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Sample records for dna adduct levels

  1. 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

  2. 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

  3. 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...

  4. 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.

  5. 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.

  6. 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...

  7. 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

  8. [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

  9. 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)

  10. 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).

  11. 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).

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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...

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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....

  8. 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.

  9. 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

  10. 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.

  11. /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.

  12. 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

  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. 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.

  15. 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...

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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...

  6. 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)

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  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. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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....

  14. 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.

  15. 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.

  16. 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.

  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. 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...

  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. 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.

  1. 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

  2. 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

  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. 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.

  5. 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.

  6. 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

  7. 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...

  8. {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.

  9. 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

  10. 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.)

  11. 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)

  12. 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

  13. 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....

  14. 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, H; Daneshvar, B; Dragsted, L O

    1999-01-01

    /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...

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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)

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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....

  10. 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

  11. 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.

  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. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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)

  2. 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.

  3. 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...

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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...

  9. 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.

  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. 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. 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.

  13. 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

  14. 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

  15. 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

  16. 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...

  17. Vitamin A and β-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA-repair activities in hamster tracheal epithelium in organ culture

    NARCIS (Netherlands)

    Wolterbeek, A.P.M.; Roggeband, R.; Moorsel, C.J.A. van; Baan, R.A.; Koeman, J.H.; Feron, V.J.; Rutten, A.A.J.J.L.

    1995-01-01

    Although most studies concerning the effect of vitamin A and β-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and β-carotene on unscheduled DNA synthesis (UDS) was

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans. The U.S. National Toxicology Program (NTP) classified riddelliine, a tumorigenic pyrrolizidine alkaloid, as "reasonably anticipated to be a human carcinogen" in the NTP 12th Report on Carcinogens in 2011. We previously determined that four DNA adducts were formed in rats dosed with riddelliine. The structures of the four DNA adducts were elucidated as (i) a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N(2)-yl)dehydrosupinidine adducts (termed as DHP-dG-3 and DHP-dG-4) as the predominant adducts; and (ii) a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N(6)-yl)dehydrosupinidine adducts (termed as DHP-dA-3 and DHP-dA-4 adducts). In this study, we selected a nontumorigenic pyrrolizidine alkaloid, platyphylliine, a pyrrolizidine alkaloid N-oxide, riddelliine N-oxide, and nine tumorigenic pyrrolizidine alkaloids (riddelliine, retrorsine, monocrotaline, lycopsamine, retronecine, lasiocarpine, heliotrine, clivorine, and senkirkine) for study in animals. Seven of the nine tumorigenic pyrrolizidine alkaloids, with the exception of lycopsamine and retronecine, are liver carcinogens. At 8-10 weeks of age, female F344 rats were orally gavaged for 3 consecutive days with 4.5 and 24 μmol/kg body weight test article in 0.5 mL of 10% DMSO in water. Twenty-four hours after the last dose, the rats were sacrificed, livers were removed, and liver DNA was isolated for DNA adduct analysis. DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts were formed in the liver of rats treated with the individual seven hepatocarcinogenic pyrrolizidine alkaloids and riddelliine N-oxide. These DNA adducts were not formed in the liver of rats administered retronecine, the nontumorigenic pyrrolizidine alkaloid, platyphylliine, or vehicle control. These results indicate that this set of DNA adducts, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, is a common biological biomarker of

  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. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  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. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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)

  13. 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.

  14. Differences in micronucleus frequency and acrylamide adduct levels with hemoglobin between vegetarians and non-vegetarians.

    Science.gov (United States)

    Kotova, Natalia; Frostne, Cecilia; Abramsson-Zetterberg, Lilianne; Tareke, Eden; Bergman, Rolf; Haghdoost, Siamak; Paulsson, Birgit; Törnqvist, Margareta; Segerbäck, Dan; Jenssen, Dag; Grawé, Jan

    2015-10-01

    Nutrients and food constituents can prevent or contribute to genotoxicity. In this study, the possible influence of a vegetarian/non-vegetarian diet on genotoxic effects was investigated in 58 non-smoking healthy vegetarians (V) and non-vegetarians (NV), age 21-37 years from the Stockholm area in Sweden. Physical activity and dietary habits were similar in both groups, with the exception of the intake of meat and fish. Using flow cytometry, we determined the formation of micronuclei (MN) in transferrin-positive immature peripheral blood reticulocytes (Trf-Ret) (Total: n = 53; V: n = 27; NV: n = 26). Dietary exposure to acrylamide was measured through hemoglobin (Hb) adducts in peripheral erythrocytes (Total: n = 53; V: n = 29; NV: n = 24). Hb adducts of both acrylamide and its genotoxic metabolite glycidamide were monitored as a measure of the corresponding in vivo doses. Our data demonstrated that compared with the non-vegetarians, the vegetarians exhibited lower frequencies of MN (fMN) in the Trf-Ret (p vegetarians and non-vegetarians. Furthermore, there were no significant relationships between the adduct levels and fMN in the individuals. The ratio of the Hb adduct levels from glycidamide and acrylamide, however, showed a significant difference (p vegetarian diet might be beneficial in lowering genomic instability in healthy individuals. The measured Hb adduct levels indicate that the total intake of acrylamide does not differ between the two studied groups and does not contribute to the observed difference in fMN, although an influence of the diet on the metabolic rates of acrylamide was indicated. In addition, the observed significant difference in the background fMN in the two groups demonstrated that the MN analysis method has a sensitivity applicable to the biomonitoring of human lifestyle factors.

  15. 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.

  16. Dynamic and Progressive Control of DNA Origami Conformation by Modulating DNA Helicity with Chemical Adducts.

    Science.gov (United States)

    Chen, Haorong; Zhang, Hanyu; Pan, Jing; Cha, Tae-Gon; Li, Shiming; Andréasson, Joakim; Choi, Jong Hyun

    2016-05-24

    DNA origami has received enormous attention for its ability to program complex nanostructures with a few nanometer precision. Dynamic origami structures that change conformation in response to environmental cues or external signals hold great promises in sensing and actuation at the nanoscale. The reconfiguration mechanism of existing dynamic origami structures is mostly limited to single-stranded hinges and relies almost exclusively on DNA hybridization or strand displacement. Here, we show an alternative approach by demonstrating on-demand conformation changes with DNA-binding molecules, which intercalate between base pairs and unwind DNA double helices. The unwinding effect modulates the helicity mismatch in DNA origami, which significantly influences the internal stress and the global conformation of the origami structure. We demonstrate the switching of a polymerized origami nanoribbon between different twisting states and a well-constrained torsional deformation in a monomeric origami shaft. The structural transformation is shown to be reversible, and binding isotherms confirm the reconfiguration mechanism. This approach provides a rapid and reversible means to change DNA origami conformation, which can be used for dynamic and progressive control at the nanoscale.

  17. 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

  18. 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.

  19. 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

    more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.

  20. 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

    more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.

  1. 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 ...

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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...

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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...

  17. 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.

  18. 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.

  19. 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)...

  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. 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

  2. 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

  3. 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.

  4. 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...

  5. 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...

  6. 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

  7. 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).

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. CYP1A2 and NAT2 phenotyping and 3-aminobiphenyl and 4-aminobiphenyl hemoglobin adduct levels in smokers and non-smokers

    International Nuclear Information System (INIS)

    Sarkar, Mohamadi; Stabbert, Regina; Kinser, Robin D.; Oey, Jan; Rustemeier, Klaus; Holt, Klaus von; Schepers, Georg; Walk, Roger A.; Roethig, Hans J.

    2006-01-01

    Some aromatic amines are considered to be putative bladder carcinogens. Hemoglobin (Hb) adducts of 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP) have been used as biomarkers of exposure to aromatic amines from cigarette smoke. One of the goals of this study was to determine intra- and inter-individual variability in 3-ABP and 4-ABP Hb adducts and to explore the predictability of ABP Hb adduct levels based on caffeine phenotyping. The study was conducted in adult smokers (S, n = 65) and non-smokers (NS, n 65). The subjects were phenotyped for CYP1A2 and NAT2 using urinary caffeine metabolites. Blood samples were collected twice within 6 weeks and adducts measured by GC/MS. The levels of 4-ABP Hb adducts were significantly (p < 0.0001) greater in S (34.5 ± 21.06 pg/g Hb) compared to NS (6.3 ± 3.02 pg/g Hb). The levels of 3-ABP Hb adducts were below the limit of quantification (BLOQ) in most (82%) of the NS and about 10-fold lower in S (3.6 ± 3.29 pg/g Hb) compared to 4-ABP Hb adducts. No differences were observed in the adduct levels between weeks 1 and 6 in the smokers, suggesting that a single sample would be adequate to monitor cigarette smoke exposure. The regression model developed with CYP1A2, NAT2 phenotype and number of cigarettes smoked (NCIG) accounted for 47% of the variability in 3-ABP adducts, whereas 32% variability in 4-ABP adducts was accounted by CYP1A2 and NCIG. The ratio of 4-ABP Hb adducts in adult S:NS was ∼ 5:1, whereas 3-ABP Hb adducts levels were BLOQ in some S, exhibited large interindividual variability (∼ 91% compared to 57% for 4-ABP Hb) and poor dose response relationship. Therefore, 4-ABP Hb adduct levels may be a more useful biomarker of aminobiphenyl exposure from cigarette smoke

  14. 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.

  15. 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...

  16. The thermodynamics of translesion DNA synthesis past major adducts of enantiomeric analogues of antitumor cisplatin

    Czech Academy of Sciences Publication Activity Database

    Malina, Jaroslav; Nováková, Olga; Natile, G.; Brabec, Viktor

    2012-01-01

    Roč. 7, č. 5 (2012), s. 1026-1031 ISSN 1861-4728 R&D Projects: GA ČR(CZ) GAP205/11/0856; GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : thermodynamics * DNA * translesion Subject RIV: BO - Biophysics Impact factor: 4.572, year: 2012

  17. Synthesis, characterization and DNA cleavage activity of nickel(II adducts with aromatic heterocyclic bases

    Directory of Open Access Journals (Sweden)

    G. H. PHILIP

    2010-01-01

    Full Text Available Mixed ligand complexes of nickel(II with 2,4-dihydroxyaceto-phenone oxime (DAPO and 2,4-dihydroxybenzophenone oxime (DBPO as primary ligands, and pyridine (Py and imidazole (Im as secondary ligands were synthesized and characterized by molar conductivity, magnetic moments measurements, as well as by electronic, IR, and 1H-NMR spectroscopy. Electrochemical studies were performed by cyclic voltammetry. The active signals are assignable to the NiIII/II and NiII/I redox couples. The binding interactions between the metal complexes and calf thymus DNA were investigated by absorption and thermal denaturation. The cleavage activity of the complexes was determined using double-stranded pBR322 circular plasmid DNA by gel electrophoresis. All complexes showed increased nuclease activity in the presence of the oxidant H2O2. The nuclease activities of mixed ligand complexes were compared with those of the parent copper(II complexes.

  18. Thermodynamics of translesion synthesis across a major DNA adduct of antitumor oxaliplatin: Differential scanning calorimetric study

    Czech Academy of Sciences Publication Activity Database

    Florian, Jakub; Brabec, Viktor

    2012-01-01

    Roč. 18, č. 6 (2012), s. 1634-1639 ISSN 0947-6539 R&D Projects: GA ČR(CZ) GD301/09/H004; GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : antitumor platinum * DNA * calorimetry Subject RIV: BO - Biophysics Impact factor: 5.831, year: 2012

  19. 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

  20. 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.

  1. Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases

    Science.gov (United States)

    Sherrer, Shanen M.; Taggart, David J.; Pack, Lindsey R.; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2012-01-01

    N- (deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolkk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans. PMID:22917544

  2. DNA adducts of antitumor cisplatin preclude telomeric sequences from forming G quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Heringová, Pavla; Kašpárková, Jana; Brabec, Viktor

    2009-01-01

    Roč. 14, č. 6 (2009), s. 959-968 ISSN 0949-8257 R&D Projects: GA MZd(CZ) NR8562; GA MŠk(CZ) LC06030; GA MŠk(CZ) ME08017; GA MŠk(CZ) OC08003; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA AV ČR(CZ) IAA400040803 Grant - others:GA MŠk(CZ) OC09018 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : cisplatin * DNA quadruplex * telomere Subject RIV: BO - Biophysics Impact factor: 3.415, year: 2009

  3. Bifunctional amine-tethered ruthenium(II) arene complexes form monofunctional adducts on DNA

    Czech Academy of Sciences Publication Activity Database

    Melchart, M.; Habtemariam, A.; Nováková, Olga; Moggach, S.A.; Fabbiani, F.P.A.; Parsons, S.; Brabec, Viktor; Sadler, P.J.

    2007-01-01

    Roč. 46, č. 21 (2007), s. 8950-8962 ISSN 0020-1669 R&D Projects: GA ČR(CZ) GA305/05/2030; GA ČR(CZ) GA203/06/1239; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * ruthenium * cancer Subject RIV: BO - Biophysics Impact factor: 4.123, year: 2007

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

    Science.gov (United States)

    Bose, Arindam; Pande, Paritosh; Jasti, Vijay P.; Millsap, Amy D.; Hawkins, Edward K.; Rizzo, Carmelo J.; Basu, Ashis K.

    2015-01-01

    The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5′-G1G2CG3CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass. PMID:26220181

  12. 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

  13. 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

  14. 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,

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Direct dissolution of g-level U metal and U-6 % Zr alloy bits by TBP-nitric acid adduct and in situ extraction at ambient pressures

    International Nuclear Information System (INIS)

    Shekhar Kumar; Bijendra Kumar; Gelatar, J.K.; Pranay Kumar Sinha; Alok Kumar Mishra; Kamachi Mudali, U.

    2016-01-01

    A study on direct dissolution of g-level metallic U and U-6 % Zr alloy bits by TBP-nitric acid adduct followed by in situ extraction at ambient pressures under batch and dynamic conditions was performed. The product organic solution was adjusted to 30 % TBP in dodecane and from it, U(VI) could be stripped quantitatively with 0.01 N nitric acid. Experimental results of the study are presented in this paper. (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. 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

  2. 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

  3. Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: Dose–response, mechanisms, and clinical implications

    International Nuclear Information System (INIS)

    McGill, Mitchell R.; Lebofsky, Margitta; Norris, Hye-Ryun K.; Slawson, Matthew H.; Bajt, Mary Lynn; Xie, Yuchao; Williams, C. David; Wilkins, Diana G.; Rollins, Douglas E.; Jaeschke, Hartmut

    2013-01-01

    At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose–response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia–reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter. - Highlights: • Extensive GSH depletion is not required for APAP-protein binding in the liver. • APAP-protein adducts appear in plasma at subtoxic doses. • Proteins are adducted in the cell and secreted out. • Coincidental liver injury increases plasma APAP-protein adducts at subtoxic doses

  4. 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

  5. Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse

    Energy Technology Data Exchange (ETDEWEB)

    Siddens, Lisbeth K. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331 (United States); Superfund Research Center, Oregon State University, Corvallis, OR 97331 (United States); Bunde, Kristi L. [College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331 (United States); Harper, Tod A. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331 (United States); Linus Pauling Institute, Oregon State University, Corvallis, OR 97331 (United States); Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 (United States); McQuistan, Tammie J. [Superfund Research Center, Oregon State University, Corvallis, OR 97331 (United States); Linus Pauling Institute, Oregon State University, Corvallis, OR 97331 (United States); Löhr, Christiane V. [Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 (United States); College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331 (United States); Bramer, Lisa M. [Applied Statistics and Computational Modeling, Pacific Northwest National Laboratory, Richland, WA 99352 (United States); Waters, Katrina M. [Superfund Research Center, Oregon State University, Corvallis, OR 97331 (United States); Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352 (United States); Tilton, Susan C. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331 (United States); Superfund Research Center, Oregon State University, Corvallis, OR 97331 (United States); Krueger, Sharon K. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331 (United States); Superfund Research Center, Oregon State University, Corvallis, OR 97331 (United States); Linus Pauling Institute, Oregon State University, Corvallis, OR 97331 (United States); and others

    2015-09-01

    FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8 h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8 h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4 h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8 h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators. - Highlights: • Cyp1b1 null mice exhibit lower skin cancer sensitivity to DBC but not BaP or CTE. • Cyp1b1 expression impacts expression of other PAH metabolizing enzymes. • cis/trans-DBCDE-dA ratio significantly higher in the skin than the spleen, lung or liver • Potency of DBC and CTE in mouse skin is higher than predicted by RPFs.

  6. 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

  7. DNA adducts, mutant frequencies and mutation spectra in λlacZ transgenic mice treated with N-nitrosodimethylamine

    NARCIS (Netherlands)

    Souliotis, V.L.; Delft, J.H.M. van; Steenwinkel, M.-J.S.T.; Baan, R.A.; Kyrtopoulos, S.A.

    1998-01-01

    Groups of λlacZ transgenic mice were treated i.p. with N-nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in

  8. 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.

  9. Kin3 protein, a NIMA-related kinase of Saccharomyces cerevisiae, is involved in DNA adduct damage response.

    Science.gov (United States)

    Moura, Dinara J; Castilhos, Bruna; Immich, Bruna F; Cañedo, Andrés D; Henriques, João A P; Lenz, Guido; Saffi, Jenifer

    2010-06-01

    Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.

  10. Structural insight into dynamic bypass of the major cisplatin-DNA adduct by Y-family polymerase Dpo4

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Jimson H.Y.; Brown, Jessica A.; Suo, Zucai; Blum, Paul; Nohmi, Takehiko; Ling, Hong (OSU); (NINA-Japan); (UNL); (UWO)

    2010-08-23

    Y-family DNA polymerases bypass Pt-GG, the cisplatin-DNA double-base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y-family DNA polymerase, Dpo4, in complex with Pt-GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3{prime}G (first insertion) and 5{prime}G (second insertion) of Pt-GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation-coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt-GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4-mediated Pt-GG bypass was addressed by a dpo-4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.

  11. METABOLISM AND DNA ADDUCT FORMATION OF 2-ACETYLAMINOFLUORENE BY BLADDER EXPLANTS FROM HUMAN, DOG, MONKEY, HAMSTER AND RAT

    Science.gov (United States)

    It is concluded that bladder explants of the human, dog, monkey, hamster, and rat metabolize AAF mainly to ring-hydroxylated products, but also form small amounts of the proximate carcinogenic metabolite N-hydroxy-AAF. Neither the overall binding of AAF to bladder DNA, nor the fo...

  12. Diazido Mixed-Amine Platinum(IV) Anticancer Complexes Activatable by Visible-Light Form Novel DNA Adducts

    Czech Academy of Sciences Publication Activity Database

    Zhao, Y.; Woods, J.; Farrer, N.J.; Robinson, K.S.; Prachařová, J.; Kašpárková, J.; Nováková, Olga; Li, H.L.; Salassa, L.; Pizarro, A.M.; Clarkson, G.J.; Song, L.J.; Brabec, Viktor; Sadler, P. J.

    2013-01-01

    Roč. 19, č. 29 (2013), s. 9578-9591 ISSN 0947-6539 R&D Projects: GA ČR(CZ) GAP301/10/0598 Grant - others:GA AV(CZ) M200041201 Institutional support: RVO:68081707 Keywords : antitumor agents * DNA binding * medicinal chemistry Subject RIV: BO - Biophysics Impact factor: 5.696, year: 2013

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Analysis of Low Level DNA Mixtures

    Czech Academy of Sciences Publication Activity Database

    Slovák, Dalibor; Zvárová, Jana

    2013-01-01

    Roč. 1, č. 1 (2013), s. 63-63 ISSN 1805-8698. [EFMI 2013 Special Topic Conference. 17.04.2013-19.04.2013, Prague] Institutional support: RVO:67985807 Keywords : forensic DNA interpretation * low level samples * allele peak heights * dropout probability Subject RIV: IN - Informatics, Computer Science

  18. Stochastic Models for Low Level DNA Mixtures

    Czech Academy of Sciences Publication Activity Database

    Slovák, Dalibor; Zvárová, Jana

    2012-01-01

    Roč. 8, č. 5 (2012), s. 25-30 ISSN 1801-5603 Grant - others:GA UK(CZ) SVV-2012-264513 Institutional support: RVO:67985807 Keywords : forensic DNA interpretation * low level samples * allele peak areas * dropout probability Subject RIV: IN - Informatics, Computer Science http://www.ejbi.org/img/ejbi/2012/5/Slovak_en.pdf

  19. Stochastic Models for Low Level DNA Mixtures

    Czech Academy of Sciences Publication Activity Database

    Slovák, Dalibor; Zvárová, Jana

    2013-01-01

    Roč. 1, č. 1 (2013), s. 28-28 ISSN 1805-8698. [EFMI 2013 Special Topic Conference. 17.04.2013-19.04.2013, Prague] Institutional support: RVO:67985807 Keywords : forensic DNA interpretation * low level samples * allele peak heights * dropout probability Subject RIV: IN - Informatics, Computer Science

  20. Metabolism of aflatoxin B1 and identification of the major aflatoxin B1-DNA adducts formed in cultured human bronchus and colon

    DEFF Research Database (Denmark)

    Autrup, Herman; Essigmann, John M.; Croy, Robert G.

    1979-01-01

    Aflatoxin B1 and benzo(a)pyrene were activated by both cultured human bronchus and human colon as measured by binding to cellular DNA and protein. The binding of aflatoxin B1 to DNA was dose dependent, and the level of binding was higher in cultured human bronchus than it was in the colon. When c...

  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. 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

  3. 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

  4. 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.

  5. 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)

  6. 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

  7. 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)

  8. Replacement of a thiourea with an amidine group in a monofunctional platinum-acridine antitumor agent. Effect on DNA interactions, DNA adduct recognition and repair

    Czech Academy of Sciences Publication Activity Database

    Kostrhunová, Hana; Malina, Jaroslav; Pickard, A.J.; Štěpánková, Jana; Vojtíšková, Marie; Kašpárková, Jana; Muchová, T.; Rohlfing, M.L.; Bierbach, U.; Brabec, Viktor

    2011-01-01

    Roč. 8, č. 5 (2011), s. 1941-1954 ISSN 1543-8384 R&D Projects: GA ČR(CZ) GAP205/11/0856 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : platinum * DNA binding * anticancer effects Subject RIV: BO - Biophysics Impact factor: 4.782, year: 2011

  9. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

    Czech Academy of Sciences Publication Activity Database

    Shing, R.; Šrám, Radim; Binková, Blanka; Kalina, I.; Popov, T. A.; Georgieva, T.; Garte, S.; Taioli, E.; Farmer, P. B.

    2007-01-01

    Roč. 620, - (2007), s. 83-92 ISSN 0027-5107 Grant - others:EU(GB) 2000-00091; EU(GB) G0100873 Institutional research plan: CEZ:AV0Z50390512 Source of funding: R - rámcový projekt EK ; R - rámcový projekt EK Keywords : air pollution * PAHs * oxidative DNA damage Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 4.159, year: 2007

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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)

  16. 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.

  17. Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts.

    Science.gov (United States)

    Jędrak, Paulina; Krygier, Magdalena; Tońska, Katarzyna; Drozd, Małgorzata; Kaliszewska, Magdalena; Bartnik, Ewa; Sołtan, Witold; Sitek, Emilia J; Stanisławska-Sachadyn, Anna; Limon, Janusz; Sławek, Jarosław; Węgrzyn, Grzegorz; Barańska, Sylwia

    2017-08-01

    Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.

  18. Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes

    DEFF Research Database (Denmark)

    Jeppesen, A N; Hvas, A-M; Grejs, A M

    2017-01-01

    Background Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone...... after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus. The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. Results We found no difference...... in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P

  19. 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

  20. Temporal Variation and Association of Aflatoxin B₁ Albumin-Adduct Levels with Socio-Economic and Food Consumption Factors in HIV Positive Adults.

    Science.gov (United States)

    Jolly, Pauline E; Akinyemiju, Tomi F; Jha, Megha; Aban, Inmaculada; Gonzalez-Falero, Andrea; Joseph, Dnika

    2015-11-30

    The association between aflatoxin exposure and alteration in immune responses observed in humans suggest that aflatoxin could suppress the immune system and work synergistically with HIV to increase disease severity and progression to AIDS. No longitudinal study has been conducted to assess exposure to aflatoxin (AF) among HIV positive individuals. We examined temporal variation in AFB₁ albumin adducts (AF-ALB) in HIV positive Ghanaians, and assessed the association with socioeconomic and food consumption factors. We collected socioeconomic and food consumption data for 307 HIV positive antiretroviral naive adults and examined AF-ALB levels at recruitment (baseline) and at six (follow-up 1) and 12 (follow-up 2) months post-recruitment, by age, gender, socioeconomic status (SES) and food consumption patterns. Generalized linear models were used to examine the influence of socioeconomic and food consumption factors on changes in AF-ALB levels over the study period, adjusting for other covariates. AF-ALB levels (pg/mg albumin) were lower at baseline (mean AF-ALB: 14.9, SD: 15.9), higher at six months (mean AF-ALB: 23.3, SD: 26.6), and lower at 12 months (mean AF-ALB: 15.3, SD: 15.4). Participants with the lowest SES had the highest AF-ALB levels at baseline and follow up-2 compared with those with higher SES. Participants who bought less than 20% of their food and who stored maize for less than two months had lower AF-ALB levels. In the adjusted models, there was a statistically significant association between follow up time and season (dry or rainy season) on AF-ALB levels over time (p = 0.04). Asymptomatic HIV-positive Ghanaians had high plasma AF-ALB levels that varied according to season, socioeconomic status, and food consumption patterns. Steps need to be taken to ensure the safety and security of the food supply for the population, but in particular for the most vulnerable groups such as HIV positive people.

  1. Temporal Variation and Association of Aflatoxin B1 Albumin-Adduct Levels with Socio-Economic and Food Consumption Factors in HIV Positive Adults

    Directory of Open Access Journals (Sweden)

    Pauline E. Jolly

    2015-11-01

    Full Text Available The association between aflatoxin exposure and alteration in immune responses observed in humans suggest that aflatoxin could suppress the immune system and work synergistically with HIV to increase disease severity and progression to AIDS. No longitudinal study has been conducted to assess exposure to aflatoxin (AF among HIV positive individuals. We examined temporal variation in AFB1 albumin adducts (AF-ALB in HIV positive Ghanaians, and assessed the association with socioeconomic and food consumption factors. We collected socioeconomic and food consumption data for 307 HIV positive antiretroviral naive adults and examined AF-ALB levels at recruitment (baseline and at six (follow-up 1 and 12 (follow-up 2 months post-recruitment, by age, gender, socioeconomic status (SES and food consumption patterns. Generalized linear models were used to examine the influence of socioeconomic and food consumption factors on changes in AF-ALB levels over the study period, adjusting for other covariates. AF-ALB levels (pg/mg albumin were lower at baseline (mean AF-ALB: 14.9, SD: 15.9, higher at six months (mean AF-ALB: 23.3, SD: 26.6, and lower at 12 months (mean AF-ALB: 15.3, SD: 15.4. Participants with the lowest SES had the highest AF-ALB levels at baseline and follow up-2 compared with those with higher SES. Participants who bought less than 20% of their food and who stored maize for less than two months had lower AF-ALB levels. In the adjusted models, there was a statistically significant association between follow up time and season (dry or rainy season on AF-ALB levels over time (p = 0.04. Asymptomatic HIV-positive Ghanaians had high plasma AF-ALB levels that varied according to season, socioeconomic status, and food consumption patterns. Steps need to be taken to ensure the safety and security of the food supply for the population, but in particular for the most vulnerable groups such as HIV positive people.

  2. Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

    Science.gov (United States)

    McGreal, Steven R; Bhushan, Bharat; Walesky, Chad; McGill, Mitchell R; Lebofsky, Margitta; Kandel, Sylvie E; Winefield, Robert D; Jaeschke, Hartmut; Zachara, Natasha E; Zhang, Zhen; Tan, Ee Phie; Slawson, Chad; Apte, Udayan

    2018-04-01

    Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

  3. Parental epigenetic difference in DNA methylation-level may play ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... We found that a specific type of DNA methylation-level difference, that is, relative CHG (H ... eukaryotes and is particularly abundant in higher plants, ..... characterization of a set of disease resistance-gene analogs (RGAs).

  4. 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.

  5. 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.

  6. How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene.

    Science.gov (United States)

    Sholder, Gabriel; Creech, Amanda; Loechler, Edward L

    2015-11-01

    To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N(2)-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N(2)-dG. To investigate structural differences between Y-Family-classes, regions are swapped between DNAP IV (a κ/IV-class-member) and Dpo4 (a η/V-class-member); the kinetic consequences are evaluated via primer-extension studies with a BP-N(2)-dG-containing template. Four key structural elements are revealed. (1) Y-Family DNAPs have discreet non-covalent contacts between their little finger-domain (LF-Domain) and their catalytic core-domain (CC-Domain), which we call "non-covalent bridges" (NCBs). Arg37 and Arg38 in DNAP IV's CC-Domain near the active site form a non-covalent bridge (AS-NCB) by interacting with Glu251 and Asp252, respectively, in DNAP IV's LF-Domain. Without these interactions dATP/dGTP/dTTP misinsertions increase. DNAP IV's AS-NCB suppresses misinsertions better than Dpo4's equivalent AS-NCB. (2) DNAP IV also suppresses dATP/dGTP/dTTP misinsertions via a second non-covalent bridge, which is ∼8Å from the active site (Distal-NCB). Dpo4 has no Distal-NCB, rendering it inferior at dATP/dGTP/dTTP suppression. (3) dCTP insertion is facilitated by the larger minor groove opening near the active site in DNAP IV versus Dpo4, which is sensible given that Watson/Crick-like [dCTP:BP-N(2)-dG] pairing requires the BP-moiety to be in the minor groove. (4) Compared to Dpo4, DNAP IV has a smaller major groove opening, which suppresses dGTP misinsertion, implying BP-N(2)-dG bulk in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. In summary, DNAP IV has a large minor groove opening to enhance dCTP insertion, a plugged major groove opening to suppress dGTP misinsertion, and two non-covalent bridges (near and distal

  7. DNA methylation levels associated with race and childhood asthma severity.

    Science.gov (United States)

    Chan, Marcia A; Ciaccio, Christina E; Gigliotti, Nicole M; Rezaiekhaligh, Mo; Siedlik, Jacob A; Kennedy, Kevin; Barnes, Charles S

    2017-10-01

    Asthma is a common chronic childhood disease worldwide. Socioeconomic status, genetic predisposition and environmental factors contribute to its incidence and severity. A disproportionate number of children with asthma are economically disadvantaged and live in substandard housing with potential indoor environmental exposures such as cockroaches, dust mites, rodents and molds. These exposures may manifest through epigenetic mechanisms that can lead to changes in relevant gene expression. We examined the association of global DNA methylation levels with socioeconomic status, asthma severity and race/ethnicity. We measured global DNA methylation in peripheral blood of children with asthma enrolled in the Kansas City Safe and Healthy Homes Program. Inclusion criteria included residing in the same home for a minimum of 4 days per week and total family income of less than 80% of the Kansas City median family income. DNA methylation levels were quantified by an immunoassay that assessed the percentage of 5-methylcytosine. Our results indicate that overall, African American children had higher levels of global DNA methylation than children of other races/ethnicities (p = 0.029). This difference was more pronounced when socioeconomic status and asthma severity were coupled with race/ethnicity (p = 0.042) where low-income, African American children with persistent asthma had significantly elevated methylation levels relative to other races/ethnicities in the same context (p = 0.006, Hedges g = 1.14). Our study demonstrates a significant interaction effect among global DNA methylation levels, asthma severity, race/ethnicity, and socioeconomic status.

  8. 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.

  9. Parental epigenetic difference in DNA methylation-level may play ...

    African Journals Online (AJOL)

    Parental epigenetic difference in DNA methylation-level may play contrasting roles for different agronomic traits related to yield heterosis in maize. ... or hybrid vigor has been exploited to nearly the fullest extent, the molecular and genetic basis underlying this remarkable biological phenomenon remains largely an enigma.

  10. DNA damage and plasma homocysteine levels are associated with ...

    African Journals Online (AJOL)

    This study describes the association between levels of DNA damage and homocysteine (Hcy) in persistent diarrheic (PD) patients and correlates them with serum biochemical metabolites and mineral components. PD patients (n = 36) age 4 - 6 years from Faisalabad hospitals were examined for anthropometric factors, ...

  11. 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.

  12. 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.

  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. 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.

  15. Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples.

    Science.gov (United States)

    Peñarrubia, Luis; Alcaraz, Carles; Vaate, Abraham Bij de; Sanz, Nuria; Pla, Carles; Vidal, Oriol; Viñas, Jordi

    2016-12-14

    The zebra mussel (Dreissena polymorpha Pallas, 1771) and the quagga mussel (D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebra and quagga mussels in environmental DNA samples. Our results show specific dreissenid DNA present in filtered water samples for which microscopic diagnostic identification for larvae failed. Monitoring a large number of locations for invasive dreissenid species based on a highly specific environmental DNA qPCR assay may prove to be an essential tool for management and control plans focused on prevention of establishment of dreissenid mussels in new locations.

  16. [Laryngeal adduction reflex].

    Science.gov (United States)

    Ptok, M; Bonenberger, S; Miller, S; Kühn, D; Jungheim, M

    2014-07-01

    Laryngeal Adductor Reflex Background: A rapid closure of the vocal folds is necessary, whenever foreign materials or food particles penetrate into the larynx. Otherwise a passage of these particles into the trachea or the lower respiratory tract would be imminent. An aspiration could mechanically block the respiratory tract and cause severe dyspnoea or cause aspiration pneumonia. For this systematic review a selective literature research in PubMed and Scopus using the keywords "laryngeal adductor reflex" and "vocal fold closure" has been carried out. Apart from the oesophago-glottal and pharyngo-glottal closure reflexes, the laryngeal adductor reflex (LAR) has been investigated in particular. The LAR qualifies as a reflectory laryngeal adductor mechanism and involves early, presumably di- or oligosynaptic ipsilateral LAR1 as well as late polysynaptic ipsi- and contralateral LAR2 components. In clinical routine diagnostic settings of dysphagia, LAR is only assessed qualitatively and usually triggered by air pulses or tactile stimulation. Dysphagiologists often find that not only the laryngeal sensibility in general is impaired, but especially the protective laryngeal adduction mechanism, which results in a higher risk of aspiration. Thus, it appears mandatory to test the LAR not only qualitatively but also quantitatively. Unfortunately a valid and reliable method that can be employed in clinical practice has not yet been put forward. © Georg Thieme Verlag KG Stuttgart · New York.

  17. 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

  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. Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

    Science.gov (United States)

    Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

    2015-11-24

    Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

  20. Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

    Science.gov (United States)

    Meroni, Alice; Nava, Giulia M; Sertic, Sarah; Plevani, Paolo; Muzi-Falconi, Marco; Lazzaro, Federico

    2018-01-01

    Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.

  1. NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (εdA) opposite thymidine in a DNA duplex. Nonplanar alignment of εdA(anti) and dT(anti) at the lesion site

    International Nuclear Information System (INIS)

    Kouchakdjian, M.; Patel, D.J.; Eisenberg, M.; Yarema, K.; Basu, A.; Essigmann, J.

    1991-01-01

    Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C)·d(G-T-A-C-εA-C-A-T-G) nonanucleotide duplex (designated εdA·dT 9-mer duplex) containing 1,N 6 -ethenodeoxyadenosine (εdA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. The authors NMR studies have focused on the conformation of the εdA·dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5·εdA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and εdA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4·dC15 and dG6·dC13 pairs. Furthermore, the d(G4-T5-G6)·d(C13-εA14-C15) trinucleotide segment centered about the dT5·εdA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and εdA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the εdA·dT 9-mer duplex. The NMR data are consistent with a nonplanar alignment of εdA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4·dC15 base pair within the d(G4-T5-G6)·d(C13-εA14-C15) segment of the εdA·dT 9-mer duplex

  2. 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

  3. DNA adducts and human atherosclerotic lesions

    Czech Academy of Sciences Publication Activity Database

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

    2001-01-01

    Roč. 204, - (2001), s. 49-54 ISSN 1438-4639 R&D Projects: GA MŽP SI/340/00 Institutional research plan: CEZ:AV0Z5039906 Keywords : atherosclerosis * autopsy thoracic aortas Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 0.480, year: 2001

  4. 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

  5. Modelling DNA origami self-assembly at the domain level

    Energy Technology Data Exchange (ETDEWEB)

    Dannenberg, Frits; Kwiatkowska, Marta [Department of Computer Science, University of Oxford, Wolfson Building, Parks Road, Oxford OX1 3QD (United Kingdom); Dunn, Katherine E. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Department of Electronics, University of York, York YO10 5DD (United Kingdom); Bath, Jonathan; Turberfield, Andrew J. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Ouldridge, Thomas E. [Department of Physics, University of Oxford, Rudolf Peierls Centre for Theoretical Physics, 1 Keble Road, Oxford OX1 3NP (United Kingdom); Department of Mathematics, Imperial College, 180 Queen’s Gate, London SW7 2AZ (United Kingdom)

    2015-10-28

    We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

  6. Modelling DNA origami self-assembly at the domain level

    International Nuclear Information System (INIS)

    Dannenberg, Frits; Kwiatkowska, Marta; Dunn, Katherine E.; Bath, Jonathan; Turberfield, Andrew J.; Ouldridge, Thomas E.

    2015-01-01

    We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami

  7. Modelling DNA origami self-assembly at the domain level

    Science.gov (United States)

    Dannenberg, Frits; Dunn, Katherine E.; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J.; Ouldridge, Thomas E.

    2015-10-01

    We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

  8. Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB)

    OpenAIRE

    van Loon, Barbara; Samson, Leona D.

    2013-01-01

    Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known...

  9. Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

    Science.gov (United States)

    Cai, Yi; Tsai, Hsing-Chen; Yen, Ray-Whay Chiu; Zhang, Yang W; Kong, Xiangqian; Wang, Wei; Xia, Limin; Baylin, Stephen B

    2017-04-01

    Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential. © 2017 Cai et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Including the Copenhagen Adduction Exercise in the FIFA 11+ Provides Missing Eccentric Hip Adduction Strength Effect in Male Soccer Players: A Randomized Controlled Trial.

    Science.gov (United States)

    Harøy, Joar; Thorborg, Kristian; Serner, Andreas; Bjørkheim, André; Rolstad, Linn E; Hölmich, Per; Bahr, Roald; Andersen, Thor Einar

    2017-11-01

    The FIFA 11+ was developed as a complete warm-up program to prevent injuries in soccer players. Although reduced hip adduction strength is associated with groin injuries, none of the exercises included in the FIFA 11+ seem to specifically target hip adduction strength. To investigate the effect on eccentric hip adduction strength of the FIFA 11+ warm-up program with or without the Copenhagen adduction exercise. Randomized controlled trial; Level of evidence, 1. We recruited 45 eligible players from 2 U19 elite male soccer teams. Players were randomized into 2 groups; 1 group carried out the standard FIFA 11+ program, while the other carried out the FIFA 11+ but replaced the Nordic hamstring exercise with the Copenhagen adduction exercise. Both groups performed the intervention 3 times weekly for 8 weeks. Players completed eccentric strength and sprint testing before and after the intervention. Per-protocol analyses were performed, and 12 players were excluded due to low compliance (<67% of sessions completed). The main outcome was eccentric hip adduction strength (N·m/kg). Between-group analyses revealed a significantly greater increase in eccentric hip adduction strength of 0.29 Nm/kg (8.9%; P = .01) in favor of the group performing the Copenhagen adduction exercise, whereas no within-group change was noted in the group that used the standard FIFA 11+ program (-0.02 N·m/kg [-0.7%]; P = .69). Including the Copenhagen adduction exercise in the FIFA 11+ program increases eccentric hip adduction strength, while the standard FIFA 11+ program does not. Registration: Registration: ISRCTN13731446 (International Standard Randomised Controlled Trial Number registry).

  11. Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in colon and liver of Big Blue Rats: role of DNA adducts, strand breaks, DNA repair and oxidative stress

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Håkan; Vogel, Ulla

    2002-01-01

    , indicating a higher rate of protein oxidation in the liver following IQ administration. In plasma and erythrocytes there were unaltered levels of oxidized protein, malondialdehyde, and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase) indicating...

  12. Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in colon and liver of Big Blue rats: role of DNA adducts, strand breaks, DNA repair and oxidative stress

    DEFF Research Database (Denmark)

    Moller, P.; Wallin, H.; Vogel, U.

    2002-01-01

    , indicating a higher rate of protein oxidation in the liver following IQ administration. In plasma and erythrocytes there were unaltered levels of oxidized protein, malondialdehyde, and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase) indicating...

  13. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  14. S-(3-Aminobenzanthron-2-yl)cysteine in the globin of rats as a novel type of adduct and possible biomarker of exposure to 3-nitrobenzanthrone, a potent environmental carcinogen.

    Science.gov (United States)

    Linhart, Igor; Hanzlíková, Iveta; Mráz, Jaroslav; Dušková, Šárka

    2017-10-01

    3-Nitrobenzanthrone (3-NBA), a potent environmental mutagen and carcinogen, is known to be activated in vivo to 3-benzanthronylnitrenium ion which forms both NH and C2-bound adducts with DNA and also reacts with glutathione giving rise to urinary 3-aminobenzanthron-2-ylmercapturic acid. In this study, acid hydrolysate of globin from rats dosed intraperitoneally with 3-NBA was analysed by HPLC/MS to identify a novel type of cysteine adduct, 3-aminobenzanthron-2-ylcysteine (3-ABA-Cys), confirmed using a synthesised standard. The 3-ABA-Cys levels in globin peaked after single 3-NBA doses of 1 and 2 mg/kg on day 2 to attain 0.25 and 0.49 nmol/g globin, respectively, thereafter declining slowly to 70-80% of their maximum values during 15 days. After dosing rats for three consecutive days with 1 mg 3-NBA/kg a significant cumulation of 3-ABA-Cys in globin was observed. 3-ABA-Cys was also found in the plasma hydrolysate. Herein, after dosing with 1 and 2 mg 3-NBA/kg the adduct levels peaked on day 1 at 0.15 and 0.51 nmol/ml plasma, respectively, thereafter declining rapidly to undetectable levels on day 15. In addition, sulphinamide adducts were also found in the exposed rats, measured indirectly as 3-aminobenzanthrone (3-ABA) split off from globin by mild acid hydrolysis. Levels of both types of adducts in the globin samples parallelled very well with 3-ABA/3-ABA-Cys ratio being around 1:8. In conclusion, 3-ABA-Cys is the first example of arylnitrenium-cysteine adduct in globin representing a new promising class of biomarkers to assess cumulative exposures to aromatic amines, nitroaromatics and heteroaromatic amines.

  15. DNA damage and plasma homocysteine levels are associated with ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-01-18

    Jan 18, 2010 ... (Fluitest Glu, Biocon Solutions Pte Ltd, Singapore). Cholesterol, ... migration in the comet tail was taken as an estimate of DNA damage and is ..... fever, and dietary energy intake on weight gain in rural Bangladeshi children.

  16. Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Lorenzo, Y.; Karlsen, A.; Carlsen, M. H.; Novosadová, Vendula; Blomhoff, R.; Vodička, Pavel; Collins, A. R.

    2014-01-01

    Roč. 16, APR 2014 (2014), s. 66-73 ISSN 1568-7864 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : DNA damage * DNA repair capacity * diet Subject RIV: EB - Genetics ; Molecular Biology; EI - Biotechnology ; Bionics (BTO-N) Impact factor: 3.111, year: 2014

  17. 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.)

  18. 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.)

  19. 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

  20. Decreased Circulating mtDNA Levels in Professional Male Volleyball Players.

    Science.gov (United States)

    Nasi, Milena; Cristani, Alessandro; Pinti, Marcello; Lamberti, Igor; Gibellini, Lara; De Biasi, Sara; Guazzaloca, Alessandro; Trenti, Tommaso; Cossarizza, Andrea

    2016-01-01

    Exercise exerts various effects on the immune system, and evidence is emerging on its anti-inflammatory effects; the mechanisms on the basis of these modifications are poorly understood. Mitochondrial DNA (mtDNA) released from damaged cells acts as a molecule containing the so-called damage-associated molecular patterns and can trigger sterile inflammation. Indeed, high plasma levels of mtDNA are associated to several inflammatory conditions and physiological aging and longevity. The authors evaluated plasma mtDNA in professional male volleyball players during seasonal training and the possible correlation between mtDNA levels and clinical parameters, body composition, and physical performance. Plasma mtDNA was quantified by real-time PCR every 2 mo in 12 professional volleyball players (PVPs) during 2 consecutive seasons. As comparison, 20 healthy nonathlete male volunteers (NAs) were analyzed. The authors found lower levels of mtDNA in plasma of PVPs than in NAs. However, PVPs showed a decrease of circulating mtDNA only in the first season, while no appreciable variations were observed during the second season. No correlation was observed among mtDNA, hematochemical, and anthropometric parameters. Regular physical activity appeared associated with lower levels of circulating mtDNA, further confirming the protective, anti-inflammatory effect of exercise.

  1. Changes in DNA methylation levels during seed development in ...

    Indian Academy of Sciences (India)

    User

    flowering and seeds were collected every week starting from 6thday after fertilization (DAP) till ... (Mitutoyo, Japan).Seeds for DNA isolation were lyophilized at -55 0C for 24 hours in a ... phase(stages 4, 5, 6 and7),seed length increased to 21.14 mm.During the .... Government of India, for providing fellowship for this work.

  2. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S.A.; Oijen, A.M. van

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or

  3. Revisiting the stability of endo/exo Diels-Alder adducts between cyclopentadiene and 1,4-benzoquinone

    International Nuclear Information System (INIS)

    Tormena, Claudio F.; Lacerda Junior, Valdemar; Oliveira, Kleber T. de

    2010-01-01

    In this work it is presented a detailed theoretical analysis of the relative stability of endo/exo Diels-Alder adducts formed by the reaction between cyclopentadiene (1) and 1,4-benzoquinone (2). The intrinsic reaction coordinate (IRC) showed the existence of only one transition state for the reaction studied, for both endo 3 and exo 4 adducts. The energies of both adducts were obtained at high level of theory (CBS-Q) confirming that the endo adduct is more stable than exo, which is in the opposite way to the observed in reactions that usually follow Alder's rule. An electronic structure analysis was performed through NBO methodology, indicating that the attractive delocalization interaction predominates over the steric repulsive interaction in the endo adducts. In summary, for the studied cycloaddition reaction the endo adduct is the thermodynamic and kinetic product, which can be also confirmed by experimental data mentioned in this work. (author)

  4. Revisiting the stability of endo/exo Diels-Alder adducts between cyclopentadiene and 1,4-benzoquinone

    Energy Technology Data Exchange (ETDEWEB)

    Tormena, Claudio F. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil). Inst. de Quimica. Dept. de Quimica Organica; Lacerda Junior, Valdemar [Universidade Federal do Espirito Santo (UFES), Vitoria, ES (Brazil). Centro de Ciencias Exatas. Dept. de Quimica; Oliveira, Kleber T. de [Universidade Federal do ABC (UFABC), Santo Andre, SP (Brazil). Centro de Ciencias Naturais e Humanas

    2010-07-01

    In this work it is presented a detailed theoretical analysis of the relative stability of endo/exo Diels-Alder adducts formed by the reaction between cyclopentadiene (1) and 1,4-benzoquinone (2). The intrinsic reaction coordinate (IRC) showed the existence of only one transition state for the reaction studied, for both endo 3 and exo 4 adducts. The energies of both adducts were obtained at high level of theory (CBS-Q) confirming that the endo adduct is more stable than exo, which is in the opposite way to the observed in reactions that usually follow Alder's rule. An electronic structure analysis was performed through NBO methodology, indicating that the attractive delocalization interaction predominates over the steric repulsive interaction in the endo adducts. In summary, for the studied cycloaddition reaction the endo adduct is the thermodynamic and kinetic product, which can be also confirmed by experimental data mentioned in this work. (author)

  5. 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...

  6. Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    John P Grady

    Full Text Available Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied MT-ND1/MT-ND4 multiplex mtDNA deletion assay and a recently developed MT-ND1/B2M singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.

  7. Mass spectrometric analyses of organophosphate insecticide oxon protein adducts.

    Science.gov (United States)

    Thompson, Charles M; Prins, John M; George, Kathleen M

    2010-01-01

    Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. A number of OP-based insecticides share common structural elements that result in predictable OP-protein adducts. The resultant OP-protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure.

  8. EFFECT OF SHORT-TERM ART INTERRUPTION ON LEVELS OF INTEGRATED HIV DNA.

    Science.gov (United States)

    Strongin, Zachary; Sharaf, Radwa; VanBelzen, D Jake; Jacobson, Jeffrey M; Connick, Elizabeth; Volberding, Paul; Skiest, Daniel J; Gandhi, Rajesh T; Kuritzkes, Daniel R; O'Doherty, Una; Li, Jonathan Z

    2018-03-28

    Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group (ACTG) ATI studies were assayed for integrated HIV DNA levels. Cryopreserved PBMCs were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV Integrated DNA size-Exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu- gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in levels of integrated HIV DNA between the pre- and post-ATI time points, with the median ratio of post:pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on levels of integrated proviral DNA in the peripheral blood. IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated

  9. Protein adducts of malondialdehyde and 4-hydroxynonenal contribute to trichloroethene-mediated autoimmunity via activating Th17 cells: Dose– and time–response studies in female MRL+/+ mice

    International Nuclear Information System (INIS)

    Wang, Gangduo; Wang, Jianling; Fan, Xiuzhen; Ansari, G.A.S.; Khan, M. Firoze

    2012-01-01

    Highlights: ► TCE exposure led to dose- and time-related increases in MDA-/HNE-protein adducts and their antibodies. ► Increased MDA-/HNE-adducts were associated with increases in serum autoantibodies. ► MDA-/HNE-albumin adducts trigger greater release of IL-17 and IL-21 from splenocytes of TCE-treated mice. ► Results support that MDA-/HNE-modified proteins could contribute to an autoimmune response. -- Abstract: Trichloroethene (TCE), a common occupational and environmental toxicant, is known to induce autoimmunity. Previous studies in our laboratory showed increased oxidative stress in TCE-mediated autoimmunity. To further establish the role of oxidative stress and to investigate the mechanisms of TCE-mediated autoimmunity, dose– and time–response studies were conducted in MRL+/+ mice by treating them with TCE via drinking water at doses of 0.5, 1.0 or 2.0 mg/ml for 12, 24 or 36 weeks. TCE exposure led to dose-related increases in malondialdehyde (MDA)-/hydroxynonenal (HNE)-protein adducts and their corresponding antibodies in the sera and decreases in GSH and GSH/GSSG ratio in the kidneys at 24 and 36 weeks, with greater changes at 36 weeks. The increases in these protein adducts and decreases in GSH/GSSG ratio were associated with significant elevation in serum anti-nuclear- and anti-ssDNA-antibodies, suggesting an association between TCE-induced oxidative stress and autoimmune response. Interestingly, splenocytes from mice treated with TCE for 24 weeks secreted significantly higher levels of IL-17 and IL-21 than did splenocytes from controls after stimulation with MDA-mouse serum albumin (MSA) or HNE-MSA adducts. The increased release of these cytokines showed a dose-related response and was more pronounced in mice treated with TCE for 36 weeks. These studies provide evidence that MDA- and or HNE-protein adducts contribute to TCE-mediated autoimmunity, which may be via activation of Th17 cells.

  10. 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

  11. DNA endoreplication level in endosperm during seed development in three monocotyledonous species

    Directory of Open Access Journals (Sweden)

    Kazimierz Marciniak

    2014-01-01

    Full Text Available The DNA content after the Feulgen reaction in the endosperm of three monocotyledonous plant species (Asparagus officinalis, Muscari comosom, Haemanthus kurharinae differing in their 2C DNA content, was cytophotometrically measured. During endosperm development 1-6 endoreplication cycles take place, depending on the species. Differences in nuclear DNA endoreplication dynamics in the tested species are similar to those occurring in root parenchyma, but the endoreplication level in the endosperm is higher.

  12. Physiologically based in silico modelling to examine DNA adduct formation by different food-borne a,ß-unsaturated aldehydes at realistic low dietary exposure levels

    NARCIS (Netherlands)

    Kiwamoto, R.

    2015-01-01

    Abstract (R.Kiwamoto ISBN 978-94-6257-284-3)

    Various α,β-unsaturated aldehydes are present in fruits, vegetables, spices, or processed products containing these items as natural constituents or as added food flavouring agents. Because of the α,β-unsaturated aldehyde moiety the β carbon in

  13. Aminoazo dye-protein-adduct enhances inhibitory effect on digestibility and damages to Gastro-Duodenal-Hepatic axis.

    Directory of Open Access Journals (Sweden)

    Li-Yun Lin

    Full Text Available 4-Dimethylaminoazobenzene (DAB, methyl yellow, or butter yellow, a human carcinogen, has been banned for use in foods since 1988. In 2014, DAB adulteration in Tofu occurred in Taiwan. We hypothesize that DAB can form [DAB•SBP]adduct adduct with soybean protein (SBP which could damage Gastro-Duodenal-Hepatic axis. Sprague-Dawley rats gavage fed [DAB•SBP]adduct adduct revealed severely reduced body weight and damaged duodenum, liver, hepatic mitochondria, and spleen. Hepatic levels of glutathione and ATP were severely reduced. Serum GOT and GPT were substantially elevated. Analysis by the adsorption isotherm clearly revealed DAB formed very stable [DAB•SBP]adduct adduct at 1:1 molar ration (Phase A. The equilibrium constant of this colloidal adduct [DAB•SBP]adduct was KeqA = ∝, behaving as the most stable and toxic species. At higher protein concentration (Phase C it formed conjugate [DAB×SBPgross]conjugate, with KeqC = 3.23×10-2 mg/mL, implicating a moderately strong adsorption. The in vitro pepsin digestibility test showed apparently reduced digestibility by 27% (by Ninhydrin assay or 8% (by Bradford assay. Conclusively, this is the first report indicating that [DAB•SBP]adduct potentially is capable to damage the Gastro-Duodenal-Hepatic axis.

  14. Mitochondrial DNA (mtDNA haplogroups and serum levels of anti-oxidant enzymes in patients with osteoarthritis

    Directory of Open Access Journals (Sweden)

    Fernandez-Moreno Mercedes

    2011-11-01

    Full Text Available Abstract Background Oxidative stress play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. To prevent this, the chondrocytes possess a well-coordinated enzymatic antioxidant system. Besides, the mitochondrial DNA (mtDNA haplogroups are associated with the OA disease. Thus, the main goal of this work is to assess the incidence of the mtDNA haplogroups on serum levels of two of the main antioxidant enzymes, Manganese Superoxide Dismutase (Mn-SOD or SOD2 and catalase, and to test the suitability of these two proteins for potential OA-related biomarkers. Methods We analyzed the serum levels of SOD2 and catalase in 73 OA patients and 77 healthy controls carrying the haplogroups J, U and H, by ELISA assay. Knee and hip radiographs were classified according to Kellgren and Lawrence (K/L scoring from Grade 0 to Grade IV. Appropriate statistical analyses were performed to test the effects of clinical variables, including gender, body mass index (BMI, age, smoking status, diagnosis, haplogroups and radiologic K/L grade on serum levels of these enzymes. Results Serum levels of SOD2 appeared statistically increased in OA patients when compared with healthy controls (p Conclusions The increased levels of SOD2 in OA patients indicate an increased oxidative stress OA-related, therefore this antioxidant enzyme could be a suitable candidate biomarker for diagnosis of OA. Mitochondrial haplogroups significantly correlates with serum levels of catalase

  15. 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

  16. 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)

  17. Quantification of trace-level DNA by real-time whole genome amplification.

    Science.gov (United States)

    Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

    2011-01-01

    Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

  18. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

    2014-01-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

  19. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were...... not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also......High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates...

  20. Serum ALT levels as a surrogate marker for serum HBV DNA levels in HBeAg-negative pregnant women.

    Science.gov (United States)

    Sangfelt, Per; Von Sydow, Madeleine; Uhnoo, Ingrid; Weiland, Ola; Lindh, Gudrun; Fischler, Björn; Lindgren, Susanne; Reichard, Olle

    2004-01-01

    In Stockholm, Sweden, the majority of pregnant women positive for hepatitis B surface antigen (HBsAg) are hepatitis Be antigen (HBeAg) negative. Newborns to HBeAg positive mothers receive vaccination and hepatitis B immunoglobulin (HBIg). Newborns to HBeAg negative mothers receive vaccine and HBIg only if the mothers have elevated ALT levels. The aim of this study was to retrospectively evaluate ALT levels as a surrogate marker for HBV DNA levels in HBeAg negative carrier mothers. Altogether 8947 pregnant women were screened for HBV markers from 1999 to 2001 at the Virology Department, Karolinska Hospital. Among mothers screened 192 tested positive for HBsAg (2.2%). 13 of these samples could not be retrieved. Of the remaining 179 sera, 8 (4%) tested positive for HBeAg and 171 (95.5%) were HBeAg negative. Among the HBeAg negative mothers, 9 had HBV DNA levels > 10(5) copies/ml, and of these 7 had normal ALT levels indicating low sensitivity of an elevated ALT level as a surrogate marker for high HBV DNA level. Furthermore, no correlation was found between ALT and HBV DNA levels. Hence, it is concluded that the use of ALT as a surrogate marker for high viral replication in HBeAg negative mothers could be questioned.

  1. Correlation between residual level of DNA double-strand breaks and the radiosensitivity of cancer cells

    International Nuclear Information System (INIS)

    Sun Jianxiang; Sun Weijian; Sui Jianli; Zhou Pingkun

    2008-01-01

    Objective: To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients, and to explore the predictor of radiotherapy responses of cancers. Methods: DNA double-strand breaks (DSBs) were induced by 60 Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results: A wide variation of radiosensitivity, e.g. the survival parameter of Do varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radiosensitivity (D 0 or SF 2 ). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions: The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy. (authors)

  2. 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...

  3. Hypoxia and oxidation levels of DNA and lipids in humans and animal experimental models

    DEFF Research Database (Denmark)

    Møller, Peter; Risom, Lotte; Lundby, Carsten

    2008-01-01

    The objective of this review was to evaluate the association between hypoxia and oxidative damage to DNA and lipids. Evaluation criteria encompassed specificity and validation status of the biomarkers, study design, strength of the association, dose-response relationship, biological plausibility......, analogous exposures, and effect modification by intervention. The collective interpretation indicates persuasive evidence from the studies in humans for an association between hypoxia and elevated levels of oxidative damage to DNA and lipids. The levels of oxidatively generated DNA lesions and lipid...... in subjects at high altitude. Most of the animal experimental models should be interpreted with caution because the assays for assessment of lipid peroxidation products have suboptimal validity....

  4. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Science.gov (United States)

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. PMID:23566956

  5. Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

    Directory of Open Access Journals (Sweden)

    Nino Guy Cassuto

    2016-01-01

    Full Text Available Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p<10-6, OR = 2.4; and p<0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI.

  6. Vitamin C for DNA damage prevention

    International Nuclear Information System (INIS)

    Sram, Radim J.; Binkova, Blanka; Rossner, Pavel

    2012-01-01

    The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

  7. Vitamin C for DNA damage prevention

    Energy Technology Data Exchange (ETDEWEB)

    Sram, Radim J., E-mail: sram@biomed.cas.cz [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic); Binkova, Blanka; Rossner, Pavel [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic)

    2012-05-01

    The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2 Prime -deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 {mu}mol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with {gamma}-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 {mu}mol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 {mu}mol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

  8. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    International Nuclear Information System (INIS)

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates

  9. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  10. 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

  11. Circulating DNA and its methylation level in inflammatory bowel disease and related colon cancer.

    Science.gov (United States)

    Bai, Xuming; Zhu, Yaqun; Pu, Wangyang; Xiao, Li; Li, Kai; Xing, Chungen; Jin, Yong

    2015-01-01

    Both of chronic inflammation and abnormal immune in inflammatory bowel disease can induce colon cancer. Previous research showed that cell apoptosis and necrosis become the main source of circulating DNA in the peripheral blood during tumorigenesis that reduced along with methylation degree. However, its role in the process of colitis transforming to colon cancer is not clarified. Drinking 3% DSS was used to establish colitis model, while 3% dextran sodium sulfate (DSS) combined with azo oxidation methane (AOM) intraperitoneal injection was applied to establish colitis related colon cancer model. Circulating DNA and its methylation level in peripheral blood were tested. Morphology observation, HE staining, and p53 and β-catenin expression detection confirmed that drinking 3% DSS and 3% DSS combined with AOM intraperitoneal injection can successfully establish colitis and colitis associated colorectal cancer models. Circulating DNA level in colitis and colon cancer mice increased by gradient compared with control, while significant difference was observed between each other. Circulating DNA methylation level decreased obviously in colitis and colon cancer, and significant difference was observed between each other. Abnormal protein expression, circulating DNA and its methylation level in ulcerative colitis associated colorectal tissues change in gradient, suggesting that circulating DNA and its methylation level can be treated as new markers for colitis cancer transformation that has certain significance to explore the mechanism of human ulcerative colitis canceration.

  12. Elucidation of polymer induced DNA condensation. Visualisation at the single molecular level

    International Nuclear Information System (INIS)

    Martin, Alison Laura

    2002-01-01

    DNA condensation is a phenomenon that has stimulated interest from biologists, physicists, and polymer chemists for decades. At the cellular level, this process is key to the packing of DNA within the nuclear envelope, and the exposure of the appropriate nucleic acid sequences in order for transcription to occur, and proteins to be produced. The advent of gene therapy has led to an invigoration of this subject area. In order to successfully deliver to, and transfect target cells, many delivery vectors condense the therapeutic DNA into small compact particles. The nature of these particles have a considerable influence on the ultimate expression of the administered nucleic acid material. In addition, at its most fundamental, DNA itself is a classical polyelectrolyte polymer, the behaviour of which has applicability to other charged polymeric systems. There are two core interwound themes to this investigation; the visualisation of DNA condensate morphology at ultra-resolution, and the elucidation of the mechanisms of formation of these structures. The technique of atomic force microscopy is central to these investigations. Methodologies have been devised allowing the visualisation of the tertiary structure and conformational behaviour of individual DNA condensates in near in situ conditions. Condensation of the nucleic acid material has been induced by two classes of cation; small molecular cations, like those found within eukaryotic cells, and a range of cationic polymers. The cationic polymers investigated all have considerable potential as gene delivery vectors. The resultant DNA condensates have been assessed and contrasted in terms of their tertiary morphology, lateral dimensions, and structural volume. Assessments have also been made regarding the influence of the molecular architecture of the cationic moiety and the nature of the input nucleic acid material on the resultant DNA condensates. With regard to the elucidation of the mechanisms of DNA condensate

  13. Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

    Directory of Open Access Journals (Sweden)

    Lucette A Cysique

    Full Text Available To determine the contribution of peripheral blood mononuclear cells' (PBMCs HIV DNA levels to HIV-associated dementia (HAD and non-demented HIV-associated neurocognitive disorders (HAND in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART.Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points.At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03. Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05. Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC. Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04, and higher in those with no ART treatment during HIV infection 1st year (p = .03. Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03, and motor-coordination (p = .02-.12 to a lesser extent.PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

  14. The prognostic significance of whole blood global and specific DNA methylation levels in gastric adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Mansour S Al-Moundhri

    Full Text Available BACKGROUND: Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC. METHODS: Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ(2 tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival. RESULTS: Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR = 2.0, 95% CI: 1.1-3.8; p = 0.02 and high methylation mean values across p16 promoter sites 1-7 were associated with better survival with HR of 0.3 (95% CI, 0.1-0.8; p = 0.02 respectively. CONCLUSIONS: Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important

  15. Increased Plasma Cell-Free DNA Level during HTNV Infection: Correlation with Disease Severity and Virus Load

    Directory of Open Access Journals (Sweden)

    Jing Yi

    2014-07-01

    Full Text Available Cell-free DNA (cf-DNA in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS and Hantavirus cardiopulmonary syndrome (HCPS, depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.

  16. Flow cytometric measurement of DNA level and steroid hormone receptor assay in breast cancer

    International Nuclear Information System (INIS)

    Zubrikhina, G.N.; Kuz'mina, Eh.V.; Bassalyk, L.S.; Murav'eva, N.I.

    1989-01-01

    DNA level measured by flow cytometry and estrogen and progesteron receptors assayed in tissue samples obtained from 85 malignant and 16 benign lesions of the breast. All the benign tumors revealed 2c DNA content and most of them were receptor-negative, while 74.1% of breast carcinomas displayed aneuploidy. Three patients (3.5%) had two lines of aneuploid cells. Many aneuploid tumors were receptor-negative. Preoperative radiation treatmet (14-20 Gy) did not significantly influence the level of steroid hormone receptors in tumors. Estrogen receptor level was higher in menopausal patients than in premenopausal ones

  17. 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.

  18. Hepatitis B virus (HBV) DNA levels and the management of HBV-infected health care workers

    NARCIS (Netherlands)

    van der Eijk, A A; de Man, R A; Niesters, H G M; Schalm, S W; Zaaijer, H L

    Different guidelines exist for the management of hepatitis B virus (HBV)-infected health care workers (HCWs). Various HBV DNA levels are used as a cutoff level to determine whether an HBV-infected HCW is allowed to perform exposure-prone procedures (EPPs) or not. In this paper we discuss the factors

  19. Hepatitis B virus (HBV) DNA levels and the management of HBV-infected health care workers

    NARCIS (Netherlands)

    van der Eijk, A. A.; de Man, R. A.; Niesters, H. G. M.; Schalm, S. W.; Zaaijer, H. L.

    2006-01-01

    Different guidelines exist for the management of hepatitis B virus (HBV)-infected health care workers (HCWs). Various HBV DNA levels are used as a cutoff level to determine whether an HBV-infected HCW is allowed to perform exposure-prone procedures (EPPs) or not. In this paper we discuss the factors

  20. Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples

    OpenAIRE

    Peñarrubia Lozano, Luis; Alcaraz Cazorla, Carles; Vaate, Abraham bij de; Sanz Ball-llosera, Núria; Pla Zanuy, Carles; Vidal Fàbrega, Oriol; Viñas de Puig, Jordi

    2016-01-01

    The zebra mussel (Dreissena polymorpha Pallas, 1771) and the quagga mussel (D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebr...

  1. o-Toluidine blood protein adducts: HPLC analysis with fluorescence detection after a single dose in the adult male rat

    International Nuclear Information System (INIS)

    Cheever, K.L.; DeBord, G.D.; Swearengin, T.F.

    1991-01-01

    Hemoglobin (Hb) and albumin (Alb) adducts of the suspect human carcinogen o-toluidine (OT) were quantified in blood samples collected from rats after a single i.p. injection. Mild alkaline hydrolysis of Hb-adducted [ 14 C]OT followed by extraction with ethylacetate resulted in recovery of 66% of the bound radioactivity. HPLC analysis revealed a single radiolabeled peak which was identified as OT by GC-MS. In subsequent experiments the Hb and Alb adduct levels were determined by HPLC analysis of this split product using fluorescence detection. 4-Ethylaniline was used as internal standard. The detection limit for OT was approximately 450 pg/injection of 5 pmol. mg Hb. Mean adduct levels for Hb increased rapidly over the first 4 hr with the highest (ng/mg Hb ± SD) 3.7 ± 0.5 detected 24 hr after OT (50 mg/kg body wt). In contrast, adduct levels for pooled Alb samples increased from 0.7 ng/mg Alb at 2 hr to 2.5 ng/mg Alb at 4 hr, but were not detectable 24 hr after OT (50 mg/kg body wt). In contrast, adduct levels for pooled Alb samples increased from 0.7 ng/mg Alb at 2 hr to 2.5 ng/mg Alb at 4 hr, but were not detectable 24 hr after dosing. Hb adducts showed a linear relationship for OT doses of 10, 20, 40, 50, and 100 mg/kg body wt. The Hb adduct t 1/2 (11.2 days) was determined after a single 100 mg/kg OT dose. Hb adduct levels were quantifiable (1.3 ± 0.2 ng/mg Hb) by HPLC/fluorescence 28 days after 100 mg/kg OT

  2. Variation of global DNA methylation levels with age and in autistic children.

    Science.gov (United States)

    Tsang, Shui-Ying; Ahmad, Tanveer; Mat, Flora W K; Zhao, Cunyou; Xiao, Shifu; Xia, Kun; Xue, Hong

    2016-09-23

    The change in epigenetic signatures, in particular DNA methylation, has been proposed as risk markers for various age-related diseases. However, the course of variation in methylation levels with age, the difference in methylation between genders, and methylation-disease association at the whole genome level is unclear. In the present study, genome-wide methylation levels in DNA extracted from peripheral blood for 2116 healthy Chinese in the 2-97 age range and 280 autistic trios were examined using the fluorescence polarization-based genome-wide DNA methylation quantification method developed by us. Genome-wide or global DNA methylation levels proceeded through multiple phases of variation with age, consisting of a steady increase from age 2 to 25 (r = 0.382) and another rise from age 41 to 55 to reach a peak level of ~80 % (r = 0.265), followed by a sharp decrease to ~40 % in the mid-1970s (age 56 to 75; r = -0.395) and leveling off thereafter. Significant gender effect in methylation levels was observed only for the 41-55 age group in which methylation in females was significantly higher than in males (p = 0.010). In addition, global methylation level was significantly higher in autistic children than in age-matched healthy children (p < 0.001). The multiphasic nature of changes in global methylation levels with age was delineated, and investigation into the factors underlying this profile will be essential to a proper understanding of the aging process. Furthermore, this first report of global hypermethylation in autistic children also illustrates the importance of age-matched controls in characterization of disease-associated variations in DNA methylation.

  3. Association of postmenopausal endogenous sex hormones with global methylation level of leukocyte DNA among Japanese women

    Directory of Open Access Journals (Sweden)

    Iwasaki Motoki

    2012-07-01

    Full Text Available Abstract Background Although global hypomethylation of leukocyte DNA has been associated with an increased risk of several sites of cancer, including breast cancer, determinants of global methylation level among healthy individuals remain largely unexplored. Here, we examined whether postmenopausal endogenous sex hormones were associated with the global methylation level of leukocyte DNA. Methods A cross-sectional study was conducted using the control group of a breast cancer case–control study in Nagano, Japan. Subjects were postmenopausal women aged 55 years or over who provided blood samples. We measured global methylation level of peripheral blood leukocyte DNA by luminometric methylation assay; estradiol, estrone, androstenedione, dehydroepiandrosterone sulfate, testosterone and free testosterone by radioimmunoassay; bioavailable estradiol by the ammonium sulfate precipitation method; and sex-hormone binding globulin by immunoradiometric assay. A linear trend of association between methylation and hormone levels was evaluated by regression coefficients in a multivariable liner regression model. A total of 185 women were included in the analyses. Results Mean global methylation level (standard deviation was 70.3% (3.1 and range was from 60.3% to 79.2%. Global methylation level decreased 0.27% per quartile category for estradiol and 0.39% per quartile category for estrone while it increased 0.41% per quartile category for bioavailable estradiol. However, we found no statistically significant association of any sex hormone level measured in the present study with global methylation level of leukocyte DNA. Conclusions Our findings suggest that endogenous sex hormones are not major determinants of the global methylation level of leukocyte DNA.

  4. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

  5. 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.

  6. Oxidative stress generated damage to DNA by gastrointestinal exposure to insoluble particles

    DEFF Research Database (Denmark)

    Møller, Peter; Folkmann, J K; Danielsen, P H

    2012-01-01

    that gastrointestinal exposure to single-walled carbon nanotubes (SWCNT), fullerenes C60, carbon black, titanium dioxide and diesel exhaust particles generates oxidized DNA base lesions in organs such as the bone marrow, liver and lung. Oral exposure to nanosized carbon black has also been associated with increased...... level of lipid peroxidation derived exocyclic DNA adducts in the liver, suggesting multiple pathways of oxidative stress for particle-generated damage to DNA. At equal dose, diesel exhaust particles (SRM2975) generated larger levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in rat liver than carbon black...

  7. Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans

    International Nuclear Information System (INIS)

    Park, Yoo Kyoung; Park, Eunju; Kim, Jung-Shin; Kang, Myung-Hee

    2003-01-01

    Grape contains flavonoids with antioxidant properties which are believed to be protective against various types of cancer. This antioxidative protection is possibly provided by the effective scavenging of reactive oxygen species (ROS), thus defending cellular DNA from oxidative damage and potential mutations. This study of healthy adults tested whether a daily regimen of grape juice supplementation could reduce cellular DNA damage in peripheral lymphocytes and reduce the amount of free radicals released. Sixty-seven healthy volunteers (16 women and 51 men) aged 19-57 years were given 480 ml of grape juice daily for 8 weeks in addition to their normal diet, and blood samples were drawn before and after the intervention. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length (TL). Levels of free radicals were determined by reading the lucigenin-perborate ROS generating source, using the Ultra-Weak Chemiluminescence Analyzer System. Grape juice consumption resulted in a significant decrease in lymphocyte DNA damage expressed by TL (before supplementation: 88.75±1.55 μm versus after supplementation: 70.25±1.31 μm; P=0.000 by paired t-test). Additionally, grape juice consumption for 8 weeks reduced the ROS/photon count by 15%, compared to the beginning of the study. The preventive effect of grape juice against DNA damage was simultaneously shown in both sexes. These results indicate that the consumption of grape juice may increase plasma antioxidant capacity, resulting in reduced DNA damage in peripheral lymphocytes achieved at least partially by a reduced release of ROS. Our findings support the hypothesis that polyphenolic compounds contained in grape juice exert cancer-protective effects on lymphocytes, limiting oxidative DNA damage possibly via a decrease in free radical levels

  8. Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoo Kyoung; Park, Eunju; Kim, Jung-Shin; Kang, Myung-Hee

    2003-08-28

    Grape contains flavonoids with antioxidant properties which are believed to be protective against various types of cancer. This antioxidative protection is possibly provided by the effective scavenging of reactive oxygen species (ROS), thus defending cellular DNA from oxidative damage and potential mutations. This study of healthy adults tested whether a daily regimen of grape juice supplementation could reduce cellular DNA damage in peripheral lymphocytes and reduce the amount of free radicals released. Sixty-seven healthy volunteers (16 women and 51 men) aged 19-57 years were given 480 ml of grape juice daily for 8 weeks in addition to their normal diet, and blood samples were drawn before and after the intervention. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length (TL). Levels of free radicals were determined by reading the lucigenin-perborate ROS generating source, using the Ultra-Weak Chemiluminescence Analyzer System. Grape juice consumption resulted in a significant decrease in lymphocyte DNA damage expressed by TL (before supplementation: 88.75{+-}1.55 {mu}m versus after supplementation: 70.25{+-}1.31 {mu}m; P=0.000 by paired t-test). Additionally, grape juice consumption for 8 weeks reduced the ROS/photon count by 15%, compared to the beginning of the study. The preventive effect of grape juice against DNA damage was simultaneously shown in both sexes. These results indicate that the consumption of grape juice may increase plasma antioxidant capacity, resulting in reduced DNA damage in peripheral lymphocytes achieved at least partially by a reduced release of ROS. Our findings support the hypothesis that polyphenolic compounds contained in grape juice exert cancer-protective effects on lymphocytes, limiting oxidative DNA damage possibly via a decrease in free radical levels.

  9. 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....

  10. 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

  11. Seroprevalence, molecular epidemiology and quantitation of parvovirus B19 DNA levels in Iranian blood donors.

    Science.gov (United States)

    Zadsar, Maryam; Aghakhani, Arezoo; Banifazl, Mohammad; Kazemimanesh, Monireh; Tabatabaei Yazdi, Seyed Morteza; Mamishi, Setareh; Bavand, Anahita; Sadat Larijani, Mona; Ramezani, Amitis

    2018-04-16

    Human parvovirus B19 (B19) infection is common among blood donors, and healthy blood donors can transmit virus via transfusion. Due to resistance of B19 to viral inactivation methods, there is a potential concern regarding transfusion safety in blood products. We aimed to determine the seroprevalence, molecular epidemiology, and quantitation of B19 DNA levels in blood donors in Tehran, Iran. A total of 500 blood donors from Blood Transfusion Research Center were studied. ELISA was used for detection of B19 IgG and IgM and nested PCR was carried out for detection of B19 DNA. PCR products were subjected to direct sequencing. B19 viral load was determined by real time PCR. B19 IgG, IgM, and DNA were detected in 27.6, 2.6, and 1.2% of donors respectively. Ten samples (2%) were positive for both antibodies while in four cases (0.8%), B19 IgG and DNA detected simultaneously. One case had B19 IgM, IgG, and viremia concurrently. The titers of B19 DNA in four of six donors were more than 10 6  IU/mL (high level viremia) and all four cases had IgG simultaneously. All B19 isolates categorized in genotype 1A. Our findings indicated that prevalence of B19 DNA in Iranian blood donors was comparable with previous studies throughout the world. High level B19 viremia found in 0.8% of our donors and all viremic donors revealed neutralizing B19 antibody. Therefore implementation of a B19 screening test for each volunteer blood donor does not appear to be necessary but B19 testing for plasma-derived products seems important in Iranian donors. © 2018 Wiley Periodicals, Inc.

  12. Role of plasma EBV DNA levels in predicting recurrence of nasopharyngeal carcinoma in a western population

    International Nuclear Information System (INIS)

    Ferrari, Daris; Alterio, Daniela; Foa, Paolo; Codecà, Carla; Bertuzzi, Cecilia; Broggio, Francesca; Crepaldi, Francesca; Luciani, Andrea; Floriani, Irene; Ansarin, Mohssen; Chiesa, Fausto

    2012-01-01

    Loco-regionally advanced nasopharyngeal carcinomas can be cured by the combination of chemotherapy and radiotherapy. In Eastern countries, plasma levels of viral Epstein-Barr deoxyribonucleic acid (DNA) are accurate in predicting recurrence, but few data are available in Western populations. The aim of this prospective study was to evaluate the relationship between viral Epstein-Barr DNA copy numbers in plasma and the response rate, progression-free survival and overall survival in a cohort of Western patients with stage IIb-IVb nasopharyngeal cancer. We evaluated plasma samples from 36 consecutive patients treated with induction chemotherapy followed by chemoradiation. EBV copy numbers were determined after DNA extraction using real-time quantitative polymerase chain reaction. Survival curves were estimated using the Kaplan–Meier method. Circulating Epstein-Barr virus DNA levels were measured before treatment, at the end of concomitant chemo- and radiotherapy, and during the follow-up period. Pre-treatment levels significantly correlated with the initial stage and probability of relapse. Their increase was 100% specific and 71.3% sensitive in detecting loco-regional or metastatic recurrence (an overall accuracy of 94.4%). Three-year progression-free and overall survival were respectively 78.2% and 97.1%. The results of this study confirm that patients from a Western country affected by loco-regionally advanced nasopharyngeal carcinoma have high plasma Epstein-Barr virus DNA levels at diagnosis. The monitoring of plasma levels is sensitive and highly specific in detecting disease recurrence and metastases

  13. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    International Nuclear Information System (INIS)

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M.

    1990-01-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts

  14. 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.

  15. 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

  16. Radiation sensitivity of organisms of different organization level: an approach including DNA strand breakage

    International Nuclear Information System (INIS)

    Kampf, G.

    1983-01-01

    The mean numbers of DNA double-strand breaks (DSB) suggested to be necessary to lead to the loss of reproductive capacity are compared with bacteriophages, bacteria, and cells of the Chinese hamster after the influence of several radiation qualities. The results suggest that the critical target for the inactivating action of radiations may not be the entire DNA of all organisms but a structure unit of it designed as membrane-attached super structure unit. With organisms having only one of these structures (bacteria) the inactivation probability of one DSB will be near unity, with their multiplication in higher cells it will become lower. This means, eukaryotic cells are able to tolerate more DSB before being inactivated than organisms of a lower organization level, and consequently are more ''lesion resistant''. This behavior represents an evolutionary stabilization of higher cells towards the lethal action of severe DNA lesions such as DSB. (author)

  17. Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

    Science.gov (United States)

    Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

    2017-08-10

    Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Growth and DNA Methylation Level of Triticum Aestivum Seedlings Treated with 5-Azacytidine

    International Nuclear Information System (INIS)

    Yingduan, H.; Liu, W. X.; Li, J. Y.; Ding, W. K.; Zhu, Y.; Wang, H.; Jiang, L. N. A.; Zhou, Y. Q.

    2016-01-01

    In this study, two wheat varieties were used to study effects of DNA methylation inhibitor 5-azacytidine on wheat seedling growth, and found that the high concentration of 5-azacytidine (over 100 meu m) significantly affected growth of wheat seedling root, especially for wheat AK58. When the concentration of 5-azacytidine was between 50 to 250 meu m, plant height of wheat AK58 significantly reduced, and the minimum dwarf phenotype was obtained when treated by 250 meu m 5-azacytidine. Different with wheat AK58, plant height of wheat XM13 increased after being treated by 5-azacytidine. In addition, leaf area and chlorophyll content of two wheat varieties both increased under low concentration of 5-azacytidine (10-50 meu m), and the increase magnitude of wheat AK58 was far more compared with wheat XM13, indicating that moderate methylation could promote development of wheat leaf and photosynthesis, and photosynthesis of wheat AK58 might be closely related with methylation status of genomic DNA. This study also found that proline content of wheat AK58 and soluble sugar content of two wheat varieties increased after being treated by 5-azacytidine, and showed a concentration-dependent increase, further more, soluble sugar content of wheat AK58 was far higher than that of wheat XM13 under normal conditions, thus 5-azacytidine might be conducive to accumulation of soluble sugar in wheat and could obviously influence osmotic adjustment ability of wheat AK58. Further analysis showed that DNA methylation level of wheat AK58 was higher compared with wheat XM13, and was both lower in leaf genomic DNA of two wheat varieties than that in root genomic DNA. Although 5-azacytidine significantly reduced DNA methylation level of leaf and root genome, the decrease amplitude of leaf was more obvious. Collectively, these results suggest that there are some differences in seedling growth, physiological characteristics and DNA methylation level of two wheat varieties, furthermore, DNA

  19. A decrease in cyclin B1 levels leads to polyploidization in DNA damage-induced senescence.

    Science.gov (United States)

    Kikuchi, Ikue; Nakayama, Yuji; Morinaga, Takao; Fukumoto, Yasunori; Yamaguchi, Naoto

    2010-05-04

    Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration-dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin-induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated beta-galactosidase activity. In DNA damage-induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage-induced senescence.

  20. Seasonal and PAH impact on DNA strand-break levels in gills of transplanted zebra mussels.

    Science.gov (United States)

    Michel, Cécile; Bourgeault, Adeline; Gourlay-Francé, Catherine; Palais, Frédéric; Geffard, Alain; Vincent-Hubert, Françoise

    2013-06-01

    Genotoxicity endpoints are useful tools to biomonitor the physicochemical and biological quality of aquatic ecosystems. A caging study on the freshwater bivalve Dreissena polymorpha was planned to run over four seasons in the Seine River basin in order to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. Three sites were chosen along the Seine River, one upstream from Paris and two downstream, corresponding to a chemical gradient of water contamination. The DNA strand break (comet assay) and chromosomal damage (micronucleus test) were measured in caged mussels at each site and in winter, spring and summer, along with PAH water contamination, PAH bioaccumulation, the mussel condition index (CI), the gonado-somatic index (GSI) and the filtration rate (FR). The level of DNA strand break measured in winter was low and increased in spring, concomitantly with FR and GSI. Over the same period, micronucleus (MN) frequency and PAH bioaccumulation decreased significantly in caged mussels, with both parameters positively correlated to each other. DNA strand-break levels and MN frequencies showed inter-site variations corresponding to the chemical contamination gradient. These two genotoxicity endpoints usefully complement each other in field studies. These results show that the MN test and comet assay, when applied to gill cells of caged zebra mussels, are sensitive tools for freshwater genotoxicity monitoring. Copyright © 2013. Published by Elsevier Inc.

  1. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    Science.gov (United States)

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  2. Gender differences in the knee adduction moment after anterior cruciate ligament reconstruction surgery.

    Science.gov (United States)

    Webster, Kate E; McClelland, Jodie A; Palazzolo, Simon E; Santamaria, Luke J; Feller, Julian A

    2012-04-01

    The external knee adduction moment during gait has previously been associated with knee pain and osteoarthritis (OA). Recently, the knee adduction moment has been shown to be increased following anterior cruciate ligament (ACL) reconstruction surgery and has been suggested as a potential mechanism for the progression of early onset knee OA in this population. No study has investigated the gender differences in gait biomechanics following ACL reconstruction. To examine gender differences in gait biomechanics following ACL reconstruction surgery. 36 subjects (18 females, 18 males) who had previously undergone ACL reconstruction surgery (mean time since surgery 20 months) underwent gait analysis at a self-selected walking speed. Males and females were well matched for age, time since surgery and walking speed. Maximum flexion and adduction angles and moments were recorded during the stance phase of level walking and compared between the male and female groups. The knee adduction moment was 23% greater in the female compared with the male ACL group. No gender differences were seen in the sagittal plane. No differences were seen between the reconstructed and contralateral limb. The higher knee adduction moment seen in females compared with males may suggest an increased risk for the development of OA in ACL-reconstructed females.

  3. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known...... an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...

  4. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

  5. Effect of methionine load on homocysteine levels, lipid peroxidation and DNA damage in rats receiving ethanol

    Directory of Open Access Journals (Sweden)

    Alceu Afonso Jordao Júnior

    2009-12-01

    Full Text Available Changes in the metabolism of methionine can cause hyperhomocysteinemia, inducing a triad of atherosclerosis, hypertension, and increased oxidative stress. The generation of free radicals and oxidative damage to DNA is important in the liver damage caused by ethanol. In this study, the effect of methionine overload associated or otherwise with acute administration of ethanol on homocysteine values, damage to DNA, lipoperoxidation and vitamin E was evaluated. Thirty rats were divided into 3 groups: Group Ethanol 24 hours (EG24, Group Methionine 24 hours (MG24, and Group Methionine and Ethanol 24 hours (MEG24. TBARS, vitamin E, GS and, homocysteine values were determined and the Comet assay was carried out. Increased GSH, vitamin E and homocysteine levels were observed for MEG24, and increased TBARS were observed in EG24. The Comet assay showed an increase in DNA damage in EG24 and DNA protection in MEG24. The administration of ethanol decreased antioxidant levels and increased TBARS, indicating the occurrence of oxidative stress with possible DNA damage. The combination of methionine and ethanol had a protective effect against the ethanol-induced damage, but increased the levels of homocysteine.Alterações no metabolismo da metionina podem ocasionar hiper-homocisteinemia, quadro indutivo de aterosclerose, hipertensão e aumento do estresse oxidativo. A geração de radicais livres e dano oxidativo ao DNA são importantes na injúria hepática provocada pelo etanol. Neste estudo avaliaram-se os efeitos da sobrecarga de metionina associada ou não à administração aguda de etanol sobre valores de homocisteína, dano ao DNA, lipoperoxidação e vitamina E. Foram utilizados 30 ratos Wistar distribuídos em 3 Grupos: Grupo Etanol 24 horas (GE24, Grupo Metionina 24 horas (GM24 e Grupo Metionina e Etanol 24 horas (GME24. Realizaram-se determinações hepáticas de SRATB, vitamina E, GSH, homocisteína e Teste do Cometa e determinações plasm

  6. 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

  7. Calculation of direct effects of {sup 60}Co gamma rays on the different DNA structural levels: A simulation study using the Geant4-DNA toolkit

    Energy Technology Data Exchange (ETDEWEB)

    Tajik, Marjan; Rozatian, Amir S.H. [Department of Physics, University of Isfahan, Hezar Jarib Street, Isfahan 81746-73441 (Iran, Islamic Republic of); Semsarha, Farid, E-mail: Semsarha@ibb.ut.ac.ir [Institute of Biochemistry and Biophysics (IBB), University of Tehran, P.O. Box: 13145-1384, Tehran (Iran, Islamic Republic of)

    2015-03-01

    In this study, simple single strand breaks (SSB) and double strand breaks (DSB) due to direct effects of the secondary electron spectrum of {sup 60}Co gamma rays on different organizational levels of a volume model of the B-DNA conformation have been calculated using the Geant4-DNA toolkit. Result of this study for the direct DSB yield shows a good agreement with other theoretical and experimental results obtained by both photons and their secondary electrons; however, in the case of SSB a noticeable difference can be observed. Moreover, regarding the almost constant yields of the direct strand breaks in the different structural levels of the DNA, calculated in this work, and compared with some theoretical studies, it can be deduced that the direct strand breaks yields depend mainly on the primary double helix structure of the DNA and the higher-order structures cannot have a noticeable effect on the direct DNA damage inductions by {sup 60}Co gamma rays. In contrast, a direct dependency between the direct SSB and DSB yields and the volume of the DNA structure has been found. Also, a further study on the histone proteins showed that they can play an important role in the trapping of low energy electrons without any significant effect on the direct DNA strand breaks inductions, at least in the range of energies used in the current study.

  8. Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

    International Nuclear Information System (INIS)

    Barfknecht, T.R.; Mecca, D.J.; Naismith, R.W.

    1988-01-01

    The results presented in this report demonstrate that an 18-20 hour exposure/ 3 H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin B1 and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18-20-hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18-20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10(-3) M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 X 10(-4) M when the 18-20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 X 10(-5) M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10(-5) M when the 18-20 hour exposure/labeling period was used

  9. Low levels of methylmercury induce DNA damage in rats: protective effects of selenium

    Energy Technology Data Exchange (ETDEWEB)

    Grotto, Denise; Barcelos, Gustavo R.M.; Antunes, Lusania M.G.; Barbosa, Fernando [Universidade de Sao Paulo, Departamento de Analises Clinicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Ribeirao Preto, Sao Paulo (Brazil); Valentini, Juliana [Universidade de Sao Paulo, Departamento de Analises Clinicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Ribeirao Preto, Sao Paulo (Brazil); Universidade Federal de Santa Maria, Departamento de Analises Clinicas e Toxicologicas, Santa Maria, Rio Grande do Sul (Brazil); Angeli, Jose Pedro F. [Universidade de Sao Paulo, Departamento de Bioquimica, Instituto de Quimica, Sao Paulo (Brazil); Garcia, Solange C. [Universidade Federal de Santa Maria, Departamento de Analises Clinicas e Toxicologicas, Santa Maria, Rio Grande do Sul (Brazil)

    2009-03-15

    In this study we examined the possible antigenotoxic effect of selenium (Se) in rats chronically exposed to low levels of methylmercury (MeHg) and the association between glutathione peroxidase (GSH-Px) activity and DNA lesions (via comet assay) in the same exposed animals. Rats were divided into six groups as follows: (Group I) received water; (Group II) received MeHg (100 {mu}g/day); (Group III) received Se (2 mg/L drinking water); (Group IV) received Se (6 mg/L drinking water); (Group V) received MeHg (100 {mu}g/day) and Se (2 mg/L drinking water); (Group VI) received MeHg (100 {mu}g/day) and Se (6 mg/L drinking water). Total treatment time was 100 days. GSH-Px activity was determined spectrophotometrically and DNA damage was determined by comet assay. Mean GSH-Px activity in groups I, II, III, IV, V and VI were, respectively: 40.19{+-}17.21; 23.63{+-}6.04; 42.64{+-}5.70; 38.50{+-}7.15; 34.54{+-}6.18 and 41.39{+-}11.67 nmolNADPH/min/gHb. DNA damage was represented by a mean score from 0 to 300; the results for groups I, II, III, IV, V and VI were, respectively: 6.87{+-}3.27; 124.12{+-}13.74; 10.62{+-}3.81; 13.25{+-}1.76; 86.87{+-}11.95 and 76.25{+-}7.48. There was a significant inhibition of GSH-Px activity in group II compared with group I (P<0.05). Groups V and VI did not show a difference in enzyme activity compared with groups III and IV, showing the possible protective action of Se. Comet assay presented a significant difference in DNA migration between group II and group I (P<0.0001). Groups V and VI showed a significant reduction in MeHg-induced genotoxicity (P < 0.001) when compared with group II. A negative correlation (r = -0.559, P<0.05) was found between GSH-Px activity and DNA lesion, showing that the greater the DNA damage, the lower the GSH-Px activity. Our findings demonstrated the oxidative and genotoxic properties of MeHg, even at low doses. Moreover, Se co-administration reestablished GSH-Px activity and reduced DNA damage. (orig.)

  10. DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast.

    Directory of Open Access Journals (Sweden)

    Ulrika Norman-Axelsson

    Full Text Available Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A(Cnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A(Cnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A(Cnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A(Cnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A(Cnp1 nucleosomes.

  11. Are we at risk from level radiation - DNA repair capacity studies

    International Nuclear Information System (INIS)

    Riklis, E.; Kol, R.; Heimer, Y.M.

    1979-01-01

    A new biochemical method, based on determination of DNA repair capability, which will enable predetermination of radiation sensitivity and further - an indication of inherent sensitivity which may be expressed only in the future when a cell will be faced with a situation in which its repair capacity will have to function to its full capability was developed. Cells are treated with trioxalen (trimethylpsoralen, TMP) and near ultraviolet light (NUV), bringing about an almost complete cessation of semiconservative DNA synthesis, 99.5 to 99.8 % inhibition. This method enabled the accurate measurement of incorporation of labelled thymidine into DNA following assaults by radiation or chemicals, indicating that repair synthesis is occurring. The method has been found suitable for the following cells: human fibroblasts, human breast cancer cells, chinese hamster V-79 cells, human lymphocytes. Since the method is applicable also for lymphocytes, it will enable carrying out a world-wide interlaboratory comparative study in which the range of 'repair capacity' of 'normal healthy' humans will be established. Individuals showing no repair capacity will not be permitted to be exposed to any level of radiation above natural background. These are the persons 'at risk' from radiation, while the general public, showing normal repair capacity may be considered safe from the effects of low-level radiations. (B.G.)

  12. Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level

    KAUST Repository

    Shirbini, Afnan

    2017-05-01

    Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.

  13. 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....

  14. Circulating bacterial-derived DNA fragment level is a strong predictor of cardiovascular disease in peritoneal dialysis patients.

    Directory of Open Access Journals (Sweden)

    Cheuk-Chun Szeto

    Full Text Available Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001. After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 - 42.5% excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001 and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001. Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV, but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005.Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.

  15. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    International Nuclear Information System (INIS)

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-01-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with [ 14 C]aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment

  16. Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

    Science.gov (United States)

    Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

    2018-01-01

    The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

  17. LINE-1 methylation levels in leukocyte DNA and risk of renal cell cancer.

    Directory of Open Access Journals (Sweden)

    Linda M Liao

    Full Text Available Leukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1 methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk.LINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1 on age(±5years, sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated.LINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84-83.47 compared to 81.67% (IQR: 80.35-83.03 among controls (p = 0.003, Wilcoxon. Compared to the lowest LINE-1 methylation quartile(Q1, the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2 = 1.84(1.20-2.81, OR(Q3 = 1.72(1.11-2.65 and OR(Q4 = 2.06(1.34-3.17, with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001 than former or never smokers (p-interaction = 0.03. To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well.Higher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis.

  18. 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

  19. Association between serum organochlorines and global methylation level of leukocyte DNA among Japanese women: a cross-sectional study

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Hiroaki [Department of Epidemiology and Environmental Health, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421 Japan (Japan); Iwasaki, Motoki, E-mail: moiwasak@ncc.go.jp [Epidemiology Division, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104–0045 Japan (Japan); Kasuga, Yoshio [Department of Surgery, Nagano Matsushiro General Hospital, 183 Matsushiro, Matsushiro-cho, Nagano City, Nagano Prefecture 381–1231 Japan (Japan); Yokoyama, Shiro; Onuma, Hiroshi [Department of Breast and Thyroid Surgery, Nagano Red Cross Hospital, 5-22-1 Wakasato, Nagano City, Nagano Prefecture 380–8582 Japan (Japan); Nishimura, Hideki [Department of Respiratory Surgery and Breast Surgery, Nagano Municipal Hospital, 1333–1 Tomitake, Nagano City, Nagano Prefecture 381–8551 Japan (Japan); Kusama, Ritsu [Department of Surgery, Hokushin General Hospital, 1-5-63 Nishi, Nakano City, Nagano Prefecture 383–8505 Japan (Japan); Yoshida, Teruhiko [Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104–0045 Japan (Japan); Yokoyama, Kazuhito [Department of Epidemiology and Environmental Health, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421 Japan (Japan); Tsugane, Shoichiro [Dierctor Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104–0045 Japan (Japan)

    2014-08-15

    While the global methylation level of leukocyte DNA may be a suitable biomarker for cancer risk, the level may be influenced by multiple factors, both environmental and host-related, one of which is exposure to environmental pollutants. To date, three epidemiologic studies have examined associations between serum organochlorine levels and global DNA methylation level, but their findings are not fully consistent, and the associations thus require confirmation in other well-characterized populations. We tested the association between organochlorine exposure and the global DNA methylation level of leukocytes in Japanese women. We conducted a cross-sectional study using the control group of a breast cancer case–control study in Japan. Subjects were 403 Japanese women who provided blood samples. Serum polychlorinated biphenyls (PCBs) and nine pesticide-related organochlorines were measured by gas chromatography isotope-dilution high-resolution mass spectrometry. Further, global methylation level of peripheral leukocyte DNA among 399 women was measured by luminometric methylation assay. Linear trends in the association between methylation and quartile levels of organochlorines were evaluated by regression coefficients in a multivariable linear regression model. We found significant inverse associations between the global methylation level in leukocyte DNA and many of the organochlorine levels measured. Global methylation level was significantly decreased by 0.33–0.83% per quartile category for serum o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT), p,p′-DDT, p,p′-dichlorodiphenyldichloroethylene, trans-nonachlor, oxychlordane, hexachlorobenzene, β-hexachlorocyclohexane, PCB17, PCB52/69, PCB74, PCB114, and PCB183. Serum organochlorine levels were inversely associated with the global methylation level of leukocyte DNA in a relatively large sample of Japanese women. - Highlights: • Many serum organochlorine pesticides were inversely associated with the global

  20. Antibody Drug Conjugates Differentiate Uptake and DNA Alkylation of Pyrrolobenzodiazepines in Tumors from Organs of Xenograft Mice.

    Science.gov (United States)

    Ma, Yong; Khojasteh, S Cyrus; Hop, Cornelis E C A; Erickson, Hans K; Polson, Andrew; Pillow, Thomas H; Yu, Shang-Fan; Wang, Hong; Dragovich, Peter S; Zhang, Donglu

    2016-12-01

    Pyrrolobenzodiazepine (PBD)-dimer is a DNA minor groove alkylator, and its CD22 THIOMAB antibody drug conjugate (ADC) demonstrated, through a disulfide linker, an efficacy in tumor reduction for more than 7 weeks with minimal body weight loss in xenograft mice after a single 0.5-1 mg/kg i.v. dose. The DNA alkylation was investigated here in tumors and healthy organs of mice to understand the sustained efficacy and tolerability. The experimental procedures included the collection of tumors and organ tissues of xenograft mice treated with the ADC followed by DNA isolation/hydrolysis/quantitation and payload recovery from reversible DNA alkylation. PBD-dimer formed a considerable amount of adducts with tissue DNA, representing approximately 98% (at 24 hours), and 99% (at 96 hours) of the total PBD-dimer in tumors, and 78-89% in liver and lung tissues, suggesting highly efficient covalent binding of the released PBD-dimer to tissue DNA. The amount of PBD-DNA adducts in tumor tissues was approximately 24-fold (at 24 hours) and 70-fold (at 96 hours) greater than the corresponding amount of adducts in liver and lung tissues. In addition, the DNA alkylation levels increased 3-fold to 4-fold from 24 to 96 hours in tumors [41/10 6 base pairs (bp) at 96 hours] but remained at the same level (1/10 6 bp) in livers and lungs. These results support the typical target-mediated cumulative uptake of ADC into tumors and payload release that offers an explanation for its sustained antitumor efficacy. In addition, the low level of DNA alkylation in normal tissues is consistent with the tolerability observed in mice. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  1. 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.

  2. Pooled analysis of studies on DNA adducts and dietary vitamins

    Czech Academy of Sciences Publication Activity Database

    Ragin, C.; Minor, A.; Agudo, A.; Farmer, P.; Garte, S.; Gonzales, C.; Kalina, I.; Matullo, G.; Popov, T.; Palli, D.; Peluso, M.; Ricceri, F.; Šrám, Radim; Vineis, P.; Taioli, E.

    2010-01-01

    Roč. 705, č. 2 (2010), s. 77-82 ISSN 1383-5742 Grant - others:EU(XE) FOOD -CT-2005-513943 Institutional research plan: CEZ:AV0Z50390512 Keywords : biomarkers * carcinogenicity * molecular epidemiology Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 8.741, year: 2010

  3. 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

  4. 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

  5. Synthesis and physicochemical investigation of adducts of rare earth thenoyltrifluoroacetonates

    International Nuclear Information System (INIS)

    Anufrieva, S.I.; Snezhko, N.I.; Martynenko, L.I.; Pechurova, N.I.

    1982-01-01

    Adducts of rare earth thenoyltrifluoroacetonates (3) have been synthesized with tributylphosphate (TBP), trioctylphosphenoxide (TOPO), triphenylphosphenoxide (TPO) of 1:1 and 1:2 composition as well as with α, α'-dipyridine (Dipy), o-phenanthroline (Phen) of 1:1 composition. The separated adducts have been studied by methods of element analysis, X-ray phase and derivatographic analyses and IR spectroscopy. It is shown that the adducts are more thermostable compared to the corresponding rare earth thenoyltrifluoroacetonate hydrates

  6. Evidence for phosphorus bonding in phosphorus trichloride-methanol adduct: a matrix isolation infrared and ab initio computational study.

    Science.gov (United States)

    Joshi, Prasad Ramesh; Ramanathan, N; Sundararajan, K; Sankaran, K

    2015-04-09

    The weak interaction between PCl3 and CH3OH was investigated using matrix isolation infrared spectroscopy and ab initio computations. In a nitrogen matrix at low temperature, the noncovalent adduct was generated and characterized using Fourier transform infrared spectroscopy. Computations were performed at B3LYP/6-311++G(d,p), B3LYP/aug-cc-pVDZ, and MP2/6-311++G(d,p) levels of theory to optimize the possible geometries of PCl3-CH3OH adducts. Computations revealed two minima on the potential energy surface, of which, the global minimum is stabilized by a noncovalent P···O interaction, known as a pnictogen bonding (phosphorus bonding or P-bonding). The local minimum corresponded to a cyclic adduct, stabilized by the conventional hydrogen bonding (Cl···H-O and Cl···H-C interactions). Experimentally, 1:1 P-bonded PCl3-CH3OH adduct in nitrogen matrix was identified, where shifts in the P-Cl modes of PCl3, O-C, and O-H modes of CH3OH submolecules were observed. The observed vibrational frequencies of the P-bonded adduct in a nitrogen matrix agreed well with the computed frequencies. Furthermore, computations also predicted that the P-bonded adduct is stronger than H-bonded adduct by ∼1.56 kcal/mol. Atoms in molecules and natural bond orbital analyses were performed to understand the nature of interactions and effect of charge transfer interaction on the stability of the adducts.

  7. The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

    Science.gov (United States)

    Falconer, A. C.; Hayes, L. J.

    1986-01-01

    Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

  8. Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy

    Directory of Open Access Journals (Sweden)

    Ilko Bald

    2014-09-01

    Full Text Available DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates.

  9. Determination of albumin adducts of 4,4'-methylenediphenyl diisocyanate after specific inhalative challenge tests in workers.

    Science.gov (United States)

    Sabbioni, Gabriele; Dongari, Nagaraju; Kumar, Anoop; Baur, Xaver

    2016-10-17

    4,4'-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate used in the industry. Lung sensitization with bronchial asthma is the main disorder in exposed workers. Albumin adducts of MDI might be involved in specific immunological reactions. MDI adducts with lysine (MDI-Lys) of albumin have been found in MDI-workers and construction workers. MDI-Lys is an isocyanate-specific adduct of MDI with albumin. In the present study, we report MDI-adducts in workers undergoing diagnostic MDI challenge tests. The workers were exposed for 2h to 5ppb of MDI. The adduct levels increase significantly after the exposure to MDI in the challenge chamber. About 0.6% of the dose was bound to albumin. So far, only urinary metabolites of MDI were measured to monitor isocyanate workers. However, such urinary metabolites are not isocyanate specific. Therefore, we propose to measure albumin adducts for monitoring MDI exposed subjects. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Science.gov (United States)

    Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

    2017-01-01

    Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  11. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Directory of Open Access Journals (Sweden)

    Matthias Christen

    Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  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. DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

    Directory of Open Access Journals (Sweden)

    Dimos Gaidatzis

    2014-02-01

    Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

  14. Moderate variations in CDC25B protein levels modulate the response to DNA damaging agents

    International Nuclear Information System (INIS)

    Aressy, B.; Bugler, B.; Valette, A.; Ducommun, B.; Biard, D.

    2008-01-01

    CDC25B, one of the three members of the CDC25 dual-specificity phosphatase family, plays a critical role in the control of the cell cycle and in the checkpoint response to DNA damage. CDC25B is responsible for the initial dephosphorylation and activation of the cyclin-dependent kinases, thus initiating the train of events leading to entry into mitosis. The critical role played by CDC25B is illustrated by the fact that it is specifically required for checkpoint recovery and that unscheduled accumulation of CDC25B is responsible for illegitimate entry into mitosis. Here, we report that in p53 colon carcinoma cells, a moderate increase in the CDC25B level is sufficient to impair the DNA damage checkpoint, to increase spontaneous mutagenesis, and to sensitize cells to ionising radiation and genotoxic agents. Using a tumour cell spheroid assay as an alternative to animal studies, we demonstrate that the level of CDC25B expression modulates growth inhibition and apoptotic death. Since CDC25B overexpression has been observed in a significant number of human cancers, including colon carcinoma, and is often associated with high grade tumours and poor prognosis, our work suggests that the expression level of CDC25B might be a potential key parameter of the cellular response to cancer therapy. (authors)

  15. Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate

    International Nuclear Information System (INIS)

    Williams, J.I.; Friedberg, E.C.

    1982-01-01

    Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m 2 . The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine. (author)

  16. EXTRACELLULAR DNA AND THE LEVEL OF ITS METHYLATION IN DIFFERENT RHEUMATIC DISEASES

    Directory of Open Access Journals (Sweden)

    N O Shubayeva

    2012-01-01

    Conclusion. RDs are characterized by the higher concentration of apoptotic and necrotic DNA, impaired exDNA methylation, varying complexification of exDNA with monometinic proteins, which is associated with the hyperproduction of autoantibodies (including anti-exDNA antibodies and inflammatory markers.

  17. High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery

    DEFF Research Database (Denmark)

    Jakobsen, Tanja R; Clausen, Frederik B; Rode, Line

    2012-01-01

    To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation.......To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation....

  18. Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level

    KAUST Repository

    Shirbini, Afnan

    2017-01-01

    and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image

  19. Low Melatonin Production During Adulthood - Phase 2: Association with Levels of Hydroxyl Radical Scavenging and DNA Damage

    National Research Council Canada - National Science Library

    Sobel, Eugene L; Davanipour, Zoreh; Poulsen, Henrik

    2004-01-01

    The primary purpose of the proposed study is to develop cross-sectional evidence concerning whether or not lower melatonin production levels are associated with increased oxidative DNA guanine damage...

  20. Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay

    Science.gov (United States)

    Jackson, Petra

    2013-01-01

    The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor. PMID:24136994

  1. Resource base influences genome-wide DNA methylation levels in wild baboons (Papio cynocephalus)

    Science.gov (United States)

    Lea, Amanda J.; Altmann, Jeanne; Alberts, Susan C.; Tung, Jenny

    2015-01-01

    Variation in resource availability commonly exerts strong effects on fitness-related traits in wild animals. However, we know little about the molecular mechanisms that mediate these effects, or about their persistence over time. To address these questions, we profiled genome-wide whole blood DNA methylation levels in two sets of wild baboons: (i) ‘wild-feeding’ baboons that foraged naturally in a savanna environment and (ii) ‘Lodge’ baboons that had ready access to spatially concentrated human food scraps, resulting in high feeding efficiency and low daily travel distances. We identified 1,014 sites (0.20% of sites tested) that were differentially methylated between wild-feeding and Lodge baboons, providing the first evidence that resource availability shapes the epigenome in a wild mammal. Differentially methylated sites tended to occur in contiguous stretches (i.e., in differentially methylated regions or DMRs), in promoters and enhancers, and near metabolism-related genes, supporting their functional importance in gene regulation. In agreement, reporter assay experiments confirmed that methylation at the largest identified DMR, located in the promoter of a key glycolysis-related gene, was sufficient to causally drive changes in gene expression. Intriguingly, all dispersing males carried a consistent epigenetic signature of their membership in a wild-feeding group, regardless of whether males dispersed into or out of this group as adults. Together, our findings support a role for DNA methylation in mediating ecological effects on phenotypic traits in the wild, and emphasize the dynamic environmental sensitivity of DNA methylation levels across the life course. PMID:26508127

  2. Estrogen-Induced Depurination of DNA: A Novel Target for Breast Cancer Prevention

    National Research Council Canada - National Science Library

    Cavalieri, Ercole L

    2008-01-01

    ... and their reaction with DNA. Compelling evidence obtained in the various specific aims of this COE will be decisive for determining the risk of breast cancer by using the depurinating estrogen-DNA adducts as biomarkers...

  3. Estrogen-Induced Depurination of DNA: A Novel Target for Breast Cancer Prevention

    National Research Council Canada - National Science Library

    Cavalieri, Ercole L

    2007-01-01

    ... and their reaction with DNA. Compelling evidence obtained in the various specific aims of this COE will be decisive for determining the risk of breast cancer by using the depurinating estrogen-DNA adducts as biomarkers...

  4. Determination of the level of DNA modification with cisplatin by catalytic hydrogen evolution at mercury-based electrodes.

    Science.gov (United States)

    Horáková, Petra; Tesnohlídková, Lucie; Havran, Ludek; Vidláková, Pavlína; Pivonková, Hana; Fojta, Miroslav

    2010-04-01

    Electrochemical methods proved useful as simple and inexpensive tools for the analysis of natural as well as chemically modified nucleic acids. In particular, covalently attached metal-containing groups usually render the DNA well-pronounced electrochemical activity related to redox processes of the metal moieties, which can in some cases be coupled to catalytic hydrogen evolution at mercury or some types of amalgam electrodes. In this paper we used voltammetry at the mercury-based electrodes for the monitoring of DNA modification with cis-diamminedichloroplatinum (cisplatin), a representative of metallodrugs used in the treatment of various types of cancer or being developed for such purpose. In cyclic voltammetry at the mercury electrode, the cisplatin-modified DNA yielded catalytic currents the intensity of which reflected DNA modification extent. In square-wave voltammetry, during anodic polarization after prereduction of the cisplatinated DNA, a well-developed, symmetrical signal (peak P) was obtained. Intensity of the peak P linearly responded to the extent of DNA modification at levels relevant for biochemical studies (rb = 0.01-0.10, where rb is the number of platinum atoms bound per DNA nucleotide). We demonstrate a correlation between the peak P intensity and a loss of sequence-specific DNA binding by tumor suppressor protein p53, as well as blockage of DNA digestion by a restriction endonuclease Msp I (both caused by the DNA cisplatination). Application of the electrochemical technique in studies of DNA reactivity with various anticancer platinum compounds, as well as for an easy determination of the extent of DNA platination in studies of its biochemical effects, is discussed.

  5. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

  6. Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals.

    Directory of Open Access Journals (Sweden)

    Shashi Bala

    Full Text Available Binge drinking, the most common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its biological consequences are poorly defined. Previous studies demonstrated that chronic alcohol use results in increased gut permeability and increased serum endotoxin levels that contribute to many of the biological effects of chronic alcohol, including alcoholic liver disease. In this study, we evaluated the effects of acute binge drinking in healthy adults on serum endotoxin levels. We found that acute alcohol binge resulted in a rapid increase in serum endotoxin and 16S rDNA, a marker of bacterial translocation from the gut. Compared to men, women had higher blood alcohol and circulating endotoxin levels. In addition, alcohol binge caused a prolonged increase in acute phase protein levels in the systemic circulation. The biological significance of the in vivo endotoxin elevation was underscored by increased levels of inflammatory cytokines, TNFα and IL-6, and chemokine, MCP-1, measured in total blood after in vitro lipopolysaccharide stimulation. Our findings indicate that even a single alcohol binge results in increased serum endotoxin levels likely due to translocation of gut bacterial products and disturbs innate immune responses that can contribute to the deleterious effects of binge drinking.

  7. Determination of isocyanate specific albumin-adducts in workers exposed to toluene diisocyanates.

    Science.gov (United States)

    Sabbioni, Gabriele; Gu, Qi; Vanimireddy, Lakshiminiranjan Reddy

    2012-03-01

    Toluene diisocyanates (2,4-TDI and 2,6-TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. It is therefore necessary to have sensitive and specific methods to monitor isocyanate exposure of workers. Urinary metabolites or protein adducts have been used as biomarkers in workers exposed to TDI. However, with these methods it was not possible to determine if the biomarkers result from exposure to TDI or to the corresponding toluene diamines (TDA). This work presents a new procedure for the determination of isocyanate-specific albumin adducts. Isotope dilution mass spectrometry was used to measure the adducts in albumin present in workers exposed to TDI. 2,4-TDI and 2,6-TDI formed adducts with lysine: N(ϵ)-[({3-amino-4-methylphenyl}amino)carbonyl]-lysine, N(ϵ)-[({5-amino-2-methylphenyl}amino)carbonyl]-lysine, and N(ϵ)- [({3-amino-2-methylphenyl}amino)carbonyl]-lysine. In future studies, this new method can be applied to measure TDI-exposures in workers.

  8. Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sergio E Palma-Vera

    Full Text Available Ovarian steroid hormones (mainly E2 and P4 regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.A cell line derived from the porcine oviductal epithelium (CCLV-RIE270 was characterized (lineage markers, proliferation characteristics and transformation status. Primary porcine oviduct epithelial cells (POEC were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml. Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR and inflammation was quantified.CCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8. Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.CCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

  9. Transfer and persistence of non-self DNA on hands over time: Using empirical data to evaluate DNA evidence given activity level propositions.

    Science.gov (United States)

    Szkuta, Bianca; Ballantyne, Kaye N; Kokshoorn, Bas; van Oorschot, Roland A H

    2018-03-01

    Questions relating to how DNA from an individual got to where it was recovered from and the activities associated with its pickup, retention and deposition are increasingly relevant to criminal investigations and judicial considerations. To address activity level propositions, investigators are typically required to assess the likelihood that DNA was transferred indirectly and not deposited through direct contact with an item or surface. By constructing a series of Bayesian networks, we demonstrate their use in assessing activity level propositions derived from a recent legal case involving the alleged secondary transfer of DNA to a surface following a handshaking event. In the absence of data required to perform the assessment, a set of handshaking simulations were performed to obtain probabilities on the persistence of non-self DNA on the hands following a 40min, 5h or 8h delay between the handshake and contact with the final surface (an axe handle). Variables such as time elapsed, and the activities performed and objects contacted between the handshake and contact with the axe handle, were also considered when assessing the DNA results. DNA from a known contributor was transferred to the right hand of an opposing hand-shaker (as a depositor), and could be subsequently transferred to, and detected on, a surface contacted by the depositor 40min to 5h post-handshake. No non-self DNA from the known contributor was detected in deposits made 8h post-handshake. DNA from the depositor was generally detected as the major or only contributor in the profiles generated. Contributions from the known contributor were minor, decreasing in presence and in the strength of support for inclusion as the time between the handshake and transfer event increased. The construction of a series of Bayesian networks based on the case circumstances provided empirical estimations of the likelihood of direct or indirect deposition. The analyses and conclusions presented demonstrate both the

  10. C-14 bomb peak dating of human DNA samples at the microgram level

    International Nuclear Information System (INIS)

    Liebl, J. C.

    2011-01-01

    Radiocarbon (14C, t 1/2 = 5700 ± 30 years) is probably the radionuclide with the most versatile applications, spanning from archaeology to geoscience and medicine. Many of these applications are finally limited by the minimum amount of carbon in which the isotopic ratio 14C/12C can be measured. The required carbon sample size has dramatically decreased with the development of Accelerator Mass Spectrometry (AMS), typically from gram amounts for the classical beta counting method to about 1 milligram for AMS. The current work presents a further reduction into the few-μg carbon range. This means a decrease by a factor of one million compared to classical beta counting and is essential for the field of retrospective birth dating of human cells by means of radiocarbon from above-ground nuclear weapons testing between 1955 and 1963. The determination of 14C levels in genomic DNA can be used to retrospectively establish the birth date of cells in the human body. The main motive of the current work was to reduce the amount of carbon required for reliable 14C measurements to such an extent that investigations of neurons of particularly interesting small sections of the human brain (e.g. the olfactory bulb, bulbus olfactorius) were possible. In-depth investigations and development of 14C AMS sample preparation and measurement methods for μg-size DNA samples were carried out in close collaboration with the Department of Cell and Molecular Biology of the Karolinska Institute in Stockholm. As the most significant result, 14C measurements of 4.6 μg carbon DNA samples were performed with an overall precision of 2.3%. This allowed to study neurogenesis in the human olfactory bulb, which turned out to take place primarily at birth. Assuming throughout life a constant annual renewal rate of neurons in the human olfactory bulb, an upper limit of 0.34% for the renewal rate (95% confidence) was determined. At the Vienna Environmental Research Accelerator (VERA) the μg carbon

  11. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  12. dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

    Science.gov (United States)

    Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

    2015-05-12

    Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

  13. Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

    Science.gov (United States)

    Manzanares-Palenzuela, C Lorena; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

    2015-06-15

    Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is fina