, a two-dimensional total correlation spectroscopy spectrum was acquired on 1.6 μg in 18 h. This work helps to define the utility of NMR in combination with other analytical methods for the structural characterization of a small amount of a DNA adduct.

  8. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    Science.gov (United States)

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. Copyright © 2015 Elsevier B

  9. Biomarkers for exposure to ambient air pollution - Comparison of carcinogen-DNA adduct levels with other exposure markers and markers for oxidative stress

    DEFF Research Database (Denmark)

    Autrup, Herman; Daneshvar, Bahram; Dragsted, Lars Ove

    1999-01-01

    Human exposure to genotoxic compounds present in ambient air has been studied using selected biomarkers in nonsmoking Danish bus drivers and postal workers. A large interindividual variation in biomarker levels was observed. Significantly higher levels of bulky carcinogen-DNA adducts (75.42 adducts...... correlations were observed between bulky carcinogen-DNA adduct and PAM-albumin levels (p = 0.005), and between DNA adduct and gamma-glutamyl semialdehyde (GGS) in hemoglobin (p = 0.11). Highly significant correlations were found between PAM-albumin adducts and AAS in plasma (r = 0.001) and GGS in hemoglobin (p...... in the combined group. A significant negative correlation was only observed between bulky carcinogen-DNA adducts and PAM-albumin adducts (p = 0.02) and between DNA adduct and urinary mutagenic activity (p = 0.02) in the GSTM1 null group, bur not in the workers who were homozygotes or heterozygotes for GSTM1. Our...

  10. Effect of Increased Water Intake on Urinary DNA Adduct Levels and Mutagenicity in Smokers: A Randomized Study

    Directory of Open Access Journals (Sweden)

    Inmaculada Buendia Jimenez

    2015-01-01

    Full Text Available The association between fluid intake and bladder cancer risk remains controversial. Very little is known about to which extent the amount of water intake influences the action of excreting toxics upon the urinary system. This proof of concept trial investigates the effect of water intake on mutagenesis in smokers, a high risk population for bladder cancer. Methods. Monocentric randomized controlled trial. Inclusion Criteria. Male subjects aged 2045–45 y/o, smokers, and small drinkers (24-hour urinary volume 700 mOsmol/kg. Outcomes. 4-ABP DNA adducts formation in exfoliated bladder cells in 24-hour urine collection and urinary mutagenicity in 24-hour urine. Test Group. Subjects consumed 1.5 L daily of the study product (EVIAN on top of their usual water intake for 50 days. Control Group. Subjects continued their usual lifestyle habits. Results. 65 subjects were randomized. Mean age was 30 y/o and mean cigarettes per day were 20. A slight decrease in adducts formation was observed between baseline and last visit but no statistically significant difference was demonstrated between the groups. Urinary mutagenicity significantly decreased. The study shows that increasing water intake decreases urinary mutagenicity. It is not confirmed by urinary adducts formation. Further research would be necessary.

  11. Detection of Pyrrolizidine Alkaloid DNA Adducts in Livers of Cattle Poisoned with Heliotropium europaeum.

    Science.gov (United States)

    Fu, Peter P; Xia, Qingsu; He, Xiaobo; Barel, Shimon; Edery, Nir; Beland, Frederick A; Shimshoni, Jakob A

    2017-03-20

    Pyrrolizidine alkaloids are among the most common poisonous plants affecting livestock, wildlife, and humans. Exposure of humans and livestock to toxic pyrrolizidine alkaloids through the intake of contaminated food and feed may result in poisoning, leading to devastating epidemics. During February 2014, 73 mixed breed female beef cows from the Galilee region of Israel were accidently fed pyrrolizidine alkaloid contaminated hay for 42 days, resulting in the sudden death of 24 cows over a period of 63 days. The remaining cows were slaughtered 2.5 months after the last ingestion of the contaminated hay. In this study, we report the histopathological analysis of the livers from five of the slaughtered cows and quantitation of pyrrolizidine alkaloid-derived DNA adducts from their livers and three livers of control cows fed with feed free of weeds producing pyrrolizidine alkaloids. Histopathological examination revealed that the five cows suffered from varying degrees of bile duct proliferation, fibrosis, and megalocytosis. Selected reaction monitoring HPLC-ES-MS/MS analysis indicated that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts were formed in all five livers. The livers from the three control cows did not have any liver damage nor any indication of DHP-DNA adduct formed. These results confirm that the toxicity observed in these cattle was caused by pyrrolizidine alkaloid poisoning and that pyrrolizidine alkaloid-derived DNA adducts could still be detected and quantified in the livers of the chronically poisoned cows 2.5 months after their last exposure to the contaminated feed, suggesting that DHP-derived DNA adducts can serve as biomarkers for pyrrolizidine alkaloid exposure and poisoning.

  12. Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by 1H and 15N NMR studies

    International Nuclear Information System (INIS)

    Lin, Chin Hsiung; Hurley, L.H.

    1990-01-01

    (+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. The [6- 15 N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1 H and 15 N NMR. One-dimensional NOE difference and two-dimensional NOESY 1 H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6- 15 N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15 N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. The authors conclude that the covalently modified adenine N6 of the (+)-CC-1065-12-mer duplex adduct is predominantly in the doubly protonated form, in which calculations predict that the C6-N6 bond is shortened and the positive charge is delocalized over the entire adenine molecule

  13. Mechanisms of the different DNA adduct forming potentials of the urban air pollutants 2-nitrobenzanthrone and carcinogenic 3-nitrobenzanthrone.

    Science.gov (United States)

    Stiborová, Marie; Martínek, Václav; Svobodová, Martina; Sístková, Jana; Dvorák, Zdenek; Ulrichová, Jitka; Simánek, Vilím; Frei, Eva; Schmeiser, Heinz H; Phillips, David H; Arlt, Volker M

    2010-07-19

    2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. We compared the efficiencies of human enzymatic systems [hepatic microsomes and cytosols, NAD(P)H:quinone oxidoreductase 1 (NQO1), xanthine oxidase, NADPH:cytochrome P450 reductase, N,O-acetyltransferases, and sulfotransferases] and human primary hepatocytes to activate 2-NBA and its isomer 3-NBA to species forming DNA adducts. In contrast to 3-NBA, 2-NBA was not metabolized at detectable levels by the tested human enzymatic systems and enzymes expressed in human hepatocytes, and no DNA adducts detectable by (32)P-postlabeling were generated by 2-NBA. Even NQO1, the most efficient human enzyme to bioactive 3-NBA, did not activate 2-NBA. Molecular docking of 2-NBA and 3-NBA to the active site of NQO1 showed similar binding affinities; however, the binding orientation of 2-NBA does not favor the reduction of the nitro group. This was in line with the inhibition of 3-NBA-DNA adduct formation by 2-NBA, indicating that 2-NBA can compete with 3-NBA for binding to NQO1, thereby decreasing the metabolic activation of 3-NBA. In addition, the predicted equilibrium conditions favor a 3 orders of magnitude higher dissociation of N-OH-3-ABA in comparison to N-OH-2-ABA. These findings explain the very different genotoxicity, mutagenicity, and DNA adduct forming potential of the two compounds. Collectively, our results suggest that 2-NBA possesses a relatively lower risk to humans than 3-NBA.

  14. Bulky carcinogen-DNA adducts and exposure to environmental and occupational sources of polycyclic aromatic hydrocarbons. Influence of susceptibility genotypes on adduct level

    International Nuclear Information System (INIS)

    Sabro Nielsen, P.

    1996-01-01

    PAH exposure, whether it is of occupational or environmental origin, is thought to result in an elevated risk of cancer especially in the lungs. DNA damage is considered an important step in the carcinogenic effect of PAH. Hence, methods that elucidate the steps in the carcinogenic process are important to understand the action of PAH. It may prove useful in the exposure assessment and in combination with classical epidemiological methods give better basis for risk estimation. The objective in this thesis was to evaluate the feasibility of the 32 P-postlabeling method to detect carcinogen-DNA adducts for assessing exposure to DNA damaging compounds in different occupationally and environmentally exposed groups. The studies included groups, that have an elevated cancer risk due to occupational exposure to PAH. Exposure levels were supposed to be relatively low according to reports on occupational and environmental air quality programs. Another aim was to evaluate the influence of polymorphisms in metabolizing enzyme genes on DNA adduct levels. A third objective was to establish some kind of baseline DNA adduct level for individuals with supposed low exposure, and compare it to the more exposed groups. A fourth aim in these studies was to examine if biomarkers of genotoxic exposure could be useful in epidemiological studies to identify groups at risk and thereby contribute with better exposure estimates in the study of PAH related cancer risk. (EG)

  15. Bulky carcinogen-DNA adducts and exposure to environmental and occupational sources of polycyclic aromatic hydrocarbons. Influence of susceptibility genotypes on adduct level

    Energy Technology Data Exchange (ETDEWEB)

    Sabro Nielsen, P

    1997-12-31

    PAH exposure, whether it is of occupational or environmental origin, is thought to result in an elevated risk of cancer especially in the lungs. DNA damage is considered an important step in the carcinogenic effect of PAH. Hence, methods that elucidate the steps in the carcinogenic process are important to understand the action of PAH. It may prove useful in the exposure assessment and in combination with classical epidemiological methods give better basis for risk estimation. The objective in this thesis was to evaluate the feasibility of the {sup 32}P-postlabeling method to detect carcinogen-DNA adducts for assessing exposure to DNA damaging compounds in different occupationally and environmentally exposed groups. The studies included groups, that have an elevated cancer risk due to occupational exposure to PAH. Exposure levels were supposed to be relatively low according to reports on occupational and environmental air quality programs. Another aim was to evaluate the influence of polymorphisms in metabolizing enzyme genes on DNA adduct levels. A third objective was to establish some kind of baseline DNA adduct level for individuals with supposed low exposure, and compare it to the more exposed groups. A fourth aim in these studies was to examine if biomarkers of genotoxic exposure could be useful in epidemiological studies to identify groups at risk and thereby contribute with better exposure estimates in the study of PAH related cancer risk. (EG).

  16. Bulky carcinogen-DNA adducts and exposure to environmental and occupational sources of polycyclic aromatic hydrocarbons. Influence of susceptibility genotypes on adduct level

    Energy Technology Data Exchange (ETDEWEB)

    Sabro Nielsen, P.

    1996-12-31

    PAH exposure, whether it is of occupational or environmental origin, is thought to result in an elevated risk of cancer especially in the lungs. DNA damage is considered an important step in the carcinogenic effect of PAH. Hence, methods that elucidate the steps in the carcinogenic process are important to understand the action of PAH. It may prove useful in the exposure assessment and in combination with classical epidemiological methods give better basis for risk estimation. The objective in this thesis was to evaluate the feasibility of the {sup 32}P-postlabeling method to detect carcinogen-DNA adducts for assessing exposure to DNA damaging compounds in different occupationally and environmentally exposed groups. The studies included groups, that have an elevated cancer risk due to occupational exposure to PAH. Exposure levels were supposed to be relatively low according to reports on occupational and environmental air quality programs. Another aim was to evaluate the influence of polymorphisms in metabolizing enzyme genes on DNA adduct levels. A third objective was to establish some kind of baseline DNA adduct level for individuals with supposed low exposure, and compare it to the more exposed groups. A fourth aim in these studies was to examine if biomarkers of genotoxic exposure could be useful in epidemiological studies to identify groups at risk and thereby contribute with better exposure estimates in the study of PAH related cancer risk. (EG).

  17. Effect of acetylator genotype on the levels of carcinogen-DNA adducts in inbred hamsters treated with 2-aminofluorene

    International Nuclear Information System (INIS)

    Flammang, T.J.; Yerokun, T.; Hein, D.W.; Talaska, G.; Kirlin, W.G.; Ogolla, F.; Ferguson, R.J.

    1990-01-01

    A genetic polymorphism in N-acetyltransferase has been described previously in humans and in animal models that is known to affect an individual's susceptibility to certain drug toxicities and diseases including bladder cancer. In hamsters, the polymorphism is known to regulate the conversion of carcinogenic 2-aminofluorene to its amide and of N-hydroxy-2-aminofluorene to a reactive electrophile that forms a covalently-bound adduct with DNA; an event thought to initiate the tumorigenic process. A single dose of 2-aminofluorene (60 mg/kg body wt., i.p) was administered to homozygous rapid- (rr) and homozygous slow-acetylator (ss) hamsters, and the levels of aminofluorene-DNA adducts in bladder and liver were evaluated by a 32 P-postlabeling assay. Only a non-acetylated aminofluorene-DNA adduct was detected in the DNA samples. In this study, no differences were detected between the levels of hepatic 2-aminofluorene-DNA adducts in males or females or between the rr or ss hamsters. In contrast, the levels of 2-amino-fluorene-adducts in bladder DNA were 5-fold higher in the male rr than in the ss hamsters, and were 2-fold higher in the male rr than in the female rr animals

  18. Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

    Science.gov (United States)

    Yu, Rui; Lai, Yongquan; Hartwell, Hadley J.; Moeller, Benjamin C.; Doyle-Eisele, Melanie; Kracko, Dean; Bodnar, Wanda M.; Starr, Thomas B.; Swenberg, James A.

    2015-01-01

    Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N2-hydroxymethyl-dG (N2-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N2-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [13CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N2-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency’s Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair. PMID:25904104

  19. Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.

    Science.gov (United States)

    Carmichael, P L; Sardar, S; Crooks, N; Neven, P; Van Hoof, I; Ugwumadu, A; Bourne, T; Tomas, E; Hellberg, P; Hewer, A J; Phillips, D H

    1999-02-01

    Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen.

  20. Tamoxifen Forms DNA Adducts In Human Colon After Administration Of A Single [14C]-Labeled Therapeutic Dose.

    Energy Technology Data Exchange (ETDEWEB)

    Brown, K; Tompkins, E M; Boocock, D J; Martin, E A; Farmer, P B; Turteltaub, K W; Ubick, E; Hemingway, D; Horner-Glister, E; White, I H

    2007-05-23

    Tamoxifen is widely prescribed for the treatment of breast cancer and is also licensed in the U.S. for the prevention of this disease. However, tamoxifen therapy is associated with an increased occurrence of endometrial cancer in women and there is also evidence that it may elevate the risk of colorectal cancer. The underlying mechanisms responsible for tamoxifen-induced carcinogenesis in women have not yet been elucidated but much interest has focussed on the role of DNA adduct formation. We investigated the propensity of tamoxifen to bind irreversibly to colorectal DNA when given to ten women as a single [{sup 14}C]-labeled therapeutic (20 mg) dose, {approx}18 h prior to undergoing colon resections. Using the sensitive technique of accelerator mass spectrometry, coupled with HPLC separation of enzymatically digested DNA, a peak corresponding to authentic dG-N{sup 2}-tamoxifen adduct was detected in samples from three patients, at levels ranging from 1-7 adducts/10{sup 9} nucleotides. No [{sup 14}C]-radiolabel associated with tamoxifen or its major metabolites was detected. The presence of detectable CYP3A4 protein in all colon samples suggests this tissue has the potential to activate tamoxifen to {alpha}-hydroxytamoxifen, in addition to that occurring in the systemic circulation, and direct interaction of this metabolite with DNA could account for the binding observed. Although the level of tamoxifeninduced damage displayed a degree of inter-individual variability, when present it was {approx}10-100 times higher than that reported for other suspect human colon carcinogens such as PhIP. These findings provide a mechanistic basis through which tamoxifen could increase the incidence of colon cancers in women.

  1. 32P-postlabeling analysis of DNA adducts in liver of wild English sole (Parophrys vetulus) and winter flounder (Pseudopleuronectes americanus)

    International Nuclear Information System (INIS)

    Varanasi, U.; Reichert, W.L.; Stein, J.E.

    1989-01-01

    The 1-butanol adduct enhancement version of the 32P-postlabeling assay was used to measure the levels of hepatic DNA adducts in the marine flatfish, English sole (Parophrys vetulus), sampled from the Duwamish Waterway and Eagle Harbor, Puget Sound, WA, where they are exposed to high concentrations of sediment-associated chemical contaminants and exhibit an elevated prevalence of hepatic neoplasms. Hepatic DNA was also analyzed from English sole from a reference area (Useless Bay, WA) and from reference English sole treated with organic-solvent extracts of sediments from the two contaminated sites. Autoradiograms of thin-layer chromatograms of 32P-labeled hepatic DNA digests from English sole from the contaminated sites exhibited up to three diagonal radioactive zones, which were not present in autoradiograms of thin-layer chromatogram maps of 32P-labeled DNA digests from English sole from the reference site. These diagonal radioactive zones contained several distinct spots as well as what appeared to be multiple overlapping adduct spots. The levels (nmol of adducts/mol of nucleotides) of total DNA adducts for English sole from Duwamish Waterway and Eagle Harbor were 26 +/- 28 (DS) and 17 +/- 9.6, respectively. All autoradiograms of DNA from fish from the contaminated sites exhibited a diagonal radioactive zone where DNA adducts of chrysene, benzo(a)pyrene, and dibenz(a,h)anthracene, formed in vitro using English sole hepatic microsomes, were shown to chromatograph. English sole treated with extracts of the contaminated sediments had adduct profiles generally similar to those for English sole from the respective contaminated sites

  2. Whole body exposure of mice to secondhand smoke induces dose-dependent and persistent promutagenic DNA adducts in the lung

    International Nuclear Information System (INIS)

    Kim, Sang-In; Arlt, Volker M.; Yoon, Jae-In; Cole, Kathleen J.; Pfeifer, Gerd P.; Phillips, David H.; Besaratinia, Ahmad

    2011-01-01

    Secondhand smoke (SHS) exposure is a known risk factor for lung cancer in lifelong nonsmokers. However, the underlying mechanism of action of SHS in lung carcinogenesis remains elusive. We have investigated, using the 32 P-postlabeling assay, the genotoxic potential of SHS in vivo by determining the formation and kinetics of repair of DNA adducts in the lungs of mice exposed whole body to SHS for 2 or 4 months (5 h/day, 5 days/week), and an ensuing one-month recovery period. We demonstrate that exposure of mice to SHS elicits a significant genotoxic response as reflected by the elevation of DNA adduct levels in the lungs of SHS-exposed animals. The increases in DNA adduct levels in the lungs of SHS-exposed mice are dose-dependent as they are related to the intensity and duration of SHS exposure. After one month of recovery in clean air, the levels of lung DNA adducts in the mice exposed for 4 months remain significantly higher than those in the mice exposed for 2 months (P < 0.0005), levels in both groups being significantly elevated relative to controls (P < 0.00001). Our experimental findings accord with the epidemiological data showing that exposure to smoke-derived carcinogens is a risk factor for lung cancer; not only does the magnitude of risk depend upon carcinogen dose, but it also becomes more irreversible with prolonged exposure. The confirmation of epidemiologic data by our experimental findings is of significance because it strengthens the case for the etiologic involvement of SHS in nonsmokers' lung cancer. Identifying the etiologic factors involved in the pathogenesis of lung cancer can help define future strategies for prevention, early detection, and treatment of this highly lethal malignancy.

  3. Fat Content and Nitrite-Curing Influence the Formation of Oxidation Products and NOC-Specific DNA Adducts during In Vitro Digestion of Meat

    Science.gov (United States)

    Van Hecke, Thomas; Vossen, Els; Vanden Bussche, Julie; Raes, Katleen; Vanhaecke, Lynn; De Smet, Stefaan

    2014-01-01

    The effects of fat content and nitrite-curing of pork were investigated on the formation of cytotoxic and genotoxic lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal, volatile simple aldehydes), protein oxidation products (protein carbonyl compounds) and NOC-specific DNA adducts (O6-carboxy-methylguanine) during in vitro digestion. The formation of these products during digestion is suggested to be responsible for the association between red meat and processed meat consumption and colorectal cancer risk. Digestion of uncured pork to which fat was added (total fat content 5 or 20%), resulted in significantly higher lipid and protein oxidation in the mimicked duodenal and colonic fluids, compared to digestion of pork without added fat (1% fat). A higher fat content also significantly favored the formation of O6-carboxy-methylguanine in the colon. Nitrite-curing of meat resulted in significantly lower lipid and protein oxidation before and after digestion, while an inconsistent effect on the formation of O6-carboxy-methylguanine was observed. The presented results demonstrate that haem-Fe is not solely responsible for oxidation and nitrosation reactions throughout an in vitro digestion approach but its effect is promoted by a higher fat content in meat. PMID:24978825

  4. Fat content and nitrite-curing influence the formation of oxidation products and NOC-specific DNA adducts during in vitro digestion of meat.

    Science.gov (United States)

    Van Hecke, Thomas; Vossen, Els; Vanden Bussche, Julie; Raes, Katleen; Vanhaecke, Lynn; De Smet, Stefaan

    2014-01-01

    The effects of fat content and nitrite-curing of pork were investigated on the formation of cytotoxic and genotoxic lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal, volatile simple aldehydes), protein oxidation products (protein carbonyl compounds) and NOC-specific DNA adducts (O6-carboxy-methylguanine) during in vitro digestion. The formation of these products during digestion is suggested to be responsible for the association between red meat and processed meat consumption and colorectal cancer risk. Digestion of uncured pork to which fat was added (total fat content 5 or 20%), resulted in significantly higher lipid and protein oxidation in the mimicked duodenal and colonic fluids, compared to digestion of pork without added fat (1% fat). A higher fat content also significantly favored the formation of O6-carboxy-methylguanine in the colon. Nitrite-curing of meat resulted in significantly lower lipid and protein oxidation before and after digestion, while an inconsistent effect on the formation of O6-carboxy-methylguanine was observed. The presented results demonstrate that haem-Fe is not solely responsible for oxidation and nitrosation reactions throughout an in vitro digestion approach but its effect is promoted by a higher fat content in meat.

  5. Fat content and nitrite-curing influence the formation of oxidation products and NOC-specific DNA adducts during in vitro digestion of meat.

    Directory of Open Access Journals (Sweden)

    Thomas Van Hecke

    Full Text Available The effects of fat content and nitrite-curing of pork were investigated on the formation of cytotoxic and genotoxic lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal, volatile simple aldehydes, protein oxidation products (protein carbonyl compounds and NOC-specific DNA adducts (O6-carboxy-methylguanine during in vitro digestion. The formation of these products during digestion is suggested to be responsible for the association between red meat and processed meat consumption and colorectal cancer risk. Digestion of uncured pork to which fat was added (total fat content 5 or 20%, resulted in significantly higher lipid and protein oxidation in the mimicked duodenal and colonic fluids, compared to digestion of pork without added fat (1% fat. A higher fat content also significantly favored the formation of O6-carboxy-methylguanine in the colon. Nitrite-curing of meat resulted in significantly lower lipid and protein oxidation before and after digestion, while an inconsistent effect on the formation of O6-carboxy-methylguanine was observed. The presented results demonstrate that haem-Fe is not solely responsible for oxidation and nitrosation reactions throughout an in vitro digestion approach but its effect is promoted by a higher fat content in meat.

  6. DNA adducts of 2,3-epoxy-4-hydroxynonanal: detection of 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N6-ethenoadenine by gas chromatography/negative ion chemical ionization/mass spectrometry.

    Science.gov (United States)

    Chen, H J; Zhang, L; Cox, J; Cunningham, J A; Chung, F L

    1998-12-01

    2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids. EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EH in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1, N6-ethenoadenine (epsilonAde) and 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine (DHH-epsilonAde) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [15N5]epsilonAde and [15N5]DHH-epsilonAde to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE), (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilonAde and PFB-DHH-epsilonAde on a Si SPE column, (6) acetonide (ACT) formation of PFB-DHH-epsilonAde, and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilonAde monitoring of the (M - PFB)- ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilonAde monitoring of the (M - PFB)- ion at m/z 328 was 0.4 fmol; the detection limits for the entire assay were 6.3 fmol for epsilonAde and 36 fmol for DHH-epsilonAde. In calf thymus DNA modified with EH at 37 degreesC for 50 h, both epsilonAde and DHH-epsilonAde were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis, indicating that EH extensively reacts

  7. [Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust].

    Science.gov (United States)

    Shen, M L; He, Z N; Zhang, X; Duan, H W; Niu, Y; Bin, P; Ye, M; Meng, T; Dai, Y F; Yu, S F; Chen, W; Zheng, Y X

    2017-06-06

    Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median ( P (25)- P (75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group ( P 0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95 %CI ) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95 %CI ) was -0.094 (-0.179--0.008), P= 0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A

  8. Lifestyle, Environmental, and Genetic Predictors of Bulky DNA Adducts in a Study Population Nested within a Prospective Danish Cohort

    DEFF Research Database (Denmark)

    Eriksen, K. T.; Sørensen, M.; Autrup, H.

    2010-01-01

    Danish cohort. At enrollment, blood samples were collected and information on lifestyle, including dietary and smoking habits, obtained. Previously, bulky DNA adducts were measured in 245 individuals who developed lung cancer and 255 control members of the cohort. Of these 500 individuals, data on 375...... of bulky DNA adduct levels were analyzed by univariate and multivariate regression analyses. Women tended to have higher adduct levels than men. Living in central Copenhagen and surface darkness of fried meat and fish were associated with quantitative higher adduct levels. No significant associations were...

  9. Maternal diet and dioxin-like activity, bulky DNA adducts and micronuclei in mother–newborns

    International Nuclear Information System (INIS)

    Pedersen, Marie; Halldorsson, Thorhallur I.; Autrup, Herman; Brouwer, Abraham; Besselink, Harrie; Loft, Steffen; Knudsen, Lisbeth E.

    2012-01-01

    Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006–2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet was estimated on the basis of maternal food frequency questionnaire (FFQ) completed by the end of pregnancy. Biomarkers were detected in paired blood samples through the dioxin-responsive chemical-activated luciferase expression (CALUX) ® bioassay, 32 P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal (β 95%CI; 0.46 (0.08, 0.84)) and cord blood (β 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size.

  10. Maternal diet and dioxin-like activity, bulky DNA adducts and micronuclei in mother-newborns

    Energy Technology Data Exchange (ETDEWEB)

    Pedersen, Marie, E-mail: mpedersen@creal.cat [Section of Environmental Health, Department of Public Health, University of Copenhagen, CSS, Oester Farimagsgade, Copenhagen K (Denmark); Halldorsson, Thorhallur I., E-mail: lur@ssi.dk [Faculty of Food Science and Nutrition, School of Health Sciences, University of Iceland Reykjavik (Iceland); Center for Fetal Programming, Department of Epidemiology, Statens Serum Institute, Copenhagen (Denmark); Autrup, Herman, E-mail: ha@mil.au.dk [School of Public Health, Department of Environmental and Occupational Medicine, Aarhus University, Aarhus (Denmark); Brouwer, Abraham, E-mail: Bram.Brouwer@bds.nl [BioDetection Systems B.V., Amsterdam (Netherlands); Besselink, Harrie, E-mail: Harrie.Besselink@bds.nl [BioDetection Systems B.V., Amsterdam (Netherlands); Loft, Steffen, E-mail: stl@sund.ku.dk [Section of Environmental Health, Department of Public Health, University of Copenhagen, CSS, Oester Farimagsgade, Copenhagen K (Denmark); Knudsen, Lisbeth E., E-mail: liek@sund.ku.dk [Section of Environmental Health, Department of Public Health, University of Copenhagen, CSS, Oester Farimagsgade, Copenhagen K (Denmark)

    2012-06-01

    Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006-2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet was estimated on the basis of maternal food frequency questionnaire (FFQ) completed by the end of pregnancy. Biomarkers were detected in paired blood samples through the dioxin-responsive chemical-activated luciferase expression (CALUX){sup Registered-Sign} bioassay, {sup 32}P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal ({beta} 95%CI; 0.46 (0.08, 0.84)) and cord blood ({beta} 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size.

  11. Formation of formaldehyde adducts in the reactions of DNA and deoxyribonucleosides with alpha-acetates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-nitrosodimethylamine (NDMA).

    Science.gov (United States)

    Cheng, Guang; Wang, Mingyao; Upadhyaya, Pramod; Villalta, Peter W; Hecht, Stephen S

    2008-03-01

    The cytochrome P450-mediated alpha-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) as stable precursors to the alpha-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These alpha-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 degrees C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatography-electrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4-hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl- N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde.

  12. Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage.

    Science.gov (United States)

    Yu, Rui; Lai, Yongquan; Hartwell, Hadley J; Moeller, Benjamin C; Doyle-Eisele, Melanie; Kracko, Dean; Bodnar, Wanda M; Starr, Thomas B; Swenberg, James A

    2015-07-01

    Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N(2-)hydroxymethyl-dG (N(2)-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N(2)-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [(13)CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N(2)-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair. © The Author 2015

  13. Serum Level of Antibody against Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA Adducts in People Dermally Exposed to PAHs

    Directory of Open Access Journals (Sweden)

    Lenka Borska

    2014-01-01

    Full Text Available Some specific antibodies indicate the presence of antigenic structures on DNA (DNA adducts that can play an important role in the process of mutagenesis and/or carcinogenesis. They indicate the presence of increased genotoxic potential (hazard prior to the formation of disease (primary prevention. The present study was focused on the serum level of benzo[a]pyrene 7,8-diol-9,10-epoxide-DNA adducts antibodies (anti-BPDE-DNA in psoriatic patients (n=55 dermally exposed to different levels of polycyclic aromatic hydrocarbons (PAHs. The general goal of the study was to contribute to better understanding of the value of the assumed biomarker (anti-BPDE-DNA for evaluation of the organism's answer to genotoxic exposure to PAHs. Elevated level of exposure to PAHs resulted in the increased level of anti-BPDE-DNA. However, almost all levels of anti-BPDE-DNA ranged within the field of low values. Both variants of GT (CCT-3% and CCT-5% induced higher expression of anti-BPDE-DNA in the group of nonsmokers. Significant relations between the level of anti-BPDE-DNA and PASI score, total duration of the therapy, or time of UVR exposure were not found. Further studies are needed to reduce interpretation uncertainty of this promising bioindicator.

  14. A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP.

    Science.gov (United States)

    Kafle, Amol; Klaene, Joshua; Hall, Adam B; Glick, James; Coy, Stephen L; Vouros, Paul

    2013-07-15

    There is continued interest in exploring new analytical technologies for the detection and quantitation of DNA adducts, biomarkers which provide direct evidence of exposure and genetic damage in cells. With the goal of reducing clean-up steps and improving sample throughput, a Differential Mobility Spectrometry/Mass Spectrometry (DMS/MS) platform has been introduced for adduct analysis. A DMS/MS platform has been utilized for the analysis of dG-ABP, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP). After optimization of the DMS parameters, each sample was analyzed in just 30 s following a simple protein precipitation step of the digested DNA. A detection limit of one modification in 10^6 nucleosides has been achieved using only 2 µg of DNA. A brief comparison (quantitative and qualitative) with liquid chromatography/mass spectrometry is also presented highlighting the advantages of using the DMS/MS method as a high-throughput platform. The data presented demonstrate the successful application of a DMS/MS/MS platform for the rapid quantitation of DNA adducts using, as a model analyte, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene

    Energy Technology Data Exchange (ETDEWEB)

    DeMarini, David M., E-mail: demarini.david@epa.gov [Integrated Systems Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Hanley, Nancy M.; Warren, Sarah H.; Adams, Linda D.; King, Leon C. [Integrated Systems Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States)

    2011-09-01

    Highlights: {yields} Benzo[a]pyrene (BP) induces stable DNA adducts and mutations primarily at guanine. {yields} Dibenzo[a,l]pyrene (DBP) induces them primarily at adenine. {yields} BP induces abasic sites, but DBP does not in the Salmonella mutagenicity assay. {yields} Stable DNA adducts alone appear to account for the mutation spectrum of DBP. {yields} Stable DNA adducts and possibly abasic sites account for the mutation spectrum of BP. - Abstract: Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts ({sup 32}P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine

  16. Quantitative strategies to determine cisplatin adducts with DNA nucleotides in drosofila larvae and tumoral cell cultures

    International Nuclear Information System (INIS)

    Garcia Sar, D.; Montes-Bayon, M.; Hann, S.; Koellensperger, G.; Blanco-Gonzalez, E.; Sanz-Medel, A.

    2009-01-01

    Full text: The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. A novel sensitive and selective method is proposed to quantify cisplatin-DNA adducts induced in vivo in somatic cells of Drosophila melanogaster at biologically relevant concentrations. The method uses HPLC-ICPMS in combination with species-specific isotope dilution analysis (cisplatin enriched in 194 Pt). For the first time, a cisplatin-DNA adduct is quantified by this approach. The obtained results show the great potential of this system to advance our molecular understanding of the biological effects of cisplatin. (author)

  17. Bulky DNA adducts in white blood cells: a pooled analysis of 3,600 subjects.

    NARCIS (Netherlands)

    Ricceri, F.; Godschalk, R.W.; Peluso, M.; Phillips, D.H.; Agudo, A; Georgiadis, P.; Loft, S.; Tjonneland, A.; Raaschou-Nielsen, O.; Palli, D.; Perera, F.; Vermeulen, R.; Taioli, E.; Sram, R.J.; Munnia, A.; Rosa, F.; Allione, A.; Matullo, G.; Vineis, P.

    2010-01-01

    BACKGROUND: Bulky DNA adducts are markers of exposure to genotoxic aromatic compounds, which reflect the ability of an individual to metabolically activate carcinogens and to repair DNA damage. Polycyclic aromatic hydrocarbons (PAHs) represent a major class of carcinogens that are capable of forming

  18. Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-family DNA Polymerase

    Science.gov (United States)

    Gadkari, Varun V.; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2014-01-01

    3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dGC8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dGC8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dGC8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dGC8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dGC8-N-ABA bypass catalyzed by Dpo4. PMID:25048879

  19. Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate

    International Nuclear Information System (INIS)

    Chen, Kun-Ming; Sacks, Peter G.; Spratt, Thomas E.; Lin, Jyh-Ming; Boyiri, Telih; Schwartz, Joel; Richie, John P.; Calcagnotto, Ana; Das, Arunangshu; Bortner, James; Zhao, Zonglin; Amin, Shantu; Guttenplan, Joseph; El-Bayoumy, Karam

    2009-01-01

    Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

  20. Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells.

    Science.gov (United States)

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Hu, Yu; Chen, Wei-sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A; Tang, Moon-shong

    2014-06-15

    Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.

  1. Genotoxicity induced by xenobiotics:the role of DNA adducts, individual susceptibility and DNA repair

    Czech Academy of Sciences Publication Activity Database

    Vodička, Pavel; Koskinen, M.; Štětina, R.; Vodičková, L.; Kuricová, M.; Hemminki, K.

    2002-01-01

    Roč. 10, č. 1 (2002), s. 322 ISSN 1107-3756. [World Congress on Advances in Oncology /7./ and International Symposium on Molecular Medicine /5./. Hersonissos, 10.10.2002-12.10.2002] R&D Projects: GA ČR GA310/01/0802 Institutional research plan: CEZ:AV0Z5039906 Keywords : DNA adducts Subject RIV: FM - Hygiene Impact factor: 2.063, year: 2002

  2. {sup 32}P-postlabeling determination of DNA adducts in the earthworm Lumbricus terrestris exposed to PAH-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, P. [Laval Univ. Research Center, Quebec (Canada)]|[Ministere de l`Environnement et de la Faune du Quebec (Canada); El Adlouni, C.; Mukhopadhyay, M.J.; Nadeau, D.; Poirier, G.G. [Laval Univ. Research Center, Quebec (Canada); Viel, G. [CreaLab., Quebec (Canada)

    1995-05-01

    The importance of the search for reliable biomarkers of DNA damage in environmental health assessment is well recognized by the scientific community and regulatory agencies. Among the major biomarkers of DNA damage is the measurement of DNA adducts in target cells or tissues. Up to now, DNA adduct determinations have been directed mostly toward human exposure to toxic substances from the workplace and environment. Moreover, techniques for measuring DNA adducts, and in particular the {sup 32}P-postlabelling technique, presented also the possibility of determining DNA adduct levels in endogenous animal populations exposed to polluted environments as early warning monitors of ecotoxicity. Soil contamination is becoming a major environmental issue. Therefore, numerous contaminated sites must now be remediated to protect human health and to permit new uses of these sites as agricultural, residential, or industrial areas. Fulfillment of this task requires standardized and sensitive bioassays to carry out site evaluations and to establish scientifically defensible soil quality criteria. To that effect, the earthworm appears to be one of the best organisms for use in soil toxicity evaluation. Earthworms are probably the most relevant soil species, representing 60 to 80% of the total animal biomass in soil. Present soil bioassays focus mostly on plant species with end points like seed germination, root elongation, seedling growth and seedling emergence, and on acute toxicity evaluation (re: LC 50) on the earthworm Eisenia fetida. As yet, a standardized soil invertebrate test for teratogenic or mutagenic end points has not been developed. In this paper, we report the feasibility of DNA adduct determination by {sup 32}P-postlabelling in the earthworm Lumbricus terrestris as a way to detect the presence of genotoxic substances in soils. 20 refs., 1 fig., 1 tab.

  3. Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging

    Directory of Open Access Journals (Sweden)

    Matthew C. Zimmerman

    2017-10-01

    Full Text Available Methotrexate (MTX is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD. Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA and acetaldehyde (AA, known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.

  4. Risk assessment of DNA-reactive carcinogens in food.

    Science.gov (United States)

    Jeffrey, A M; Williams, G M

    2005-09-01

    Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. A single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B(1) (AFB(1)), a limit of 20 ppb or approximately 30 microg/day has been set and is considered a tolerable daily intake (TDI). Since AFB(1) is considered a potent carcinogen, doses of carcinogens is made.

  5. Drug-DNA adducts as biomarkers for metabolic activation of the nitro-aromatic nitrogen mustard prodrug PR-104A.

    Science.gov (United States)

    Stornetta, Alessia; Deng, Kai-Cheng Kieren; Danielli, Sara; Liyanage, H D Sarath; Sturla, Shana J; Wilson, William R; Gu, Yongchuan

    2018-04-07

    PR-104A is a clinical-stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR-104H) or amine (PR-104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo-ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR-104A have not been used in the clinical evaluation of PR-104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR-104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR-104A itself, and from its reduced metabolites, accumulated over 4 h in AKR1C3-expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR-104H and PR-104M reached steady state within 1 h. We then varied rates of PR-104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overexpression induced large increases in PR-104A sensitivity (inhibition of proliferation), DNA damage response (γH2AX formation), steady-state concentrations of PR-104H/M and formation of reduced drug-DNA adducts but not DNA adducts retaining the dinitro groups of PR-104A. The fold-change in the sum of PR-104H and PR-104M correlated with the fold-change in reduced crosslinks or monoadducts (R 2  = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR-104A. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Risk assessment of DNA-reactive carcinogens in food

    International Nuclear Information System (INIS)

    Jeffrey, A.M.; Williams, G.M.

    2005-01-01

    Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. A single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B 1 (AFB 1 ), a limit of 20 ppb or ∼30 μg/day has been set and is considered a tolerable daily intake (TDI). Since AFB 1 is considered a potent carcinogen, doses of 32 P-postlabeling or the use of surrogates such as hemoglobin adducts, together with approaches to evaluate the results. A discussion of approaches to estimating possible threshold effects for DNA-reactive carcinogens is made

  7. Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part I: Bulky DNA adducts

    International Nuclear Information System (INIS)

    Topinka, J.; Milcova, A.; Libalova, H.; Novakova, Z.; Rossner, P.; Balascak, I.; Sram, R.J.

    2009-01-01

    32 P-postlabelling and PAH-ELISA using the antiserum no. 29 were employed to analyze DNA adducts in venous and umbilical cord blood and the placenta of 79 mothers giving birth to 80 living babies in Prague (Czech Republic). Ambient air exposure was measured by stationary measurements of basic air pollutants (PM2.5, c-PAHs) during the entire pregnancy. Tobacco smoke exposure was assessed by questionnaire data and by plasma cotinine levels. The total DNA adduct levels in the lymphocytes of mothers and newborns were elevated by 30-40% (p 8 nucleotides vs. 0.15 ± 0.06 adducts/10 8 nucleotides) with newborns indicated a 30-40% increase of adducts in mothers. Almost equal PAH-DNA adduct levels were detected by anti-BPDE-DNA ELISA in the placenta of tobacco smoke-exposed and -unexposed mothers. Our results suggest a protective effect of the placental barrier against the genotoxic effect of some tobacco smoke components between the circulation of mother and child. We found a correlation between adduct levels in the blood of mothers and newborns.

  8. Effect of adduct formation on valent state of cerium in its ν-diketonates

    International Nuclear Information System (INIS)

    Spitsyn, V.I.; Martynenko, L.I.; Pechurova, N.I.; Snezhko, N.I.; Murav'eva, I.A.; Anufrieva, S.I.

    1982-01-01

    Physicochemical investigation of the system cerium (III, IV)-ν-diketone-additional ligand shows that ν-diketonate ability to adduct formation decreases in the series tenoyltrifluoro-acetonate > acetylacetonate > dibenzoylmethanate > benzoylmethanate. Adduct formation of the cerium (III, IV) ν-diketonates stabilizes cerium in trivalent condition, while oxidation degree 4+ is stable in tetrakis-ν-diketonates. The additional ligands are arranged in the series: tributhylphosphate < trioctyl-phosphineoxide < triphenylphosphineoxide < α, α'-dipyridyl < o-phenanthroline by the effect on cerium (III) stabilization in its ν-diketonates

  9. Increased levels of etheno-DNA adducts and genotoxicity biomarkers of long-term exposure to pure diesel engine exhaust.

    Science.gov (United States)

    Shen, Meili; Bin, Ping; Li, Haibin; Zhang, Xiao; Sun, Xin; Duan, Huawei; Niu, Yong; Meng, Tao; Dai, Yufei; Gao, Weimin; Yu, Shanfa; Gu, Guizhen; Zheng, Yuxin

    2016-02-01

    Etheno-DNA adducts are biomarkers for assessing oxidative stress. In this study, the aim was to detect the level of etheno-DNA adducts and explore the relationship between the etheno-DNA adducts and genotoxicity biomarkers of the diesel engine exhaust (DEE)-exposed workers. We recruited 86 diesel engine testing workers with long-term exposure to DEE and 99 non-DEE-exposed workers. The urinary mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and etheno-DNA adducts (εdA and εdC) were detected by HPLC-MS/MS and UPLC-MS/MS, respectively. Genotoxicity biomarkers were also evaluated by comet assay and cytokinesis-block micronucleus assay. The results showed that urinary εdA was significantly higher in the DEE-exposed workers (p<0.001), exhibited 2.1-fold increase compared with the non-DEE-exposed workers. The levels of urinary OH-PAHs were positively correlated with the level of εdA among all the study subjects (p<0.001). Moreover, we found that the increasing level of εdA was significantly associated with the increased olive tail moment, percentage of tail DNA, or frequency of micronucleus in the study subjects (p<0.01). No significant association was observed between the εdC level and any measured genotoxicity biomarkers. In summary, εdA could serve as an indicator for DEE exposure in the human population. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity.

    Science.gov (United States)

    Kathuria, Preetleen; Sharma, Purshotam; Manderville, Richard A; Wetmore, Stacey D

    2017-08-21

    Exposure to ochratoxin A (OTA), a possible human carcinogen, leads to many different DNA mutations. As a first step toward understanding the structural basis of OTA-induced mutagenicity, the present work uses a robust computational approach and a slipped mutagenic intermediate model previously studied for C 8 -dG aromatic amine adducts to analyze the conformational features of postreplication two-base deletion DNA duplexes containing OT-dG, the major OTA lesion at the C 8 position of guanine. Specifically, a total of 960 ns of molecular dynamics simulations (excluding trial simulations) were carried out on four OT-dG ionization states in three sequence contexts within oligomers containing the NarI recognition sequence, a known hotspot for deletion mutations induced by related adducts formed from known carcinogens. Our results indicate that the structural properties and relative stability of the competing "major groove" and "stacked" conformations of OTA adducted two-base deletion duplexes depend on both the OTA ionization state and the sequence context, mainly due to conformation-dependent deviations in discrete local (hydrogen-bonding and stacking) interactions at the lesion site, as well as DNA bending. When the structural characteristics of the OT-dG adducted two-base deletion duplexes are compared to those associated with previously studied C 8 -dG adducts, a greater understanding of the effects of the nucleobase-carcinogen linkage, and size of the carcinogenic moiety on the conformational preferences of damaged DNA is obtained. Most importantly, our work predicts key structural features for OT-dG-adducted deletion DNA duplexes, which in turn allow us to develop hypotheses regarding OT-dG replication outcomes. Thus, our computational results are valuable for the design and interpretation of future biochemical studies on the potentially carcinogenic OT-dG lesion.

  11. Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons

    DEFF Research Database (Denmark)

    Georgiadis, Panagiotis; Kovács, Katalin; Kaila, Stella

    2012-01-01

    We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated...

  12. High-performance liquid chromatography/electrospray mass spectrometry for the analysis of modified bases in DNA: 7-(2-hydroxyethyl)guanine, the major ethylene oxide-DNA adduct.

    Science.gov (United States)

    Leclercq, L; Laurent, C; De Pauw, E

    1997-05-15

    A method was developed for the analysis of 7-(2-hydroxyethyl)guanine (7HEG), the major DNA adduct formed after exposure to ethylene oxide (EO). The method is based on DNA neutral thermal hydrolysis, adduct micro-concentration, and final characterization and quantification by HPLC coupled to single-ion monitoring electrospray mass spectrometry (HPLC/SIR-ESMS). The method was found to be selective, sensitive, and easy to handle with no need for enzymatic digestion or previous sample derivatization. Detection limit was found to be close to 1 fmol of adduct injected (10(-10) M), thus allowing the detection of approximately three modified bases on 10(8) intact nucleotides in blood sample analysis. Quantification results are shown for 7HEG after calf thymus DNA and blood exposure to various doses of EO, in both cases obtaining clear dose-response relationships.

  13. Effect of adduct formation on valent state of cerium in its. beta. -diketonates

    Energy Technology Data Exchange (ETDEWEB)

    Spitsyn, V.I.; Martynenko, L.I.; Pechurova, N.I.; Snezhko, N.I.; Murav' eva, I.A.; Anufrieva, S.I. (Moskovskij Gosudarstvennyj Univ. (USSR))

    1982-04-01

    Physicochemical investigation of the system cerium (III, IV)-..beta..-diketone-additional ligand shows that ..beta..-diketonate ability to adduct formation decreases in the series tenoyltrifluoro-acetonate > acetylacetonate > dibenzoylmethanate > benzoylmethanate. Adduct formation of the cerium (III, IV) ..beta..-diketonates stabilizes cerium in trivalent condition, while oxidation degree 4+ is stable in tetrakis-..beta..-diketonates. The additional ligands are arranged in the series: tributhylphosphate < trioctyl-phosphineoxide < triphenylphosphineoxide < ..cap alpha.., ..cap alpha..'-dipyridyl < o-phenanthroline by the effect on cerium (III) stabilization in its ..beta..-diketonates.

  14. Development and application of non-invasive biomarkers for carcinogen-DNA adduct analysis in occupationally exposed populations.

    Science.gov (United States)

    Talaska, G; Cudnik, J; Jaeger, M; Rothman, N; Hayes, R; Bhatnagar, V J; Kayshup, S J

    1996-07-17

    Biological monitoring of exposures to carcinogenic compounds in the workplace can be a valuable adjunct to environmental sampling and occupational medicine. Carcinogen-DNA adduct analysis has promise as a biomarker of effective dose if target organ samples can be obtained non-invasively. We have developed non-invasive techniques using exfoliated urothelial and bronchial cells collected in urine and sputum, respectively. First morning urine samples were collected from 33 workers exposed to benzidine or benzidine-based dyes and controls matched for age, education, and smoking status. Sufficient DNA for 32P-postlabelling analysis was obtained from every sample. Mean levels of a specific DNA adduct (which co-chromatographed with standard characterized by MS) were elevated significantly in the benzidine-exposed workers relative to controls. In addition, workers exposed to benzidine had higher adduct levels than those exposed to benzidine-based dyes. This study demonstrates the usefulness of these non-invasive techniques for exposure/effect assessment. To be useful in occupational studies, biomarkers must also be sensitive to exposure interventions. We have conducted topical application studies of used gasoline engine oils in mice and found that the levels of carcinogen-DNA adducts in skin and lung can be significantly lowered if skin cleaning is conducted in a timely manner. The combination of useful, non-invasive techniques to monitor exposure and effect and industrial hygiene interventions can be used to detect and prevent exposures to a wide range of carcinogens including those found in used gasoline engine oils and jet exhausts.

  15. Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin

    Science.gov (United States)

    Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C.; Malle, Ernst; Sattler, Wolfgang

    2015-01-01

    Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl− system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood–brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120 min, decaying at a rate of 5.9 × 10−3 min−1. NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC–MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. PMID:25576489

  16. Detection of endogenous DNA adducts, O-carboxymethyl-2'-deoxyguanosine and 3-ethanesulfonic acid-2'-deoxycytidine, in the rat stomach after duodenal reflux.

    Science.gov (United States)

    Terasaki, Masaru; Totsuka, Yukari; Nishimura, Koichi; Mukaisho, Ken-Ichi; Chen, Kuan-Hao; Hattori, Takanori; Takamura-Enya, Takeji; Sugimura, Takashi; Wakabayashi, Keiji

    2008-09-01

    The endogenous DNA adducts O(6)-carboxymethyl-deoxyguanosine (O(6)-CM-dG) and 3-ethanesulfonic acid-deoxycytidine (3-ESA-dC) are produced from N-nitroso bile acid conjugates, such as N-nitrosoglycocholic acid (NO-GCA) and N-nitrosotaurocholic acid (NO-TCA), respectively. Formation of these DNA adducts in vivo was here analyzed by 32P-postlabeling in the glandular stomach of rats subjected to duodenal content reflux surgery. In this model, all duodenal contents, including bile acid conjugates, flow back from the jejunum into the gastric corpus. The levels of O(6)-CM-dG found at 4 and 8 weeks after surgery were 40.9 +/- 9.4 and 56.3 +/- 3.2 per 10(8) nucleotides, respectively, whereas the sham operation groups had values of 5.8 +/- 2.3 and 5.9 +/- 0.5 per 10(8) nucleotides. Moreover, adduct spots corresponding to 3-ESA-dC were detected in both duodenal reflux and sham operation groups and levels in the duodenal reflux groups were around four-fold elevated at 11.2 +/- 1.0 and 8.9 +/- 1.0 per 10(8) nucleotides after 4 and 8 weeks, respectively. When the duodenal reflux animals were treated with a nitrite trapping agent, thiazolidine- 4-carboxylic acid (thioproline, TPRO), the levels of O(6)-CM-dG and 3-ESA-dC were reduced to the same levels as in the sham operation animals. These observations suggest that NO-TCA and NO-GCA are formed by nitrosation of glycocholic acid and taurocholic acid, respectively, and these nitroso compounds produce DNA adducts in the glandular stomach of rats subjected to duodenal content reflux surgery.

  17. Repair of furocoumarin adducts in mammalian cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Smith, C.A.; Hanawalt, P.C.

    1984-01-01

    DNA repair was studied in cultured mammalian cells treated with the furocoumarins 8-methoxypsoralen (8-MOP), aminomethyl trioxsalen, or angelicin and irradiated with near UV light. The amount of DNA cross-linked by 8-MOP in normal human cells decreased by about one-half in 24 hours after treatment; no decrease was observed in xeroderma pigmentosum cells, group A. At present, it is not known to what extent this decrease represents complete repair events at the sites of cross-links. Furocoumarin adducts elicited excision repair in normal human and monkey cells but not in xeroderma pigmentosum group A cells. This excision repair resembled in several aspects that elicited by pyrimidine dimers, formed in DNA by irradiation with 254-nm UV light; however, it appeared that for at least 8-MOP and aminomethyl trioxsalen, removal of adducts was not as efficient as was the removal of pyrimidine dimers. A comparison was also made of repair in the 172-base-pair repetitive alpha-DNA component of monkey cells to repair in the bulk of the genome. Although repair elicited by pyrimidine dimers in alpha-DNA was the same as in the bulk DNA, that following treatment of cells with either aminomethyl trioxsalen or angelicin and near UV was markedly deficient in alpha-DNA. This deficiency reflected the removal of fewer adducts from alpha-DNA after the same initial adduct frequencies. These results could mean that each furocoumarin may produce several structurally distinct adducts to DNA in cells and that the capacity of cellular repair systems to remove these various adducts may vary greatly

  18. Levels of PAH-DNA adducts in cord blood and cord tissue and the risk of fetal neural tube defects in a Chinese population.

    Science.gov (United States)

    Yi, Deqing; Yuan, Yue; Jin, Lei; Zhou, Guodong; Zhu, Huiping; Finnell, Richard H; Ren, Aiguo

    2015-01-01

    Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) has been shown to be associated with an elevated risk for neural tube defects (NTDs). In the human body, PAHs are bioactivated and the resultant reactive epoxides can covalently bind to DNA to form PAH-DNA adducts, which may, in turn, cause transcription errors, changes in gene expression or altered patterns of apoptosis. During critical developmental phases, these changes can result in abnormal morphogenesis. We aimed to examine the relationship between the levels of PAH-DNA adducts in cord blood and cord tissue and the risk of NTDs. From 2010 to 2012, 60 NTD cases and 60 healthy controls were recruited from a population-based birth defects surveillance system in five counties of Shanxi Province in Northern China, where the emission of PAHs remains one of the highest in the country and PAHs exposure is highly prevalent. PAH-DNA adducts in cord blood of 15 NTD cases and 15 control infants, and in cord tissue of 60 NTD cases and 60 control infants were measured using the (32)P-postlabeling method. PAH-DNA adduct levels in cord blood tend to be higher in the NTD group (28.5 per 10(8) nucleotides) compared with controls (19.7 per 10(8) nucleotides), although the difference was not statistically significant (P=0.377). PAH-DNA adducts in cord tissue were significantly higher in the NTD group (24.6 per 10(6) nucleotides) than in the control group (15.3 per 10(6) nucleotides), P=0.010. A positive dose-response relationship was found between levels of PAH-DNA adducts in cord tissue and the risk of NTDs (P=0.009). When the lowest tertile was used as the referent and potential confounding factors were adjusted for, a 1.03-fold (95% CI, 0.37-2.89) and 2.96-fold (95% CI, 1.16-7.58) increase in the risk of NTDs was observed for fetuses whose cord tissue PAH-DNA adduct levels were in the second and highest tertile, respectively. High levels of PAH-DNA adducts in fetal tissues were associated with increased risks of

  19. DNA adducts and cancer risk in prospective studies: a pooled analysis and a meta-analysis

    DEFF Research Database (Denmark)

    Veglia, Fabrizio; Loft, Steffen; Matullo, Giuseppe

    2008-01-01

    in which bulky DNA adducts have been measured in blood samples collected from healthy subjects (N = 1947; average follow-up 51-137 months). In addition, we have performed a meta-analysis by identifying all articles on the same subject published up to the end of 2006, including case-control studies......). The association was evident only in current smokers and was absent in former smokers. Also the meta-analysis, which included both lung and bladder cancers, showed a statistically significant association in current smokers, whereas the results in never smokers were equivocal; in former smokers, no association......Bulky DNA adducts are biomarkers of exposure to aromatic compounds and of the ability of the individual to metabolically activate carcinogens and to repair DNA damage. Their ability to predict cancer onset is uncertain. We have performed a pooled analysis of three prospective studies on cancer risk...

  20. DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Alden C Klarer

    Full Text Available Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta, is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE, the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.

  1. Characterization of the Major Purine and Pyrimidine Adducts Formed after Incubations of 1-Chloro-3-buten-2-one with Single-/Double-Stranded DNA and Human Cells.

    Science.gov (United States)

    Liu, Ling-Yan; Zheng, Jin; Kong, Cong; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu; Elfarra, Adnan A

    2017-02-20

    We have previously shown that 1-chloro-3-buten-2-one (CBO), a potential reactive metabolite of 1,3-butadiene (BD), exhibits potent cytotoxicity and genotoxicity that have been attributed in part to its reactivity toward DNA. In an effort to identify the DNA adducts of CBO, we characterized the CBO reactions with 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), and 2'-deoxyadenosine (dA) under in vitro physiological conditions (pH 7.4, 37 °C). In the present study, we investigated the CBO reaction with 2'-deoxythymidine (dT) and compared the rate constants of the reactions of CBO with dA, dC, dG, and dT at both individual- and mixed-nucleosides levels. We also investigated the reactions of CBO with single- and double-stranded DNA using HPLC with UV detection after adducts were released by either acid or enzymatic hydrolysis of DNA. Consistent with the results from the nucleoside reactions and the rate constant experiments, 1,N 6 -(1-hydroxy-1-chloromethylpropan-1,3-diyl)adenine (A-2D) was identified as the major DNA adduct detected after acid hydrolysis, followed by N7-(4-chloro-3-oxobutyl)guanine (CG-2H) and a small amount of 1,N 6 -(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)adenine (A-1D). After enzymatic hydrolysis, 1,N 6 -(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2'-dexoyadenosine (dA-1), 3,N 4 -(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2'-deoxycytidine (dC-1/2), and 1,N 2 -(3-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-dexoyguanosine (CG-1) were detected, with dA-1 being the major product, followed by dC-1/2. When a nontoxic concentration of CBO (1 μM) was incubated with HepG2 cells, no adducts could be detected by LC-MS. However, pretreatment of cells with l-buthionine sulfoximine to deplete GSH levels allowed A-2D to be consistently detected in cellular DNA. These results may contribute to a better understanding of the role of the DNA adducts in CBO genotoxicity and mutagenicity. It also suggests that A-2D could be developed as a biomarker of CBO formation

  2. PEG and mPEG-anthracene induce DNA condensation and particle formation.

    Science.gov (United States)

    Froehlich, E; Mandeville, J S; Arnold, D; Kreplak, L; Tajmir-Riahi, H A

    2011-08-18

    In this study, we investigated the binding of DNA with poly(ethylene glycol) (PEG) of different sizes and compositions such as PEG 3350, PEG 6000, and mPEG-anthracene in aqueous solution at physiological conditions. The effects of size and composition on DNA aggregation and condensation as well as conformation were determined using Fourier transform infrared (FTIR), UV-visible, CD, fluorescence spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed moderate complex formation for PEG 3350 and PEG 6000 and weaker interaction for mPE-anthracene-DNA adducts with both hydrophilic and hydrophobic contacts. The order of ± stability of the complexes formed is K(PEG 6000) = 1.5 (±0.4) × 10(4) M(-1) > K(PEG 3350) = 7.9 (±1) × 10(3) M(-1) > K(m(PEG-anthracene))= 3.6 (±0.8) × 10(3) M(-1) with nearly 1 bound PEG molecule per DNA. No B-DNA conformational changes were observed, while DNA condensation and particle formation occurred at high PEG concentration.

  3. Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.

    Science.gov (United States)

    Rajesh, Mathur; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2005-02-15

    The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene

  4. Adduct formation of ionic and nanoparticular silver with amino acids and glutathione

    International Nuclear Information System (INIS)

    Blaske, Franziska; Stork, Lisa; Sperling, Michael; Karst, Uwe

    2013-01-01

    To investigate the interaction of ionic and nanoparticular silver with amino acids and small peptides, an electrospray ionization time-of-flight mass spectrometry method was developed. Monomeric and oligomeric silver adducts were formed with amino acids including cysteine (Cys), methionine, histidine, lysine, or the tripeptide glutathione (GSH). The obtained spectra for ionic silver show clusters in different ratios between Ag + and the reaction partners (X) including [Ag n X m − (n + 1)H] − (n = 1–4, m = 1–3). Regarding Cys, adduct clusters up to n = 5 and m = 4 were observed as well. Considering silver–GSH interactions, even doubly charged oligomers occur generating [Ag (a+1) GSH a − (a + 3)H] 2− (a = 5–7) and [Ag b GSH b − (b + 2)H] 2− (b = 4–8) ions. 1 H NMR data of free GSH compared to that after treatment with Ag + confirm sulfur–metal interactions due to changing chemical shifts for the protons located adjacent to the thiol group. Density functional theory calculations for silver–GSH clusters may explain the formation of experimentally recorded large clusters due to cooperative effects between silver and carboxylic acid side chains. Both sets of experiments indicate the presence of these adducts in the liquid phase. For silver nanoparticles, the respective data confirm the release of silver ions and the subsequent adduct formation

  5. Adduct formation of ionic and nanoparticular silver with amino acids and glutathione

    Science.gov (United States)

    Blaske, Franziska; Stork, Lisa; Sperling, Michael; Karst, Uwe

    2013-09-01

    To investigate the interaction of ionic and nanoparticular silver with amino acids and small peptides, an electrospray ionization time-of-flight mass spectrometry method was developed. Monomeric and oligomeric silver adducts were formed with amino acids including cysteine (Cys), methionine, histidine, lysine, or the tripeptide glutathione (GSH). The obtained spectra for ionic silver show clusters in different ratios between Ag+ and the reaction partners (X) including [Ag n X m - ( n + 1)H]- ( n = 1-4, m = 1-3). Regarding Cys, adduct clusters up to n = 5 and m = 4 were observed as well. Considering silver-GSH interactions, even doubly charged oligomers occur generating [Ag( a + 1)GSH a - ( a + 3)H]2- ( a = 5-7) and [Ag b GSH b - ( b + 2)H]2- ( b = 4-8) ions. 1H NMR data of free GSH compared to that after treatment with Ag+ confirm sulfur-metal interactions due to changing chemical shifts for the protons located adjacent to the thiol group. Density functional theory calculations for silver-GSH clusters may explain the formation of experimentally recorded large clusters due to cooperative effects between silver and carboxylic acid side chains. Both sets of experiments indicate the presence of these adducts in the liquid phase. For silver nanoparticles, the respective data confirm the release of silver ions and the subsequent adduct formation.

  6. Typical signature of DNA damage in white blood cells: a pilot study on etheno adducts in Danish mother-newborn child pairs

    DEFF Research Database (Denmark)

    Arab, K; Pedersen, Marie; Nair, J

    2009-01-01

    The impact of DNA damage commonly thought to be involved in chronic degenerative disease causation is particularly detrimental during fetal development. Within a multicenter study, we analyzed 77 white blood cell (WBC) samples from mother-newborn child pairs to see if imprinting of DNA damage...... in mother and newborn shows a similar pattern. Two adducts 1,N(6)-ethenodeoxyadenosine (epsilondA) and 3,N(4)-ethenodeoxycytidine (epsilondC) were measured by our ultrasensitive immunoaffinity (32)P-post-labeling method. These miscoding etheno-DNA adducts are generated by the reaction of lipid peroxidation...... arising from endogenous reactive aldehydes in WBC of both mother and newborn can be reliably assessed by epsilondA and epsilondC as biomarkers. The high correlation of etheno adduct levels in mother and child WBC suggests that a typical signature of DNA damage is induced similarly in fetus and mother...

  7. 7-Alkylguanine adduct levels in urine, lungs and liver of mice exposed to styrene by inhalation

    International Nuclear Information System (INIS)

    Vodicka, Pavel Erik; Linhart, Igor; Novak, Jan; Koskinen, Mikko; Vodickova, Ludmila; Hemminki, Kari

    2006-01-01

    This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7αG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7βG), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32 + 1.14 and 6.91 + 1.17 pmol/animal for lower and higher styrene exposure, respectively. β-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F = 13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10 8 normal nucleotides, i.e., 0.74 fmol/μg DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m 3 , while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing α-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0 x 10 -5 % of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly

  8. Methemoglobin Formation and Characterization of Hemoglobin Adducts of Carcinogenic Aromatic Amines and Heterocyclic Aromatic Amines.

    Science.gov (United States)

    Pathak, Khyatiben V; Chiu, Ting-Lan; Amin, Elizabeth Ambrose; Turesky, Robert J

    2016-03-21

    Arylamines (AAs) and heterocyclic aromatic amines (HAAs) are structurally related carcinogens formed during the combustion of tobacco or cooking of meat. They undergo cytochrome P450 mediated N-hydroxylation to form metabolites which bind to DNA and lead to mutations. The N-hydroxylated metabolites of many AAs also can undergo a co-oxidation reaction with oxy-hemolgobin (HbO2) to form methemoglobin (met-Hb) and the arylnitroso intermediates, which react with the β-Cys(93) chain of Hb to form Hb-arylsulfinamide adducts. The biochemistry of arylamine metabolism has been exploited to biomonitor certain AAs through their Hb arylsulfinamide adducts in humans. We examined the reactivity of HbO2 with the N-hydroxylated metabolites of 4-aminobiphenyl (ABP, HONH-ABP), aniline (ANL, HONH-ANL), and the HAAs 2-amino-9H-pyrido[2,3-b]indole (AαC, HONH-AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, HONH-PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx, HONH-MeIQx). HONH-ABP, HO-ANL, and HONH-AαC induced methemoglobinemia and formed Hb sulfinamide adducts. However, HONH-MeIQx and HONH-PhIP did not react with the oxy-heme complex, and met-Hb formation and chemical modification of the β-Cys(93) residue were negligible. Molecular modeling studies showed that the distances between the H-ON-AA or H-ON-HAA substrates and the oxy-heme complex of HbO2 were too far away to induce methemoglobinemia. Different conformational changes in flexible helical and loop regions around the heme pocket induced by the H-ON-AA or H-ON-HAAs may explain the different proclivities of these chemicals to induce methemoglobinemia. Hb-Cys(93β) sulfinamide and sulfonamide adducts of ABP, ANL, and AαC were identified, by Orbitrap MS, following the proteolysis of Hb with trypsin, Glu-C, or Lys-C. Hb sulfinamide and sulfonamide adducts of ABP were identified in the blood of mice exposed to ABP, by Orbitrap MS. This is the first report of the identification of intact Hb

  9. Correlation between base-excision repair gene polymorphisms and levels of in-vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes.

    Directory of Open Access Journals (Sweden)

    Hongping Yu

    Full Text Available In vitro benzo[a]pyrene diol epoxide (BPDE-induced DNA adducts in cultured peripheral lymphocytes have been shown to be a phenotypic biomarker of individual's DNA repair phenotype that is associated with cancer risk. In this study, we explored associations between genotypes of base-excision repair genes (PARP1 Val762Ala, APEX1 Asp148Glu, and XRCC1 Arg399Gln and in vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes in 706 cancer-free non-Hispanic white subjects. We found that levels of BPDE-induced DNA adducts were significantly higher in ever smokers than in never smokers and that individuals with the Glu variant genotypes (i.e., Asp/Glu and Glu/Glu exhibited lower levels of BPDE-induced DNA adducts than did individuals with the common Asp/Asp homozygous genotype (median RAL levels: 32.0 for Asp/Asp, 27.0 for Asp/Glu, and 17.0 for Glu/Glu, respectively; P(trend = 0.030. Further stratified analysis showed that compared with individuals with the common APEX1-148 homozygous Asp/Asp genotype, individuals with the APEX1-148Asp/Glu genotype or the Glu/Glu genotype had a lower risk of having higher-level adducts (adjusted OR = 0.60, 95% CI: 0.36-0.98 and adjusted OR = 0.47, 95% CI: 0.26-0.86, respectively; P(trend = 0.012 among smokers. Such an effect was not observed in non-smokers. However, there was no significant interaction between the APEX1 Asp148Glu polymorphism and smoking exposure in this study population (P = 0.512. Additional genotype-phenotype analysis found that the APEX1-148Glu allele had significantly increased expression of APEX1 mRNA in 270 Epstein-Barr virus-transformed lymphoblastoid cell lines, which is likely associated with more active repair activity. Our findings suggest that the functional APEX1-148Glu allele is associated with reduced risk of having high levels of BPDE-induced DNA adducts mediated with high levels of mRNA expression.

  10. Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

    Science.gov (United States)

    Vyas, Rajan; Efthimiopoulos, Georgia; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2015-01-01

    1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism. PMID:26327169

  11. Adduct formation of ionic and nanoparticular silver with amino acids and glutathione

    Energy Technology Data Exchange (ETDEWEB)

    Blaske, Franziska; Stork, Lisa; Sperling, Michael; Karst, Uwe, E-mail: uk@uni-muenster.de [University of Muenster, Institute of Inorganic and Analytical Chemistry (Germany)

    2013-09-15

    To investigate the interaction of ionic and nanoparticular silver with amino acids and small peptides, an electrospray ionization time-of-flight mass spectrometry method was developed. Monomeric and oligomeric silver adducts were formed with amino acids including cysteine (Cys), methionine, histidine, lysine, or the tripeptide glutathione (GSH). The obtained spectra for ionic silver show clusters in different ratios between Ag{sup +} and the reaction partners (X) including [Ag{sub n}X{sub m} - (n + 1)H]{sup -} (n = 1-4, m = 1-3). Regarding Cys, adduct clusters up to n = 5 and m = 4 were observed as well. Considering silver-GSH interactions, even doubly charged oligomers occur generating [Ag{sub (a+1)}GSH{sub a} - (a + 3)H]{sup 2-} (a = 5-7) and [Ag{sub b}GSH{sub b} - (b + 2)H]{sup 2-} (b = 4-8) ions. {sup 1}H NMR data of free GSH compared to that after treatment with Ag{sup +} confirm sulfur-metal interactions due to changing chemical shifts for the protons located adjacent to the thiol group. Density functional theory calculations for silver-GSH clusters may explain the formation of experimentally recorded large clusters due to cooperative effects between silver and carboxylic acid side chains. Both sets of experiments indicate the presence of these adducts in the liquid phase. For silver nanoparticles, the respective data confirm the release of silver ions and the subsequent adduct formation.

  12. Translesion DNA synthesis and mutation induced in a plasmid with a single adduct of the environmental contaminant 3-nitrobenzanthrone in SOS-induced Escherichia coli

    International Nuclear Information System (INIS)

    Kawanishi, M.; Kanno, T.; Yagi, T.; Enya-Takamura, T.; Fuchs, R.P.

    2003-01-01

    Full text: 3-Nitrobenzanthrone (NBA) is a powerfully mutagenic nitrated aromatic hydrocarbon found in diesel exhaust and in airborne particulate matters. NBA forms an unusual DNA adduct in vitro that has a C-C bond between the C-8 position of deoxyguanosine and the C-2 position of NBA. We previously found that this adduct is also present in the human cells treated with NBA, and induces mutations in supF shuttle vector system. In this study, we analyzed translesion DNA synthesis (TLS) over a single adduct in lacZ' gene in a plasmid in uvrAmutS Escherichia coli. The result showed that the adduct blocked DNA replication and an observed TLS frequency was 5.4% in non-SOS-induced E. coli. All progenies after the TLS had no mutation. On the other hand, TLS increased to 11.3%, and 4.8% of them had mostly G to T mutations in SOS-induced E. coli. These results suggest that this unusual adduct would be one of causes of lung cancer that is increasing in the urban areas polluted with diesel exhaust. It must be interesting to reveal which DNA polymerase is involved in this TLS

  13. Bulky DNA adducts in white blood cells: a pooled analysis of 3600 subjects

    Czech Academy of Sciences Publication Activity Database

    Ricceri, F.; Godschalk, R. W.; Peluso, M.; Philips, D. H.; Agudo, A.; Georgiadis, P.; Loft, S.; Tjonneland, A.; Raaschau-Nielsen, O.; Palli, D.; Perera, F.; Vermeulen, R.; Taioli, E.; Šrám, Radim; Munnia, A.; Rosa, F.; Allione, A.; Matullo, G.; Vineis, P.

    2010-01-01

    Roč. 19, č. 12 (2010), s. 3174-3181 ISSN 1055-9965 Grant - others:European Union ECNIS(XE) FOOD-CT-2005-513943 Institutional research plan: CEZ:AV0Z50390512 Keywords : DNA adducts * biomarkers of exposure * air pollution Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 4.190, year: 2010

  14. Separation and identification of DNA-carcinogen adduct conformers by polyacrylamide gel electrophoresis with laser-induced fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Marsch, G.A.; Jankowiak, R.; Farhat, J.H.; Small, G.J. (Ames Lab., IA (United States) Iowa State Univ., Ames (United States))

    1992-12-01

    The authors have developed a separation protocol utilizing high-resolution polyacrylamide gel electrophoresis (PAGE) to isolate stable anti-benzo[a]pyrene diol epoxide adducts of oligodeoxynucleotides. Both enantiomers produced multiple adduct species. The distribution of adduct types could be quantitated by densitometry of autoradiograms or Cerenkov counting of eluted oligomers modified by anti-BPDE isomers. Laser-induced fluorescence (LIF) spectra of eluted adducts at 4.2 K (fluorescence line-narrowing spectroscopy) and 77 K revealed that bands corresponded to pure conformers of pyrene chromophore. Carcinogen-modified oligodeoxynucleotides were single-stranded, but there were often considerable stacking interactions between the pyrenyl residues and the oligonucleotide bases, indicating that electrophoresed oligomers were single-stranded but in a native, versus random-coil conformation. The ability to identify and quantitate adducts by PAGE-LIF, coupled with the high resolution and sensitivity of both techniques, makes PAGE and LIF in tandem a potentially powerful tool in the study of chemical carcinogenesis or other ligand-DNA interactions. 43 refs., 7 figs., 1 tab.

  15. Measurement of DNA adducts and strand breaks in dab (Limanda limanda) collected in the field: effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Akcha, F.; Leday, G.; Pfohl-Leszkowicz, A

    2004-08-18

    In the Eastern English Channel, the potential application of the comet assay and post-labelling technique in dab was evaluated for genotoxicity monitoring of the marine environment. The effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA lesions were also studied. Female and male dab of two class of size (juvenile and adult) were collected by trawling in different sites in Seine Bay and Somme Bay during September 2001. Single-strand breaks and adducts were, respectively, measured in erythrocytes and the liver. Results obtained for the adult female were compared with those collected during a first cruise in March 2001 [Akcha et al., Mutat Res. 534 (1-2) (2003) 21]. Significant effects of sex and age were demonstrated on the level of strand breaks. Moreover, a significant interaction between age and sex was shown that might indicate the complex influence of other factors on the extent of DNA damage (i.e. reproduction status). In the adult dab, the level of breaks is higher in the male than in the female, whereas the opposite trend was observed for the juvenile. Whatever the sex, the number of DNA breaks is higher in the adult than in the juvenile. For the female dab, significant differences were observed with the comet assay between the Seine Bay and the Somme Bay in March but not in September. This may be due to seasonal variations in the formation of DNA lesions related to variations in lipid content and levels of biotransformation activities and/or to spawning cycles. The presence of genotoxic substances in the study areas was also confirmed by the detection of DNA adducts in each sample analysed. Whereas no effect was shown on the total level of adducts for the tested biotic and abiotic factors, qualitative differences in adduct profiles were observed for each of these factors. For the female dab, comparison of adduct profiles obtained in March and September with one generated by hepatic microsomal activation in dab of

  16. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  17. Retardation of experimental tumorigenesis and reduction in DNA adducts by turmeric and curcumin.

    Science.gov (United States)

    Krishnaswamy, K; Goud, V K; Sesikeran, B; Mukundan, M A; Krishna, T P

    1998-01-01

    Turmeric and its active principle curcumin have been extensively investigated for their antimutagenic and antioxidant effects in bacterial and animal systems. Because oral cancers are common in India, an experimental model of 7,12-dimethylbenzanthracene-induced buccal pouch tumors in Syrian Golden hamsters was used to evaluate the tumor retardation effects of turmeric and curcumin. Turmeric and/or curcumin was administered in the diet and/or applied locally for 14 weeks along with 7,12-dimethylbenzanthracene. After the experimental period, the animals were sacrificed and oral pouches were examined for tumor number and size. DNA adducts were estimated by 32P postlabel assay in the cheek pouches. Neoplastic changes were graded by histopathology. The results of the study suggest that turmeric or curcumin in the diet and/or applied locally significantly reduced DNA adducts at the target site. Tumor number and tumor burden were significantly lower (p curcumin (p curcumin administered in the diet or applied as paint may have a plausible chemopreventive effect on oral precancerous lesions.

  18. Formation and repair of physically and chemically induced DNA damage in human cells. Final report, September 1, 1976-November 30, 1978

    International Nuclear Information System (INIS)

    Cerutti, P.A.

    1979-01-01

    The major topic was the study of the formation and repair of DNA damage by energy related physical and chemical agents in cultured human cells. Two pathways of damage production were distinguished: (1) indirect action, i.e., attack of DNA by active oxygen species which are formed by the reaction of the primary agent with a non-DNA target; and (2) direct action, i.e., reaction of the primary agent or a chemical derivative of the primary agent with DNA usually resulting in the formation of a covalent adduct. Near-ultraviolet light and ionizing radiation were studied as agents which operate at least in part via indirect action and benzo(a)pyrene as chemical carcinogen operating mostly by direct action. The formation of monomeric thymine damage of the 5,6-dihydroxy-dihydrothymine type by γ-rays and ultraviolet light was investigated. Indirect action of near-ultraviolet light is also responsible for the induction of DNA single strand breaks. Their formation and repair following exposure to 313 nm light was studied in skin fibroblasts from patients with the hereditary disease Xeroderma pigmentosum (XP). Excision repair of γ-ray induced 5,6-dihydroxy-dihydrothymine type lesions was studied in fibroblasts from Ataxia telangiectasia (AT) patients. The formation and repair of covalent purine adducts was studied in actively metabolizing rodent and human cells following treatment with the procarcinogen benzo(a)pyrene and with the ultimate metabolite benzo(a)pyrene-diol-epoxide I

  19. Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine.

    Science.gov (United States)

    Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong; Prakash, Satya; Prakash, Louise

    2017-11-10

    N3-Methyladenine (3-MeA) is formed in DNA by reaction with S -adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro , we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay

    Czech Academy of Sciences Publication Activity Database

    Topinka, Jan; Rössner ml., Pavel; Milcová, Alena; Schmuczerová, Jana; Švecová, Vlasta; Šrám, Radim

    2011-01-01

    Roč. 202, č. 3 (2011), s. 186-192 ISSN 0378-4274 R&D Projects: GA MŠk 2B08005; GA MŽP(CZ) SP/1B3/8/08 Institutional research plan: CEZ:AV0Z50390512 Keywords : air pollution * genotoxicity * DNA adducts Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.230, year: 2011

  1. Miscoding properties of 1,N{sup 6}-ethanoadenine, a DNA adduct derived from reaction with antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

    Energy Technology Data Exchange (ETDEWEB)

    Hang, Bo; Guliaev, Anton B.; Chenna, Ahmed; Singer, B.

    2003-03-05

    1,N{sup 6}-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) {alpha}, {beta}, {eta} and {iota}. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using a primer extension assay, both pols {alpha} and {beta} were primarily blocked by EA or {var_epsilon}A with very minor extension. Pol {eta} a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol {eta} incorporated all four nucleotides opposite EA and {var_epsilon}A, but with differential preferences and mainly in an error-prone manner. Human pol {iota}, a paralog of human pol {eta}, was blocked by both adducts with a very small amount of synthesis past {var_epsilon}A. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. {var_epsilon}A, could affect the specificity of pol {iota} toward the template T immediately 3 feet to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or {var_epsilon}A showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to {var_epsilon}A, is a miscoding lesion.

  2. Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

    Directory of Open Access Journals (Sweden)

    Parvathi Chary

    Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

  3. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

    International Nuclear Information System (INIS)

    Yu, Shuangying; Tang, Song; Mayer, Gregory D.; Cobb, George P.; Maul, Jonathan D.

    2015-01-01

    Highlights: • Interactive effects of UVB radiation-pesticide co-exposures were examined in frogs. • Responses included induction of DNA photo-adducts and DNA damage and repair genes. • Elevated DNA adduct levels occurred for co-exposures compared to UVB alone. • One mechanism is that pesticides may alter nuclear excision repair gene expression. - Abstract: Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides

  4. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    International Nuclear Information System (INIS)

    Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

    1988-01-01

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32 P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  5. Gold Nanoparticles for the Detection of DNA Adducts as Biomarkers of Exposure to Acrylamide

    Science.gov (United States)

    Larguinho, Miguel Angelo Rodrigues

    The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in

  6. Spectrophotometric study of the formation of adducts between U(TTA)4 and neutral organosulfoxide donors

    International Nuclear Information System (INIS)

    Ramanujam, A.; Gudi, N.M.; Nadkarni, M.N.; Ramakrishna, V.V.; Patil, S.K.

    1981-01-01

    Synergistic extraction of several tetravalent actinides by mixtures of a β diketone and a neutral donor has been fairly well studied except for U(IV). Presumably, this is due to the instability of U(IV) at tracer level which does not permit the use of usual distribution methods for the study of its synergistic extraction. However, it is known that the formation of adduct between metal β diketonate and the neutral donor occurs in the organic phase and its formation is mainly responsible for the synergistic enhancement in the extraction of the metal ion. U(TTA) 4 dissolved in benzene is stable in presence of excess HTTA and addition of a neutral donor to the same results in considerable spectral changes and this has been exploited in earlier studies on the adduct formation reaction between U(TTA) 4 and several organo phosphorous neutral donors. In the present study, the adduct formation between U(TTA) 4 and few neutral sulfoxide donors: dibenzyl sulfoxide (DBSO), dimethyl sulfoxide (DMSO), dihexyl sulfoxide (DHSO) and dioctyl sulfoxide (DOSO) has been investigated in benzene medium. All the donors (S) used in the present work form 1:1 adduct with U(TTA) 4 and the equilibrium constants βsub(AB) for the reaction viz., U(TTA) 4 +S U(TTA) 4 .S have been calculated. The βsub(AB) values follow the order DBSO< DMSO< DHSP approx. DOSO. An attempt has been made to compare the values with those obtained using neutral organo phosphorous compounds and to explain the observed trends on the basis of the basicity of the neutral donors. (author)

  7. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Tetsuya, E-mail: suzukite@hiroshima-u.ac.jp [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Grúz, Petr; Honma, Masamitsu [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Adachi, Noritaka [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Nohmi, Takehiko [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan)

    2016-09-15

    Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

  8. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

    International Nuclear Information System (INIS)

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    2016-01-01

    Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

  9. Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wei [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States); College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035 (China); Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (United States); Zhu, Hong; Jia, Zhenquan [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States); Li, Jianrong [College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035 (China); Misra, Hara P. [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States); Zhou, Kequan, E-mail: kzhou@wayne.edu [Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202 (United States); Li, Yunbo, E-mail: yli@vcom.vt.edu [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States)

    2009-12-04

    Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in {phi}X-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 {mu}M SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.

  10. Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

    International Nuclear Information System (INIS)

    Chen, Wei; Zhu, Hong; Jia, Zhenquan; Li, Jianrong; Misra, Hara P.; Zhou, Kequan; Li, Yunbo

    2009-01-01

    Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in φX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 μM SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.

  11. Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene

    International Nuclear Information System (INIS)

    Xu Mian; Nelson, Garret B.; Moore, Joseph E.; McCoy, Thomas P.; Dai, Jian; Manderville, Richard A.; Ross, Jeffrey A.; Miller, Mark Steven

    2005-01-01

    Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2-8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24-48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2-5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P 32 post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically significant

  12. Protein modification by acrolein: Formation and stability of cysteine adducts

    OpenAIRE

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2009-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

  13. Serum Level of Antibodies (IgG, IgM Against Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA Adducts in Children Dermatologically Exposed to Coal Tar

    Directory of Open Access Journals (Sweden)

    Pavel Borský

    2017-06-01

    Full Text Available Crude coal tar (CCT contains polycyclic aromatic hydrocarbons (PAHs. Benzo[a]pyrene (BaP is metabolized into a highly reactive metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE that is able to bind to DNA and creates BPDE-DNA adducts. Adducted DNA becomes immunogenic and induces immune response by production of antibodies against BPDE-DNA adducts (Ab-BPDE-DNA. Circulating Ab-BPDE-DNA was proposed as potential biomarker of genotoxic exposure to BaP (PAHs. Goeckerman therapy (GT of psoriasis uses dermal application of CCT ointment (PAHs. In presented study (children with psoriasis treated by GT; n = 19 the therapy significantly increased the level of Ab-BPDE-DNA (EI = 0.29/0.19–0.34 vs. 0.31/0.25–0.40; median/lower–upper quartile; p < 0.01. The results support the idea of Ab-BPDE-DNA level as a possible tentative indicator of exposure, effects and susceptibility of the organism to the exposure of BaP (PAHs.

  14. 7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

    Science.gov (United States)

    He, Xiaobo; Xia, Qingsu; Ma, Liang; Fu, Peter P

    2016-01-01

    Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

  15. Biomonitoring of diesel exhaust-exposed workers. DNA and hemoglobin adducts and urinary 1-hydroxypyrene as markers of exposure

    DEFF Research Database (Denmark)

    Nielsen, Per Sabro; Andreassen, Åshild; Farmer, Peter B.

    1996-01-01

    Diesel exhaust-exposed workers have been shown to have an increased risk of lung cancer. A battery of biomarkers were evaluated for their ability to assess differences in exposure to genotoxic compounds in bus garage workers and mechanics and controls. Lymphocyte DNA adducts were analyzed using...... correlated with HPU but not with DNA adducts. The levels of HPU in urine were 0.11 micromol/mol creatinine compared to 0.05 in controls. All three assays applied were sensitive enough to evaluate a low level of exposure to environmental pollutants, with postlabelling and GC-MS as the most sensitive assays....... The study indicated that skin absorption of polycyclic aromatic hydrocarbons (PAH) might be an important factor to consider when studying PAH exposure from air pollution sources....

  16. Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay.

    Science.gov (United States)

    Tompkins, Elaine M; Jones, Donald J L; Lamb, John H; Marsden, Debbie A; Farmer, Peter B; Brown, Karen

    2008-01-01

    A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.

  17. Genetic polymorphisms in 19q13.3 genes associated with alteration of repair capacity to BPDE-DNA adducts in primary cultured lymphocytes.

    Science.gov (United States)

    Xiao, Mingyang; Xiao, Sha; Straaten, Tahar van der; Xue, Ping; Zhang, Guopei; Zheng, Xiao; Zhang, Qianye; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Zhu, Guolian; Lu, Xiaobo

    2016-12-01

    Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage in cells caused by BPDE is normally repaired by Nucleotide Excision Repair (NER) and Base Excision Repair (BER). Genetic variations in NER and BER can change individual DNA repair capacity to DNA damage induced by BPDE. In the present study we determined the number of in vitro induced BPDE-DNA adducts in lymphocytes, to reflect individual susceptibility to Polycyclic aromatic hydrocarbons (PAHs)-induced carcinogenesis. The BPDE-DNA adduct level in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 281 randomly selected participants. We genotyped for 9 single nucleotide polymorphisms (SNPs) in genes involved in NER (XPB rs4150441, XPC rs2228001, rs2279017 and XPF rs4781560), BER (XRCC1 rs25487, rs25489 and rs1799782) and genes located on chromosome 19q13.2-3 (PPP1R13L rs1005165 and CAST rs967591). We found that 3 polymorphisms in chromosome 19q13.2-3 were associated with lower levels of BPDE-DNA adducts (MinorT allele in XRCC1 rs1799782, minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571). In addition, a modified comet assay was performed to further confirm the above conclusions. We found both minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571 were associated with the lower levels of BPDE-adducts. Our data suggested that the variant genotypes of genes in chromosome 19q13.2-3 are associated with the alteration of repair efficiency to DNA damage caused by Benzo[a]pyrene, and may contribute to enhance predictive value for individual's DNA repair capacity in response to environmental carcinogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts of the template

    Energy Technology Data Exchange (ETDEWEB)

    Lockhart, M.L.; Deutsch, J.F.; Yamaura, I.; Cavalieri, L.F.; Rosenberg, B.H.

    1982-01-01

    The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phi X174 DNA as template and a 392-base restriction fragment as primer with E. coli polymerase I (Klenow fragment). Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough. 22 references, 3 figures, 1 table.

  19. Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

    International Nuclear Information System (INIS)

    Stiborová, Marie; Moserová, Michaela; Černá, Věra; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J.; Wolf, C. Roland; Schmeiser, Heinz H.; Phillips, David H.; Frei, Eva; Arlt, Volker M.

    2014-01-01

    In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b 5 , and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b 5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b 5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b 5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR

  20. Conformations of stereoisomeric base adducts to 4-hydroxyequilenin.

    Science.gov (United States)

    Ding, Shuang; Shapiro, Robert; Geacintov, Nicholas E; Broyde, Suse

    2003-06-01

    Exposure to estrogen through estrogen replacement therapy increases the risk of women developing cancer in hormone sensitive tissues. Premarin (Wyeth), which has been the most frequent choice for estrogen replacement therapy in the United States, contains the equine estrogens equilin and equilenin as major components. 4-Hydroxyequilenin (4-OHEN) is a phase I metabolite of both of these substances. This catechol estrogen autoxidizes to potent cytotoxic quinoids that can react with dG, dA, and dC to form unusual stereoisomeric cyclic adducts (Bolton, J. L., et al. (1998) Chem. Res. Toxicol. 11, 1113-1127). Like other bulky DNA adducts, these lesions may exhibit different susceptibilities to DNA repair and mutagenic potential, if not repaired in a structure-dependent manner. To ultimately gain insights into structure-function relationships, we computed conformations of stereoisomeric guanine, adenine, and cytosine base adducts using density functional theory. We find near mirror image conformations in stereoisomer adduct pairs for each modified base, suggesting opposite orientations with respect to the 5' --> 3' direction of the modified strand when the stereoisomer pairs are incorporated into duplex DNA. Such opposite orientations could cause stereoisomer pairs of lesions to respond differently to DNA replication and repair enzymes.

  1. Acetaldehyde Adducts in Alcoholic Liver Disease

    Directory of Open Access Journals (Sweden)

    Mashiko Setshedi

    2010-01-01

    Full Text Available Chronic alcohol abuse causes liver disease that progresses from simple steatosis through stages of steatohepatitis, fibrosis, cirrhosis, and eventually hepatic failure. In addition, chronic alcoholic liver disease (ALD, with or without cirrhosis, increases risk for hepatocellular carcinoma (HCC. Acetaldehyde, a major toxic metabolite, is one of the principal culprits mediating fibrogenic and mutagenic effects of alcohol in the liver. Mechanistically, acetaldehyde promotes adduct formation, leading to functional impairments of key proteins, including enzymes, as well as DNA damage, which promotes mutagenesis. Why certain individuals who heavily abuse alcohol, develop HCC (7.2–15% versus cirrhosis (15–20% is not known, but genetics and co-existing viral infection are considered pathogenic factors. Moreover, adverse effects of acetaldehyde on the cardiovascular and hematologic systems leading to ischemia, heart failure, and coagulation disorders, can exacerbate hepatic injury and increase risk for liver failure. Herein, we review the role of acetaldehyde adducts in the pathogenesis of chronic ALD and HCC.

  2. An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species.

    Science.gov (United States)

    Harvey, J S; Lyons, B P; Page, T S; Stewart, C; Parry, J M

    1999-04-26

    The grounding of the Sea Empress oil tanker resulted in the release of 72,000 tonnes of crude oil into Milford Haven, Wales, UK. Our initial studies indicated that this contamination resulted in elevated levels of DNA adducts in one of the area's native marine species Lipophrys pholis [B.P. Lyons, J.S. Harvey, J.M. Parry, An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis, Mutat. Res. 390 (1997) 263-268]. These original studies were extended and the genotoxic impact of the oil contamination was investigated in the invertebrates Halichondria panicea and Mytilus edulis, along with the vertebrate fish species L. pholis, Pleuronectes platessa and Limanda limanda. DNA adduct levels were assessed in these species over a period of 2-17 months after the incident. The studies indicate differences in the impact of acute oil contamination upon vertebrate and invertebrate species. The oil contamination did not induce any detectable elevations in adduct levels in the invertebrate species H. panicea and M. edulis. In contrast, the oil contamination did appear to induce adducts in the vertebrate teleost species L. pholis, P. platessa and Lim. limanda. Despite some difficulties in sampling, the data obtained 12-17 months after the spill suggested that the affected species recovered from the oil contamination. While the studies indicate that the genetic impact of the oil contamination was less severe than might have been expected, it remains possible that the DNA adducts detected in the teleosts could lead to genetic changes in these species in the future. Copyright 1999 Elsevier Science B.V.

  3. Evaluation of the metabolic fate of munitions material (TNT & RDX) in plant systems and initial assessment of material interaction with plant genetic material (DNA). Initial assessment of plant DNA adducts as biomarkers

    Energy Technology Data Exchange (ETDEWEB)

    Harvey, S.D.; Clauss, T.W.; Fellows, R.J.; Cataldo, D.A.

    1995-08-01

    Genetic damage to deoxyribonucleic acid (DNA) has long been suspected of being a fundamental event leading to cancer. A variety of causal factors can result in DNA damage including photodimerization of base pairs, ionizing radiation, specific reaction of DNA with environmental pollutants, and nonspecific oxidative damage caused by the action of highly reactive oxidizing agents produced by metabolism. Because organisms depend on an unadulterated DNA template for reproduction, DNA repair mechanisms are an important defense for maintaining genomic integrity. The objective of this exploratory project was to evaluate the potential for TNT to form DNA adducts in plants. These adducts, if they exist in sufficient quantities, could be potential biomarkers of munitions exposure. The ultimate goal is to develop a simple analytical assay for the determination of blomarkers that is indicative of munitions contamination. DNA repair exists in dynamic equilibrium with DNA damage. Repair mechanisms are capable of keeping DNA damage at remarkably low concentrations provided that the repair capacity is not overwhelmed.

  4. Formation of covalent complexes between human O sup 6 -alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligodeoxynucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Brent, T.P.; Remack, J.S. (St. Jude Children' s Research Hospital, Memphis, TN (USA))

    1988-07-25

    Repair of chloroethylnitrosourea (CENU)-induced precursors of DNA interstrand cross-links by O{sup 6}-alkylguanine-DNA alkyltransferase (GAT or GATase) appears to be a factor in tumor resistance to therapy with this class of antineoplastic drugs. Since human GAT is highly specific for O{sup 6}-guanine, yet the probably cross-link structure is N{prime}-Guanine N{sup 3}cytosine ethane, rearrangement of the initial O{sup 6}-guanine adduct via O{sup 6},N{sup 1}ethanoguanine has been proposed. The authors suggested that GAT reaction with this intermediate would produce DNA covalently linked to protein through an ethane link from N{sup 1}-guanine to the alkylacceptor site on GAT. In preliminary studies they demonstrated a covalent complex between GAT and carmustine (BCNU)-treated DNA by a precipitation assay method. They have now developed a method for isolating the reaction product of BCNU-treated synthetic 14-mer ({sup 32}P)-labeled oligodeoxynucleotide and GAT using polyacrylamide gel electrophoresis. This approach can be used to characterize the adducts induced by CENUs that lead to complex formation with GAT.

  5. Spectrophotometric studies on the formation of adducts involved in synergistic extraction of uranium (IV) by mixtures of HTTA and neutral donors

    International Nuclear Information System (INIS)

    Patil, S.K.; Ramakrishna, V.V.; Ramanujam, A.

    1979-01-01

    Adduct formation between U(TTA) 4 and several neutral donor (S) was investigated by utilizing the changes in the absorption spectra of U(IV) resulting from the addition of neutral donors to a solution of U(TTA) 4 . All the donors used in the present work from 1:1 adducts with U(TTA) 4 . From the spectral changes, the equilibrium constants βsub(AB) for the adduct formation reaction viz U(TTA) 4 S reversible U(TTA) 4 .S were calculated for a few neutral donors. The log βsub(AB) values obtained in benzene medium, are :TOPO (6.23), TBPO (6.13), TPPO (4.72), DBBP(4.04) TBP(3.04), TIOTP(1.27) and MIBK(-0.10) and a value of 3.98 for TOPO in chloroform medium. The adduct formation was found to result in increasing the coordination number of U(IV) from 8 in U(TTA) 4 to 9 in the adducts it forms with the neutral donors. Similar absorption spectral studies with U(DBM) 4 revealed that it forms much weaker adducts than the corresponding ones with U(TTA) 4 . The absorption spectra of the organic extracts of U(IV) from perchloric acid with mixtures of HTTA and a neutral donor were identical with the spectra of the adduct between U(TTA) 4 and the neutral donor. This suggested that the adduct species viz U(TTA) 4 .S is involved in the synergistic extraction of U(IV). The extraction constants relating to the synergistic extraction of U(IV), from aqueous perchloric acid into benzene solutions of HTTA and some of the neutral donors, were derived. (author)

  6. Methyl DNA adducts, DNA repair, and hypoxanthine-guanine phosphoribosyl transferase mutations in peripheral white blood cells from patients with malignant melanoma treated with dacarbazine and hydroxyurea

    NARCIS (Netherlands)

    Philip, P.A.; Souliotis, V.L.; Harris, A.L.; Salisbury, A.; Tates, A.D.; Mitchell, K.; Delft, J.H.M. van; Ganesan, T.S.; Kyrtopoulos, S.A.

    1996-01-01

    Dacarbazine (DTIC) is a DNA-methylating drug used in the treatment of malignant melanoma. Among the DNA dducts induced by DTIC are N7-methylguanine (N7-meG) and O6-methylguamne (O6-meG). The latter adduct, in particular, may be important in the mutagenic as well as the cytotoxic activity of DTIC.

  7. Bulky DNA Adducts in Cord Blood, Maternal Fruit-and-Vegetable Consumption, and Birth Weight in a European Mother-Child Study (NewGeneris)

    DEFF Research Database (Denmark)

    Pedersen, Marie; Schoket, Bernadette; Godschalk, Roger W

    2013-01-01

    , Greece, Norway, and Spain were recruited in 2006-2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires.Results: Adduct levels in maternal and cord blood samples were similar...... versus lowest tertile of adducts. The negative association with birth weight was limited to births in Norway, Denmark, and England, the countries with the lowest adduct levels, and was more pronounced in births to mothers with low intake of fruits and vegetables (-248 g; 95% CI: -405, -92 g) compared......, Kleinjans JC, Segerbäck D, Kogevinas M. 2013. Bulky DNA adducts in cord blood, maternal fruit-and-vegetable consumption, and birth weight in a European mother-child study (NewGeneris). Environ Health Perspect 121:1200-1206; http://dx.doi.org/10.1289/ehp.1206333....

  8. Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

    International Nuclear Information System (INIS)

    Pierce, J.R.; Case, R.; Tang, Moonshong

    1989-01-01

    Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both φX174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. The authors have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, they have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. φX174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-Af. However, they have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed

  9. DNA adducts and atherosclerotic: A study of accidental and sudden death males in the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Binková, Blanka; Šmerhovský, Zdeněk; Strejc, P.; Boubelík, O.; Stávková, Zdena; Chvátalová, Irena; Šrám, Radim

    č. 501 (2002), s. 115-128 ISSN 0027-5107 R&D Projects: GA MŽP SI/340/1/97 Institutional research plan: CEZ:AV0Z5039906 Keywords : atherosclerosis * DNA-adducts * GSTM1 and CYP1A1 polymorphism Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.158, year: 2002

  10. Increased micronuclei and bulky DNA adducts in cord blood after maternal exposures to traffic-related air pollution

    DEFF Research Database (Denmark)

    Pedersen, M.; Wichmann, J.; Autrup, H.

    2009-01-01

    assessed through the use of validated biomarkers in blood cells from mother-newborn pairs. A cross-sectional biomonitoring study with healthy pregnant women living in the Greater Copenhagen area, Denmark, was conducted. Bulky DNA adducts and micronuclei (MN) were measured in blood from 75 women and 69...... levels were similar and positively correlated in maternal and cord blood (1.40 vs. 1.37 n/10(8) nucleotides; r = 0.99; p cells). Adduct levels were...... highest among mother-newborn pairs who lived near medium-traffic-density (> 400-2500 vehicle km/24 h; p 2500 vehicle km/24 h) were significantly increased (p = 0.02). This trend remained after adjusting...

  11. Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Maayah, Zaid H. [Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451 (Saudi Arabia); Ghebeh, Hazem [Stem Cell & Tissue Re-Engineering, King Faisal Specialist Hospital and Research Center, Riyadh 11211 (Saudi Arabia); Alhaider, Abdulqader A. [Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451 (Saudi Arabia); Camel Biomedical Research Unit, College of Pharmacy and Medicine, King Saud University, Riyadh 11451 (Saudi Arabia); El-Kadi, Ayman O.S. [Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton (Canada); Soshilov, Anatoly A.; Denison, Michael S. [Department of Environmental Toxicology, University of California at Davis, Davis, CA 95616 (United States); Ansari, Mushtaq Ahmad [Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451 (Saudi Arabia); Korashy, Hesham M., E-mail: hkorashy@ksu.edu.sa [Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451 (Saudi Arabia)

    2015-04-15

    Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H:quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism

  12. Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway

    International Nuclear Information System (INIS)

    Maayah, Zaid H.; Ghebeh, Hazem; Alhaider, Abdulqader A.; El-Kadi, Ayman O.S.; Soshilov, Anatoly A.; Denison, Michael S.; Ansari, Mushtaq Ahmad; Korashy, Hesham M.

    2015-01-01

    Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H:quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism

  13. The regioselectivity of glutathione adduct formation with flavonoid quinone methides is pH-dependent

    NARCIS (Netherlands)

    Awad, H.M.; Boersma, M.G.; Boeren, S.; Vervoort, J.; Bladeren, van P.J.; Rietjens, I.M.C.M.

    2002-01-01

    In the present study, the formation of glutathionyl adducts from a series of 3',4'-dihydroxy flavonoid o-quinone/p-quinone methides was investigated with special emphasis on the regioselectivity of the glutathione addition as a function of pH. The flavonoid o-quinones were generated using

  14. ACCUMULATION OF M1DG DNA ADDUCTS AFTER CHRONIC EXPOSURE TO PCBS, BUT NOT FROM ACUTE EXPOSURE TO DIOXIN-LIKE COMPOUNDS

    Science.gov (United States)

    ABSTRACT: Oxidative DNA damage is one of the key events leading to mutation and cancer. The present study examined the accumulation of M1dG DNA adducts, 3-(2’-deoxy-β-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, after single or yearly exposur...

  15. Adduction of acrylamide with biomacromolecules at environmental dose level measured by accelerator mass spectrometry (AMS)

    International Nuclear Information System (INIS)

    Xie, Q.Y.; Sun, H.F.; Liu, Y.F.; Ding, X.F.; Fu, D.P.; Liu, K.X.

    2005-01-01

    Acrylamide (AA) is a well-known neurotoxin, which also has developmental, reproductive as well as genetic toxicity. AA has been classified as a probable human carcinogen by IARC in 1994 since its carcinogenic effects in animals were reported after repetitive high level dosing. Over the last 10 years, there have been a large number of studies investigating the effects of AA on rodent reproductive performance. In 2002, the Swedish Food Administration reported the presence of AA in the heat-treated food products. which again elicited great concern on the toxicity of AA. However most of these studies were investigated at a dose level of mg/kg b.w and above, which is much higher than the actual human relevant dose. In this study we investigate the adduction of environmental level AA with biomacromolecules by the ultra-sensitive AMS technique. This may provide some information on the reproductive toxicity of AA under extremely low level exposure. A series doses of [2, 3- 14 C] AA (0, 0.1, 1, 10, 100, 250, 1000 μg/kg bw) were administrated with a single intraperitoneal injection (i.p.) to ICR adult male mice. The blood and spermatozoon were collected 24 h post dosing. Hemoglobin (Hb), serum albumin (SA), protamine, spermatozoon DNA, spermatozoon head and tail were isolated respectively, and then transformed into graphite following our previous procedure, The adduct levels were determined by a 0.6 MV compact AMS facility at the Institute of Heavy Ion Physics of Peking University. The results indicate that: (1) AA adduct number increases with the doses within 0.1-1000 μg/kg b.w. range in a log/log linear mode, except for DNA within 10-1000 μg/kg b.w. range. (2) Comparing protamine, Hb, and SA adducts with that of spermatozoon DNA (see Fig. 1), AA mainly adducts to proteins. For instance, at 1000 μg/kg b.w. dose level, spermatozoon DNA adducts only account for about 0.71%, 1.36% and 0.82% of protamine, Hb and SA adducts, respectively. (3) AA-protamine adducts, AA

  16. Chemical-Biological Properties of Zinc Sensors TSQ and Zinquin: Formation of Sensor-Zn-Protein Adducts versus Zn(Sensor)2 Complexes.

    Science.gov (United States)

    Nowakowski, Andrew B; Meeusen, Jeffrey W; Menden, Heather; Tomasiewicz, Henry; Petering, David H

    2015-12-21

    Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and

  17. SYNTHESIS OF THE FULLY PROTECTED PHOSPHORAMIDITE OF THE BENZENE-DNA ADDUCT, N2- (4-HYDROXYPHENYL)-2'-DEOXYGUANOSINE AND INCORPORATION OF THE LATER INTO DNA OLIGOMERS

    Energy Technology Data Exchange (ETDEWEB)

    Chenna, Ahmed; Gupta, Ramesh C.; Bonala, Radha R.; Johnson, Francis; Huang, Bo

    2008-06-09

    N2-(4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2'-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.

  18. Effect of Green Tea Catechins and Hydrolyzable Tannins on Benzo[a]pyrene-Induced DNA Adducts and Structure–Activity Relationship

    OpenAIRE

    Cao, Pengxiao; Cai, Jian; Gupta, Ramesh C.

    2010-01-01

    Green tea catechins and hydrolyzable tannins are gaining increasing attention as chemopreventive agents. However, their mechanism of action is poorly understood. We investigated the effects of four green tea catechins and two hydrolyzable tannins on microsome-induced benzo[a]pyrene (BP)–DNA adducts and the possible structure–activity relationship. BP (1 μM) was incubated with rat liver microsomes and DNA in the presence of the test compound (1–200 μM) or vehicle. The purified DNA was analyzed...

  19. Significant interactions between maternal PAH exposure and haplotypes in candidate genes on B[a]P-DNA adducts in a NYC cohort of non-smoking African-American and Dominican mothers and newborns

    Science.gov (United States)

    Tang, Deliang

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAH) are a class of chemicals common in the environment. Certain PAH are carcinogenic, although the degree to which genetic variation influences susceptibility to carcinogenic PAH remains unclear. Also unknown is the influence of genetic variation on the procarcinogenic effect of in utero exposures to PAH. Benzo[a]pyrene (B[a]P) is a well-studied PAH that is classified as a probable human carcinogen. Within our New York City-based cohort, we explored interactions between maternal exposure to airborne PAH during pregnancy and maternal and newborn haplotypes (and in one case, a single-nucleotide polymorphism) in key B[a]P metabolism genes on B[a]P-DNA adducts in paired cord blood samples. The study subjects included non-smoking African-American (n = 132) and Dominican (n = 235) women with available data on maternal PAH exposure, paired cord adducts and genetic data who resided in the Washington Heights, Central Harlem and South Bronx neighborhoods of New York City. We selected seven maternal and newborn genes related to B[a]P metabolism, detoxification and repair for our analyses: CYP1A1, CYP1A2, CYP1B1, GSTM3, GSTT2, NQO1 and XRCC1. We found significant interactions between maternal PAH exposure and haplotype on cord B[a]P-DNA adducts in the following genes: maternal CYP1B1, XRCC1 and GSTM3, and newborn CYP1A2 and XRCC1 in African-Americans; and maternal XRCC1 and newborn NQO1 in Dominicans. These novel findings highlight differences in maternal and newborn genetic contributions to B[a]P-DNA adduct formation, as well as ethnic differences in gene–environment interactions, and have the potential to identify at-risk subpopulations who are susceptible to the carcinogenic potential of B[a]P. PMID:24177223

  20. Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

    International Nuclear Information System (INIS)

    Perrone, Carmen E.; Duan, Jian Dong; Jeffrey, Alan M.; Williams, Gary M.; Ahr, Hans-Juergen; Schmidt, Ulrich; Enzmann, Harald H.

    2004-01-01

    Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovoturkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using 32 P-postlabeling for DNA adducts. In ovoexposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had 32 P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity. (orig.)

  1. Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Perrone, Carmen E.; Duan, Jian Dong; Jeffrey, Alan M.; Williams, Gary M. [New York Medical College, Department of Pathology, Valhalla (United States); Ahr, Hans-Juergen; Schmidt, Ulrich [Bayer AG, Institute of Toxicology, Wuppertal (Germany); Enzmann, Harald H. [Federal Institute for Drugs and Medical Devices, Bonn (Germany)

    2004-10-01

    Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovoturkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using {sup 32}P-postlabeling for DNA adducts. In ovoexposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had {sup 32}P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity. (orig.)

  2. DNA adducts and liver DNA replication in rats during chronic exposure to N-nitrosodimethylamine (NDMA) and their relationships to the dose-dependence of NDMA hepatocarcinogenesis.

    Science.gov (United States)

    Souliotis, Vassilis L; Henneman, John R; Reed, Carl D; Chhabra, Saranjit K; Diwan, Bhalchandra A; Anderson, Lucy M; Kyrtopoulos, Soterios A

    2002-03-20

    Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.

  3. Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

    International Nuclear Information System (INIS)

    Kouchakdjian, M.; Patel, D.J.; Eisenberg, M.; Johnson, F.; Grollman, A.P.

    1991-01-01

    Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N 2 -propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9)·(G10-T11-A12-C13-F14-C15-A16-T17-G18) X·F 9-mer duplex. Two-dimensional NMR experiments establish that the X·F 9-mer helix is right-handed with Watson-Crick A·T and G·C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4·C15 and G6·C13 which flank the lesion site, as well as to the H1' and H1 double-prime protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4·C15 and G6·C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N 2 -propanodeoxyguanosine adduct fits into the cavity generated by the abasic site

  4. Detection and quantitation of benzo[a]pyrene-DNA adducts in brain and liver tissues of Beluga whales (Delphinapterus leucas) from the St. Lawrence and Mackenzie Estuaries

    International Nuclear Information System (INIS)

    Shugart, L.R.

    1988-01-01

    It should be noted that there are few analytical techniques available for the detection and quantitation of chemical adducts in the DNA of living organisms. The reasons for this are: the analytical technique often has to accommodate the unique chemical and/or physical properties of the individual chemical or its metabolite; the percentage of total chemical that becomes most of the parent compound is usually detoxified and excreted; not all adducts that form between the genotoxic agent and DNA are stable or are involved in the development of subsequent deleterious events in the organism; and the amount of DNA available for analysis is often quite limited. 16 refs., 1 tab

  5. In vitro synthesis and purification of PhIP-deoxyguanosine and PhIP-DNA oligomer covalent complexes

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, J.

    1994-12-01

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine compound formed when meats are cooked at high temperatures. PhIP damages DNA by forming covalent complexes with DNA carcinogen. In an effort to understand how the binding of PhIP to DNA may cause cancer, it is important to characterize the structures of PhIP-damaged DNA molecules. Our HPLC data support fluorescence and {sup 32}P Post-labeling studies which indicate the formation of several species of 2{prime}deoxyguanosine-(dG) or oligodeoxynucleotide-PhIP adducts. The reaction of PhIP with dG resulted in a reddish precipitate that was likely the major adduct, N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) adduct, with a more polar adduct fraction remaining in the supernatant. Reversed-phase HPLC analysis of the adducts in the supernatant revealed the existence of species of much shorter retention times than the dG-C8-PhIP adduct, confirming that these species are more polar than dG-C8-PhIP. At least four adducts were formed in the reaction of PhIP with DNA oligomer. HPLC analysis of the PhIP-DNA oligomer supernatant after butanol extractions revealed four unresolved peaks which spectra had maximum wavelengths between 340 and 360 nm. Though adduct peaks were not completely resolved, there was {approximately}3 minutes interval between the DNA oligomer peak and the adduct peaks. Furthermore, fluorescence emission data of the DNA oligomer-PhIP adduct solution show heterogeneous binding. The more polar PhIP adducts were fraction-collected and their structures will be solved by nuclear magnetic resonance or x-ray crystallography.

  6. NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

    Science.gov (United States)

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2012-01-01

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

  7. Conformational preferences of DNA following damage by aristolochic acids: Structural and energetic insights into the different mutagenic potential of the ALI and ALII-N(6)-dA adducts.

    Science.gov (United States)

    Kathuria, Preetleen; Sharma, Purshotam; Abendong, Minette N; Wetmore, Stacey D

    2015-04-21

    Aristolochic acids (AAI and AAII), produced by the Aristolochiaceae family of plants, are classified as group I (human) carcinogens by the International Agency for Research on Cancer. These acids are metabolized in cells to yield aristolactams (ALI and ALII, respectively), which further form bulky adducts with the purine nucleobases. Specifically, the adenine lesions are more persistent in cells and have been associated with chronic renal diseases and related carcinogenesis. To understand the structural basis of the nephrotoxicity induced by AAs, the ALI-N(6)-dA and ALII-N(6)-dA lesions are systematically studied using computational methods. Density functional theory calculations indicate that the aristolactam moiety intrinsically prefers a planar conformation with respect to adenine. Nucleoside and nucleotide models suggest that the anti and syn orientations about the glycosidic bond are isoenergetic for both adducts. Molecular dynamics simulations and free energy calculations reveal that the anti base-displaced intercalated conformation is the most stable conformer for both types of AL-N(6)-dA adducted DNA, which agrees with previous experimental work on the ALII-N(6)-dA adduct and thereby validates our approach. Interestingly, this conformer differs from the dominant conformations adopted by other N6-linked adenine lesions, including those derived from polycyclic aromatic hydrocarbons. Furthermore, the second most stable syn base-displaced intercalated conformation lies closer in energy to the anti base-displaced intercalated conformation for ALI-N(6)-dA compared to ALII-N(6)-dA. This indicates that a mixture of conformations may be detectable for ALI-N(6)-dA in DNA. If this enhanced conformational flexibility of double-stranded DNA persists when bound to a lesion-bypass polymerase, this provides a possible structural explanation for the previously observed greater nephrotoxic potential for the ALI versus ALII-N(6)-dA adduct. In addition, the structural

  8. Nitric acid adduct formation during crystallization of barium and strontium nitrates and their co-precipitation from nitric acid media

    International Nuclear Information System (INIS)

    Mishina, N.E.; Zilberman, B.Ya.; Lumpov, A.A.; Koltsova, T.I.; Puzikov, E.A.; Ryabkov, D.V.

    2015-01-01

    The molar solubilities of Ba, Sr and Pb nitrates in nitric acid as a function of total nitrate concentration is presented and described by the mass action law, indicating on formation of the adducts with nitric acid. Precipitates of Ba(NO 3 ) 2 and Sr(NO 3 ) 2 crystallized from nitric acid were studied by ISP OES and IR spectroscopy. The data obtained confirmed formation of metastable adducts with nitric acid. IR and X-ray diffraction studies of the mixed salt systems indicated conversion of the mixed salts into (Ba,Sr)(NO 3 ) 2 solid solution of discrete structure in range of total nitrate ion concentration ∼6 mol/L. (author)

  9. Electron transfer from nucleobase electron adducts to 5-bromouracil. Is guanine an ultimate sink for the electron in irradiated DNA?

    International Nuclear Information System (INIS)

    Nese, C.; Yuan, Z.; Schuchmann, M.N.; Sonntag, C. von

    1992-01-01

    Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after γ-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatom-protonated forms transfer electrons quantitatively to 5-BrU. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct. (Author)

  10. Environmental, Dietary, Maternal, and Fetal Predictors of Bulky DNA Adducts in Cord Blood

    DEFF Research Database (Denmark)

    Pedersen, Marie; Mendez, Michelle A; Schoket, Bernadette

    2015-01-01

    and drinking-water disinfection by-products, mainly trihalomethanes (THMs), were available for a large proportion of the study population. RESULTS: Greek and Spanish neonates had higher adduct levels than the northern European neonates [median, 12.1 (n = 179) vs. 6.8 (n = 332) adducts per 108 nucleotides, p...... with higher adduct levels in adjusted models. Exposure to fine particulate matter and nitrogen dioxide was associated with significantly higher adducts in the Danish subsample only. Overall, the pooled results for THMs in water show no evidence of association with adduct levels; however, there are country...

  11. Equilibrium and ab initio computational studies on the adduct formation of 1,3-diketonato-lithium(I), -sodium(I) and -potassium(I) with 1,10-phenanthroline and its 2,9-dimethyl derivatives.

    Science.gov (United States)

    Ishimori, Ken-ichiro; Mori, Seiji; Ito, Yuji; Ohashi, Kousaburo; Imura, Hisanori

    2009-06-15

    Highly effective and selective synergistic extraction of Li+ has been found using 2-naphthoyltrifluoroacetone (Hnta) as an acidic chelating agent and 2,9-dimethyl-1,10-phenanthroline (dmp) or 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (dmdpp) as a neutral ligand (denoted as L) in toluene. The synergism was ascribed to the adduct formation in the organic phase, and the composition and the formation constants of the adducts for alkali metal ions (M+) were determined by the extraction equilibrium analysis. The adducts found were M(nta)L for Li+ and Na+, while M(nta)L and M(nta)L2 for K+. To understand thermodynamics of the adduct formation with the bidentate amines, quantum chemical calculations of the 1:1 and 1:2 adduct formations with dmp and 1,10-phenanthroline (phen) were performed. The electronic and steric effects of the methyl groups at 2,9-positions of phen on the thermodynamic functions of adduct formation as well as the high lithium selectivity were quantitatively elucidated.

  12. Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase iota: Hoogsteen or Watson-Crick base pairing?

    Science.gov (United States)

    Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

    2009-01-13

    Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase iota (poliota) is a bypass polymerase of the Y family. Crystal structures of poliota suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that poliota is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in poliota for bypass of dG-AAF. In poliota with Hoogsteen-paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick-paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that poliota would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for poliota in lesion bypass.

  13. Persistence of DNA adducts, hypermutation and acquisition of cellular resistance to alkylating agents in glioblastoma.

    Science.gov (United States)

    Head, R J; Fay, M F; Cosgrove, L; Y C Fung, K; Rundle-Thiele, D; Martin, J H

    2017-12-02

    Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success of this multimodal approach is maintaining apoptotic sensitivity of tumour cells to the alkylating agent. This initial treatment likely establishes conditions contributing to development of drug resistance as alkylating agents form the O 6 -methylguanine adduct. This activates the mismatch repair (MMR) process inducing apoptosis and mutagenesis. This review describes key juxtaposed drivers in the balance between alkylation induced mutagenesis and apoptosis. Mutations in MMR genes are the probable drivers for alkylation based drug resistance. Critical to this interaction are the dose-response and temporal interactions between adduct formation and MMR mutations. The precision in dose interval, dose-responses and temporal relationships dictate a role for alkylating agents in either promoting experimental tumour formation or inducing tumour cell death with chemotherapy. Importantly, this resultant loss of chemotherapeutic selective pressure provides opportunity to explore novel therapeutics and appropriate combinations to minimise alkylation based drug resistance and tumour relapse.

  14. Maternal diet and dioxin-like activity, bulky DNA adducts and micronuclei in mother-newborns

    DEFF Research Database (Denmark)

    Pedersen, Marie; Halldorsson, Thorhallur I; Autrup, Herman

    2012-01-01

    Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei...... (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006-2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet...

  15. Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects.

    Science.gov (United States)

    Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

    2008-12-15

    Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines

  16. Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

    Science.gov (United States)

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2012-12-04

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

  17. Elucidating the Key Role of a Lewis Base Solvent in the Formation of Perovskite Films Fabricated from the Lewis Adduct Approach.

    Science.gov (United States)

    Cao, Xiaobing; Zhi, Lili; Li, Yahui; Fang, Fei; Cui, Xian; Yao, Youwei; Ci, Lijie; Ding, Kongxian; Wei, Jinquan

    2017-09-27

    High-quality perovskite films can be fabricated from Lewis acid-base adducts through molecule exchange. Substantial work is needed to fully understand the formation mechanism of the perovskite films, which helps to further improve their quality. Here, we study the formation of CH 3 NH 3 PbI 3 perovskite films by introducing some dimethylacetamide into the PbI 2 /N,N-dimethylformamide solution. We reveal that there are three key processes during the formation of perovskite films through the Lewis acid-base adduct approach: molecule intercalation of solvent into the PbI 2 lattice, molecule exchange between the solvent and CH 3 NH 3 I, and dissolution-recrystallization of the perovskite grains during annealing. The Lewis base solvents play multiple functions in the above processes. The properties of the solvent, including Lewis basicity and boiling point, play key roles in forming smooth perovskite films with large grains. We also provide some rules for choosing Lewis base additives to prepare high-quality perovskite films through the Lewis adduct approach.

  18. DNA damage in internal organs after cutaneous exposure to sulphur mustard

    Energy Technology Data Exchange (ETDEWEB)

    Batal, Mohamed [Laboratoire « Lésions des Acides Nucléiques », Université Joseph Fourier – Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France); Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP87, F-38702 La Tronche Cedex (France); Boudry, Isabelle; Mouret, Stéphane; Cléry-Barraud, Cécile; Wartelle, Julien [Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP87, F-38702 La Tronche Cedex (France); Bérard, Izabel [Laboratoire « Lésions des Acides Nucléiques », Université Joseph Fourier – Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France); Douki, Thierry, E-mail: thierry.douki@cea.fr [Laboratoire « Lésions des Acides Nucléiques », Université Joseph Fourier – Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France)

    2014-07-01

    Sulphur mustard (SM) is a chemical warfare agent that attacks mainly skin, eye and lungs. Due to its lipophilic properties, SM is also able to diffuse through the skin and reach internal organs. DNA represents one of the most critical molecular targets of this powerful alkylating agent which modifies DNA structure by forming monoadducts and biadducts. These DNA lesions are involved in the acute toxicity of SM as well as its long-term carcinogenicity. In the present work we studied the formation and persistence of guanine and adenine monoadducts and guanine biadducts in the DNA of brain, lungs, kidneys, spleen, and liver of SKH-1 mice cutaneously exposed to 2, 6 and 60 mg/kg of SM. SM-DNA adducts were detected in all studied organs, except in liver at the two lowest doses. Brain and lungs were the organs with the highest level of SM-DNA adducts, followed by kidney, spleen and liver. Monitoring the level of adducts for three weeks after cutaneous exposure showed that the lifetime of adducts were not the same in all organs, lungs being the organ with the longest persistence. Diffusion from skin to internal organs was much more efficient at the highest compared to the lowest dose investigated as the result of the loss of the skin barrier function. These data provide novel information on the distribution of SM in tissues following cutaneous exposures and indicate that brain is an important target. - Highlights: • Sulphur mustard reaches internal organs after skin exposure • Adducts are detected in the DNA of internal organs • Brain is the organ with the highest level of DNA damage • The barrier function of skin is lost at high dose of sulphur mustard • DNA adducts persist in organs for 2 or 3 weeks.

  19. DNA damage in internal organs after cutaneous exposure to sulphur mustard

    International Nuclear Information System (INIS)

    Batal, Mohamed; Boudry, Isabelle; Mouret, Stéphane; Cléry-Barraud, Cécile; Wartelle, Julien; Bérard, Izabel; Douki, Thierry

    2014-01-01

    Sulphur mustard (SM) is a chemical warfare agent that attacks mainly skin, eye and lungs. Due to its lipophilic properties, SM is also able to diffuse through the skin and reach internal organs. DNA represents one of the most critical molecular targets of this powerful alkylating agent which modifies DNA structure by forming monoadducts and biadducts. These DNA lesions are involved in the acute toxicity of SM as well as its long-term carcinogenicity. In the present work we studied the formation and persistence of guanine and adenine monoadducts and guanine biadducts in the DNA of brain, lungs, kidneys, spleen, and liver of SKH-1 mice cutaneously exposed to 2, 6 and 60 mg/kg of SM. SM-DNA adducts were detected in all studied organs, except in liver at the two lowest doses. Brain and lungs were the organs with the highest level of SM-DNA adducts, followed by kidney, spleen and liver. Monitoring the level of adducts for three weeks after cutaneous exposure showed that the lifetime of adducts were not the same in all organs, lungs being the organ with the longest persistence. Diffusion from skin to internal organs was much more efficient at the highest compared to the lowest dose investigated as the result of the loss of the skin barrier function. These data provide novel information on the distribution of SM in tissues following cutaneous exposures and indicate that brain is an important target. - Highlights: • Sulphur mustard reaches internal organs after skin exposure • Adducts are detected in the DNA of internal organs • Brain is the organ with the highest level of DNA damage • The barrier function of skin is lost at high dose of sulphur mustard • DNA adducts persist in organs for 2 or 3 weeks

  20. Sustained induction of cytochrome P4501A1 in human hepatoma cells by co-exposure to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole underlies the synergistic effects on DNA adduct formation

    Energy Technology Data Exchange (ETDEWEB)

    Gábelová, Alena, E-mail: alena.gabelova@savba.sk [Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava (Slovakia); Poláková, Veronika [Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava (Slovakia); Prochazka, Gabriela [Department of Biosciences and Nutrition, Karolinska Institute, Novum, SE-141 83 Huddinge (Sweden); Department of Medical Epidemiology and Biostatistics, Karolinska Institute, SE-171 77 Stockholm (Sweden); Kretová, Miroslava; Poloncová, Katarína; Regendová, Eva; Luciaková, Katarína [Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava (Slovakia); Segerbäck, Dan [Department of Biosciences and Nutrition, Karolinska Institute, Novum, SE-141 83 Huddinge (Sweden)

    2013-08-15

    To gain a deeper insight into the potential interactions between individual aromatic hydrocarbons in a mixture, several benzo[a]pyrene (B[a]P) and 7H-dibenzo[c,g]carbazole (DBC) binary mixtures were studied. The biological activity of the binary mixtures was investigated in the HepG2 and WB-F344 liver cell lines and the Chinese hamster V79 cell line that stably expresses the human cytochrome P4501A1 (hCYP1A1). In the V79 cells, binary mixtures, in contrast to individual carcinogens, caused a significant decrease in the levels of micronuclei, DNA adducts and gene mutations, but not in cell survival. Similarly, a lower frequency of micronuclei and levels of DNA adducts were found in rat liver WB-F344 cells treated with a binary mixture, regardless of the exposure time. The observed antagonism between B[a]P and DBC may be due to an inhibition of Cyp1a1 expression because cells exposed to B[a]P:DBC showed a decrease in Cyp1a1 mRNA levels. In human liver HepG2 cells exposed to binary mixtures for 2 h, a reduction in micronuclei frequency was also found. However, after a 24 h treatment, synergism between B[a]P and DBC was determined based on DNA adduct formation. Accordingly, the up-regulation of CYP1A1 expression was detected in HepG2 cells exposed to B[a]P:DBC. Our results show significant differences in the response of human and rat cells to B[a]P:DBC mixtures and stress the need to use multiple experimental systems when evaluating the potential risk of environmental pollutants. Our data also indicate that an increased expression of CYP1A1 results in a synergistic effect of B[a]P and DBC in human cells. As humans are exposed to a plethora of noxious chemicals, our results have important implications for human carcinogenesis. - Highlights: • B[a]P:DBC mixtures were less genotoxic in V79MZh1A1 cells than B[a]P and DBC alone. • An antagonism between B[a]P and DBC was determined in rat liver WB-F344 cells. • The inhibition of CYP1a1 expression by B[a]P:DBC mixture

  1. Impact of DNA repair on the dose-response of colorectal cancer formation induced by dietary carcinogens.

    Science.gov (United States)

    Fahrer, Jörg; Kaina, Bernd

    2017-08-01

    Colorectal cancer (CRC) is one of the most frequently diagnosed cancers, which is causally linked to dietary habits, notably the intake of processed and red meat. Processed and red meat contain dietary carcinogens, including heterocyclic aromatic amines (HCAs) and N-nitroso compounds (NOC). NOC are agents that induce various N-methylated DNA adducts and O 6 -methylguanine (O 6 -MeG), which are removed by base excision repair (BER) and O 6 -methylguanine-DNA methyltransferase (MGMT), respectively. HCAs such as the highly mutagenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) cause bulky DNA adducts, which are removed from DNA by nucleotide excision repair (NER). Both O 6 -MeG and HCA-induced DNA adducts are linked to the occurrence of KRAS and APC mutations in colorectal tumors of rodents and humans, thereby driving CRC initiation and progression. In this review, we focus on DNA repair pathways removing DNA lesions induced by NOC and HCA and assess their role in protecting against mutagenicity and carcinogenicity in the large intestine. We further discuss the impact of DNA repair on the dose-response relationship in colorectal carcinogenesis in view of recent studies, demonstrating the existence of 'no effect' point of departures (PoDs), i.e. thresholds for genotoxicity and carcinogenicity. The available data support the threshold concept for NOC with DNA repair being causally involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Competitive Deprotonation and Superoxide [O₂⁻•)] Radical-Anion Adduct Formation Reactions of Carboxamides under Negative-Ion Atmospheric-Pressure Helium-Plasma Ionization (HePI) Conditions.

    Science.gov (United States)

    Hassan, Isra; Pinto, Spencer; Weisbecker, Carl; Attygalle, Athula B

    2016-03-01

    Carboxamides bearing an N-H functionality are known to undergo deprotonation under negative-ion-generating mass spectrometric conditions. Herein, we report that N-H bearing carboxamides with acidities lower than that of the hydroperoxyl radical (HO-O(•)) preferentially form superoxide radical-anion (O2(-•)) adducts, rather than deprotonate, when they are exposed to the glow discharge of a helium-plasma ionization source. For example, the spectra of N-alkylacetamides show peaks for superoxide radical-anion (O2(-•)) adducts. Conversely, more acidic amides, such as N-alkyltrifluoroacetamides, preferentially undergo deprotonation under similar experimental conditions. Upon collisional activation, the O2(-•) adducts of N-alkylacetamides either lose the neutral amide or the hydroperoxyl radical (HO-O(•)) to generate the superoxide radical-anion (m/z 32) or the deprotonated amide [m/z (M - H)(-)], respectively. For somewhat acidic carboxamides, the association between the two entities is weak. Thus, upon mildest collisional activation, the adduct dissociates to eject the superoxide anion. Superoxide-adduct formation results are useful for structure determination purposes because carboxamides devoid of a N-H functionality undergo neither deprotonation nor adduct formation under HePI conditions.

  3. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    International Nuclear Information System (INIS)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-01-01

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 (micro)g of DNA. This sensitivity may allow the method to be used for clinical applications

  4. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-12-29

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

  5. Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Yue, Lijun; Wei, Yuxia; Chen, Jia; Shi, Huiqin; Liu, Qin; Zhang, Yajiao; He, Jun; Guo, Lei; Zhang, Tingfen; Xie, Jianwei; Peng, Shuangqing

    2014-04-21

    Sulfur mustard (SM) is a highly reactive alkylating vesicant and causes blisters upon contact with skin, eyes, and respiratory organs. It covalently links with DNAs by forming four mono- or cross-link adducts. In this article, the reference standards of SM-DNA adducts and deuterated analogues were first synthesized with simplified procedures containing only one or two steps and using less toxic chemical 2-(2-chloroethylthio)ethanol or nontoxic chemical thiodiglycol as starting materials. A sensitive and high-throughput simultaneous quantification method of N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG), N(3)-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N(3)-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) in the Sprague-Dawley rat derma samples was developed by stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) with the aim of revealing the real metabolic behaviors of four adducts. The method was validated, the limit of detection (S/N ratio greater than 10) was 0.01, 0.002, 0.04, and 0.11 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively, and the lower limit of quantification (S/N ratio greater than 20) was 0.04, 0.01, 0.12, and 0.33 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively. The accuracy of this method was determined to be 76% to 129% (n = 3), and both the interday (n = 6) and intraday (n = 7) precisions were less than 10%. The method was further applied for the quantifications of four adducts in the derma of adult male Sprague-Dawley rats exposed to SM ex vivo and in vivo, and all adducts had time- and dose-effect relationships. To the best of our knowledge, this is the first time that the real presented status of four DNA adducts was simultaneously revealed by the MS-based method, in which Bis-G showed much higher abundance than the result previously reported and N(3

  6. Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts.

    Science.gov (United States)

    Diaz-Parga, Pedro; Goto, Joy J; Krishnan, V V

    2018-01-01

    Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.

  7. DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

    Directory of Open Access Journals (Sweden)

    Fabrizio Gentile

    2017-04-01

    Full Text Available Oxidative stress and lipid peroxidation (LPO induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity.

  8. Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase ι: Hoogsteen or Watson-Crick base pairing?†

    Science.gov (United States)

    Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

    2009-01-01

    Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase ι (polι) is a bypass polymerase of the Y family. Crystal structures of polι suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that polι is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in polι for bypass of dG-AAF. In polι with Hoogsteen paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that polι would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for polι in lesion bypass. PMID:19072536

  9. Biomarkers for Exposure to Ambient Air Pollution - Comparison of Carcinogen-DNA Adduct Levels with Other Exposure Markers and Markers for Oxidative Stress

    DEFF Research Database (Denmark)

    Autrup, Herman; Daneshvar, Bahram; Dragsted, Lars Ove

    1999-01-01

    Human exposure to genotoxic compounds present in ambient air has been studied using selected biomarkers in nonsmoking Danish bus drivers and postal workers. A large interindividual variation in biomarker levels was observed. Significantly higher levels of bulky carcinogen-DNA adducts (75.42 adducts....../10(8) nucleotides) and of 2-amino-apidic semialdehyde (AAS) in plasma proteins (56.7 pmol/mg protein) were observed in bus drivers working in the central part of Copenhagen, Denmark. In contrast, significantly higher levels of AAS in hemoglobin (55.8 pmol/mg protein), malondialdehyde in plasma (0. 96...... nmol/ml plasma), and polycyclic aromatic hydrocarbon (PAH)-albumin adduct (3.38 fmol/ microg albumin) were observed in the suburban group. The biomarker levels in postal workers were similar to the levels in suburban bus drivers. In the combined group of bus drivers and postal workers, negative...

  10. Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes.

    Directory of Open Access Journals (Sweden)

    Masashi Fujita

    Full Text Available Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4 photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4 photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

  11. Positive/negative liquid secondary ion mass spectrometry of Ln-EDTA (1:1) complexes. Formation of molecular ion adducts with neutral species of the matrix or Ln-EDTA

    International Nuclear Information System (INIS)

    Plaziak, A.S.; Lis, S.; Elbanowski, M.

    1992-01-01

    The mass spectra of 1:1 complexes of EDTA with lanthanide cations (Ln=Sm, Eu, Gd, Tb or Dy) upon positive/negative LSIMS are presented. In glycerol used as a matrix, adduct-ions such as [M+H] + , [M+H+nGly] + , [2M+H] + , [2M+H+Gly] + (positive LSIMS) or [M-H] - , [M-H+nGly] - , [2M-H] - , [2M-H+Gly] - (negative LSIMS), where n=1-3, are formed. Reactions leading to the formation of adduct-ions are suggested. (authors)

  12. Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

    Science.gov (United States)

    Scalabrin, Matteo; Quintieri, Luigi; Palumbo, Manlio; Riccardi Sirtori, Federico; Gatto, Barbara

    2017-02-20

    The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

  13. DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by 32P-postlabeling

    International Nuclear Information System (INIS)

    Randerath, E.; Zhou, G.D.; Donnelly, K.C.; Safe, S.H.; Randerath, K.

    1996-01-01

    In the present study, a mouse bioassay was used in combination with 32 P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6 x 10 6 DNA nucleotides in skin (both extracts) and one adduct in 3.2 x 10 7 or 1.2 x 10 7 DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the 32 P-labeled derivative of the reaction product of 7β, 8α-dihydroxy-9α, 10α-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) with N 2 of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. (orig.) (orig.). With 5 figs., 6 tabs

  14. Interaction between CYP1A1 polymorphisms and exposure to environmental tobacco smoke in the modulation of lymphocyte bulky DNA adducts and chromosomal aberrations

    Czech Academy of Sciences Publication Activity Database

    Georgiadis, P.; Topinka, Jan; Vlachodimitropoulos, D.; Stoikidou, M.; Gioka, M.; Stephanou, G.; Autrup, H.; Demopoulos, N. A.; Katsouyanni, K.; Šrám, Radim; Kyrtopoulos, S. A.

    2005-01-01

    Roč. 26, č. 1 (2005), s. 93-101 ISSN 0143-3334 Institutional research plan: CEZ:AV0Z50390512 Keywords : DNA adducts * aberrant cells Subject RIV: DI - Air Pollution ; Quality Impact factor: 5.108, year: 2005

  15. Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

    Science.gov (United States)

    Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

    2017-11-20

    DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

  16. Dietary phenolics as anti-mutagens and inhibitors of tobacco-related DNA adduction in the urothelium of smokers.

    Science.gov (United States)

    Malaveille, C; Hautefeuille, A; Pignatelli, B; Talaska, G; Vineis, P; Bartsch, H

    1996-10-01

    Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified N-(deoxyguanosine-8-yl)-4-aminobiphenyl adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. Urinary anti-mutagenicity in vitro appears thus to be a good indicator of the anti-genotoxicity exerted by substances excreted in urine, that protect the bladder mucosal cells (and possibly other cells) against DNA damage. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n = 18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (quercetin, isorhamnetin and naringenin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited noncompetitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Several structural features of the flavonoids were identified as necessary for the inhibition of PhIP and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxiline mutagenicity. Fractionation by reverse-phase HPLC and subsequent analysis of two urinary extracts, showed the presence of several anti

  17. Comparative synchronous fluorescence spectrophotometry and 32P-postlabeling analysis of PAH-DNA adducts in human lung and the relationship to TP53 mutations

    DEFF Research Database (Denmark)

    Andreassen, Åshild; Kure, Elin H.; Nielsen, Per Sabro

    1996-01-01

    Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)...

  18. 7-N-Acetylcysteine-pyrrole conjugate-A potent DNA reactive metabolite of pyrrolizidine alkaloids.

    Science.gov (United States)

    He, Xiaobo; Ma, Liang; Xia, Qingsu; Fu, Peter P

    2016-10-01

    Plants containing pyrrolizidine alkaloids (PAs) are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs) that are capable of alkylating cellular DNA and proteins, form (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP) and DHP. Reactions of 7-NAC-DHP with 2'-deoxyguanosine (dG), 2'-deoxyadenosine (dA), and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation. Copyright © 2016. Published by Elsevier B.V.

  19. 7-N-Acetylcysteine-pyrrole conjugate—A potent DNA reactive metabolite of pyrrolizidine alkaloids

    Directory of Open Access Journals (Sweden)

    Xiaobo He

    2016-10-01

    Full Text Available Plants containing pyrrolizidine alkaloids (PAs are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs that are capable of alkylating cellular DNA and proteins, form (±-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP and DHP. Reactions of 7-NAC-DHP with 2′-deoxyguanosine (dG, 2′-deoxyadenosine (dA, and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation.

  20. Impact of phase I or phase II enzyme polymorphisms on lymphocyte DNA adducts in subjects exposed to urban air pollution and environmental tobacco smoke

    Czech Academy of Sciences Publication Activity Database

    Georgiadis, P.; Demopoulos, N. A.; Topinka, Jan; Stephanou, G.; Stoikidou, M.; Bekyrou, M.; Katsouyianni, K.; Šrám, Radim; Autrup, H.; Kyrtopoulos, S. A.

    2004-01-01

    Roč. 149, 1-3 (2004), s. 269-280 ISSN 0378-4274 Institutional research plan: CEZ:AV0Z5039906 Keywords : DNA adducts Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 2.571, year: 2004

  1. DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by {sup 32}P-postlabeling

    Energy Technology Data Exchange (ETDEWEB)

    Randerath, E. [Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX (United States); Zhou, G.D. [Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX (United States); Donnelly, K.C. [Department of Veterinary Anatomy and Public Health, Texas A and M University, College Station, TX (United States); Safe, S.H. [Department of Veterinary Physiology/Pharmacology, Texas A and M University, College Station, TX (United States); Randerath, K. [Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX (United States)

    1996-09-01

    In the present study, a mouse bioassay was used in combination with {sup 32}P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6 x 10{sup 6} DNA nucleotides in skin (both extracts) and one adduct in 3.2 x 10{sup 7} or 1.2 x 10{sup 7} DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the {sup 32}P-labeled derivative of the reaction product of 7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) with N{sup 2} of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. (orig.) (orig.). With 5 figs., 6 tabs.

  2. DNA methylation and memory formation.

    Science.gov (United States)

    Day, Jeremy J; Sweatt, J David

    2010-11-01

    Memory formation and storage require long-lasting changes in memory-related neuronal circuits. Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for an epigenetic mechanism in learning and long-term memory maintenance and raise apparent conundrums and questions. For example, it is unclear how DNA methylation might be reversed during the formation of a memory, how changes in DNA methylation alter neuronal function to promote memory formation, and how DNA methylation patterns differ between neuronal structures to enable both consolidation and storage of memories. Here we evaluate the existing evidence supporting a role for DNA methylation in memory, discuss how DNA methylation may affect genetic and neuronal function to contribute to behavior, propose several future directions for the emerging subfield of neuroepigenetics, and begin to address some of the broader implications of this work.

  3. Multiple analyte adduct formation in liquid chromatography-tandem mass spectrometry - Advantages and limitations in the analysis of biologically-related samples.

    Science.gov (United States)

    Dziadosz, Marek

    2018-05-01

    Multiple analyte adduct formation was examined and discussed in the context of reproducible signal detection in liquid chromatography-tandem mass spectrometry applied in the analysis of biologically-related samples. Appropriate infusion solutions were prepared in H 2 O/methanol (3/97, v/v) with 1 mM sodium acetate and 10 mM acetic acid. An API 4000 QTrap tandem mass spectrometer was used for experiments performed in the negative scan mode (-Q1 MS) and the negative enhanced product ion mode (-EPI). γ‑Hydroxybutyrate and its deuterated form were used as model compounds to highlight both the complexity of adduct formation in popular mobile phases used and the effective signal compensation by the application of isotope-labelled analytes as internal standards. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Imidazolidinone adducts of peptides and hemoglobin

    International Nuclear Information System (INIS)

    San George, R.C.; Hoberman, H.D.

    1986-01-01

    Acetaldehyde reacts selectively with the terminal amino groups of the α and β chains of hemoglobin to form stable adducts, the structures of which, based on 13 C NMR studies, are proposed to be diastereomeric 2-methyl imidazolidin-4-ones. In this scheme, acetaldelhyde forms a reversible Schiff base with the α-amino groups of the polypeptide chains which cyclize with the amide nitrogen of the first peptide bond to form the stable imidazolidinone adducts. In support of this mechanism, the authors found that in following the reaction of the peptide val-gly-gly with [1,2- 13 C] acetaldehyde, 13 C NMR resonances attributed to a Schiff base (δ = 170 ppm) were observed which slowly disappeared prior to appearance of resonances from a pair of stable adducts (δ = 70 and 71 ppm) believed to be the diastereomeric imidazolidinones. Schiff base formation appeared to limit the overall rate. Tetraglycine reacted in a similar manner but with a resonance from a single stable adduct observed representing the enantiomeric imidazolidinone adducts of this peptide. Peptides with proline in position 2 should be incapable of forming imidazolidinones, and the authors found that ala-pro-gly did in fact fail to form a stable adduct with acetaldehyde. The 2-methyl imidazolidin-4-one adducts of hemoglobin may be useful in determining the contribution of the amino terminal groups to the structure and functional properties of hemoglobins

  5. Detection of environmental carcinogens-DNA

    International Nuclear Information System (INIS)

    Pfohl-Leszkowicz, A.; Guillemaut, G.; Rether, B.; Masfaraud, J.F.; Haguenoer, J.M.

    1995-01-01

    It has been estimated that majority of human cancer is due to environmental factors including pollutants in air, soil, water and food, work places exposure and personal habits such as smoking. After penetration in organism, xenobiotics could be directly excreted or are bio transformed by oxidation or reduction in more hydrophilic compounds which could be conjugate and then eliminated in urine. But in some case, the biotransformation leads to electrophilic compounds which interact with macromolecules such as DNA, forming addition products named adduct. The 32 P post-labelling method, inspired by recent developments in the methodology for sequencing nucleic acids, is an extremely sensitive method for assessing and quantifying DNA adducts and is applicable to structurally diverse classes of chemicals. In the first study, we have analysed hepatic DNA from fish living in the River Rhone downstream and upstream from a polychlorinated biphenyl incineration plant. Our results suggest that fish are exposed to genotoxic chemicals. In another study, leave DNA from healthy and declining hop were analysed. The total adduct level is 3 time higher in declining hop. A comparison between DNA adducts from several vegetal cells cultured in presence of heptachlor and DNA adduct in declining hop, confirmed the implication of heptachlor. In these examples, our data indicate the usefulness of the 32 P-post labelling method to assess the contamination of the environment by genotoxic pollutants. Epidemiological data suggested that increasing exposure to airborne PAH contributes to increase risk cancer in this population. Exposure-dependent adducts were detected in while blood cells in coke oven workers. The adduct levels is function of the level of pollutant. In the last example we have analysed lung tissue from patient with cancer. We observed many adducts in peritumoral tissue, while few adducts could be detected in tumoral tissues. (author)

  6. Mono- and Di-Alkylation Processes of DNA Bases by Nitrogen Mustard Mechlorethamine.

    Science.gov (United States)

    Larrañaga, Olatz; de Cózar, Abel; Cossío, Fernando P

    2017-12-06

    The reactivity of nitrogen mustard mechlorethamine (mec) with purine bases towards formation of mono- (G-mec and A-mec) and dialkylated (AA-mec, GG-mec and AG-mec) adducts has been studied using density functional theory (DFT). To gain a complete overview of DNA-alkylation processes, direct chloride substitution and formation through activated aziridinium species were considered as possible reaction paths for adduct formation. Our results confirm that DNA alkylation by mec occurs via aziridine intermediates instead of direct substitution. Consideration of explicit water molecules in conjunction with polarizable continuum model (PCM) was shown as an adequate computational method for a proper representation of the system. Moreover, Runge-Kutta numerical kinetic simulations including the possible bisadducts have been performed. These simulations predicted a product ratio of 83:17 of GG-mec and AG-mec diadducts, respectively. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells.

    Science.gov (United States)

    Lamy, Evelyn; Kassie, Fekadu; Gminski, Richard; Schmeiser, Heinz H; Mersch-Sundermann, Volker

    2004-01-15

    3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.

  8. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    Science.gov (United States)

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  9. Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part I: Bulky DNA adducts

    Czech Academy of Sciences Publication Activity Database

    Topinka, Jan; Milcová, Alena; Líbalová, Helena; Nováková, Zuzana; Rössner ml., Pavel; Balascak, I.; Šrám, Radim

    2009-01-01

    Roč. 669, 1-2 (2009), s. 13-19 ISSN 0027-5107 R&D Projects: GA MŠk 2B06088 Institutional research plan: CEZ:AV0Z50390512 Keywords : DNA adducts * PAHs * complex mixtures Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.556, year: 2009

  10. Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA

    International Nuclear Information System (INIS)

    Paramio, J.M.; Bauluz, C.; Vidania, R. de

    1986-01-01

    Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs

  11. THE EFFECT OF ROUTE OF ADMINISTRATION OF POLYCYCLIC AROMATIC HYDROCARBONS ON DNA ADDUCTION AND CYTOGENETIC DAMAGE IN PERIPHERAL BLOOD LYMPHOCYTES OF MICE AND RATS

    Science.gov (United States)

    Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic endpoints and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[a]anthracene (B[a]A), benzo[b]fl...

  12. Probe DNA-Cisplatin Interaction with Solid-State Nanopores

    Science.gov (United States)

    Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

    2014-03-01

    Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

  13. 2'-Deoxyguanosine as a surrogate trapping agent for DNA reactive drug metabolites.

    Science.gov (United States)

    Häkkinen, Merja R; Laine, Jaana E; Juvonen, Risto O; Auriola, Seppo; Häyrinen, Jukka; Pasanen, Markku

    2011-11-10

    Drug metabolism can result in the production of highly reactive metabolites that may form adducts with cellular macromolecules, and thus initiate adverse drug reactions, cause toxicity, and even require the withdrawal of drug from the market. In this study, a 2'-deoxyguanosine (dG)-based chemical trapping test system was developed for use as a fast screening tool for DNA adducting metabolites of new drug candidates. Reactive metabolites were generated from parent compounds in in vitro incubations with phenobarbital-induced mouse liver microsomes, human liver microsomes and different recombinant human CYP enzymes in the presence of dG. The formed dG-adducts were separated, characterized and their stability was studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was evaluated with six test compounds, aflatoxin B1, estrone, clozapine, tolcapone, ticlopidine and imipramine. Estrone and aflatoxin B1 formed dG adducts with phenobarbital-induced mouse liver microsomes, human liver microsomes and human recombinant CYP enzymes. Adduct formation was also observed with tolcapone when phenobarbital-induced mouse liver microsomes were used as the enzyme source. The stability of each formed adduct was independent of the different enzyme sources. No dG-adducts were identified with ticlopidine, clozapine and imipramine. Compared to other classical DNA reactivity tests, e.g. Ames test, the present surrogate endpoint, the dG adduct, is faster, enables the characterization of the formed compounds, and also permits the investigation of more unstable adducts. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Effect of Watson-Crick and Hoogsteen base pairing on the conformational stability of C8-phenoxyl-2'-deoxyguanosine adducts.

    Science.gov (United States)

    Millen, Andrea L; Churchill, Cassandra D M; Manderville, Richard A; Wetmore, Stacey D

    2010-10-14

    Bulky DNA addition products (adducts) formed through attack at the C8 site of guanine can adopt the syn orientation about the glycosidic bond due to changes in conformational stability or hydrogen-bonding preferences directly arising from the bulky group. Indeed, the bulky substituent may improve the stability of (non-native) Hoogsteen pairs. Therefore, such adducts often result in mutations upon DNA replication. This work examines the hydrogen-bonded pairs between the Watson-Crick and Hoogsteen faces of the ortho or para C8-phenoxyl-2'-deoxyguanosine adduct and each natural (undamaged) nucleobase with the goal to clarify the conformational preference of this type of damage, as well as provide insight into the likelihood of subsequent mutation events. B3LYP/6-311+G(2df,p)//B3LYP/6-31G(d) hydrogen-bond strengths were determined using both nucleobase and nucleoside models for adduct pairs, as well as the corresponding complexes involving natural 2'-deoxyguanosine. In addition to the magnitude of the binding strengths, the R(C1'···C1') distances and ∠(N9C1'C1') angles, as well as the degree of propeller-twist and buckle distortions, were carefully compared to the values observed in natural DNA strands. Due to structural changes in the adduct monomer upon inclusion of the sugar moiety, the monomer deformation energy significantly affects the relative hydrogen-bond strengths calculated with the nucleobase and nucleoside models. Therefore, we recommend the use of at least a nucleoside model to accurately evaluate hydrogen-bond strengths of base pairs involving flexible, bulky nucleobase adducts. Our results also emphasize the importance of considering both the magnitude of the hydrogen-bond strength and the structure of the base pair when predicting the preferential binding patterns of nucleobases. Using our best models, we conclude that the Watson-Crick face of the ortho phenoxyl adduct forms significantly more stable complexes than the Hoogsteen face, which

  15. Ultrastructural morphology and localisation of cisplatin-induced platinum-DNA adducts in a cisplatin-sensitive and -resistant human small cell lung cancer cell line using electron microscopy

    NARCIS (Netherlands)

    Meijer, C; van Luyn, MJA; Nienhuis, EF; Blom, N; Mulder, NH; de Vries, EGE

    2001-01-01

    Ultrastructural morphology (transmission electron microscopy) and localisation of cisplatin-induced platinum (Pt)-DNA adducts (immunoelectron microscopy) were analysed in the human small cell lung cancer cell line GLC(4) and its 40-fold in vitro acquired cisplatin-resistant subline GLC(4)-CDDP,

  16. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    International Nuclear Information System (INIS)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-01-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.

  17. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Kumer, Sean C.; Schmitt, Timothy M. [Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Ding, Wen-Xing [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2015-12-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.

  18. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    International Nuclear Information System (INIS)

    Kim, Hyun-Suk; Guo, Chunlu; Thompson, Eric L.; Jiang, Yanlin; Kelley, Mark R.; Vasko, Michael R.; Lee, Suk-Hee

    2015-01-01

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons

  19. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Suk [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Guo, Chunlu; Thompson, Eric L. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Jiang, Yanlin [Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Lee, Suk-Hee, E-mail: slee@iu.edu [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States)

    2015-09-15

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

  20. Supercoil Formation During DNA Melting

    Science.gov (United States)

    Sayar, Mehmet; Avsaroglu, Baris; Kabakcioglu, Alkan

    2009-03-01

    Supercoil formation plays a key role in determining the structure-function relationship in DNA. Biological and technological processes, such as protein synthesis, polymerase chain reaction, and microarrays relys on separation of the two strands in DNA, which is coupled to the unwinding of the supercoiled structure. This problem has been studied theoretically via Peyrard-Bishop and Poland-Scheraga type models, which include a simple representation of the DNA structural properties. In recent years, computational models, which provide a more realtistic representaion of DNA molecule, have been used to study the melting behavior of short DNA chains. Here, we will present a new coarse-grained model of DNA which is capable of simulating sufficiently long DNA chains for studying the supercoil formation during melting, without sacrificing the local structural properties. Our coarse-grained model successfully reproduces the local geometry of the DNA molecule, such as the 3'-5' directionality, major-minor groove structure, and the helical pitch. We will present our initial results on the dynamics of supercoiling during DNA melting.

  1. Fullerene–Carbene Lewis Acid–Base Adducts

    KAUST Repository

    Li, Huaping; Risko, Chad; Seo, Jung Hwa; Campbell, Casey; Wu, Guang; Brédas, Jean-Luc; Bazan, Guillermo C.

    2011-01-01

    The reaction between a bulky N-heterocylic carbene (NHC) and C60 leads to the formation of a thermally stable zwitterionic Lewis acid-base adduct that is connected via a C-C single bond. Low-energy absorption bands with weak oscillator strengths

  2. Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O6-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Chadt, J.; Sýkora, D.; Nilsson, R.; Vodička, Pavel

    2008-01-01

    Roč. 867, č. 1 (2008), s. 43-48 ISSN 1570-0232 Grant - others:EU(SE) 505609 Source of funding: R - rámcový projekt EK Keywords : DNA adducts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.500, year: 2008

  3. Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.

    Science.gov (United States)

    Dziegielewska, Barbara; Beerman, Terry A; Bianco, Piero R

    2006-09-01

    To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.

  4. NITRO MUSK ADDUCTS OF RAINBOW TROUT ...

    Science.gov (United States)

    Rainbow trout and other fish species can serve as 'sentinel' species for the assessment of ecological status and the presence of certain environmental contaminants. As such they act as bioindicators of exposure. Here we present seminal data regarding dose-response and toxicokinetics of trout hemoglobin adduct formation from exposure to nitro musks that are frequently used as fragrance ingredients in formulations of personal care products. Hemoglobin adducts serve as biomarkers of exposure of the sentinel species as we have shown in previous studies of hemoglobin adducts formed in trout and environmental carp exposed to musk xylene (MX) and musk ketone (MK). Gas chromatography-electron capture negative ion chemical ionization-mass spectrometry (GC-NICI-MS) employing selected ion monitoring is used to measure 4-amino-MX (4-AMX), 2-amino-MX (2-AMX), and 2-amino-MK (2-AMK) released by alkaline hydrolysis from the sulfinamide adducts of hemoglobin. Dose-response and toxicokinetics were investigated using this sensitive method for analysis of these metabolites. In the dose-response investigation, the concentrations of 4-AMX and 2-2AMX are observed to pass through a maximum at 0.10 mg/g. In the case of 2-AMK, the adduct concentration is almost the same at dosages in the range of 0.030 to 0.10 mg/g. For toxicokinetics, the concentration of the metabolites in the Hb reaches a maximum in the 3-day sample after administration of MX or MK. Further elimination of the metabo

  5. Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065

    International Nuclear Information System (INIS)

    Zsido, T.J.; Woynarowski, J.M.; Baker, R.M.; Gawron, L.S.; Beerman, T.A.

    1991-01-01

    CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand break. The authors have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, They observe thermally induced breakage of [ 14 C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [ 3 H]thymidine-labeled untreated cells with the [ 14 C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10 6 base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The results of this study demonstrate that CC-1065 does generate heat-labile sites with the cellular DNA of intact cells and suggest that a mechanism of cytotoxic action of CC-1065 involves formation of covalent adducts to DNA

  6. 32P-labeling test for DNA damage

    International Nuclear Information System (INIS)

    Randerath, K.; Reddy, M.V.; Gupta, R.C.

    1981-01-01

    Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32 P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, β-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [γ- 32 P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'- 32 P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyl-eneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10 5 DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding

  7. Substrate specificity of Micrococcus luteus uv endonuclease and its overlap with DNA photolyase activity

    International Nuclear Information System (INIS)

    Patrick, M.H.

    1975-01-01

    The action of an endonuclease from Micrococcus luteus that operates on uv damage in DNA overlaps with that of DNA photolyase from yeast: homo- and heterocyclobutane dipyrimidines in DNA are substrates for both enzymes, but pyrimidine adducts or the spore photoproduct in DNA are not. As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photolyase-cyclobutane dipyrimidine complex can prevent nicking by the endonuclease

  8. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution

    International Nuclear Information System (INIS)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz; Andre, Veronique; Le Goff, Jeremie; Sichel, Francois; Roy Saint-Georges, Francoise; Mulliez, Philippe

    2012-01-01

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% 2 /g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). 32 P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC 50 = 74.63 μg/mL and L132: LC-5-0 = 75.36 μg/mL). Exposure to desorbed particles (MA: C1= 61.11 μg/mL and L132 : C2 = 61.71 μg/mL) and B[a]P (1 μM) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that they were competent in terms of metabolic activation of PAHs. The

  9. Flanking bases influence the nature of DNA distortion by platinum 1,2-intrastrand (GG cross-links.

    Directory of Open Access Journals (Sweden)

    Debadeep Bhattacharyya

    Full Text Available The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP and oxaliplatin (OX are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR spectroscopy and molecular dynamics (MD simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.

  10. Scavenging of Toxic Acrolein by Resveratrol and Hesperetin and Identification of Adducts.

    Science.gov (United States)

    Wang, Weixin; Qi, Yajing; Rocca, James R; Sarnoski, Paul J; Jia, Aiqun; Gu, Liwei

    2015-11-04

    The objective of this study was to investigate the ability of resveratrol and hesperetin to scavenge acrolein at pH 7.4 and 37 °C. About 6.4 or 5.2% of acrolein remained after reaction with resveratrol or hesperetin for 12 h at equimolar concentrations. An acrolein-resveratrol adduct and two acrolein-hesperetin adducts were isolated. Their structures were elucidated using mass and NMR spectroscopy. Acrolein reacted with resveratrol at the C-2 and C-3 positions through nucleophilic addition and formed an additional heterocyclic ring. Two similar monoacrolein-conjugated adducts were identified for hesperetin. Spectroscopic data suggested each acrolein-hesperetin adduct was a mixture of four stereoisomers due to the existence of two chiral carbon atoms. Yield of adducts was low at pH 5.4 but increased at pH 7.4 and 8.4. Higher pH also promoted the formation of diacrolein adducts. Results suggest that resveratrol and hesperetin exert health benefits in part through neutralizing toxic acrolein in vivo.

  11. Kinetics, mechanism and thermodynamics of bisulfite-aldehyde adduct formation

    Energy Technology Data Exchange (ETDEWEB)

    Olson, T.M.; Boyce, S.D.; Hoffmann, M.R.

    1986-04-01

    The kinetics and mechanism of bisulfite addition to benzaldehyde were studied at low pH in order to assess the importance of this reaction in stabilizing S(IV) in fog-, cloud-, and rainwater. Previously, the authors established that appreciable concentrations of the formaldehyde-bisulfite adduct (HMSA) are often present in fogwater. Measured HMSA concentrations in fogwater often do not fully account for observed excess S(IV) concentrations, however, so that other S(IV)-aldehyde adducts may be present. Reaction rates were determined by monitoring the disappearance of benzaldehyde by U.V. spectrophotometry under pseudo-first order conditions, (S(IV))/sub T/ >>(phi-CHO)/sub T/, in the pH range 0 - 4.4 at 25/sup 0/C. The equilibrium constant was determined by dissolving the sodium salt of the addition compound in a solution adjusted to pH 3.9, and measuring the absorbance of the equilibrated solution at 250 nm. A literature value of the extinction coefficient for benzaldehyde was used to calculate the concentration of free benzaldehyde. All solutions were prepared under an N/sub 2/ atmosphere using deoxygenated, deionized water and ionic strength was maintained at 1.0 M with sodium chloride.

  12. Mechanism for Clastogenic Activity of Naphthalene

    Energy Technology Data Exchange (ETDEWEB)

    Buchholz, Bruce A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-09-29

    Naphthalene incubations form DNA adducts in vitro in a dose dependent manner in both mouse and rat tissues. Rodent tissue incubations with naphthalene indicate that naphthalene forms as many DNA adducts as Benzo(a)pyrene, a known DNA binding carcinogen. The mouse airway has the greatest number of DNA adducts, corresponding to the higher metabolic activation of naphthalene in this location. Both rat tissues, the rat olfactory (tumor target) and the airways (non-tumor target), have similar levels of NA-DNA adducts, indicating that short term measures of initial adduct formation do not directly correlate with sites of tumor formation in the NTP bioassays.

  13. Mechanism for Clastogenic Activity of Naphthalene

    Energy Technology Data Exchange (ETDEWEB)

    Buchholz, Bruce A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-06-24

    Naphthalene incubations form DNA adducts in vitro in a dose dependent manner in both mouse and rat tissues. Rodent tissue incubations with naphthalene indicate that naphthalene forms as many DNA adducts as Benzo(a)pyrene, a known DNA binding carcinogen. The mouse airway has the greatest number of DNA adducts, corresponding to the higher metabolic activation of naphthalene in this location. Both rat tissues, the rat olfactory (tumor target) and the airways (non-tumor target), have similar levels of NA-DNA adducts, indicating that short term measures of initial adduct formation do not directly correlate with sites of tumor formation in the NTP bioassays.

  14. Oxidation and adduct formation of xenobiotics in a microfluidic electrochemical cell with boron doped diamond electrodes and an integrated passive gradient rotation mixer

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Wigger, Tina; Ma, Liwei; Odijk, Mathieu; Olthuis, Wouter; Karst, U.; van den Berg, Albert

    2016-01-01

    Reactive xenobiotic metabolites and their adduct formation with biomolecules such as proteins are important to study as they can be detrimental to human health. Here, we present a microfluidic electrochemical cell with integrated micromixer to study phase I and phase II metabolism as well as protein

  15. Characterization of model peptide adducts with reactive metabolites of naphthalene by mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Nathalie T Pham

    Full Text Available Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.

  16. Carcinogen-DNA interaction study by base sequence footprinting. Progress report, July 1, 1985-January 21, 1986

    International Nuclear Information System (INIS)

    Bases, R.

    1986-01-01

    Acetyl-aminofluorene (AAF) modified plasmid pSV 2 CAT is being studied to learn how the adducts influence expression of chloramphenicol acetyltransferase (CAT) genes. phi X-174 RF DNA exhibits specific base sequence abnormalities induced by the formation of AAF adducts. The DNAase I sensitive state of AAF modified DNA sequences could presumably lead to enhanced expression of genes since it is a well-known characteristic of active or potentially active derepressed genes. DNAase I hypersensitive sites are necessary but not sufficient for transcription. We observed enhanced expression of CAT genes in CV-1 cells after transfection with modified plasmids, using electroporation to introduce the plasmids into the cells. 34 refs., 2 figs

  17. Fullerene–Carbene Lewis Acid–Base Adducts

    KAUST Repository

    Li, Huaping

    2011-08-17

    The reaction between a bulky N-heterocylic carbene (NHC) and C60 leads to the formation of a thermally stable zwitterionic Lewis acid-base adduct that is connected via a C-C single bond. Low-energy absorption bands with weak oscillator strengths similar to those of n-doped fullerenes were observed for the product, consistent with a net transfer of electron density to the C60 core. Corroborating information was obtained using UV photoelectron spectroscopy, which revealed that the adduct has an ionization potential ∼1.5 eV lower than that of C60. Density functional theory calculations showed that the C-C bond is polarized, with a total charge of +0.84e located on the NHC framework and -0.84e delocalized on the C 60 cage. The combination of reactivity, characterization, and theoretical studies demonstrates that fullerenes can behave as Lewis acids that react with C-based Lewis bases and that the overall process describes n-doping via C-C bond formation. © 2011 American Chemical Society.

  18. Biomarkers of genotoxicity of air pollution (the AULIS project): bulky DNA adducts in subjects with moderate to low exposures to airborne polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke and other parameters

    DEFF Research Database (Denmark)

    Georgiadis, P.; Topinka, J.; Stoikidou, M.

    2001-01-01

    The levels of bulky DNA adducts were measured by (32)P-post-labelling in lymphocytes of 194 non-smoking students living in the city of Athens and the region of Halkida, Greece, once in the winter and again in the following summer. Personal exposures to particulate-bound polycyclic aromatic hydroc...... with an enhancement of adduct levels and the effect was strengthened when only individuals unexposed to ETS were taken into consideration. No significant effects were observed for other dietary parameters or factors reflecting exposure to air pollution....... surroundings with a minimal burden of urban air pollution. The remaining Halkida subjects had intermediate levels, while Athens subjects showed the lowest levels. This trend, which was observed over both monitoring seasons, consistently paralleled the variation in three markers of exposure to environmental......) positive correlations were observed between DNA adducts and (i) measured personal exposures to chrysene or benzo[a]pyrene, (ii) time of declared ETS exposure and (iii) chrysene/benzo[g,h,i] perylene ratios. These correlations suggest that, for a group suffering minimal exposure to urban air pollution...

  19. 32P-postlabeling assay for carcinogen-DNA adducts: description of beta shielding apparatus and semi-automatic spotting and washing devices that facilitate the handling of multiple samples

    International Nuclear Information System (INIS)

    Reddy, M.V.; Blackburn, G.R.

    1990-01-01

    The utilization of the 32 P-postlabeling assay in combination with TLC for the sensitive detection and estimation of aromatic DNA adducts has been increasing. The procedure consists of 32 P-labeling of carcinogen-adducted 3'-nucleotides in the DNA digests using γ- 32 P ATP and polynucleotide kinase, separation of 32 P-labeled adducts by TLC, and their detection by autoradiography. During both 32 P-labeling and initial phases of TLC, a relatively high amount of γ- 32 P ATP is handled when 30 samples are processed simultaneously. We describe the design of acrylic shielding apparatus, semi-automatic TLC spotting devices, and devices for development and washing of multiple TLC plates, which not only provide substantial protection from exposure to 32 P beta radiation, but also allow quick and easy handling of a large number of samples. Specifically, the equipment includes: (i) a multi-tube carousel rack having 15 wells to hold capless Eppendorf tubes and a rotatable lid with an aperture to access individual tubes; (ii) a pipette shielder; (iii) two semi-automatic spotting devices to apply radioactive solutions to TLC plates; (iv) a multi-plate holder for TLC plates; and (v) a mechanical device for washing multiple TLC plates. Item (i) is small enough to be held in one-hand, vortexed, and centrifuged to mix the solutions in each tube while beta radiation is shielded. Items (iii) to (iv) aid in the automation of the assay. (author)

  20. Syntheses of DNA adducts of two heterocyclic amines, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) and identification of DNA adducts in organs from rats dosed with MeA alpha C

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Frandsen, Henrik Lauritz; Pfau, W.

    2004-01-01

    2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (AalphaC) are mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. MeAalphaC and AalphaC are activated to mutagenic metabolites by cytochrome P450-mediated N-oxidation...... by reaction of the parent amines with acetylated guanine N3-oxide. N-2-OH-MeAalphaC and N-2-OH-AalphaC reacted with calf thymus DNA after addition of acetic anhydride. P-32-postlabelling analysis of modified DNA showed one major adduct co-migrating with N-2-(3',5'-diphospho-2'-deoxyguanosin-8-yl...

  1. Early models of DNA damage formation

    International Nuclear Information System (INIS)

    Śmiałek, Małgorzata A

    2012-01-01

    Quantification of DNA damage, induced by various types of incident radiation as well as chemical agents, has been the subject of many theoretical and experimental studies, supporting the development of modern cancer therapy. The primary observations showed that many factors can lead to damage of DNA molecules. It became clear that the development of experimental techniques for exploring this phenomenon is required. Another problem was simultaneously dealt with, anticipating on how the damage is distributed within the double helix of the DNA molecule and how the single strand break formation and accumulation can influence the lethal double strand break formation. In this work the most important probabilistic models for DNA strand breakage and damage propagation are summarized and compared.

  2. Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from a Drug Acyl Glucuronide Metabolite: Multiple Albumin Adductions in Diclofenac Patients

    Science.gov (United States)

    Hammond, Thomas G.; Meng, Xiaoli; Jenkins, Rosalind E.; Maggs, James L.; Castelazo, Anahi Santoyo; Regan, Sophie L.; Bennett, Stuart N. L.; Earnshaw, Caroline J.; Aithal, Guruprasad P.; Pande, Ira; Kenna, J. Gerry; Stachulski, Andrew V.; Park, B. Kevin

    2014-01-01

    Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-related hypersensitivities had either a single drug-derived adduct or one of five combinations of 2–8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein’s 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug’s AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association. PMID:24902585

  3. Base sequence effects on DNA replication influenced by bulky adducts. Final report, March 1, 1995--February 28, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Geacintov, N.E.

    1997-05-31

    Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that are present in air, food, and water. While PAH compounds are chemically inert and are sparingly soluble in aqueous solutions, in living cells they are metabolized to a variety of oxygenated derivatives, including the high mutagenic and tumorigenic diol epoxide derivatives. The diol epoxides of the sterically hindered fjord region compound benzo[c]phenanthrene (B[c]PhDE) are among the most powerful tumorigenic compounds in animal model test systems. In this project, site-specifically modified oligonucleotides containing single B[c]PhDE-N{sup 6}-dA lesions derived from the reactions of the 1S,2R,3R,4S and 1R,2S,3S,4R diol epoxides of B[c]PhDE with dA residues were synthesized. The replication of DNA catalyzed by a prokaryotic DNA polymerase (the exonuclease-free Klenow fragment E. Coli Po1 I) in the vicinity of the lesion at base-specific sites on B[c]PhDE-modified template strands was investigated in detail. The Michaelis-Menten parameters for the insertion of single deoxynucleotide triphosphates into growing DNA (primer) strands using the modified dA* and the bases just before and after the dA* residue as templates, depend markedly on the stereochemistry of the B[c]PhDE-modified dA residues. These observations provide novel insights into the mechanisms by which bulky PAH-DNA adducts affect normal DNA replication.

  4. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

    Science.gov (United States)

    Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

  5. Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2/d(GpG)/], built into a specific site in a viral genome

    International Nuclear Information System (INIS)

    Naser, L.J.; Pinto, A.L.; Lippard, S.J.; Essigmann, J.M.

    1988-01-01

    A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH 3 ) 2 /d(GpG)/] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA) x d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. Characterization by pH-dependent 1 H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [γ- 32 P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH 3 ) 2 /d)GpG)/] cross-link induces localized weakening of the DNA double helix. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH 3 ) 2 /d(GpG)/] cross-link is lethal to the single-stranded bacteriophage

  6. Formation of a Hydroxymethylfurfural-Cysteine Adduct and Its Absorption and Cytotoxicity in Caco-2 Cells.

    Science.gov (United States)

    Zhao, Qianzhu; Zou, Yueyu; Huang, Caihuan; Lan, Ping; Zheng, Jie; Ou, Shiyi

    2017-11-15

    Adducts of 5-hydroxymethylfurfural (HMF)-amino acids are formed during food processing and digestion; the elimination capacity of in vitro intestinal digests of biscuits, instant noodles, and potato crisps for HMF is 652, 727, and 540 μg/g, respectively. However, the safety of these adducts is unknown. In this study, an HMF-cysteine adduct named 1-dicysteinethioacetal-5-hydroxymehtylfurfural (DCH), which was found to be produced in the gastrointestinal tract after HMF intake, was prepared to test its effect toward Caco-2 cells. Compared with HMF, the adduct displayed lower cytotoxicity against Caco-2 cells with an IC 50 value of 31.26 mM versus 14.95 mM (HMF). The DCH did not induce cell apoptosis, whereas HMF significantly increased the apoptosis rate after incubation at concentrations of 16, 32, and 48 mM for 72 h. DCH showed an absorption rate considerably lower than that of HMF by Caco-2 cells. Lower absorption of DCH may result in lower toxicity compared with HMF against Caco-2 cells. Intracellular transformation of DCH has been observed.

  7. High Level of Tobacco Carcinogen-Derived DNA Damage in Oral Cells Is an Independent Predictor of Oral/Head and Neck Cancer Risk in Smokers.

    Science.gov (United States)

    Khariwala, Samir S; Ma, Bin; Ruszczak, Chris; Carmella, Steven G; Lindgren, Bruce; Hatsukami, Dorothy K; Hecht, Stephen S; Stepanov, Irina

    2017-09-01

    Exposure to tobacco-specific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH) is recognized to play an important role in the development of oral/head and neck squamous cell cancer (HNSCC). We recently reported higher levels of TSNA-associated DNA adducts in the oral cells of smokers with HNSCC as compared with cancer-free smokers. In this study, we further investigated the tobacco constituent exposures in the same smokers to better understand the potential causes for the elevated oral DNA damage in smokers with HNSCC. Subjects included cigarette smokers with HNSCC (cases, n = 30) and cancer-free smokers (controls, n = 35). At recruitment, tobacco/alcohol use questionnaires were completed, and urine and oral cell samples were obtained. Analysis of urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N '-Nitrosonornicotine (NNN; TSNA biomarkers), 1-hydroxypyrene (1-HOP, a PAH), cotinine, 3'-hydroxycotinine, and the nicotine metabolite ratio (NMR) were performed. Cases and controls differed in mean age, male preponderance, and frequency of alcohol consumption (but not total alcoholic drinks). Univariate analysis revealed similar levels of NNN, 1-HOP, and cotinine between groups but, as reported previously, significantly higher DNA adduct formation in the cases. Multiple regression adjusting for potential confounders showed persistent significant difference in DNA adduct levels between cases and controls [ratio of geometric means, 20.0; 95% CI, 2.7-148.6). Our cohort of smokers with HNSCC demonstrates higher levels of TSNA-derived oral DNA damage in the setting of similar exposure to nicotine and tobacco carcinogens. Among smokers, DNA adduct formation may act as a predictor of eventual development of HNSCC that is independent of carcinogen exposure indicators. Cancer Prev Res; 10(9); 507-13. ©2017 AACR See related editorial by Johnson and Bauman, p. 489 . ©2017 American Association for Cancer Research.

  8. The relevance of monitoring of antibodies against the polycyclic aromatic hydrocarbon (PAH) and PAH-DNA adducts in serum in relation to lung cancer and chronic obstructive pulmonary disease (COPD)

    Czech Academy of Sciences Publication Activity Database

    Pauk, N.; Klimešová, Š.; Kára, J.; Topinka, Jan; Lábaj, J.

    2013-01-01

    Roč. 60, č. 2 (2013), s. 182-187 ISSN 0028-2685 R&D Projects: GA MŠk 2B06150 Institutional support: RVO:68378041 Keywords : polycyclic aromatic hydrocarbons * anti-PAH antibodies * DNA adducts Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 1.642, year: 2013

  9. Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents.

    Science.gov (United States)

    Beranek, D T

    1990-07-01

    Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.

  10. In vivo induction of 4-thiouridine-cytidine adducts in tRNA of E. coli B/r by near-ultraviolet radiation

    International Nuclear Information System (INIS)

    Ramabhadran, T.V.; Fossum, T.; Jagger, J.

    1976-01-01

    Near-ultraviolet (near-UV; 320 to 405 nm) irradiation of Escherichia coli B/r induced the formation in vivo of 4 Srd-Cyd adducts in transfer RNA, as evidenced by (1) fluorescence spectrum changes of tRNA extracted from irradiated cells and reduced with NaBH 4 , (2) thin-layer chromatography on cellulose of hydrolysates of trichloroacetic acid-precipitable extracts of irradiated cells, and (3) comparison of these findings with adduct formation induced by near-UV irradiation of purified mixed tRNA from E.coli. The kinetics of induction of the 4 Srd-Cyd adduct in vivo, and the near-UV fluences required, provided strong support for our earlier hypothesis that formation of these adducts was responsible for near-UV-induced growth delay in E.coli. (author)

  11. A pulse radiolysis study of the electron reaction with DNA in aqueous solution and ice

    International Nuclear Information System (INIS)

    Verberne, J.B.

    1981-01-01

    Research is described into some aspects of the interaction of ionizing radiation with DNA, the main emphasis being on the indirect effect where the radiation causes the formation of intermediates in the surrounding medium which then interact with the DNA. Experiments are described which have been carried out to examine the dependence of the reaction rate constant on ionic strength and counterion charge. They show that for double stranded DNA (dsDNA) the reaction rate constant increases with ionic strength. To see whether quantitative agreement can be obtained between the observations and a physical interpretation the author combines a model for reaction kinetics and a refined model for the electrostatic potential into one to obtain a theoretical rate constant for reaction with a cylindrical polyelectrolyte. Optical absorption spectra of the electron adducts of ss- and dsDNA are shown to exist and they differ significantly from the spectra of the H and OH adducts. The spectra of the electron adducts of the nucleotides have been measured. Experiments are also described devoted to the precursor of the hydrated electron: the thermalized, dry electron. Although from a radiation chemical point of view the experiments yielded a lot of information (a direct observation of the dry electron, the kinetics of electron localization, the role of ice defects in the solvation process, etc.), they proved also that the dry electron does not react with DNA in an ice matrix. (Auth.)

  12. Reaction of misonidazole with DNA radicals and its effect on the template activity of DNA

    International Nuclear Information System (INIS)

    Endoh, Daiji; Kuwabara, Mikinori; Sato, Fumiaki; Yoshii, Giichi.

    1985-01-01

    After calf thymus DNA was gamma-irradiated in the solid state in vacuo and subsequently dissolved in aqueous solution containing misonidazole (3 mM) under hypoxic condition, the frequency of single-strand breaks and alkali-labile sites in DNA and the amount of misonidazole bound to DNA were measured. The presence of misonidazole converted the precursor radicals, which otherwise results in single-strand breaks, to alkali-labile sites, and the amount of alkali-labile sites increased linearly with increasing radiation dose. The amount of misonidazole bound to DNA also increased linearly with increasing radiation dose. The biological meaning of the changes in the frequency of single-strand breaks and alkali-labile sites by the reaction of misonidazole with DNA radicals and of binding misonidazole with DNA was examined using a model system to measure the template activity of DNA for RNA synthesis in vitro. The conversion of DNA radicals to alkali-labile sites protected the radiation-induced decrease in the template activity of DNA, while the adduct formation of misonidazole had no effect on it. (author)

  13. A survey of the sequence-specific interaction of damaging agents with DNA: emphasis on antitumor agents.

    Science.gov (United States)

    Murray, V

    1999-01-01

    This article reviews the literature concerning the sequence specificity of DNA-damaging agents. DNA-damaging agents are widely used in cancer chemotherapy. It is important to understand fully the determinants of DNA sequence specificity so that more effective DNA-damaging agents can be developed as antitumor drugs. There are five main methods of DNA sequence specificity analysis: cleavage of end-labeled fragments, linear amplification with Taq DNA polymerase, ligation-mediated polymerase chain reaction (PCR), single-strand ligation PCR, and footprinting. The DNA sequence specificity in purified DNA and in intact mammalian cells is reviewed for several classes of DNA-damaging agent. These include agents that form covalent adducts with DNA, free radical generators, topoisomerase inhibitors, intercalators and minor groove binders, enzymes, and electromagnetic radiation. The main sites of adduct formation are at the N-7 of guanine in the major groove of DNA and the N-3 of adenine in the minor groove, whereas free radical generators abstract hydrogen from the deoxyribose sugar and topoisomerase inhibitors cause enzyme-DNA cross-links to form. Several issues involved in the determination of the DNA sequence specificity are discussed. The future directions of the field, with respect to cancer chemotherapy, are also examined.

  14. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    Science.gov (United States)

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. In vivo induction of 4-thiouridine-cytidine adducts in tRNA of E. coli B/r by near-ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ramabhadran, T V; Fossum, T; Jagger, J [Texas Univ., Dallas (USA)

    1976-05-01

    Near-ultraviolet (near-UV; 320 to 405 nm) irradiation of Escherichia coli B/r induced the formation in vivo of /sup 4/Srd-Cyd adducts in transfer RNA, as evidenced by (1) fluorescence spectrum changes of tRNA extracted from irradiated cells and reduced with NaBH/sub 4/, (2) thin-layer chromatography on cellulose of hydrolysates of trichloroacetic acid-precipitable extracts of irradiated cells, and (3) comparison of these findings with adduct formation induced by near-UV irradiation of purified mixed tRNA from E.coli. The kinetics of induction of the /sup 4/Srd-Cyd adduct in vivo, and the near-UV fluences required, provided strong support for our earlier hypothesis that formation of these adducts was responsible for near-UV-induced growth delay in E.coli.

  16. Zinc acetylacetonate hydrate adducted with nitrogen donor ligands: Synthesis, spectroscopic characterization, and thermal analysis

    Science.gov (United States)

    Brahma, Sanjaya; Shivashankar, S. A.

    2015-12-01

    We report synthesis, spectroscopic characterization, and thermal analysis of zinc acetylacetonate complex adducted by nitrogen donor ligands, such as pyridine, bipyridine, and phenanthroline. The pyridine adducted complex crystallizes to monoclinic crystal structure, whereas other two adducted complexes have orthorhombic structure. Addition of nitrogen donor ligands enhances the thermal property of these complexes as that with parent metal-organic complex. Zinc acetylacetonate adducted with pyridine shows much higher volatility (106 °C), decomposition temperature (202 °C) as that with zinc acetylacetonate (136 °C, 220 °C), and other adducted complexes. All the adducted complexes are thermally stable, highly volatile and are considered to be suitable precursors for metal organic chemical vapor deposition. The formation of these complexes is confirmed by powder X-ray diffraction, Fourier transform infrared spectroscopy, mass spectroscopy, and elemental analysis. The complexes are widely used as starting precursor materials for the synthesis of ZnO nanostructures by microwave irradiation assisted coating process.

  17. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

    Science.gov (United States)

    Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

    2016-01-08

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Metabolic Toxicity Screening Using Electrochemiluminescence Arrays Coupled with Enzyme-DNA Biocolloid Reactors and Liquid Chromatography–Mass Spectrometry

    Science.gov (United States)

    Hvastkovs, Eli G.; Schenkman, John B.; Rusling, James F.

    2012-01-01

    New chemicals or drugs must be guaranteed safe before they can be marketed. Despite widespread use of bioassay panels for toxicity prediction, products that are toxic to a subset of the population often are not identified until clinical trials. This article reviews new array methodologies based on enzyme/DNA films that form and identify DNA-reactive metabolites that are indicators of potentially genotoxic species. This molecularly based methodology is designed in a rapid screening array that utilizes electrochemiluminescence (ECL) to detect metabolite-DNA reactions, as well as biocolloid reactors that provide the DNA adducts and metabolites for liquid chromatography–mass spectrometry (LC-MS) analysis. ECL arrays provide rapid toxicity screening, and the biocolloid reactor LC-MS approach provides a valuable follow-up on structure, identification, and formation rates of DNA adducts for toxicity hits from the ECL array screening. Specific examples using this strategy are discussed. Integration of high-throughput versions of these toxicity-screening methods with existing drug toxicity bioassays should allow for better human toxicity prediction as well as more informed decision making regarding new chemical and drug candidates. PMID:22482786

  19. Iminium ion chemistry of mitosene DNA alkylating agents. Enriched 13C NMR and isolation studies.

    Science.gov (United States)

    Ouyang, A; Skibo, E B

    2000-05-16

    Described herein is a study of the reductive alkylation chemistry of mitosene antitumor agents. We employed a 13C-enriched electrophilic center to probe the fate of the iminium ion resulting from reductive activation. The 13C-labeled center permitted the identification of complex products resulting from alkylation reactions. In the case of DNA reductive alkylation, the type and number of alkylation sites were readily assessed by 13C NMR. Although there has been much excellent work done in the area of mitosene chemistry and biochemistry, the present study provides a number of new findings: (1) The major fate of the iminium ion is head-to-tail polymerization, even in dilute solutions. (2) Dithionite reductive activation results in the formation of mitosene sulfite esters as well as the previously observed sulfonate adducts. (3) The mitosene iminium ion alkylates the adenosine 6-amino group as well as the guanosine 2-amino group. The identification of the latter adduct was greatly facilitated by the 13C-label at the electrophilic center. (4) The mitosene iminium ion alkylates DNA at both nitrogen and oxygen centers without any apparent base selectivity. The complexity of mitosene reductive alkylation of DNA will require continued adduct isolation studies.

  20. Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B2a and Biological Amines.

    Science.gov (United States)

    Rushing, Blake R; Selim, Mustafa I

    2017-06-19

    Aflatoxin B 2a has been shown to bind to proteins through a dialdehyde intermediate under physiological conditions. The proposed structure of this adduct has been published showing a Schiff base interaction, but adequate verification using structural elucidation instrumental techniques has not been performed. In this work, we synthesized the aflatoxin B 2a amino acid adduct under alkaline conditions, and the formation of a new product was determined using high performance liquid chromatography-time-of-flight mass spectrometry. The resulting accurate mass was used to generate a novel proposed chemical structure of the adduct in which the dialdehyde forms a pyrrole ring with primary amines rather than the previously proposed Schiff base interaction. The pyrrole structure was confirmed using 1 H, 13 C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation NMR and tandem mass spectrometry. Reaction kinetics show that the reaction is overall second order and that the rate increases as pH increases. Additionally, this study shows for the first time that aflatoxin B 2a dialdehyde forms adducts with phosphatidylethanolamines and does so through pyrrole ring formation, which makes it the first aflatoxin-lipid adduct to be structurally identified. Furthermore, oxidation of the pyrrole adduct produced a product that was 16 m/z heavier. When the aflatoxin B 2a -lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and 1 H NMR spectrum that match aflatoxin B 1 -lysine, which suggest the transformation of the pyrrole ring to a pyrrolin-2-one ring. These data give new insight into the fate and chemical properties of biological adducts formed from aflatoxin B 2a as well as possible interferences with known aflatoxin B 1 exposure biomarkers.

  1. DNA adductomics to study the genotoxic effects of red meat consumption with and without added animal fat in rats.

    Science.gov (United States)

    Hemeryck, Lieselot Y; Van Hecke, Thomas; Vossen, Els; De Smet, Stefaan; Vanhaecke, Lynn

    2017-09-01

    Digestion of red and processed meat has been linked to the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs) in the gut. In this study, rats were fed a meat based diet to compare the possible genotoxic effects of red vs. white meat, and the interfering role of dietary fat. To this purpose, liver, duodenum and colon DNA adductomes were analyzed with UHPLC-HRMS. The results demonstrate that the consumed meat type alters the DNA adductome; the levels of 22 different DNA adduct types significantly increased upon the consumption of beef (compared to chicken) and/or lard supplemented beef or chicken. Furthermore, the chemical constitution of the retrieved DNA adducts hint at a direct link with an increase in NOCs and LPOs upon red (and processed) meat digestion, supporting the current hypotheses on the causal link between red and processed meat consumption and the development of colorectal cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II)

    International Nuclear Information System (INIS)

    Sundquist, W.I.; Lippard, S.J.; Stollar, B.D.

    1987-01-01

    Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemothrapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)/sub b/, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations of cis-DDP bound to DNA. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl 2 ], [Pt(dach)Cl 2 ], and [Pt(NH 3 ) 2 (cbdca)] (carboplatin) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA. This antibody crossreacted with unmodified, denatured DNA. The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities

  3. Thermochemistry of adducts of some bivalent transition metal bromides with aniline

    International Nuclear Information System (INIS)

    Dunstan, Pedro Oliver

    2006-01-01

    The compounds [MBr 2 (an) 2 ] (where M is Mn(II), Fe(II), Co(II), Ni(II), Cu(II) or Zn(II); an=aniline) were synthesized and characterized by melting points, elemental analysis, thermal studies, and electronic and IR spectroscopy. The enthalpies of dissolution of the adducts, metal(II) bromides and aniline in methanol, aqueous 1.2M HCl or 25% (v/v) aqueous 1.2M HCl in methanol were measured. The following thermochemical parameters for the adducts have been determined by thermochemical cycles: the standard enthalpies for the Lewis acid/base reactions (Δ r H o ), the standard enthalpies of formation (Δ f H o ), the standard enthalpies of decomposition (Δ D H o ), the lattice standard enthalpies (Δ M H o ) and the standard enthalpies of the Lewis acid/base reactions in the gaseous phase (Δ r H o (g)). The mean bond dissociation enthalpies of the M(II)-nitrogen bonds (D-bar (M?N) ) and the enthalpies of formation of the adducts from the ions in the gaseous phase: M 2+ (g) +Br - (g) +an (g) ->[MBr 2 (an) 2 ] (g) (Δ fi H o ) have been estimated

  4. DNA damage in lung after oral exposure to diesel exhaust particles in Big Blue (R) rats

    DEFF Research Database (Denmark)

    Müller, Anne Kirstine; Farombi, E.O.; Møller, P.

    2004-01-01

    Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts....... Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated...

  5. UV and ionizing radiations induced DNA damage, differences and similarities

    Science.gov (United States)

    Ravanat, Jean-Luc; Douki, Thierry

    2016-11-01

    Both UV and ionizing radiations damage DNA. Two main mechanisms, so-called direct and indirect pathways, are involved in the degradation of DNA induced by ionizing radiations. The direct effect of radiation corresponds to direct ionization of DNA (one electron ejection) whereas indirect effects are produced by reactive oxygen species generated through water radiolysis, including the highly reactive hydroxyl radicals, which damage DNA. UV (and visible) light damages DNA by again two distinct mechanisms. UVC and to a lesser extend UVB photons are directly absorbed by DNA bases, generating their excited states that are at the origin of the formation of pyrimidine dimers. UVA (and visible) light by interaction with endogenous or exogenous photosensitizers induce the formation of DNA damage through photosensitization reactions. The excited photosensitizer is able to induce either a one-electron oxidation of DNA (type I) or to produce singlet oxygen (type II) that reacts with DNA. In addition, through an energy transfer from the excited photosensitizer to DNA bases (sometime called type III mechanism) formation of pyrimidine dimers could be produced. Interestingly it has been shown recently that pyrimidine dimers are also produced by direct absorption of UVA light by DNA, even if absorption of DNA bases at these wavelengths is very low. It should be stressed that some excited photosensitizers (such as psoralens) could add directly to DNA bases to generate adducts. The review will described the differences and similarities in terms of damage formation (structure and mechanisms) between these two physical genotoxic agents.

  6. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: A review

    International Nuclear Information System (INIS)

    Xue Weiling; Warshawsky, David

    2005-01-01

    Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic compounds (HACs) constitute a major class of chemical carcinogens present in the environment. These compounds require activation to electrophilic metabolites to exert their mutagenic or carcinogenic effects. There are three principal pathways currently proposed for metabolic activation of PAH and HAC: the pathway via bay region dihydrodiol epoxide by cytochrome P450 enzymes (CYPs), the pathway via radical cation by one-electron oxidation, and the ortho-quinone pathway by dihydrodiol dehydrogenase (DD). In addition to these major pathways, a brief description of a minor metabolic activation pathway, sulfonation, for PAHs that contain a primary benzylic alcoholic group or secondary hydroxyl group(s) is included in this review. The DNA damages caused through the reactive metabolites of PAH/HAC are described involving the DNA covalent binding to form stable or depurinating adducts, the formation of apurinic sites, and the oxidative damage. The review emphasizes the chemical/biochemical reactions involved in the metabolic processes and the chemical structures of metabolites and DNA adducts

  7. Identification and quantification of drug-albumin adducts in serum samples from a drug exposure study in mice

    NARCIS (Netherlands)

    Switzar, L.; Kwast, L.M.; Lingeman, H.; Giera, M.; Pieters, R.H.H.; Niessen, W.M.A.

    2013-01-01

    The formation of drug-protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug-protein adducts in vivo may lead to a better

  8. A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

    Science.gov (United States)

    Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

    2012-01-01

    Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

  9. Studies in human skin epithelial cell carcinogenesis

    International Nuclear Information System (INIS)

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo[a]pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and 32 P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the 32 P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts

  10. New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation With Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons

    Energy Technology Data Exchange (ETDEWEB)

    Rezaee, Mohammad, E-mail: Mohammad.Rezaee@USherbrooke.ca; Hunting, Darel John; Sanche, Léon

    2013-11-15

    Purpose: To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Methods and Materials: Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure–response curves. Results: The presence of an average of 2 Pt-drug–DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10{sup −4} Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. Conclusion: Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances.

  11. New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation With Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons

    International Nuclear Information System (INIS)

    Rezaee, Mohammad; Hunting, Darel John; Sanche, Léon

    2013-01-01

    Purpose: To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Methods and Materials: Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure–response curves. Results: The presence of an average of 2 Pt-drug–DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10 −4 Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. Conclusion: Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances

  12. DNA dosimetry applied to problems in genetic toxicology

    International Nuclear Information System (INIS)

    Rahn, R.O.; Sellin, H.

    1984-01-01

    Studies have been conducted using uv, metal ions, and polyaromatic hydrocarbons as DNA damaging agents. A method has been devised for removing Pt-base adducts from DNA and for separating these adducts chromatographically. This method has been applied to DNA isolated from tissue culture cells treated with cisplatin. The results indicate that a significant (approx. 35%) portion of the cisplatin binds in a form which is the same as that found in DNA treated in vitro. This adduct consists of two guanine molecules connected by a platinum atom. A very useful tool in photobiological research has been the substitution of BrdUrd for Thd in DNA. Following radiation, debromination and damage to the sugar phosphate backbone results. However, the actual chemical event responsible for the observed enhanced cell killing is not known. Attempts to answer this question have employed the use of IdUrd instead of BrdUrd, because of certain spectroscopic and chemical advantages. Current research deals with the mechanisms by which the uracil radical formed upon dehalogenation reacts with its environment to pick up a hydrogen atom and form uracil

  13. Capturing Labile Sulfenamide and Sulfinamide Serum Albumin Adducts of Carcinogenic Arylamines by Chemical Oxidation

    Science.gov (United States)

    Peng, Lijuan; Turesky, Robert J.

    2013-01-01

    Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by N-oxidation of the exocyclic amine group to produce N-hydroxylated metabolites, which are critical intermediates implicated in toxicity and DNA damage. The arylhydroxylamines and their oxidized arylnitroso derivatives can also react with cysteine (Cys) residues of glutathione or proteins to form, respectively, sulfenamide and sulfinamide adducts. However, sulfur-nitrogen linked adducted proteins are often difficult to detect because they are unstable and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine present in tobacco smoke were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts at the Cys34 residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve as biomarkers of these tobacco and dietary carcinogens. PMID:23240913

  14. Formation of reactive metabolites from benzene

    International Nuclear Information System (INIS)

    Snyder, R.; Jowa, L.; Witz, G.; Kalf, G.; Rushmore, T.

    1986-01-01

    Rat liver mitoplasts were incubated first with [ 3 H]dGTP, to form DNA labeled in G, and then with [ 14 C]benzene. The DNA was isolated and upon isopycnic density gradient centrifugation in CsCl yielded a single fraction of DNA labeled with both [ 3 H] and [ 14 C]. These data are consistent with the covalent binding of one or more metabolites of benzene to DNA. The DNA was enzymatically hydrolyzed to deoxynucleosides and chromatographed to reveal at least seven deoxyguanosine adducts. Further studies with labeled deoxyadenine revealed one adduct on deoxyadenine. [ 3 H]Deoxyguanosine was reacted with [ 14 C]hydroquinone or benzoquinone. The product was characterized using uv, fluorescence, mass and NMR spectroscopy. A proposed structure is described. (orig.)

  15. Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

    Science.gov (United States)

    Châtel, Amélie; Faucet-Marquis, Virginie; Gourlay-Francé, Catherine; Pfohl-Leszkowicz, Annie; Vincent-Hubert, Françoise

    2015-04-01

    The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Novel approach to integrated DNA adductomics for the assessment of in vitro and in vivo environmental exposures.

    Science.gov (United States)

    Chang, Yuan-Jhe; Cooke, Marcus S; Hu, Chiung-Wen; Chao, Mu-Rong

    2018-06-25

    Adductomics is expected to be useful in the characterization of the exposome, which is a new paradigm for studying the sum of environmental causes of diseases. DNA adductomics is emerging as a powerful method for detecting DNA adducts, but reliable assays for its widespread, routine use are currently lacking. We propose a novel integrated strategy for the establishment of a DNA adductomic approach, using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS), operating in constant neutral loss scan mode, screening for both known and unknown DNA adducts in a single injection. The LC-QqQ-MS/MS was optimized using a representative sample of 23 modified 2'-deoxyribonucleosides reflecting a range of biologically relevant DNA lesions. Six internal standards (ISTDs) were evaluated for their ability to normalize, and hence correct, possible variation in peak intensities arising from matrix effects, and the quantities of DNA injected. The results revealed that, with appropriate ISTDs adjustment, any bias can be dramatically reduced from 370 to 8.4%. Identification of the informative DNA adducts was achieved by triggering fragmentation spectra of target ions. The LC-QqQ-MS/MS method was successfully applied to in vitro and in vivo studies to screen for DNA adducts formed following representative environmental exposures: methyl methanesulfonate (MMS) and five N-nitrosamines. Interestingly, five new DNA adducts, induced by MMS, were discovered using our adductomic approach-an added strength. The proposed integrated strategy provides a path forward for DNA adductomics to become a standard method to discover differences in DNA adduct fingerprints between populations exposed to genotoxins, and facilitate the field of exposomics.

  17. [Sigma]-Adducts of pteridines and 3-deazapteridines, structure and reactivity

    NARCIS (Netherlands)

    Nagel, A.

    1978-01-01

    In the introduction of this thesis the reactions of pteridines and pyrido[2,3- b ]-pyrazines with nucleophiles are reviewed. In the following chapters the results of an NMR investigation on the formation of σ-adducts between these azaaromatic ring systems and nitrogen

  18. Oxidative stress and DNA damage caused by the urban air pollutant 3-NBA and its isomer 2-NBA in human lung cells analyzed with three independent methods.

    Science.gov (United States)

    Nagy, Eszter; Johansson, Clara; Zeisig, Magnus; Möller, Lennart

    2005-11-15

    The air pollutant 3-nitrobenzanthrone (3-NBA), emitted in diesel exhaust, is a potent mutagen and genotoxin. 3-NBA can isomerise to 2-nitrobenzanthrone (2-NBA), which can become more than 70-fold higher in concentration in ambient air. In this study, three independent methods have been employed to evaluate the oxidative stress and genotoxicity of 2-NBA compared to 3-NBA in the human A549 lung cell line. HPLC-EC/UV was applied for measurements of oxidative damage in the form of 8-oxo-2'-deoxyguanosine (8-oxodG), (32)P-HPLC for measurements of lipophilic DNA-adducts, and the Comet assay to measure a variety of DNA lesions, including oxidative stress. No significant oxidative damage from either isomer was found regarding formation of 8-oxodG analysed using HPLC-EC/UV. However, the Comet assay (with FPG-treatment), which is more sensitive and detects more types of damages compared to HPLC-EC/UV, showed a significant effect from both 3-NBA and 2-NBA. (32)P-HPLC revealed a strong DNA-adduct formation from both 3-NBA and 2-NBA, and also a significant difference between both isomers compared to negative control. These results clearly show that 2-NBA has a genotoxic potential. Even if the DNA-adduct forming capacity and the amount of DNA lesions measured with the (32)P-HPLC and Comet assay is about one third of 3-NBA, the high abundance of 2-NBA in ambient air calls for further investigation and evaluation of its health hazard.

  19. Chemical repair activity of free radical scavenger edaravone. Reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA

    International Nuclear Information System (INIS)

    Hata, Kuniki; Katsumura, Yosuke; Urushibara, Ayumi; Yamashita, Shinichi; Lin Mingzhang; Muroya, Yusa; Shikazono, Naoya; Yokoya, Akinari; Fu Haiying

    2015-01-01

    Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10 8 dm 3 mol -1 s -1 and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10–1000 μmol dm -3 ) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid. (author)

  20. Mutagenicity, stable DNA adducts, and abasic sites induced in Salmonella by phenanthro[3,4-b]- and phenanthro[4,3-b]thiophenes, sulfur analogs of benzo[c]phenanthrene

    Energy Technology Data Exchange (ETDEWEB)

    Swartz, Carol D. [Department of Environmental Science and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States); King, Leon C.; Nesnow, Stephen [Environmental Carcinogenesis Division, US Environmental Protection Agency, Research Triangle Park, NC, 27711 (United States); Umbach, David M. [Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709 (United States); Kumar, Subodh [Environmental Toxicology and Chemistry Laboratory, Great Lakes Center, State University of New York College at Buffalo, Buffalo, NY 14222 (United States); DeMarini, David M. [Environmental Carcinogenesis Division, US Environmental Protection Agency, Research Triangle Park, NC, 27711 (United States)], E-mail: demarini.david@epa.gov

    2009-02-10

    Sulfur-containing polycyclic aromatic hydrocarbons (thia-PAHs or thiaarenes) are common constituents of air pollution and cigarette smoke, but only a few have been studied for health effects. We evaluated the mutagenicity in Salmonella TA98, TA100, and TA104 of two sulfur-containing derivatives of benzo[c]phenanthrene, phenanthro[3,4-b]thiophene (P[3,4-b]T), and phenanthro[4,3-b]thiophene (P[4,3-b]T) as well as their dihydrodiol and sulfone derivatives. In addition, we assessed levels of stable DNA adducts (by {sup 32}P-postlabeling) as well as abasic sites (by an aldehydic-site assay) produced by six of these compounds in TA100. P[3,4-b]T and its 6,7- and 8,9-diols, P[3,4-b]T sulfone, P[4,3-b]T, and its 8,9-diol were mutagenic in TA100. P[3,4-b]T sulfone, the most potent mutagen, was approximately twice as potent as benzo[a]pyrene in both TA98 and TA100. Benzo-ring dihydrodiols were much more potent than K-region dihydrodiols, which had little or no mutagenic activity in any strain. P[3,4-b]T sulfone produced abasic sites and not stable DNA adducts; the other five compounds examined, B[c]P, B[c]P 3,4-diol, P[3,4-b]T, P[3,4-b]T 8,9-diol, and P[4,3-b]T 8,9-diol, produced only stable DNA adducts. P[3,4-b]T sulfone was the only compound that produced significant levels of frameshift mutagenicity and induced mutations primarily at GC sites. In contrast, B[c]P, its 3,4-diol, and the 8,9 diols of the phenanthrothiophenes induced mutations primarily at AT sites. P[3,4-b]T was not mutagenic in TA104, whereas P[3,4-b]T sulfone was. The two isomeric forms (P[3,4-b]T and P[4,3-b]T) are apparently activated differently, with the latter, but not the former, involving a diol pathway. This study is the first illustrating the potential importance of abasic sites in the mutagenicity of thia-PAHs.

  1. Translesion Synthesis of the N2-2′-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1

    Science.gov (United States)

    2016-01-01

    Translesion synthesis (TLS) of the N2-2′-deoxyguanosine (dG-N2-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5′-CG1G2CG3CC-3′). TLS efficiency was 38%, 29%, and 25% for the dG-N2-IQ located at G1, G2, and G3, respectively, which suggests that dG-N2-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8–35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N2-IQ bypass. Mutation frequencies (MFs) of dG-N2-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ((2015) Nucleic Acids Res.43, 8340−835126220181). The major type of mutation induced by dG-N2-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N2-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N2-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between

  2. Carcinogen-DNA interaction study by base sequence footprinting. Final report, July 1, 1983-June 30, 1986

    International Nuclear Information System (INIS)

    Bases, R.

    1986-01-01

    Our previous studies on acetylaminofluorene (AAF) modified DNA demonstrated three kinds of structural changes in DNA of defined base sequence. For example, adduct formation by N-Aco-AAF was found at each guanine. We studied the interaction of IgG specific for AAF guanosine in an in vitro system using AAF modified phi X-174 rf DNA. We had expected to find protection against DNAase I digestion. Instead, when the DNA was immunobound to an inert matrix via the IgG, DNAase I digestion was enhanced 20 fold without changing the base sequence pattern of digestion. DNAase I hypersensitive sites are a necessary but not a sufficient condition for transcription. Moreover, some hypersensitive sites are stably propagated, independent of the continued presence of the inducer. Stability of these hypersensitive sites in the absence of their inducer suggests that they can be propagated. It appeared likely that distortion of DNA by a carcinogen adduct such as AAF, and the interaction of this modified DNA with a specific protein such as IgG or cellular proteins might inappropriately enhance the transcription of specific genes. That hypothesis will be tested; surprisingly, little is known about the early action of carcinogens on expression of specific genes. 34 refs., 2 figs., 1 tab

  3. Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark

    DEFF Research Database (Denmark)

    von Stedingk, Hans; Vikström, Anna C; Rydberg, Per

    2011-01-01

    The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method.......20-0.73) for glycidamide, and 0.43 (range 0.17-1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38-0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together...... for analysis of Hb adducts by liquid chromatography-mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide...

  4. Sequence-Dependent Diastereospecific and Diastereodivergent Crosslinking of DNA by Decarbamoylmitomycin C.

    Science.gov (United States)

    Aguilar, William; Paz, Manuel M; Vargas, Anayatzinc; Clement, Cristina C; Cheng, Shu-Yuan; Champeil, Elise

    2018-04-20

    Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA; Implicacion de los genes uvrA de E. coli K12 en la reparacion de monoaductos y entrecruzamien tos inducidos en DNA plasmidico por 8-metoxipso raleno mas luz ultravioleta A

    Energy Technology Data Exchange (ETDEWEB)

    Paramio, J M; Bauluz, C; Vidania, R de

    1986-07-01

    Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs.

  6. Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of N-alkyl and N-alkenyl heme adduct formation

    International Nuclear Information System (INIS)

    Grab, L.A.; Swanson, B.A.; Ortiz de Montellano, P.R.

    1988-01-01

    Incubation of 3-(2-phenylethyl)-4-methylsydnone (PMS) with liver microsomes from phenobarbital-pretreated rats or with reconstituted cytochrome P-450b results in loss of the enzyme chromophore. Chromophore loss is NADPH-dependent even though the sydnone decomposes by an oxygen- but not enzyme-dependent process to give pyruvic acid and, presumably, the (2-phenylethyl)diazonium cation. N-(2-Phenylethyl)protoporphyrin IX and N-(2-phenylethenyl)protoporphyrin IX have been isolated from the livers of rats treated with PMS. Both deuteriums are retained in the N-(2-phenylethyl) adduct derived from 3-(2-phenyl[1,1- 2 H]ethyl)-4-methylsydnone, but one deuterium is lost in the N-(2-phenylethenyl) adduct. The N-(2-phenylethyl) to N-(2-phenylethenyl) adduct ratio is increased by deuterium substitution. Electron paramagnetic resonance (EPR)-spin trapping studies show that carbon radicals are formed in incubations of the sydnones with liver microsomes but by a process that is independent of chromophore destruction. It is proposed that the 2-phenylethyl radical formed by electron transfer to the sydnone-derived (2-phenylethyl)diazonium cation adds to the prosthetic heme group to give the N-(2-phenylethyl) adduct. This alkylation reaction is similar to that observed with (2-phenylethyl)hydrazine. Autoxidation of the Fe-CH(CH 2 Ph)-N bridged species expected from insertion of 2-phenyldiazoethane into one of the heme Fe-N bonds is proposed to explain the unprecedented introduction of a double bond into the N-(2-phenylethenyl)adduct

  7. In vitro adduct formation of phosgene with albumin and hemoglobin in human blood

    NARCIS (Netherlands)

    Noort, D.; Hulst, A.G.; Fidder, A.; Gurp, R.A. van; Jong, L.P.A. de; Benschop, H.P.

    2000-01-01

    The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [14C]phosgene (0-750 μM), a significant part of radioactivity (0-13%) became associated with globin and

  8. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Braddock, M.

    1985-07-01

    The hydroxyl radical (OH radical) is the most damaging radical produced by the effect of ionizing radiation in water. The rate of reaction of the OH radical with purified, native and isodisperse DNA has been determined as compared with calf thymus DNA. This has been achieved by direct observation of the rate of formation of the DNA-OH radical adduct, and by competition with SCN - . Results obtained from direct observation are consistent with calculations which have been performed using the encounter frequency model of Braams and Ebert. However, results obtained for OH radical with DNA derived from competition plots suggest a rate constant somewhat lower than that obtained from direct observation. The relative merits of both techniques are discussed. In order to study the effect of energy deposited directly in the DNA, dry films of purified plasmid DNA have been irradiated in a system where the indirect effects of radical interaction have been minimized. The present results indicate that with different molecular lengths of plasmid DNA, non-random breakage may occur, and that additional damage may be brought about at sites of previously existing damage. Differences in the sensitivity of plasmid DNA molecules of varying lengths to radiation induced double strand breaks have been demonstrated. (author)

  9. In utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns

    Energy Technology Data Exchange (ETDEWEB)

    Perera, F.; Hemminki, K.; Jedrychowski, W.; Whyatt, R.; Campbell, U.; Hsu, Y.Z.; Santella, R.; Albertini, R.; O' Neill, J.P. [Columbia University, New York, NY (United States). School of Public Health

    2002-10-01

    Transplacental exposure to carcinogenic air pollutants from the combustion of fossil fuels is a growing health concern, given evidence of the heightened susceptibility of the fetus. These mutagenic/carcinogenic pollutants include aromatic compounds such as polycyclic aromatic hydrocarbons that bind to DNA, forming chemical-DNA adducts. The genotoxic effects of transplacental exposure in humans has been investigated by analyzing aromatic-DNA adducts and the frequency of gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in umbilical cord and maternal blood samples. Here the authors show, in a cross-sectional study of 67 mothers and 64 newborns from the Krakow Region of Poland, that aromatic-DNA adducts measured by P-32-postlabeling are positively associated with HPRT mutant frequency in the newborns (beta = 0.56, P = 0.03) after controlling for exposure to tobacco smoke, diet, and socioeconomic status. In contrast to the fetus, HPRT mutations and DNA adducts do not reflect similar exposure periods in the mother, and the maternal biomarkers were not correlated. Adducts were higher in the newborn than the mother, indicating differential susceptibility of the fetus to DNA damage; but HPRT mutation frequency was 4-fold lower, consistent with the long lifetime of the biomarker. These results provide the first demonstration of a molecular link between somatic mutation in the newborn and transplacental exposure to common air pollutants, a finding that is relevant to cancer risk assessment.

  10. Mutagenesis and repair induced by the DNA advanced glycation end product N2-1-(carboxyethyl)-2'-deoxyguanosine in human cells.

    Science.gov (United States)

    Tamae, Daniel; Lim, Punnajit; Wuenschell, Gerald E; Termini, John

    2011-03-29

    Glycation of biopolymers by glucose-derived α-oxo-aldehydes such as methylglyoxal (MG) is believed to play a major role in the complex pathologies associated with diabetes and metabolic disease. In contrast to the extensive literature detailing the formation and physiological consequences of protein glycation, there is little information about the corresponding phenomenon for DNA. To assess the potential contribution of DNA glycation to genetic instability, we prepared shuttle vectors containing defined levels of the DNA glycation adduct N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and transfected them into isogenic human fibroblasts that differed solely in the capacity to conduct nucleotide excision repair (NER). In the NER-compromised fibroblasts, the induced mutation frequencies increased up to 18-fold relative to background over a range of ∼10-1400 CEdG adducts/10(5) dG, whereas the same substrates transfected into NER-competent cells induced a response that was 5-fold over background at the highest adduct density. The positive linear correlation (R(2) = 0.998) of mutation frequency with increasing CEdG level in NER-defective cells suggested that NER was the primary if not exclusive mechanism for repair of this adduct in human fibroblasts. Consistent with predictions from biochemical studies using CEdG-substituted oligonucleotides, guanine transversions were the predominant mutation resulting from replication of MG-modified plasmids. At high CEdG levels, significant increases in the number of AT → GC transitions were observed exclusively in NER-competent cells (P involvement of an NER-dependent mutagenic process in response to critical levels of DNA damage, possibly mediated by error-prone Y-family polymerases.

  11. Assessment of Multiple Types of DNA Damage in Human Placentas from Smoking and Non-smoking Women in the Czech Republic

    Science.gov (United States)

    Margaret Pratt, M.; King, Leon C.; Adams, Linda D.; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A.; Manchester, David K.; Sram, Radim J.; DeMarini, David M.; Poirier, Miriam C.

    2010-01-01

    Three classes of DNA damage were assessed in human placentas collected (in 2000-4) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by 32P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49–312 PAH-DNA adducts/108 nucleotides, were found by IHC/ACIS in 14 immediately-fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7 – 8.6 stable/bulky DNA adducts/108 nucleotides and 0.6 – 47.2 AB sites/105 nucleotides. For all methods there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and non-smokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. PMID:20839217

  12. RPA coordinates DNA end resection and prevents formation of DNA hairpins.

    Science.gov (United States)

    Chen, Huan; Lisby, Michael; Symington, Lorraine S

    2013-05-23

    Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11Δ to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA.

    Science.gov (United States)

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Lin, Mingzhang; Muroya, Yusa; Shikazono, Naoya; Yokoya, Akinari; Fu, Haiying; Katsumura, Yosuke

    2015-01-01

    Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10(8) dm(3) mol(-1) s(-1) and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10-1000 μmol dm(-3)) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  14. Cross-linking and relaxation of supercoiled DNA by psoralen and light

    International Nuclear Information System (INIS)

    Yoakum, G.H.; Cole, R.S.

    1978-01-01

    Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA. The rate of relaxation of superhelical turns by psoralen photobinding appeared to be directly proportional to the number of superhelical turns remaining. A unique reaction mechanism is presented to explain these results. By this interpretation the initial rate of psoralen photobinding to superhelical DNA was estimated to be 3 times that for linear DNA, and the ratio of cross-linking to monofunctional adducts appears to be dependent on the superhelical conformation of the DNA. The estimated ratio of psoralen molecules bound to DNA strand breaks was 1.7 . 10 4 :1, and 70% of this breakage is caused by the light alone. (Auth.)

  15. Hydrogen abstraction reactions by amide electron adducts

    International Nuclear Information System (INIS)

    Sevilla, M.D.; Sevilla, C.L.; Swarts, S.

    1982-01-01

    Electron reactions with a number of peptide model compounds (amides and N-acetylamino acids) in aqueous glasses at low temperature have been investigated using ESR spectroscopy. The radicals produced by electron attachment to amides, RC(OD)NDR', are found to act as hydrogen abstracting agents. For example, the propionamide electron adduct is found to abstract from its parent propionamide. Electron adducts of other amides investigated show similar behavior except for acetamide electron adduct which does not abstract from its parent compound, but does abstract from other amides. The tendency toward abstraction for amide electron adducts are compared to electron adducts of several carboxylic acids, ketones, aldehydes and esters. The comparison suggests the hydrogen abstraction tendency of the various deuterated electron adducts (DEAs) to be in the following order: aldehyde DEA > acid DEA = approximately ester DEA > ketone DEA > amide DEA. In basic glasses the hydrogen abstraction ability of the amide electron adducts is maintained until the concentration of base is increased sufficiently to convert the DEA to its anionic form, RC(O - )ND 2 . In this form the hydrogen abstracting ability of the radical is greatly diminished. Similar results were found for the ester and carboxylic acid DEA's tested. (author)

  16. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution; Adduits encombrants a l'ADN dans des cocultures de cellules pulmonaires humaines exposees a une pollution atmospherique particulaire

    Energy Technology Data Exchange (ETDEWEB)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz [Universite Lille Nord de France - Lille (France); Unite de Chimie Environnementale et Interactions sur le Vivant, MREI, Universite du Littoral Cote d' Opale, Dunkerque (France); Andre, Veronique; Le Goff, Jeremie; Sichel, Francois [GRECAN, Universite de Caen Basse-Normandie et centre Francois Baclesse, Caen (France); Roy Saint-Georges, Francoise; Mulliez, Philippe [Service de Pneumologie, Hopital Saint-Philibert, GHICL, Lille (France)

    2012-01-15

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% < 2.5{mu}.m), specific surface area (1 m{sup 2}/g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). {sup 32}P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC{sub 50} = 74.63 {mu}g/mL and L132: LC-5-0 = 75.36 {mu}g/mL). Exposure to desorbed particles (MA: C1= 61.11 {mu}g/mL and L132 : C2 = 61.71 {mu}g/mL) and B[a]P (1 {mu}M) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that

  17. Structural basis for recognition of 5'-phosphotyrosine adducts by TDP2

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Ke; Kurahash, Kayo; Gao, Rui; Tsutakawa, Susan E.; Tainer, John A.; Pommier, Yves; Aihara, Hideki

    2012-12-19

    The DNA repair enzyme TDP2 resolves 5'-phosphotyrosyl-DNA adducts, and is responsible for resistance to anti-cancer drugs that target covalent topoisomerase-DNA complexes. TDP2 also participates in key signaling pathways during development and tumorigenesis, and cleaves a protein-RNA linkage during picornavirus replication. The crystal structure of zebrafish TDP2 bound to DNA reveals a deep and narrow basic groove that selectively accommodates the 5'-end of single-stranded DNA in a stretched conformation. The crystal structure of the full-length C. elegans TDP2 shows that this groove can also accommodate an acidic peptide stretch in vitro, with Glu and Asp sidechains occupying the DNA backbone phosphate binding sites. This extensive molecular mimicry suggests a potential mechanism for auto-regulation and how TDP2 may interact with phosphorylated proteins in signaling. Our study provides a framework to interrogate functions of TDP2 and develop inhibitors for chemotherapeutic and antiviral applications.

  18. Electrophilic properties of patulin. Adduct structures and reaction pathways with 4-bromothiophenol and other model nucleophiles.

    Science.gov (United States)

    Fliege, R; Metzler, M

    2000-05-01

    The mycotoxin patulin (PAT) is believed to exert its cytotoxic and chromosome-damaging effects by forming covalent adducts with essential cellular thiols. Since the chemical structures of such adducts are unknown to date, we have studied the reaction of PAT and its O-acetylated derivative with the monofunctional thiol model compound 4-bromothiophenol (BTP), which was chosen due to analytical advantages. By means of analytical and preparative high-performance liquid chromatography, 16 adducts of PAT and 3 adducts of acetyl-PAT were isolated and their chemical structures elucidated by (1)H and (13)C NMR, IR, and UV spectroscopy. Time course studies and analysis of daughter product formation from isolated intermediate adducts led to a detailed scheme for the reaction of PAT with BTP. The structures of adducts of PAT formed with other model nucleophiles, e. g., the aliphatic thiol 2-mercaptoethanol and the aromatic amine 4-bromoaniline, were also elucidated and found to corroborate the reaction scheme. In addition, one further reaction pathway was observed with 2-mercaptoethanol, which appears to be independent from those found for BTP. Our study with model nucleophiles provides insights into the electrophilic reactivity of PAT and proved to be useful for the structure elucidation of PAT adducts with biological nucleophiles of toxicological relevance, as will be reported by Fliege and Metzler [(2000) Chem. Res. Toxicol. 13, 373-381].

  19. Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA.

    Science.gov (United States)

    Konda, Shyam K; Kelso, Celine; Pumuye, Paul P; Medan, Jelena; Sleebs, Brad E; Cutts, Suzanne M; Phillips, Don R; Collins, J Grant

    2016-05-18

    The ability of a bis-amino mitoxantrone anticancer drug (named WEHI-150) to form covalent adducts with DNA, after activation by formaldehyde, has been studied by electrospray ionisation mass spectrometry and HPLC. Mass spectrometry results showed that WEHI-150 could form covalent adducts with d(ACGCGCGT)2 that contained one, two or three covalent links to the octanucleotide, whereas the control drugs (daunorubicin and the anthracenediones mitoxantrone and pixantrone) only formed adducts with one covalent link to the octanucleotide. HPLC was used to examine the extent of covalent bond formation of WEHI-150 with d(CGCGCG)2 and d(CG(5Me)CGCG)2. Incubation of WEHI-150 with d(CG(5Me)CGCG)2 in the presence of formaldehyde resulted in the formation of significantly greater amounts of covalent adducts than was observed with d(CGCGCG)2. In order to understand the observed increase of covalent adducts with d(CG(5Me)CGCG)2, an NMR study of the reversible interaction of WEHI-150 at both CpG and (5Me)CpG sites was undertaken. Intermolecular NOEs were observed in the NOESY spectra of d(ACGGCCGT)2 with added WEHI-150 that indicated that the drug selectively intercalated at the CpG sites and from the major groove. In particular, NOEs were observed from the WEHI-150 H2,3 protons to the H1' protons of G3 and G7 and from the H6,7 protons to the H5 protons of C2 and C6. By contrast, intermolecular NOEs were observed between the WEHI-150 H2,3 protons to the H2'' proton of the (5Me)C3 in d(CG(5Me)CGCG)2, and between the drug aliphatic protons and the H1' proton of G4. This demonstrated that WEHI-150 preferentially intercalates at (5Me)CpG sites, compared to CpG sequences, and predominantly via the minor groove at the (5Me)CpG site. The results of this study demonstrate that WEHI-150 is likely to form interstrand DNA cross-links, upon activation by formaldehyde, and consequently exhibit greater cytotoxicity than other current anthracenedione drugs.

  20. Combination of ICP-MS, capillary electrophoresis, and their hyphenation for probing Ru(III) metallodrug-DNA interactions.

    Science.gov (United States)

    Foteeva, Lidia S; Matczuk, Magdalena; Pawlak, Katarzyna; Aleksenko, Svetlana S; Nosenko, Sergey V; Karandashev, Vasily K; Jarosz, Maciej; Timerbaev, Andrei R

    2017-03-01

    Determination of the DNA-binding reactivity and affinity is an important part of a successful program for the selection of metallodrug candidates. For such assaying, a range of complementary analytical techniques was proposed and tested here using one of few anticancer metal-based drugs that are currently in clinical trials, indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III), and a DNA oligonucleotide. A high reactivity of the Ru drug was confirmed in affinity capillary electrophoresis (CE) mode, where adduct formation takes place in situ (i.e., in the capillary filled with an oligonucleotide-containing electrolyte). To further characterize the binding kinetics, a drug-oligonucleotide mixture was incubated for a different period of time, followed by ultrafiltration separation into two different in molecular weight fractions (>3 and ICP-MS), revealing that at least two DNA adducts exist at equilibrium conditions. Using standalone ICP-MS, dominant equilibrium amount of the bound ruthenium was found to occur in a fraction of 5-10 kDa, which includes the oligonucleotide (ca. 6 kDa). Importantly, in all three assays, the drug was used for the first time in in-vitro studies, not in the intact form but as its active species released from the transferrin adduct at simulated cancer cytosolic conditions. This circumstance makes the established analytical platform promising to provide a detailed view on metallodrug targeting, including other possible biomolecules and ex vivo samples.

  1. Investigating the adduct formation of organic mercury species with carbonic anhydrase and hemoglobin from human red blood cell hemolysate by means of LC/ESI-TOF-MS and LC/ICP-MS.

    Science.gov (United States)

    Hogeback, Jens; Schwarzer, Miriam; Wehe, Christoph A; Sperling, Michael; Karst, Uwe

    2016-01-01

    The interaction of mercury species with human erythrocytes is studied to investigate possible high molecular binding partners for mercury species. Human blood hemolysate was spiked with methylmercury and investigated by means of liquid chromatography (LC) coupled to electrospray ionization time of flight mass spectrometry (ESI-ToF-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Beside adduct formation of mercury species with hemoglobin, the main compound of the erythrocytes, mercury binding to the enzyme carbonic anhydrase was revealed. Due to an enzymatic digest of the protein-mercury adduct, the binding site at the free thiol group of the protein was identified. These results indicate that carbonic anhydrase might play a role in mercury toxicity.

  2. Experimental and theoretical studies of the reaction of the OH radical with alkyl sulfides: 1. Direct observations of the formation of the OH-DMS adduct-pressure dependence of the forward rate of addition and development of a predictive expression at low temperature.

    Science.gov (United States)

    Williams, M B; Campuzano-Jost, P; Cossairt, B M; Hynes, A J; Pounds, A J

    2007-01-11

    A pulsed laser photolysis-pulsed laser-induced fluorescence (PLP-PLIF) system was employed to study the kinetics and mechanisms of reactions (1) OH + h6-DMS --> products and (2) OH + d6-DMS --> products. We report direct observations of the rate coefficients for the formation and dissociation of the h6-OHDMS and d6-OHDMS adducts over the pressure range 50-650 Torr and between 240 and 245 K, together with measurements of the oxygen dependence of the effective rate coefficients for reactions 1 and 2 under similar conditions. The effective rate coefficients increased as a function of O2 concentration reaching their limiting values in each case. The values of the adduct formation rate, obtained from the O2 dependencies, were in excellent agreement with values determined from direct observation of adduct equilibration in N2. OH regeneration is insignificant. The rate coefficients for the formation of the adduct isotopomers showed slight differences in their falloff behavior and do not approach the high-pressure limit in either case. The equilibrium constants obtained show no dependence on isotopomer and are in good agreement with previous work. A "second-law" analysis of the temperature dependence of the equilibrium constant gives an adduct bond strength (DeltaH degrees =-10.9 +/- 1.0 kcal mol(-1)), also in good agreement with previously reported values. Using the entropy calculated from the ab initio vibrational frequencies, we obtain a "third-law" value for the reaction enthalpy at 240 K, DeltaH(240K) degrees = -10.5 kcal mol(-1) in good agreement with the other approach. The rate coefficient for the reactions of the adducts with O2 was obtained from an analysis of the O2 dependence and was determined to be 6.3 +/- 1.2 x 10(-13) cm3 molecule(-1) s(-1), with no dependence on pressure or isotopomer. The pressure and temperature dependence for all of the elementary processes in the initial steps of the dimethylsulfide (DMS) oxidation mechanism have been characterized in

  3. Dynamic maps of UV damage formation and repair for the human genome.

    Science.gov (United States)

    Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

    2017-06-27

    Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

  4. A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

    DEFF Research Database (Denmark)

    Lappann, M.; Claus, H.; van Alen, T.

    2010-01-01

    formation, whereas biofilm formation of cc with low point prevalence (ST-8 cc and ST-11 cc) was eDNA-independent. For initial biofilm formation, a ST-32 cc type strain, but not a ST-11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N......-acetylmuramyl-l-alanine amidase genes. In late biofilms, outer membrane phospholipase A-dependent autolysis, which was observed in most cc, but not in ST-8 and ST-11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an e....... On the contrary, spreaders (ST-11 and ST-8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates....

  5. Limonene protects osteoblasts against methylglyoxal-derived adduct formation by regulating glyoxalase, oxidative stress, and mitochondrial function.

    Science.gov (United States)

    Suh, Kwang Sik; Chon, Suk; Choi, Eun Mi

    2017-12-25

    Methylglyoxal (MG) is a potent protein glycating agent and an important precursor of advanced glycation end products, which are involved in the pathogenesis of diabetic osteopathy. In this study, we investigated the effects of limonene on MG-induced damage in osteoblastic MC3T3-E1 cells. Pretreating cells with limonene prevented MG-induced protein adduct formation, tumor necrosis factor alpha and interleukin-6 release, mitochondrial superoxide production, and cardiolipin peroxidation. In addition, limonene increased glyoxalase I activity, and glutathione and heme oxygenase-1 levels in the presence of MG. Pretreatment with limonene prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss, and reduced the levels of adenosine monophosphate-activated protein kinase, peroxisome proliferator activated receptor γ coactivator 1α, and nitric oxide. These results demonstrate that limonene may prevent the development of diabetic osteopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Non-random alkylation of DNA sequences induced in vivo by chemical mutagens

    Energy Technology Data Exchange (ETDEWEB)

    Durante, M.; Geri, C.; Bonatti, S.; Parenti, R. (Universita di Pisa (Italy))

    1989-08-01

    Previous studies of the interaction of alkylating agents on the eukaryotic genome support the idea that induction of DNA adducts is at specific genomic sites. Here we show molecular and cytological evidence that alkylation is rather specific. Mammalian cell cultures were exposed to different doses of mutagens and the DNA was analyzed by density gradient ultracentrifugation, hydroxylapatite fractionation, and by restriction enzyme analysis. Studies with the labelled mutagens N-ethyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine show that there is a non-random distribution of the adducts. The adducts are found more frequently in A-T, G-C rich satellite DNA and highly repetitive sequences. Analysis with restriction enzymes shows that both methyl and ethyl groups influence the restriction patterns of the enzymes HpaII and MspI that recognize specific endogenous DNA methylation. These data suggest, as a subsequent mechanism, a modification in the pattern of the normal endogenous methylation of 5-methylcytosine.

  7. Probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site

    International Nuclear Information System (INIS)

    Lasko, D.D.; Basu, A.K.; Kadlubar, F.F.; Evans, F.E.; Lay, J.O. Jr.; Essigmann, J.M.

    1987-01-01

    The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG/sup 8-ABP/), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG/sup 8-ABP/ residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoroacetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG/sup 8-ABP/pCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1 H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Experiments in which the tetranucleotides were 5' end labeled with [ 32 P]phosphate revealed the following: 1)the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; 2)radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; 3) the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; 4)in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. On the basis of these and other data, the dG/sup 8-ABP/-modified genome was judged to be a useful probe for investigation of site-specific mutagenesis in E. coli

  8. Absolute configuration, stability, and interconversion of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine valine adducts and their phenylthiohydantoin derivatives

    Directory of Open Access Journals (Sweden)

    Xiao Jiang

    2015-06-01

    Full Text Available Pyrrolizidine alkaloid-containing plants are widespread in the world and probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids require metabolic activation to form dehydropyrrolizidine alkaloids that bind to cellular proteins and DNA leading to hepatotoxicity, genotoxicity, and tumorigenicity. At present, it is not clear how dehydropyrrolizidine alkaloids bind to cellular amino acids and proteins to induced toxicity. We previously reported that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP-derived valine (DHP-valine adducts that upon reaction with phenyl isothiocyanate (PITC formed four DHP-valine-PITC adduct isomers. In this study, we report the absolute configuration and stability of DHP-valine and DHP-valine-PITC adducts, and the mechanism of interconversion between DHP-valine-PITC adducts.

  9. DNA repair in spermatocytes and spermatids of the mouse

    International Nuclear Information System (INIS)

    Sega, G.A.

    1982-01-01

    When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced

  10. Environmental carcinogens in human target tissues in culture: Progress report

    International Nuclear Information System (INIS)

    Hsu, I.C.

    1987-01-01

    We have accumulated more experimental evidences that demonstrated the comparative approaches with human cells will allow us to predict human risk with good accuracy following exposure to toxic chemicals. We also synthesized several carcinogenic DNA adducts, i.e., the major benzo[a]pyrene DNA adduct, 0 6 -methyldeoxyguanosine, 7-methyl- deoxyguanosine and 2-methyl-deoxyguanosine to be used as standards for quantitating DNA adduct formation in carcinogen exposed cells. A simple synthetic method was developed for preparation of the major B[a]p DNA adduct with yields better than those reported. The main accomplishments related to the originally stated objectives are summarized. 8 refs., 2 figs., 1 tab

  11. Relationship between ambient air pollution and DNA damage in Polish mothers and newborns

    International Nuclear Information System (INIS)

    Whyatt, R.M.; Santella, R.M.; Jedrychowski, W.; Garte, S.J.; Bell, D.A.; Ottman, R.; Gladek-Yarborough, A.; Cosma, G.; Young, T.L.; Cooper, T.B.; Randall, M.C.; Manchester, D.K.; Perera, F.P.

    1998-01-01

    Industrialized regions in Poland are characterized by high ambient pollution, including polycyclic aromatic hydrocarbons (PAHs) from coal burning for industry and home heating. In experimental bioassays, certain PAHs are transplacental carcinogens and developmental toxicants. The amount of PAHs bound to DNA (PAH-DNA adducts) in maternal and umbilical white blood cells were measured in 70 mothers and newborns from Krakow, Poland. Modulation of adduct levels by genotypes previously linked to risk of lung cancer, specifically glutathione S-transferase M1(GSTM1) and cytochrome P4501A1 (CYP1A1). There was a dose-related increase in maternal and newborn adduct levels with ambient pollution at the women's place of residence among subjects who were not employed away from home (p less than or equal to 0.05). Maternal smoking (active and passive) significantly increased maternal (p less than or equal to 0.01) but not newborn adduct levels. Results indicate that PAH-induced DNA damage in mothers and newborns is increased by ambient air pollution

  12. 7-Glutathione-pyrrole and 7-cysteine-pyrrole are potential carcinogenic metabolites of pyrrolizidine alkaloids.

    Science.gov (United States)

    He, Xiaobo; Xia, Qingsu; Fu, Peter P

    2017-04-03

    Many pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Metabolism of PAs in vivo generates four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts that have been proposed to be responsible for PA-induced liver tumor formation in rats. In this present study, we determined that the same set of DHP-DNA adducts was formed upon the incubation of 7-glutathione-DHP and 7-cysteine-DHP with cultured human hepatocarcinoma HepG2 cells. These results suggest that 7-glutathione-DHP and 7-cysteine-DHP are reactive metabolites of PAs that can bind to cellular DNA to form DHP-DNA adducts in HepG2 cells, and can potentially initiate liver tumor formation.

  13. Directed Formation of DNA Nanoarrays through Orthogonal Self-Assembly

    Directory of Open Access Journals (Sweden)

    Eugen Stulz

    2011-06-01

    Full Text Available We describe the synthesis of terpyridine modified DNA strands which selectively form DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. The short DNA strands are designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as Zn(II and Ni(II induces the formation of tubular arrays consisting of DNA bundles which are 50-200 nm wide and 2-50 nm high. TEM shows additional long distance ordering of the terpy-DNA complexes into fibers.

  14. Hip adduction and abduction strength profiles in elite soccer players

    DEFF Research Database (Denmark)

    Thorborg, Kristian; Serner, Andreas; Petersen, Jesper

    2011-01-01

    An ipsilateral hip adduction/abduction strength ratio of more than 90%, and hip adduction strength equal to that of the contralateral side have been suggested to clinically represent adequate strength recovery of hip adduction strength in athletes after groin injury. However, to what extent side-......-to-side symmetry in isometric hip adduction and abduction strength can be assumed in soccer players remains uncertain.......An ipsilateral hip adduction/abduction strength ratio of more than 90%, and hip adduction strength equal to that of the contralateral side have been suggested to clinically represent adequate strength recovery of hip adduction strength in athletes after groin injury. However, to what extent side...

  15. Accelerated repair and reduced mutagenicity of DNA damage induced by cigarette smoke in human bronchial cells transfected with E.coli formamidopyrimidine DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Mara Foresta

    Full Text Available Cigarette smoke (CS is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP. Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na(+K(+-ATPase locus (oua(r were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells' capacity to repair damaged DNA.

  16. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

    Science.gov (United States)

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

    2016-01-01

    Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  17. Molecular structures of five adducts assembled from p-dimethylaminobenzaldehyde and organic acids

    Science.gov (United States)

    Jin, Shouwen; Wang, Lanqing; Liu, Hui; Liu, Li; Zhang, Huan; Wang, Daqi; Li, Minghui; Guo, Jianzhong; Guo, Ming

    2016-07-01

    Five adducts 1-5 derived from p-dimethylaminobenzaldehyde have been prepared and characterized by X-ray diffraction analysis, IR, mp, and elemental analysis. Of the five adducts two are organic salts (1, and 2) and the other three (3-5) are cocrystals. In salts 1, and 2, the L molecules are protonated. The supramolecular architectures of the adducts 1-5 involve extensive intermolecular N-H⋯O, O-H⋯O, O-H⋯S, and C-H⋯O hydrogen bonds as well as other non-covalent interactions. The role of weak and strong non-covalent interactions in the crystal packing is ascertained. The complexes displayed 2D/3D framework structure for the synergistic effect of the various non-covalent interactions. The results presented herein tell that the strength and directionality of the N-H⋯O, O-H⋯O, and O-H⋯S hydrogen bonds between organic acids and p-dimethylaminobenzaldehyde are sufficient to bring about the formation of binary cocrystals or organic salts.

  18. DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics, hydration, sequence, and ionic effects.

    Science.gov (United States)

    Kankia, Besik I; Soto, Ana Maria; Burns, Nicole; Shikiya, Ronald; Tung, Chang-Shung; Marky, Luis A

    2002-11-05

    The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG

  19. The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity

    International Nuclear Information System (INIS)

    Stiborova, Marie; Dracinska, Helena; Mizerovska, Jana; Frei, Eva; Schmeiser, Heinz H.; Hudecek, Jiri; Hodek, Petr; Phillips, David H.; Arlt, Volker M.

    2008-01-01

    3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the 32 P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40 mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential

  20. Polymerization by DNA polymerase eta is blocked by cis-diamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells.

    Science.gov (United States)

    Chijiwa, Shotaro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori; Kuraoka, Isao

    2010-03-01

    cis-Diamminedichloroplatinum(II) (cisplatin) forms DNA adducts that interfere with replication and transcription. The most common adducts formed in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and d(ApG) cross-links (Pt-AG), with minor amounts of 1,3-d(GpNpG) cross-links (Pt-GNG), interstrand cross-links and monoadducts. Although the relative contribution of these different adducts to toxicity is not known, literature implicates that Pt-GG and Pt-AG adducts block replication. Thus, nucleotide excision repair (NER), by which platinum adducts are excised, and translesion DNA synthesis (TLS), which permits adduct bypass, are thought to be associated with cisplatin resistance. Recent studies have reported that the clinical benefit from platinum-based chemotherapy is high if tumor cells express low levels of NER factors. To investigate the role of platinum-DNA adducts in mediating tumor cell survival by TLS, we examined whether 1,3-intrastrand d(GpTpG) platinum cross-links (Pt-GTG), which probably exist in NER-negative tumor cells but not in NER-positive tumor cells, are bypassed by the translesion DNA polymerase eta (pol eta), which is known to bypass Pt-GG. We show that pol eta can incorporate the correct deoxycytidine triphosphate opposite the first 3'-cross-linked G of Pt-GTG but cannot insert any nucleotides opposite the second intact T or the third 5'-cross-linked G of the adducts, thereby suggesting that TLS does not facilitate replication past Pt-GTG adducts. Thus, our findings implicate Pt-GNG adducts as mediating the cytotoxicity of platinum-DNA adducts in NER-negative tumors in vivo.

  1. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

    Directory of Open Access Journals (Sweden)

    Yonghe Qi

    2016-10-01

    Full Text Available Hepatitis B virus (HBV infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP, followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK, a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

  2. Investigation of benzo(a)pyrene-globin adducts

    Energy Technology Data Exchange (ETDEWEB)

    Wallin, H; Jeffre, A M; Santella, R M

    1987-05-01

    The nature of the adducts formed between benzo(a)pyrene (BP) and globin were investigated in animals treated with (/sup 3/H)BP. Modification levels on globin were determined by radioactivity measurements. Since BP tetraols can be released from benzo(a)pyrene diol epoxide modified protein and DNA by acid treatment, globin samples were treated with acid, released tetraols separated by HPLC and quantitated by scintillation counting. In addition, acid released material was measured in competitive enzyme linked immunosorbent assay (ELISA) using antibodies which recognize BP tetraols. Both measurements indicate that only 2% of bound radioactivity could be released as free BP tetraols. These studies indicate that benzo(a)pyrene diol epoxide may not be the major metabolite of BP involved in globin binding. (author). 14 refs.

  3. Effect of dietary factors on mutagenesis, metabolism, and binding to DNA of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol

    International Nuclear Information System (INIS)

    Vance, R.E.

    1988-01-01

    Ellagic acid (EA), a naturally occurring plant phenol, at concentrations of 5 to 50 μg/plate, inhibited rate liver S9 protein dependent benzo[a]pyrene (B[a]P)-induced mutagenesis in Salmonella typhimurium TA 100 by 30-81% and B[a]P 7,8-dihydrodiol (DHD)-induced mutagenesis by 29 to 75%. EA did not significantly affect the metabolism of B[a]P or B[a]P 7,8-DHD as determined by high performance liquid chromatographic analysis of the organosoluble fraction and by the quantification of water-soluble conjugates. At these concentrations EA inhibited the covalent binding of [ 3 H] B[a]P and [ 3 H] B[a]P 7,8-DHD metabolites to calf thymus DNA by 5 to 42% and 27 to 64%, respectively. Formation of benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide:deoxyguanosine (BPDE:dG) adducts was inhibited by 13 to 56% for B[a]P for B[a]P and 11 to 38% for B[a]P 7,8-DHD. These results suggest that the antimutagenic effect of EA and its inhibition of B[a]P and B[a]P 7,8-DHD metabolite-binding to DNA is not due to the inhibition of S9-mediated metabolism of these compounds. The inhibitory effect may be by previously described scavenging mechanism or by a DNA-affinity binding mechanism that prevents BPDE:DNA adduct formation

  4. Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation

    International Nuclear Information System (INIS)

    Williams, Kurt J.; Cruikshank, Michael K.; Plopper, Charles G.

    2003-01-01

    Heat shock proteins/stress proteins (Hsps) participate in regulation of protein synthesis and degradation and serve as general cytoprotectants, yet their role in lethal Clara cell injury is not clear. To define the pattern of Hsp expression in acute lethal Clara cell injury, mice were treated with the Clara cell-specific toxicant naphthalene (NA), and patterns of expression compared to electrophilic protein adduction and previously established organellar degradation and gluathione (GSH) depletion. In sites of lethal injury (distal bronchiole), prior to organellar degradation (1 h post-NA), protein adduction is detectable and ubiquitin, Hsp 25, Hsp 72, and heme-oxygenase 1 (HO-1) are increased. Maximal Hsp expression, protein adduction, and GSH depletion occur simultaneous (by 2-3 h) with early organelle disruption. Hsp expression is higher later (6-24 h), only in exfoliating cells. In airway sites (proximal bronchiole) with nonlethal Clara cell injury elevation of Hsp 25, 72, and HO-1 expression follows significant GSH depletion (greater than 50% 2 h post-NA). This data build upon our previous studies and we conclude that (1) in lethal (terminal bronchiole) and nonlethal (proximal bronchiole) Clara cell injury, Hsp induction is associated with the loss of GSH and increased protein adduction, and (2) in these same sites, organelle disruption is not a prerequisite for Hsp induction

  5. Close encounters for the first time: Helicase interactions with DNA damage.

    Science.gov (United States)

    Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

    2015-09-01

    DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

  6. Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

    Science.gov (United States)

    Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

    2014-01-29

    We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

  7. Time course evaluation of N-nitrosodialkylamines-induced DNA alkylation and oxidation in liver of mosquito fish

    International Nuclear Information System (INIS)

    Chao, M.-R.; Chang, Y.-Z.; Wong, R.-H.; Hu, C.-W.

    2009-01-01

    Here we simultaneously measured N7-alkylguanines and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in liver of small fish, respectively, to assess the time course of the formation and removal of alkylation and oxidative damage to DNA caused by N-nitrosodialkylamines. Mosquito fish (Gambusia affinis) were killed at various times during (4 days) and post-exposure (16 days) to N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) alone or their combination with concentrations of 10 and 50 mg/l. The modified guanine adducts were sensitively and selectively quantitated by isotope-dilution LC-MS/MS methods. During exposure, N7-methylguanine (N7-MeG) and N7-ethylguanine (N7-EtG) in liver DNA increased with the duration and dose of N-nitrosodialkylamine exposure, while 8-oxodG was dose-dependently induced within 1 day. It was found that NDMA formed substantially more N7-alkylated guanines and 8-oxodG than NDEA on the basis of adducts formed per micromolar concentration, suggesting that NDMA can be more easily bioactivated than NDEA to form reactive alkylating agents with the concomitant formation of oxygen radicals. After cessation of exposure, N7-alkylguanines remained elevated for 1 day and then gradually decreased over time but still higher than the background levels, even at day 16 (half-lives of 7-8 days). However, 8-oxodG was excised quickly from liver DNA and returned to the background level within 4 days post-exposure (half-lives less than 2 days). Taken together, this study firstly demonstrated that in addition to alkylation, N-nitrosodialkylamines can concurrently cause oxidative damage to DNA in vivo

  8. Time course evaluation of N-nitrosodialkylamines-induced DNA alkylation and oxidation in liver of mosquito fish

    Energy Technology Data Exchange (ETDEWEB)

    Chao, M -R [Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan (China); Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung 402, Taiwan (China); Chang, Y -Z [Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung 402, Taiwan (China); Wong, R -H [Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan (China); Hu, C.-W. [Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan (China)], E-mail: windyhu@csmu.edu.tw

    2009-01-15

    Here we simultaneously measured N7-alkylguanines and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in liver of small fish, respectively, to assess the time course of the formation and removal of alkylation and oxidative damage to DNA caused by N-nitrosodialkylamines. Mosquito fish (Gambusia affinis) were killed at various times during (4 days) and post-exposure (16 days) to N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) alone or their combination with concentrations of 10 and 50 mg/l. The modified guanine adducts were sensitively and selectively quantitated by isotope-dilution LC-MS/MS methods. During exposure, N7-methylguanine (N7-MeG) and N7-ethylguanine (N7-EtG) in liver DNA increased with the duration and dose of N-nitrosodialkylamine exposure, while 8-oxodG was dose-dependently induced within 1 day. It was found that NDMA formed substantially more N7-alkylated guanines and 8-oxodG than NDEA on the basis of adducts formed per micromolar concentration, suggesting that NDMA can be more easily bioactivated than NDEA to form reactive alkylating agents with the concomitant formation of oxygen radicals. After cessation of exposure, N7-alkylguanines remained elevated for 1 day and then gradually decreased over time but still higher than the background levels, even at day 16 (half-lives of 7-8 days). However, 8-oxodG was excised quickly from liver DNA and returned to the background level within 4 days post-exposure (half-lives less than 2 days). Taken together, this study firstly demonstrated that in addition to alkylation, N-nitrosodialkylamines can concurrently cause oxidative damage to DNA in vivo.

  9. Time course evaluation of N-nitrosodialkylamines-induced DNA alkylation and oxidation in liver of mosquito fish

    Energy Technology Data Exchange (ETDEWEB)

    Chao, M.-R. [Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan (China); Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung 402, Taiwan (China); Chang, Y.-Z. [Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung 402, Taiwan (China); Wong, R.-H. [Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan (China); Hu, C.-W. [Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan (China)], E-mail: windyhu@csmu.edu.tw

    2009-01-15

    Here we simultaneously measured N7-alkylguanines and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in liver of small fish, respectively, to assess the time course of the formation and removal of alkylation and oxidative damage to DNA caused by N-nitrosodialkylamines. Mosquito fish (Gambusia affinis) were killed at various times during (4 days) and post-exposure (16 days) to N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) alone or their combination with concentrations of 10 and 50 mg/l. The modified guanine adducts were sensitively and selectively quantitated by isotope-dilution LC-MS/MS methods. During exposure, N7-methylguanine (N7-MeG) and N7-ethylguanine (N7-EtG) in liver DNA increased with the duration and dose of N-nitrosodialkylamine exposure, while 8-oxodG was dose-dependently induced within 1 day. It was found that NDMA formed substantially more N7-alkylated guanines and 8-oxodG than NDEA on the basis of adducts formed per micromolar concentration, suggesting that NDMA can be more easily bioactivated than NDEA to form reactive alkylating agents with the concomitant formation of oxygen radicals. After cessation of exposure, N7-alkylguanines remained elevated for 1 day and then gradually decreased over time but still higher than the background levels, even at day 16 (half-lives of 7-8 days). However, 8-oxodG was excised quickly from liver DNA and returned to the background level within 4 days post-exposure (half-lives less than 2 days). Taken together, this study firstly demonstrated that in addition to alkylation, N-nitrosodialkylamines can concurrently cause oxidative damage to DNA in vivo.

  10. Photoreactions of ruthenium(II) and osmium(II) complexes with deoxyribonucleic acid (DNA).

    Science.gov (United States)

    Moucheron, C; Kirsch-De Mesmaeker, A; Kelly, J M

    1997-09-01

    The design of Ru(II) and Os(II) complexes which are photoreactive with deoxyribonucleic acid (DNA) represents one of the main targets for the development of novel molecular tools for the study of DNA and, in the future, for the production of new, metal-based, anti-tumor drugs. In this review, we explain how it is possible to make a complex photoreactive with nucleobases and nucleic acids. According to the photophysical behaviour of the Ru(II) compounds, two types of photochemistry are expected: (1) photosubstitution of a ligand by a nucleobase and another monodentate ligand, which takes place from the triplet, metal-centred (3MC) state; this state is populated thermally from the lowest lying triplet metal to ligand charge transfer (3MLCT) state; (2) photoreaction from the 3MLCT state, corresponding to photoredox processes with DNA bases. The two photoreactivities are in competition. By modulating appropriately the redox properties of the 3MLCT state, an electron transfer process from the base to the excited complex takes place, and is directly correlated with DNA cleavage or the formation of an adduct of the complex to DNA. In this adduct, guanine is linked by N2 to the alpha-position of a non-chelating nitrogen of the polyazaaromatic ligand without destruction of the complex. Different strategies are explained which increase the affinity of the complexes for DNA and direct the complex photoreactivity to sites of special DNA topology or targeted sequences of bases. Moreover, the replacement of the Ru(II) ion by the Os(II) ion in the photoreactive complexes leads to an increased specificity of photoreaction. Indeed, only one type of photoreactivity (from the 3MLCT state) is present for the Os(II) complexes because the 3MC state is too high in energy to be populated at room temperature.

  11. Aflatoxin B1-lysine adduct in dried blood spot samples of animals and humans.

    Science.gov (United States)

    Xue, Kathy S; Cai, Wenjie; Tang, Lili; Wang, Jia-Sheng

    2016-12-01

    Dried blood spots (DBS) were proposed as potentially viable method for exposure assessment of environmental toxicants in infant and young children. For this study, we validated an experimental protocol to quantify AFB 1 -lysine adduct in DBS samples of AFB 1 -treated F344 rats, as well as samples from human field study. Significant dose-response relationships in AFB 1 -lysine adduct formation were found in DBS samples of rats treated with single- and repeated-dose AFB 1 . AFB 1 -lysine levels in DBS samples were highly correlated with corresponding serum sample levels. The Person coefficients were 0.997 for the single-dose exposure, and 0.996 for the repeated-dose exposure. Levels of AFB 1 -lysine adduct had also good agreement between DBS and serum samples as shown by Bland-Altman plot analysis. For human field study samples (n = 36), a Pearson correlation coefficient of 0.784 was found between AFB 1 -lysine adduct levels of DBS and corresponding serum samples. Bland-Altman plots showed the distribution of the log differences between DBS and serum AFB 1 -lysine levels are within 95% confidence intervals. These results showed AFB 1 -lysine adduct levels in DBS cards and serum samples from animals and human samples are comparable, and the DBS technique and analytical protocol is a good means to assess AFB 1 exposure in infant and children populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs

    International Nuclear Information System (INIS)

    Bounaix Morand du Puch, Ch

    2010-10-01

    DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

  13. Inhibition of DNA glycosylases via small molecule purine analogs.

    Directory of Open Access Journals (Sweden)

    Aaron C Jacobs

    Full Text Available Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.

  14. Effect of benzimidazol-derivatives on the DNA-protein binding formation after UV-radiation of chromatin

    International Nuclear Information System (INIS)

    Mil', E.M.; Binyukov, V.I.; Zhil'tsova, V.M.; Stolyarova, L.G.; Kuznetsov, Yu.V.

    1991-01-01

    Effect of benzimidazol-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazol interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA, whilt aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1

  15. Redshift or adduct stabilization -- a computational study of hydrogen bonding in adducts of protonated carboxylic acids

    DEFF Research Database (Denmark)

    Olesen, Solveig Gaarn; Hammerum, Steen

    2009-01-01

    It is generally expected that the hydrogen bond strength in a D-H-A adduct is predicted by the difference between the proton affinities of D and A, measured by the adduct stabilization, and demonstrated by the IR redshift of the D-H bond stretching vibrational frequency. These criteria do...... not always yield consistent predictions, as illustrated by the hydrogen bonds formed by the E and Z OH groups of protonated carboxylic acids. The delta-PA and the stabilization of a series of hydrogen bonded adducts indicate that the E OH group forms the stronger hydrogen bonds, whereas the bond length...... carboxylic acids are different. The OH bond length and IR redshift afford the better measure of hydrogen bond strength....

  16. Effects of pesticides on DNA and protein of shrimp larvae Litopenaeus stylirostris of the California Gulf.

    Science.gov (United States)

    Galindo Reyes, J Guillermo; Leyva, Nancy R; Millan, Olivia A; Lazcano, Guadalupe A

    2002-10-01

    Recently, diverse pathologies and massive mortalities have been presented in shrimp hatcheries located along the California Gulf; therefore, toxic responses of shrimp larvae were used as biomarkers of pesticide pollution, because in this region intensive agriculture is practiced. Shrimp larvae were exposed to DDT, azinphosmethyl, permethrine, parathion, chlorpyrifos, malathion, endosulfan, and carbaryl, in order to determine LC50, DNA adducts and/or breaks, and total protein in larvae. The results indicate reductions in protein and DNA in larvae exposed to these pesticides, and in those exposed to DDT, breaks and/or adducts were registered. It is possible that pesticide pollution is a cause of these problems, because reduction in protein indicates a decrease in larvae growth rate and DNA breaks or adducts have been related to pathologies and carcinogenesis in many aquatic organisms.

  17. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  18. Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria.

    Science.gov (United States)

    Aitken, R John; De Iuliis, Geoffry N; Finnie, Jane M; Hedges, Andrew; McLachlan, Robert I

    2010-10-01

    DNA damage in human spermatozoa is known to be associated with a variety of adverse clinical outcomes affecting both reproductive efficiency and the health and wellbeing of the offspring. However, the origin of this damage, its biochemical nature and strategies for its amelioration, still await resolution. Using novel methods to simultaneously assess DNA fragmentation (modified TUNEL assay), DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine [8OHdG]) and cell vitality, spermatozoa from a cohort of 50 assisted conception patients were examined and compared with a group of donors. Receiver operating characteristic (ROC) curve analysis was then used to examine the frequency distribution of the data and to determine optimized thresholds for identifying patients exhibiting abnormally high levels of DNA damage. 8OHdG formation and DNA fragmentation were highly correlated with each other and frequently associated with cell death. Percoll centrifugation improved sperm quality but, unexpectedly, increased 8OHdG formation in live cells, as did sperm fractionation using Puresperm gradients. ROC analysis indicated that the frequency distribution of 8OHdG and DNA fragmentation data were significantly different between patients and donors (P live cells. However, the development of novel methods and optimized thresholds for diagnosing oxidative DNA damage in human spermatozoa should assist in the clinical management of this pathology.

  19. The application of psoralens to the study of DNA structure, function and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Spielmann, Peter Hans [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1991-04-01

    A series of six nitroxide spin-labeled psoralens were designed, synthesized and tested as probes for DNA dynamics. The synthesis of these spin-labeled psoralen derivatives and their photoreactivity with double-stranded DNA fragments is described. The spin labels (nitroxides) were demonstrated to survive the uv irradiation required to bind the probe to the target DNA. EPR spectra of the photobound spin-labels indicate that they do not wobble with respect to the DNA on the time-scales investigated. The author has used psoralen modified DNA as a model for the study of DNA repair enzyme systems in human cell free extracts. He has shown that damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is "repair synthesis" and not an aberrant DNA synthesis reaction potentiated by deformation of the DNA by adducts. He has found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach he has obtained evidence that this in vitro system is capable of removing psoralen cross-links as well. Reported here are synthetic methods that make use of high intensity lasers coupled with HPLC purification to make homogeneous and very pure micromole quantities of furan-side monoadducted, cross-linked, and pyrone-side monoadducted DNA oligonucleotide. These molecules are currently being studied by NMR and X-ray crystallography. The application of the site-specifically psoralen modified oligonucleotide synthesized by these methods to the construction of substrates for the investigation of DNA repair is also discussed.

  20. A biomarker model of sublethal genotoxicity (DNA single-strand breaks and adducts) using the sentinel organism Aporrectodea longa in spiked soil

    International Nuclear Information System (INIS)

    Martin, Francis L.; Piearce, Trevor G.; Hewer, Alan; Phillips, David H.; Semple, Kirk T.

    2005-01-01

    There is a need to develop risk biomarkers during the remediation of contaminated land. We employed the earthworm, Aporrectodea longa (Ude), to determine whether genotoxicity measures could be applied to this organism's intestinal tissues. Earthworms were added, for 24 h or 7 days, to soil samples spiked with benzo[a]pyrene (B[a]P) and/or lindane. After exposure, intestinal tissues (crop/gizzard or intestine) were removed prior to the measurement in disaggregated cells of DNA single-strand breaks (SSBs) by the alkaline comet assay. Damage was quantified by comet tail length (CTL, μm). B[a]P 24-h exposure induced dose-related increases (P 32 P-postlabelling, showed a two-adduct-spot pattern. This preliminary investigation suggests that earthworm tissues may be incorporated into genotoxicity assays to facilitate hazard identification within terrestrial ecosystems. - Sublethal genotoxicity in the sentinel organism A. longa can be used to monitor the effects of contaminants in soil

  1. Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage*

    Science.gov (United States)

    Comeaux, Evan Q.; Cuya, Selma M.; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C.; Mobley, James A.; Bjornsti, Mary-Ann; van Waardenburg, Robert C. A. M.

    2015-01-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

  2. Understanding the Effect of Carbonate Ion on Cisplatin Binding to DNA

    Science.gov (United States)

    Todd, Ryan C.; Lovejoy, Katherine S.; Lippard, Stephen J.

    2008-01-01

    The role of carbonate in the binding of cis-diamminedichloroplatinum(II) to DNA was investigated in order to understand the potential involvement of carbonato-cisplatin species in the mechanism of action of platinum anticancer agents. Cisplatin was allowed to react with both double- and single-stranded DNA in carbonate, phosphate, and HEPES