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Sample records for bpde-like dna adduct

  1. DNA adducts as molecular dosimeters

    International Nuclear Information System (INIS)

    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 32P-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-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.

  3. DNA adducts-chemical addons.

    Science.gov (United States)

    Rajalakshmi, T R; AravindhaBabu, N; Shanmugam, K T; Masthan, K M K

    2015-04-01

    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. PMID:26015708

  4. Sperm DNA oxidative damage and DNA adducts.

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  5. DNA adducts-chemical addons

    OpenAIRE

    T. R. Rajalakshmi; N AravindhaBabu; Shanmugam, K. T.; Masthan, K. M. K.

    2015-01-01

    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 b...

  6. [DNA adducts in human female genital organs].

    Science.gov (United States)

    Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Monist, Marta; Baranowski, Włodzimierz

    2007-12-01

    DNA adducts, one of genetic damages markers, precede and finally can lead to oncogenic mutations. They appear in genome as a result of DNA bases damages caused by various and numerous environmental factors eg. ultraviolet light, ionic radiation, toxins and also endogenic substances, for example estrogens. It is believed that the creation of DNA adducts is a necessary but insufficient process for the neoplastic transformation of the cell. The following review presents concise knowledge about the DNA adducts creation and their sequels served in healthy and cancerous tissues of the female genital organs, on the base of the available data. PMID:18411923

  7. STUDY ON GMA-DNA ADDUCTS

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Objective. DNA modification fixed as mutations in the cells may be an essential factor in the initiation step of chemical carcinogenesis. In order to explore the mechanism of gene mutation and cell transformation induced by glycidyl methacrylate (GMA), the current test studied the characteristics of GMA-DNA adducts formation in vitro.Methods. In vitro test, dAMP, dCMP, dGMP, dTMP and calf thymus DNA were allowed to react with GMA (Glycidyl Methacrylate). After the reaction, the mixtures were detected by UV and subjected to reversed-phase HPLC on ultrasphere ODS reversed-phase column, the reaction products were eluted with a linear gradients of methanol (solvent A) and 10mmol/L ammonium formate, pH5.0 (solvent B). The synthesized adducts were then characterized by UV spectroscopy in acid (pH1.0), neutral (pH7.2), alkaline (pH11.0) and by mass spectroscopy.Results. The results showed that GMA could bind with dAMP, dCMP, dGMP and calf thymus DNA by covalent bond, and the binding sites were specific (N6 of adenine, N3 of cytosine). Meanwhile, a main GMA-DNA adduct in the reaction of GMA with calf thymus DNA was confirmed as N3-methacrylate-2-hydroxypropy1-dCMP.Conclusions. GMA can react with DNA and /or deoxynucleotide monophosphate and generate some adducts such as N6-methacrylate-2-hydroxypropyl-dAMP and N3-methacrylate-2-hydroxypropyl-dCMP, ets. Formation of GMA-DNA adducts is an important molecular event in gene mutation and cell transformation induced by GMA.

  8. DNA adduct formation by alachlor metabolites

    International Nuclear Information System (INIS)

    The extent of DNA adduct formation by alachlor [ArN(CH2OCH3)C(O)CH2Cl wherein Ar is 2,6-diethylphenyl] and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. [14C-phenyl]Alachlor is compared to its two metabolic cleavage products, [14C-phenyl] 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) [ArNHC(O)CH2Cl] and [14C-phenyl]2,6-diethylaniline (DEA) (ArNH2), and to [14C-methoxy]alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CEDPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from [14C-methoxy]- than from [14C-phenyl]alachlor. This 4-fold preferential DNA labeling from the 14C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with [14C-phenyl]alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with [14C-methoxy]alachlor

  9. 32P-postlabelling methods for cyclic DNA adducts.

    Science.gov (United States)

    Watson, W P; Crane, A E; Steiner, S

    1993-01-01

    32P-Postlabelling procedures coupled with HPLC have been developed to detect and measure a range of cyclic DNA adducts formed by bifunctional genotoxic agents. The methods are based on reverse-phase HPLC, particularly column-switching HPLC, to enrich adduct 3'-monophosphates before labelling. Following 3'-dephosphorylation of the 3'5'-[5'-32P]bisphosphates with nuclease P1, the resulting 5'-[32P]monophosphate adducts are resolved, identified and characterized by co-chromatography with synthetic reference standards. The procedures have been applied to a number of cyclic adducts including those formed by chloroacetaldehyde, glycidaldehyde and malonaldehyde. In general, labelling efficiencies measured as chromatographed 5'-[32P]monophosphates were in the range 30-40%. However, the values for the malonaldehyde deoxyguanosine adduct were much lower. The techniques have been applied to studies on the formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde. The HPLC-32P-postlabelling analysis of epidermal DNA hydrolysates indicated that a single major cyclic adduct was formed by reaction with deoxyadenosine residues in mouse skin DNA. The adduct was identified as a hydroxymethyl ethenodeoxyadenosine adduct by comparison with a synthetic standard. This adduct was highly fluorescent and it was possible to make quantitative comparisons of the amounts of adduct determined by either HPLC-32P-postlabelling or HPLC-fluorescence detection. PMID:8225493

  10. Fluorescence spectroscopy in the analysis of deoxyribonucleic acid (DNA) adducts

    International Nuclear Information System (INIS)

    An important first step in the initiation of carcinogenesis by polycyclic aromatic hydrocarbons (PAH), is the formation of a covalently bound adduct between the metabolized PAH and one or more deoxyribonucleic acid bases. In vivo concentrations of these adducts are typically ∼1 adducted base per 108 normal DNA base pairs. In this paper methods of generating high resolution fluorescence spectra of adducts at these levels are described. Methods of overcoming factors such as photochemistry and nonphotochemical hole burning which limit detection limits are described. By generating line narrowed fluorescence spectra, it is possible to spectrally distinguish between various adduct possibilities

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

    OpenAIRE

    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...

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

    International Nuclear Information System (INIS)

    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

  13. 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.

  14. 32P-adduct assay: Comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures

    International Nuclear Information System (INIS)

    A (32)P-adduct assay for the measurement of low levels (1 adduct per 10(sup 7) nucleotides) of binding of carcinogens to DNA has been reported previously. In this procedure, DNA is enzymatically hydrolyzed to 3'-monophosphates of normal nucleosides and adducts, which are 5'-(32)P-labeled by T4 polynucleotide kinase and (lambda(32)P)ATP. Labeled adducts are resolved by TLC. Enrichment of adducts by extraction in 1-butanol or digestion with nuclease P1 prior to (32)P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 10(sup 9-10) nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. The observation that chemical structure of an adduct may be detrimental in its recovery in the enzyme- and extraction-mediated enrichment procedures may serve as a probe in the structural characterization of adducts of unknown carcinogens

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

    International Nuclear Information System (INIS)

    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 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 4.0/108 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 1.13/108 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 biomarker to monitor PAH

  16. Quantitation of DNA Adducts Induced by 1,3-Butadiene

    Science.gov (United States)

    Sangaraju, Dewakar; Villalta, Peter W.; Wickramaratne, Susith; Swenberg, James; Tretyakova, Natalia

    2014-07-01

    Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI+-HRMS3 analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 106 nucleotides in HT1080 cells treated with 0.5-10 μM DEB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 106 nucleotides, respectively. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI+-HRMS3 Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

  17. Mutagenesis by site-specific arylamine adducts in plasmid DNA: Enhancing replication of the adducted strand alters mutation frequency

    International Nuclear Information System (INIS)

    Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by >7-fold to 2.9% and of the AAF adduct by >12-fold to 0.75%. The AF adduct produced primarily single-base deletions in the absence of uracil but only base substitutions in the uracil-containing constructs. The AAF adduct produced mutations only in the uracil-containing DNA, which included both frame shifts and base substitutions. Mutations produced by both adducts were SOS dependent

  18. Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis

    International Nuclear Information System (INIS)

    Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. In the study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease 32P-post-labeling technique. Thin layer chromatography (TLC) maps from both groups revealed multiple DNA adducts which ranged from no adducts for one individual to six adducts for a different individual. The total DNA adduct concentrations were approximately one adduct in 10 to the seventh-10 to the eighth power normal nucleotides. Comparison of the adduct TLC profiles revealed individual variation in both pattern and level of DNA adducts. The type and amount of adduct was not influenced by smoking history and remained unchanged in four out of six subjects who were resampled after a one month interval. One adduct detected in lymphocyte DNA co-migrated on TLC with an adduct derived by in vitro incubation of lymphocytes with benzo(a)pyrene (B(a)P). The 3H-nucloside values were consistent with values obtained by 32P-postlabeling of the same sample (correlation coefficient of 0.88). No relationship was apparent between the capacity of lymphocytes to form a (3H)-B(a)P-derived adduct in vitro and the concentration of the adduct, or total adducts present in untreated lymphocytes

  19. CARCINOGEN-DNA ADDUCTS: INTRODUCTION, LITERATURE SUMMARY, AND RECOMMENDATIONS

    Science.gov (United States)

    The report summarizes the literature concerning adducts formed by xenobiotics with DNA and/or protein and discusses their feasibility as a monitoring tool for use in exposure and risk assessment. The report is divided into three segments. The first segment provides an introductio...

  20. Mutational properties of the primary aflatoxin B1-DNA adduct.

    OpenAIRE

    Bailey, E A; Iyer, R S; Stone, M. P.; Harris, T M; Essigmann, J M

    1996-01-01

    The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believ...

  1. Chemistry and Biology of Aflatoxin-DNA Adducts

    Energy Technology Data Exchange (ETDEWEB)

    Stone, Michael P.; Banerjee, Surajit; Brown, Kyle L.; Egli, Martin (Vanderbilt)

    2012-03-27

    Aspergillus flavus is a fungal contaminant of stored rice, wheat, corn, and other grainstuffs, and peanuts. This is of concern to human health because it produces the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}), which is genotoxic and is implicated in the etiology of liver cancer. AFB{sub 1} is oxidized in vivo by cytochrome P450 to form aflatoxin B{sub 1} epoxide, which forms an N7-dG adduct (AFB{sub 1}-N7-dG) in DNA. The latter rearranges to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative that equilibrates between {alpha} and {beta} anomers of the deoxyribose. In DNA, both the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts intercalate above the 5'-face of the damaged guanine. Each produces G {yields} T transversions in Escherichia coli, but the AFB{sub 1}-{beta}-FAPY adduct is more mutagenic. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) provides a model for understanding error-prone bypass of the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts. It bypasses the AFB{sub 1}-N7-dG adduct, but it conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including mis-insertion of dATP, consistent with the G {yields} T mutations characteristic of AFB{sub 1} mutagenesis in E. coli. Crystallographic analyses of a series of binary and ternary complexes with the Dpo4 polymerase revealed differing orientations of the N7-C8 bond of the AFB{sub 1}-N7-dG adduct as compared to the N{sup 5}-C8 bond in the AFB{sub 1}-{beta}-FAPY adduct, and differential accommodation of the intercalated AFB{sub 1} moieties within the active site. These may modulate AFB{sub 1} lesion bypass by this polymerase.

  2. 32P-postlabeling DNA adduct assay: cigarette smoke-induced dna adducts in the respiratory and nonrespiratory rat tissues. Book chapter

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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=5-30x108 dm3 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

  4. Formation and persistence of arylamine DNA adducts in vivo.

    OpenAIRE

    Beland, F A; Kadlubar, F F

    1985-01-01

    Aromatic amines are urinary bladder carcinogens in man and induce tumors at a number of sites in experimental animals including the liver, mammary gland, intestine, and bladder. In this review, the particular pathways involved in the metabolic activation of aromatic amines are considered as well as the specific DNA adducts formed in target and nontarget tissue. Particular emphasis is placed on the following compounds: 1-naphthylamine, 2-naphthylamine, 4-aminobiphenyl, 4-acetylaminobiphenyl, 4...

  5. Cisplatin–DNA adducts inhibit translocation of the Ku subunits of DNA-PK

    OpenAIRE

    Turchi, John J.; Henkels, Karen M.; Zhou, Yun

    2000-01-01

    We have determined the effect of cisplatin–DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin–DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku–DNA complex. In addition...

  6. Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

    OpenAIRE

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

    2008-01-01

    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/104 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...

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Myungkoo

    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 7{beta}, 8{alpha}-dihydoxy-9{alpha}, l0{alpha}-epoxy-7,8,9, 10-tetrahydrobenzo[{alpha}]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, ({minus})-trans-, (+)-cis- and ({minus})-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 ( {approximately} 25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant {pi}-{pi} 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{sub 2} or G{sub 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{sup 2}-dG in DNA isolated from the skin of mice treated topically with benzo[{alpha}]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{sup 2}-dG.

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

    International Nuclear Information System (INIS)

    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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    BACKGROUND: Bulky DNA adducts reflect genotoxic exposures, have been associated with lower birth weight, and may predict cancer risk. OBJECTIVE: We selected factors known or hypothesized to affect in utero adduct formation and repair and examined their associations with adduct levels in neonates....

  11. Metabolites and DNA adduct formation from flavoenzyme-activated porfiromycin.

    Science.gov (United States)

    Pan, S S; Iracki, T

    1988-08-01

    Porfiromycin was reductively metabolized by NADPH cytochrome P-450 reductase and xanthine oxidase under anaerobic conditions. The production of metabolites varied with the pH and the contents of the reaction buffer. In Tris buffer, two major metabolites were produced at pH 7.5 and above, whereas one major metabolite was produced at pH 6.5. The three major metabolites were separated and isolated by HPLC. Identification by californium-252 plasma desorption mass spectrometry showed that the two major metabolites from pH 7.5 were (trans) and (cis)-forms of 7-amino-1-hydroxyl-2-methylaminomitosene and the major metabolite from pH 6.5 was 7-amino-2-methylaminomitosene. All three major metabolites showed substitutions at the C-1 position. DNA was alkylated readily by enzyme-activated porfiromycin. Digestion of porfiromycin-alkylated DNA by DNase, snake venom phosphodiesterase, and alkaline phosphatase resulted in an insoluble nuclease-resistant fraction and a soluble fraction. The nuclease-resistant fraction reflected a high content of cross-linked adducts. Upon HPLC analysis, the solubilized fraction contained two monofunctionally linked porfiromycin adducts and a possibly cross-linked dinucleotide. The major adduct was isolated by HPLC and identified by NMR, as N2-(2'-deoxyguanosyl)-7-amino-2-methylaminomitosene. The N2 position of deoxyguanosine appeared as the major monofunctional alkylating site for DNA alkylation by porfiromycin. Thus, mitomycin C and porfiromycin (which differs from mitomycin C only by the addition of a methyl group to the aziridine nitrogen) share the same enzymatic activating mechanism that leads to the formation of the same types of metabolites and the same specificity of DNA alkylation. PMID:3412325

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

    International Nuclear Information System (INIS)

    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. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    Science.gov (United States)

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  14. High-performance liquid chromatography for analysis of 32P-Postlabeled DNA adducts

    OpenAIRE

    Zeisig, Magnus

    1996-01-01

    High-Performance Liquid Chromatography for Analysis of 32P-Postlabeled DNA Adducts Magnus Zeisig Center for Nutrition and Toxicology, Department of Bioscience at Novum, Karolinska Institutet, Novum, S-141 57 Huddinge, SwedenThe formation of DNA adducts, i.e. the covalent binding of chemicals and chemical groups to DNA,isbelieved to be an important step in chemical carciwg...

  15. Human Biomonitoring of DNA Adducts by Ion Trap Multistage Mass Spectrometry.

    Science.gov (United States)

    Guo, Jingshu; Turesky, Robert J

    2016-01-01

    Humans are continuously exposed to hazardous chemicals in the environment. These chemicals or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. The identification of DNA adducts is required for understanding exposure and the etiological role of a genotoxic chemical in cancer risk. The analytical chemist is confronted with a great challenge because the levels of DNA adducts generally occur at spectrometry has emerged as an important technique to screen for DNA adducts because of the high level sensitivity and selectivity, particularly when employing multi-stage scanning (MS(n) ). The product ion spectra provide rich structural information and corroborate the adduct identities even at trace levels in human tissues. Ion trap technology represents a significant advance in measuring DNA adducts in humans. © 2016 by John Wiley & Sons, Inc. PMID:27584705

  16. Formation and persistence of DNA adducts of anticancer drug ellipticine in rats

    International Nuclear Information System (INIS)

    Ellipticine is an antineoplastic agent, whose mode of antitumor and/or toxic side effects is based on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochromes P450 and peroxidases. We investigated the formation and persistence of DNA adducts generated in rat, the animal model mimicking the bioactivation of ellipticine in human. Using 32P-postlabeling, ellipticine-DNA adducts were found in liver, kidney, lung, spleen, heart and brain of female and male rats exposed to ellipticine (4, 40 and 80 mg/kg body weight, i.p.). The two major adducts were identical to the deoxyguanosine adducts generated in DNA by 13-hydroxy- and 12-hydroxyellipticine in vitro as confirmed by HPLC of the isolated adducts. At four post-treatment times (2 days, 2, 10 and 32 weeks) DNA adducts in rats treated with 80 mg/kg of ellipticine were analyzed in each tissue to study their long-term persistence. In all organs maximal adduct levels were found 2 days after administration. At all time points highest total adduct levels were in liver (402 adducts/108 nucleotides after 2 days and 3.6 adducts/108 nucleotides after 32 weeks), kidney and lung followed by spleen, heart and brain. Total adduct levels decreased over time to 0.8-8.3% of the initial levels till the latest time point and showed a biphasic profile, a rapid loss during the first 2 weeks was followed by a much slower decline till 32 weeks. These results, the first characterization of persistence of ellipticine-DNA adducts in vivo, are necessary to evaluate genotoxic side effects of ellipticine

  17. Characterization of DNA adducts with fourier transform mass spectrometry

    International Nuclear Information System (INIS)

    The sensitive detection and unambiguous structural characterization of modified nucleic acid constituents is vital for understanding the nature of DNA modification induced by carcinogenic agents. Fourier transform mass spectrometry (FTMS) combined with matrix-assisted laser desorption provides a powerful technique for examining picomole quantities of modified nucleosides, nucleotides, and oligonucleotides. The structures of these modified biomolecules can be probed in detail with a variety of gas phase processes, including collision-induced dissociation, ion-molecule reactions such as deuterium exchange, and selective cationization reactions. Each of these processes provides a wealth of structural information which can be used to not only identify the adduct present, but also determine its exact site of attachment to the nucleic acid constituent, thereby providing isomeric differentiation. This FTMS technique has been applied to the examination of DNA damage induced by high energy (x-rays) as well as low energy radiation (far ultraviolet)

  18. Depurinating estrogen-DNA adducts, generators of cancer initiation: their minimization leads to cancer prevention.

    Science.gov (United States)

    Cavalieri, Ercole L; Rogan, Eleanor G

    2016-12-01

    Estrogens can initiate cancer by reacting with DNA. Specific metabolites of endogenous estrogens, the catechol estrogen-3,4-quinones, react with DNA to form depurinating estrogen-DNA adducts. Loss of these adducts leaves apurinic sites in the DNA, generating mutations that can lead to the initiation of cancer. A variety of endogenous and exogenous factors can disrupt estrogen homeostasis, which is the normal balance between estrogen activating and protective enzymes. In fact, if estrogen metabolism becomes unbalanced and generates excessive catechol estrogen 3,4-quinones, formation of depurinating estrogen-DNA adducts increases and the risk of initiating cancer is greater. The levels of depurinating estrogen-DNA adducts are high in women diagnosed with breast cancer and those at high risk for the disease. High levels of depurinating estrogen-DNA adducts before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Women with thyroid or ovarian cancer also have high levels of estrogen-DNA adducts, as do men with prostate cancer or non-Hodgkin lymphoma. Depurinating estrogen-DNA adducts are initiators of many prevalent types of human cancer. These findings and other discoveries led to the recognition that reducing the levels of estrogen-DNA adducts could prevent the initiation of human cancer. The dietary supplements N-acetylcysteine and resveratrol inhibit formation of estrogen-DNA adducts in cultured human breast cells and in women. These results suggest that the two supplements offer an approach to reducing the risk of developing various prevalent types of human cancer. Graphical abstract Major metabolic pathway in cancer initiation by estrogens. PMID:26979321

  19. Seasonal variations in levels of DNA adducts and X-spots in human populations living in different parts of Poland.

    OpenAIRE

    Grzybowska, E; Hemminki, K; Choraźy, M

    1993-01-01

    White blood cell DNA adducts were measured in coke oven workers, in residents from the area next to the coke oven in Silesia, Poland (highly industrialized region), and in residents from the rural area of Poland using the 32P-postlabeling technique. This method detected aromatic adducts including adducts formed by polycyclic aromatic hydrocarbons (PAHs). Highest levels of adducts in DNA were seen in the group of coke battery workers (6.9 adducts/10(8) nucleotides). Seasonal variations in leve...

  20. Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa.

    Science.gov (United States)

    Musser, S M; Pan, S S; Callery, P S

    1989-07-14

    High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated. PMID:2504760

  1. DNA adducts in marine mussel Mytilus galloprovincialis living in polluted and unpolluted environments

    International Nuclear Information System (INIS)

    A generally applicable 32-P post-labelling assay was used to examine for the presence of DNA adducts in mussels experimentally exposed to known carcinogens and in mussels collected from sites impacted by wastewaters. Mussels exposed to seawater artificially polluted with 2-aminofluorene showed exclusively an adduct which was identified as dG-C8-2-aminofluorene. Under the same experimental conditions, Diesel-2 oil did not induce any detectable adducts. When mussel digestive gland DNA was collected and analyzed from one unpolluted site, two moderately impacted sites, and one site heavily impacted by cannery wastewaters, mussel DNA from the unpolluted and only one moderately polluted site showed the presence of 6 to 10 adducts. This indicates they were not related to the pollution. This was further supported by the absence of dose-related adducts. Clear evidence for the presence of pollution-related DNA adducts was, however, found in juvenile mussels collected from an oil refinery site. One major and three minor adducts were detected in these mussels with no adducts detected in juvenile mussels from an unpolluted site. Results indicate that while mussels are capable of metabolizing 2-aminofluorene to a DNA binding species, the environmental pollutants may be metabolized only selectively. Further, the mussel digestive gland DNA also contained DNA modifications which are unrelated to pollutants and their presence appeared to be sex and season dependent

  2. Isolation, identification, and assay of [3H]-porfiromycin adducts of EMT6 mouse mammary tumor cell DNA: effects of hypoxia and dicumarol on adduct patterns.

    Science.gov (United States)

    Tomasz, M; Hughes, C S; Chowdary, D; Keyes, S R; Lipman, R; Sartorelli, A C; Rockwell, S

    1991-07-01

    [3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf-thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono-adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed. PMID:1714285

  3. Seasonal and intertidal impact on DNA adduct levels in gills of blue mussels (Mytilus edulis L.)

    International Nuclear Information System (INIS)

    The aim of this study was to elucidate possible seasonal variation in DNA adduct levels in blue mussels (Mytilus edulis), and to investigate the impact of intertidal exposure on the DNA adduct levels, i.e. to explore if DNA adduct levels in mussels in the intertidal zone differ from those in the subtidal zone. Blue mussels were deployed separately in the intertidal and subtidal zone at a contaminated and a reference site in Iceland, and sampled regularly during one year. Gill DNA adduct levels were found to be higher in mussels in the intertidal zone compared to the subtidal zone at the contaminated site, the difference being largest in winter. Total PAH tissue levels were also higher in mussels in the intertidal zone. Seasonal variation was observed in both DNA adduct and PAH tissue levels in mussels at the contaminated site, with lower levels from the time of transplantation in summer to autumn, maximum levels in winter, which decreased to lower levels again in spring and summer the following year. DNA adducts and PAH levels were low or below the detection limits in mussels at the reference site at all times, both in the intertidal and subtidal zone. - Gill DNA adduct and total PAH tissue levels were higher in mussels in the intertidal than subtidal zone, and higher in winter than summer

  4. Seasonal and intertidal impact on DNA adduct levels in gills of blue mussels (Mytilus edulis L.)

    Energy Technology Data Exchange (ETDEWEB)

    Skarpheoinsdottir, Halldora [Institute of Applied Environmental Research, Laboratory for Aquatic Ecotoxicology, Stockholm University, 106 91 Stockholm (Sweden)]. E-mail: halldora@itm.su.se; Ericson, Gunilla [Institute of Applied Environmental Research, Laboratory for Aquatic Ecotoxicology, Stockholm University, 106 91 Stockholm (Sweden); Halldorsson, Halldor P. [Institute of Biology, University of Iceland, Askja, Sturlugata 7, 101 Reykjavik (Iceland); University of Iceland, Sandgeroei Marine Centre, Garoevegur 1, 245 Sandgeroei (Iceland); Svavarsson, Joerundur [Institute of Biology, University of Iceland, Askja, Sturlugata 7, 101 Reykjavik (Iceland); University of Iceland, Sandgeroei Marine Centre, Garoevegur 1, 245 Sandgeroei (Iceland)

    2005-07-15

    The aim of this study was to elucidate possible seasonal variation in DNA adduct levels in blue mussels (Mytilus edulis), and to investigate the impact of intertidal exposure on the DNA adduct levels, i.e. to explore if DNA adduct levels in mussels in the intertidal zone differ from those in the subtidal zone. Blue mussels were deployed separately in the intertidal and subtidal zone at a contaminated and a reference site in Iceland, and sampled regularly during one year. Gill DNA adduct levels were found to be higher in mussels in the intertidal zone compared to the subtidal zone at the contaminated site, the difference being largest in winter. Total PAH tissue levels were also higher in mussels in the intertidal zone. Seasonal variation was observed in both DNA adduct and PAH tissue levels in mussels at the contaminated site, with lower levels from the time of transplantation in summer to autumn, maximum levels in winter, which decreased to lower levels again in spring and summer the following year. DNA adducts and PAH levels were low or below the detection limits in mussels at the reference site at all times, both in the intertidal and subtidal zone. - Gill DNA adduct and total PAH tissue levels were higher in mussels in the intertidal than subtidal zone, and higher in winter than summer.

  5. DNA adducts in marine mussel and fresh water fishes living in polluted and unpolluted environments

    International Nuclear Information System (INIS)

    32P-postlabeling analysis of DNA adducts in the digestive gland of marine mussel Mytilus galloprovincialis from polluted and unpolluted sites near Rovinj, Northern Adriatic, revealed that majority of adducts are caused by natural environmental factors rather than by man-made chemicals. The only pollutant-specific adducts were observed in a mussel exposed to seawater experimentally polluted with aminofluorene, and in a population of mussel living at a site heavily polluted with a waste waters of an oil refinery. Fresh water fish species Leuciscus cephalus, Barbus barbus, Abramis brama and Rutilus pigus virgo living in a polluted Sava River, Yugoslavia, or in its unpolluted tributary Korana River, have induced in their livers qualitatively identical and quantitatively similar DNA adducts. These DNA adducts had a species-specific patterns and their appearance was seasonally-dependent

  6. 32P analysis of DNA adducts in tissues of benzene-treated rats

    International Nuclear Information System (INIS)

    Solid tumors have been reported in the Zymbal gland, oral and nasal cavities, liver, and mammary gland of Sprague-Dawley rats following chronic, high-dose administration of benzene. The carcinogenic activity of benzene is thought to be caused by activation to toxic metabolites that can interact with DNA, forming covalent adducts. A nuclease P1-enhanced 32P-postlabeling assay, having a sensitivity limit of 1 adduct in 10(9-10) DNA nucleotides, was found suitable for measuring aromatic DNA adducts derived in vitro from catechol, benzenetriol (BT), phenol, hydroquinone (HQ), and benzoquinone (BQ), potential metabolites of benzene. When DNA specimens isolated from tissues of female Sprague-Dawley rats at 24 hr after an oral gavage dose of 200 to 500 mg/kg, 5 days/week, in olive oil (3 mL/kg) for 1 day, 1 week, 5 weeks, and 10 weeks were analyzed by the 32P-postlabeling procedure, no aromatic adducts were detected unequivocally with DNA samples of liver, kidney, bone marrow, and mammary gland. With Zymbal gland DNA, three weak spots at levels totaling four lesions per 10(9) DNA nucleotides were seen only after 10 weeks of treatment, and these adducts did not correspond chromatographically to major adducts in vitro from the above specified compounds. Consequently, this finding requires confirmatory experiments. This distinct adduct pattern may relate to tumor induction in this organ following benzene administration. Our results also indicate that DNA adducts derived from catechol, BT, phenol, HQ, and BQ are either not formed in vivo with benzene or formed at levels below the detection limit of 1 adduct per 10(9-10) DNA nucleotides

  7. Comparison of DNA adducts from exposure to complex mixtures in various human tissues and experimental systems

    OpenAIRE

    Lewtas, Joellen; Mumford, Judy; Everson, Richard B; Hulka, Barbara; Wilcosky, Tim; Kozumbo, Walter; Thompson, Claudia; George, Michael; Dobiáš, Lubomir; Šrám, Radim; Li, Xueming; Gallagher, Jane

    1993-01-01

    DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in ...

  8. DNA adducts as a measure of lung cancer risk in humans exposed to polycyclic aromatic hydrocarbons.

    OpenAIRE

    Kriek, E; van Schooten, F.J.; Hillebrand, M J; van Leeuwen, F E; Den Engelse, L; De Looff, A J; Dijkmans, A P

    1993-01-01

    Workers in the coking, foundry, and aluminum industry can be exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and are at increased risk for lung cancer, as are cigarette smokers. In recent years several studies on workers in the foundry and coking industries have been reported. In these studies, white blood cell(WBC) DNA was used for analysis of PAH-DNA adducts. Theoretically, DNA adduct formation is a more relevant biological parameter for assessing exposure risk tha...

  9. Determinants of 4-aminobiphenyl-DNA adducts in bladder cancer biopsies.

    Science.gov (United States)

    Airoldi, Luisa; Orsi, Federica; Magagnotti, Cinzia; Coda, Renato; Randone, Donato; Casetta, Giovanni; Peluso, Marco; Hautefeuille, Agnes; Malaveille, Christian; Vineis, Paolo

    2002-05-01

    Exposure to 4-aminobiphenyl (4-ABP) is an important determinant of urinary bladder cancer in humans. We have analyzed by gas chromatography-mass spectrometry the DNA adducts of 4-ABP in 75 bladder cancer biopsies. The purpose was to understand whether smoking, N-acetyltransferase 2 (NAT2) polymorphism, diet or tumor grade were determinants of 4-ABP-DNA levels. 4-ABP-DNA adducts were above the detection limit of 0.1 fmol/microg DNA for 37/75 patients. Overall the level of adducts was 2.7 +/- 0.7 (mean +/- SE) fmol/microg DNA (86 +/- 22 adducts/10(8) normal nucleotides, mean +/- SE). A strong association with grade was observed. In the group of patients with detectable 4-ABP-DNA adducts the odds ratio for having a tumor grade of 2 or 3 was respectively 4.3 (95% CI 0.8-21.9) and 6 (1.3-27.5), compared with grade 1. A non-statistically significant association was found between adduct levels and the deduced slow acetylator phenotype in grades 2 and 3. The intake of fruit and vegetables produced a lower frequency of detectable adducts, though the association was not statistically significant. Detectable 4-ABP-DNA adducts were clearly associated with current smoking in higher tumor grades (grade 3 versus grades 1 + 2, odds ratios 10.4; 95% CI 1.7-63.1). Overall, our findings indicate that higher levels of DNA adducts characterize more invasive tumors (higher tumor grades). This seems to be facilitated by smoking and contrasted by the intake of fruit and vegetables. PMID:12016161

  10. 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 subu...

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

    International Nuclear Information System (INIS)

    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

  12. 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.

  13. Polycyclic aromatic hydrocarbon-DNA adducts and survival among women with breast cancer

    International Nuclear Information System (INIS)

    Polycyclic aromatic hydrocarbons (PAH) are mammary carcinogens in animal studies, and a few epidemiologic studies have suggested a link between elevated levels of PAH-DNA adducts and breast cancer incidence. An association between PAH-DNA adducts and survival among breast cancer cases has not been previously reported. We conducted a survival analysis among women with newly diagnosed invasive breast cancer between 1996 and 1997, enrolled in the Long Island Breast Cancer Study Project. DNA was isolated from blood samples that were obtained from cases shortly after diagnosis and assayed for PAH-DNA adducts using ELISA. Among the 722 cases with PAH-DNA adduct measurements, 97 deaths (13.4%) from all causes and 54 deaths (7.5%) due to breast cancer were reported to National Death Index (NDI) by December 31, 2002. Using Cox proportional hazards models and controlling for age at diagnosis, we did not find evidence that all-cause mortality (hazard ratio (HR)=0.88; 95% confidence interval (CI): 0.57-1.37), or breast cancer mortality (HR=1.20; 95% CI: 0.63-2.28) was strongly associated with detectable PAH-DNA adduct levels compared with non-detectable adducts; additionally, no dose-response association was observed. Among a subgroup with treatment data (n=520), adducts were associated with over a two-fold higher mortality among those receiving radiation, but mortality for adducts was reduced among hormone therapy users. Results from this large population-based study do not provide strong support for an association between detectable PAH-DNA adducts and survival among women with breast cancer, except perhaps among those receiving radiation treatment

  14. Inhaled cigarette smoke induces the formation of DNA adducts in lungs of rats

    International Nuclear Information System (INIS)

    Cigarette smoking causes a variety of adverse human health effects, including lung cancer. The molecular events associated with smoke-induced carcinogenesis are thought to be related in part to the genotoxic activities of the chemicals associated with smoke. The purpose of this investigation was to determine the molecular dosimetry of compounds in cigarette smoke in lungs of rats exposed by inhalation. These studies investigated the effects of exposure mode, sex, and time (adduct persistence) on the level of DNA adducts. Male and female F344/N rats were exposed 6 hr/day, 5 days/week for 22 days to cigarette smoke by nose-only intermittent (NOI), nose-only continuous (NOC), or whole-body continuous (WBC) exposures. Separate groups of rats were sham-exposed nose-only (NOS) or whole-body (WBS) to filtered air. All smoke exposure modes yielded daily smoke exposure concentration X time products of 600 mg particulate.hr/m3 for the first week and 1200 mg particulate.hour/m3 thereafter. Groups of rats were killed at 18 hr and 3 weeks after the 22-day exposure period and DNA adducts in lung tissues were quantified by the 32P-postlabeling method. There were significant (p less than 0.05) increases in levels of clearly resolved lung DNA adducts in male and female rats exposed to smoke compared to sham-exposed rats. There were no significant effects of exposure mode or sex on lung DNA adducts. Mean levels (+/- SE) of clearly resolved lung DNA adducts for both sexes combined in NOI, NOC, WBC, NOS, and WBS groups were 50 +/- 4, 52 +/- 6, 52 +/- 7, 21 +/- 6, and 22 +/- 4 adducts per 10(9) bases, respectively. Levels of clearly resolved DNA adducts were significantly less in lungs of rats killed 3 weeks after exposure and had declined to near control levels, suggesting that smoke-induced adducts are repaired by lung DNA repair enzymes

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-03-01

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

  16. Implications of acetaldehyde-derived DNA adducts for understanding alcohol-related carcinogenesis.

    Science.gov (United States)

    Balbo, Silvia; Brooks, Philip J

    2015-01-01

    Among various potential mechanisms that could explain alcohol carcinogenicity, the metabolism of ethanol to acetaldehyde represents an obvious possible mechanism, at least in some tissues. The fundamental principle of genotoxic carcinogenesis is the formation of mutagenic DNA adducts in proliferating cells. If not repaired, these adducts can result in mutations during DNA replication, which are passed on to cells during mitosis. Consistent with a genotoxic mechanism, acetaldehyde does react with DNA to form a variety of different types of DNA adducts. In this chapter we will focus more specifically on N2-ethylidene-deoxyguanosine (N2-ethylidene-dG), the major DNA adduct formed from the reaction of acetaldehyde with DNA and specifically highlight recent data on the measurement of this DNA adduct in the human body after alcohol exposure. Because results are of particular biological relevance for alcohol-related cancer of the upper aerodigestive tract (UADT), we will also discuss the histology and cytology of the UADT, with the goal of placing the adduct data in the relevant cellular context for mechanistic interpretation. Furthermore, we will discuss the sources and concentrations of acetaldehyde and ethanol in different cell types during alcohol consumption in humans. Finally, in the last part of the chapter, we will critically evaluate the concept of carcinogenic levels of acetaldehyde, which has been raised in the literature, and discuss how data from acetaldehyde genotoxicity are and can be utilized in physiologically based models to evaluate exposure risk. PMID:25427902

  17. Sperm DNA adducts impair fertilization during ICSI but not during IVF.

    Directory of Open Access Journals (Sweden)

    Piotr Widłak

    2008-04-01

    Full Text Available Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization and 118 ICSI (intracytoplasmic sperm injection first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999. DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI. The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002 and motility (p=0.0037 but not by DNA adducts concentration (p=0.30528, whereas during ICSI was positively influenced by sperm motility (p=0.04669 and negatively by DNA adducts concentration (p=0.00796. DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.

  18. Recent progress in quantitative analysis of DNA adducts of nephrotoxin aristolochic acid

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Aristolochic acid (AA), a mixture of structure-related nitrophenanthrene carboxylic acid derivatives derived from Aristolochia spp, is associated with nephrotoxin and carcinogen. AA-DNA adducts induced by reductive metabolic activation of AA were detected in tissues of animals and in patients exposed to AA. The DNA adducts were generally used as biomarkers in toxicological study of AA. In this short review, quantitative analysis of AA-DNA adducts in various in vitro and in vivo systems by using 32P-postlabelling assay, HPLC-UV, HPLC-radiation monitor, HPLC-FLD, HPLC-ESI/MS and UPLC-MS/MS methods is discussed. The distribution of AA-DNA adducts in various tissues is also summarized.

  19. DNA adducts in target and nontarget tissues of 3,2'-dimethyl-4-aminobiphenyl in rats.

    OpenAIRE

    Shirai, T; Tada, M; Kojima, M; Hasegawa, R.; Masui, T.; Ito, N.

    1994-01-01

    3,2'-Dimethyl-4-aminobiphenyl (DMAB) is a potent carcinogenic aromatic amine which demonstrates multiorgan tropism in rats. Using polyclonal antibodies against DMAB-DNA adducts, an immunohistochemical procedure as well as an ELISA were applied to investigate the relationship between DMAB-DNA adduct formation and tumorigenicity. Dose-related nuclear staining was observed 24 hr after application of the carcinogen but specificity in terms of sites of tumor development was lacking. No observable ...

  20. 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; Peluso, Marco; Munnia, Armelle; Perera, Frederica; Phillips, David H; Tang, Deliang; Autrup, Herman; Raaschou-Nielsen, Ole; Tjønneland, Anne; Vineis, Paolo

    2008-01-01

    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...... 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....... In the pooled analysis, a weakly statistically significant increase in the risk of lung cancer was apparent (14% per unit standard deviation change in adduct levels, 95% confidence interval 1-28%; using the weighted mean difference method, 0.15 SD, units higher adducts in cases than in controls). The...

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

    International Nuclear Information System (INIS)

    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 103-104 adducts/1012 nucleotides, and the adducts increased linearly with increasing dose of nicotine; the adducts number of liver DNA reached to 104-105 adducts/1012 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

  2. Transplatin-conjugated triplex-forming oligonucleotides form adducts with both strands of DNA.

    Science.gov (United States)

    Campbell, Meghan A; Miller, Paul S

    2009-12-01

    Triplex-forming oligonucleotides (TFOs) can bind to polypurine x polypyrimidine tracts in DNA and, as a consequence, perturb the normal functioning of a targeted gene. The effectiveness of such antigene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here, we report that attachment of N-7-platinated guanine nucleosides to the 3'- and/or 5'-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3',5'-bis-platinated TFOs can result in the formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37 degrees C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast, high levels (approximately 86%) of adduct formation by platinated 2'-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2'-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinum-conjugated TFOs may be good candidates for use as antigene agents. PMID:19950917

  3. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    Science.gov (United States)

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. PMID:24623226

  4. 4-Aminobiphenyl-DNA adducts and p53 mutations in bladder cancer.

    Science.gov (United States)

    Martone, T; Airoldi, L; Magagnotti, C; Coda, R; Randone, D; Malaveille, C; Avanzi, G; Merletti, F; Hautefeuille, A; Vineis, P

    1998-02-01

    Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms. PMID:9466649

  5. Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MS/MS

    OpenAIRE

    Monien, Bernhard H.; Schumacher, Fabian; Herrmann, Kristin; Glatt, Hansruedi; Turesky, Robert J.; Chesné, Christophe

    2014-01-01

    Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra per...

  6. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

    Science.gov (United States)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  7. The N(2)-Furfuryl-deoxyguanosine Adduct Does Not Alter the Structure of B-DNA.

    Science.gov (United States)

    Ghodke, Pratibha P; Gore, Kiran R; Harikrishna, S; Samanta, Biswajit; Kottur, Jithesh; Nair, Deepak T; Pradeepkumar, P I

    2016-01-15

    N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by ∼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct. PMID:26650891

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

    International Nuclear Information System (INIS)

    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 194Pt). 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)

  9. GSTM1 and XRCC3 Polymorphisms: Effects on Levels of Aflatoxin B1-DNA Adducts

    Institute of Scientific and Technical Information of China (English)

    Xi-dai Long; Yun Ma; Zhou-lin Deng

    2009-01-01

    Objective: Aflatoxin B1 (AFB1), which can cause the formation of AFB1-DNA adducts, is a known human carcinogen. AFB1-exposure individuals with inherited susceptible carcinogen-metabolizing or repairing genotypes may experience an increased risk of genotoxicity. This study was designed to investigate whether the polymorphisms of two genes, the metabolic gene Glutathione S-transferase M1 (GSTM1) and DNA repair gene x-ray repair cross-complementing group 3 (XRCC3), can affect the levels of AFB1-DNA adducts in Guangxi Population (n= 966) from an AFB1-exposure area.Methods: AFB1-DNA adducts were measured by ELISA, and GSTM1 and XRCC3 codon 241 genotypes were identified by PCR-RFLP.Results: The GSTM1-null genotype [adjusted odds ratio (OR) = 2.09; 95% confidence interval (CI) = 1.61(2.71] and XRCC3 genotypes with 241 Met alleles [i.e., XRCC3-TM and -MM, adjusted ORs (95% CI) were 1.43 (1.08(1.89) and 2.42 (1.13(5.22), respectively] were significantly associated with higher levels of AFB1-DNA adducts. Compared with those individuals who did not express any putative risk genotypes as reference (OR = 1), individuals featuring all of the putative risk genotypes did experience a significantly higher DNA-adduct levels (adjusted ORs were 2.87 for GSTM1-null and XRCC3-TM; 5.83 for GSTM1-null and XRCC3-MM). Additionally, there was a positive joint effect between XRCC3 genotypes and long-term AFB1 exposure in the formation of AFB1-DNA adducts.Conclusion: These results suggest that individuals with susceptible genotypes GSTM1-null, XRCC3-TM, or XRCC3-MM may experience an increased risk of DNA damage elicited by AFB1 exposure.

  10. Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development

    NARCIS (Netherlands)

    Hooser, S.T.; Dijk-Knijnenburg, C.M. van; Waalkens-Berendsen, I.D.H.; Smits-van Prooije, A.E.; Snoeij, N.J.; Baan, R.A.; Fichtinger-Schepman, M.J.

    2000-01-01

    Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin-DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. Th

  11. Formation of 1,4-dioxo-2-butene-derived adducts of 2'-deoxyadenosine and 2'-deoxycytidine in oxidized DNA.

    Science.gov (United States)

    Chen, Bingzi; Vu, Choua C; Byrns, Michael C; Dedon, Peter C; Peterson, Lisa A

    2006-08-01

    Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M(1)dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the beta-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5'-oxidation of deoxyribose in DNA, and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5'-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts. PMID:16918236

  12. 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; Autrup, Herman

    1996-01-01

    rural controls (0.074 fmol/microg DNA, n = 60, P < 0.001). No significant influence on adduct levels was demonstrated from potential confounders, including smoking and diet. The effect of the metabolizing enzymes, GSTM1 and NAT2, on adduct levels was investigated. No statistically significant effects...... levels of exposure to urban air pollution and indicated that these adducts might be helpful as a means of classifying better different exposure groups for epidemiological studies. Furthermore, it demonstrated the ability of 32P-postlabelling to discern small differences in low exposure to ambient air...

  13. NanoLC/ESI+ HRMS3 quantitation of DNA adducts induced by 1,3-butadiene.

    Science.gov (United States)

    Sangaraju, Dewakar; Villalta, Peter W; Wickramaratne, Susith; Swenberg, James; Tretyakova, Natalia

    2014-07-01

    Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI(+)-HRMS(3) analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 10(8) nucleotides in HT1080 cells treated with 0.5-10 μM EB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 10(8) nucleotides, respectively [corrected]. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI(+)-HRMS(3) Orbitrap methodology to quantitative analysis of DNA adducts in vivo. PMID:24867429

  14. Grilled Meat Consumption and PhIP-DNA Adducts in Prostate Carcinogenesis

    OpenAIRE

    Tang, Deliang; Liu, Jason J; Rundle, Andrew; Neslund-Dudas, Christine; Savera, Adnan T.; Bock, Cathryn H.; Nock, Nora L.; Yang, James J.; Rybicki, Benjamin A

    2007-01-01

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the major heterocyclic amine generated from cooking meats at high temperatures, and dietary exposures have been shown to induce prostate cancer in rats. PhIP derives its carcinogenic potential through the formation of PhIP-DNA adducts. The purpose of this study was to examine whether self-reported consumption and preparation doneness of grilled meats were associated with PhIP-DNA adduct levels in prostate epithelial cells. The study po...

  15. Polycyclic Aromatic Hydrocarbon–DNA Adducts and Breast Cancer: A Pooled Analysis

    OpenAIRE

    Gammon, Marilie D.; Sagiv, Sharon K.; Eng, Sybil M.; Shantakumar, Sumitra; Gaudet, Mia M.; Teitelbaum, Susan L; Britton, Julie A.; Terry, Mary Beth; WANG, LIAN WEN; Wang, Qiao; STELLMAN, STEVE D.; Beyea, Jan; Hatch, Maureen; Kabat, Geoffrey C; Wolff, Mary S.

    2004-01-01

    Polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been associated with breast cancer in several small studies. The authors’ pooled analysis included 873 cases and 941 controls from a population-based case-control study. Competitive enzyme-linked immunosorbent assay in peripheral mononuclear cells was conducted in 2 rounds, and results were pooled on the basis of round-specific quantiles. The odds ratio for breast cancer was elevated in relation to detectable PAH-DNA adducts (1.29 as comp...

  16. Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct.

    Science.gov (United States)

    Politica, Dustin A; Malik, Chanchal K; Basu, Ashis K; Stone, Michael P

    2015-12-21

    3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-d

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

    Institute of Scientific and Technical Information of China (English)

    CHENG Yan; WANG Hai-Fang; SUN Hong-Fang; LI Hong-Li

    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 b5 (CYb5) 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 CYb5, whereas curcumin and resveratrol induced GST. We 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.

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

    International Nuclear Information System (INIS)

    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 b5 (CYb5) 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 CYb5, 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)

  19. Glutathionetransferase activity and PAN-DNA adducts in human placenta as a risk factor for newborn in radioactively contaminated regions

    International Nuclear Information System (INIS)

    It was shown that the higher is the contamination of area the more decreased is GST activity and the more abundant are PAH adducts in placental DNA. Placental glutathionetransferase activity and level of PAH-DNA adducts content in placental tissue are the integral indices of environmental pollution, efficiency of maternal and placental detoxification and a prognostic factor for newborn

  20. White blood cell DNA adducts and fruit and vegetable consumption in bladder cancer.

    Science.gov (United States)

    Peluso, M; Airoldi, L; Magagnotti, C; Fiorini, L; Munnia, A; Hautefeuille, A; Malaveille, C; Vineis, P

    2000-02-01

    The 'Mediterranean diet', a diet rich in cereals, fruit and vegetables, has been associated with lowering the risk of a variety of cancers of the digestive tract and the bladder. In a previous study, we showed that the high phenolic content these dietary components produce in the urine could be associated with higher antimutagenic properties of the urine and lower arylamine-DNA adducts in exfoliated bladder cells. We have conducted a case-control study on 162 bladder cancer patients and 104 hospital controls. Total aromatic DNA adducts were measured in white blood cells (WBC) of all subjects by (32)P-post-labelling. Genetically based metabolic polymorphisms were analysed by PCR-RFLP (NAT2, GSTM1, GSTT1, GSTP1, COMT and NQO1). All subjects were interviewed about their tobacco use, dietary habits and other risk factors. The odds ratio (OR) for the risk of bladder cancer according to the presence/absence of WBC DNA adducts (detection limit 0.1 RALx10(8)) was 3.7 [95% confidence interval (CI) 2.2-6.3] and a dose-response relationship with levels of adducts was apparent. The association between case/control status and the presence of WBC DNA adducts was significantly stronger in the subjects who consumed fewer portions of fruit or vegetables per day (OR 7.80, 95% CI 3.0-20.30 for 0-1 portions of vegetables) than in the heavy consumers (OR 4.98 for consumers of 2 portions daily, OR 1.97 for consumers of > or =3 portions; similar but lower estimates were found for the intake of fruit). No association was noticed between tobacco smoking and WBC DNA adducts. Only NAT-2, among the several genotypes considered, was associated in a statistically significant way with the risk of bladder cancer (OR 1.72, 95% CI 1.03-2.87) and with the levels of WBC DNA adducts. Our report suggests that fruit and vegetables could protect against bladder cancer by inhibiting the formation of DNA adducts. PMID:10657956

  1. Noni juice reduces lipid peroxidation-derived DNA adducts in heavy smokers.

    Science.gov (United States)

    Wang, Mian-Ying; Peng, Lin; Jensen, Claude J; Deng, Shixin; West, Brett J

    2013-03-01

    Food plants provide important phytochemicals which help improve or maintain health through various biological activities, including antioxidant effects. Cigarette smoke-induced oxidative stress leads to the formation of lipid hydroperoxides (LOOHs) and their decomposition product malondialdehyde (MDA), both of which cause oxidative damage to DNA. Two hundred forty-five heavy cigarette smokers completed a randomized, double-blind, placebo-controlled clinical trial designed to investigate the effect of noni juice on LOOH- and MDA-DNA adducts in peripheral blood lymphocytes (PBLs). Volunteers drank noni juice or a fruit juice placebo every day for 1 month. DNA adducts were measured by (32)P postlabeling analysis. Drinking 29.5-118 mL of noni juice significantly reduced adducts by 44.6-57.4%. The placebo, which was devoid of iridoid glycosides, did not significantly influence LOOH- and MDA-DNA adduct levels in current smokers. Noni juice was able to mitigate oxidative damage of DNA in current heavy smokers, an activity associated with the presence of iridoids. PMID:24804023

  2. Simultaneous detection of multiple DNA adducts in human lung samples by isotope-dilution UPLC-MS/MS.

    Science.gov (United States)

    Monien, Bernhard H; Schumacher, Fabian; Herrmann, Kristin; Glatt, Hansruedi; Turesky, Robert J; Chesné, Christophe

    2015-01-01

    Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02-7.1 adducts/10(8) nucleosides). 3,N(4)-etheno-2'-deoxycytidine and 1,N(6)-etheno-2'-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9-115.3 and 27.2-179/10(8) nucleosides, respectively). The same was true for N(2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine, the major adduct of methyleugenol (1.7-23.7/10(8) nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung. PMID:25423194

  3. 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.

  4. Dosimetry by means of DNA and hemoglobin adducts in propylene oxide-exposed rats

    International Nuclear Information System (INIS)

    The main purpose of the study was to establish the relation between exposure dose of propylene oxide (PO) and dose in various tissues of male F344 rats exposed to the compound by inhalation. The animals were exposed to 0, 5, 25, 50, 300, or 500 ppm PO in the air for 3 days (6 h/day) or 4 weeks (6 h/day, 5 days/week). Blood, nasal respiratory epithelium, lung, and liver were collected. 2-Hydroxypropylvaline (HPVal) in hemoglobin was quantified using the N-alkyl Edman method and gas chromatography/tandem mass spectrometry. 7-(2-Hydroxypropyl)guanine (7-HPG) in DNA was quantified using 32P postlabeling. The levels of 7-HPG in DNA of nasal respiratory epithelium and lung increased linearly with concentration as measured both after 3 days and 4 weeks of exposure. Similarly, 7-HPG in liver DNA and HPVal in hemoglobin showed a linear increase with PO concentration in the 3-day exposure group, whereas a deviation from linearity was observed above 300 ppm in the 4-week exposure group. The new results confirm previous observations of a dose difference between tissues with the highest dose present in the nasal respiratory epithelium. The measured adduct levels were used for calculation of adduct increments and corresponding tissue doses per unit of external exposure dose. For this purpose, the buildup of adducts was modeled considering the different kinetics of formation and elimination of adducts with DNA and hemoglobin, respectively, and also considering the increasing body weight of the animals. The half-life of 7-HPG in vivo, as well as tissue doses, could be solved from DNA adduct data at the 3rd and 26th days. Within the range of concentrations where the dose-response curves for adduct formation are linear, the relationship between exposure dose and resulting tissue doses could be based equally well on adduct data from the short-term exposure as on adduct data from the prolonged exposure

  5. 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 ostatní: 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

  6. 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

  7. 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...

  8. Redox and catalytic reactions of adducts of DNA with osmium tetroxide complexes on mercury electrodes

    Czech Academy of Sciences Publication Activity Database

    Havran, Luděk; Fojta, Miroslav; Jelen, František; Paleček, Emil

    Bratislava : Comenius University, 2001. s. 22. [International Symposium on BIOELECTROCHEMISTRY and BIOENERGETICS /16./. 01.06.2001-06.06.2001, Bratislava] Institutional research plan: CEZ:AV0Z5004920 Keywords : mercury electrodes * osmium tetroxide * adducts of DNA Subject RIV: BO - Biophysics

  9. DNA adduct formation by ochratoxin A?: A review of the available evidence

    OpenAIRE

    Dekant, Wolfgang

    2005-01-01

    Abstract The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumour formation is unknown and conflicting results regarding the potential of OTA to react with DNA have been obtained. While experiments using radiolabelled (3H or 14C) OTA and liquid scintillation counting or accelerator mass spectrometry indicate lack of formation of covalent DNA-adducts, spots detected by 32P-postlabelling have been attribute...

  10. Benzo(a)pyrene diolepoxide-DNA adducts detected by synchronous fluorescence spectrophotometry.

    OpenAIRE

    Vahakangas, K.; Trivers, G; Rowe, M.; Harris, C. C.

    1985-01-01

    Using benzo(a)pyrene (BP) as a model carcinogen we are currently applying a fluorescence technique to detect the very low levels of carcinogen-DNA adducts in human populations due to environmental exposure. In synchronous fluorescence spectrophotometry for detection of BP-diol epoxide-DNA, excitation and emission wavelengths are scanned simultaneously with a fixed wavelength difference (delta lambda) of 34 nm. Compared to conventional fluorescence methods only one peak emerges because excitat...

  11. Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers.

    OpenAIRE

    Harris, C. C.; Vahakangas, K.; Newman, M J; Trivers, G E; Shamsuddin, A; Sinopoli, N; Mann, D L; Wright, W. E.

    1985-01-01

    Coke oven workers are exposed to high levels of carcinogenic polycyclic aromatic hydrocarbons, including benzo[a]pyrene (B[a]P), and are at increased risk of lung cancer. Since B[a]P is enzymatically activated to 7 beta,8 alpha-dihydroxy(9 alpha, 10 alpha)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) that forms adducts with DNA, the presence of these adducts was measured in DNA from peripheral blood lymphocytes by synchronous fluorescence spectrophotometry and enzyme radioimmunoassay. App...

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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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 32P-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

  14. Histone-DNA interactions within chromatin. Isolation of histones from DNA adducts induced in nuclei by UV light

    International Nuclear Information System (INIS)

    A method has been developed by which to isolate histones that have been crosslinked to DNA following irradiation of calf thymus nuclei by UV light. The procedure involves separation of protein-DNA adducts from uncrosslinked protein by Sepharose 4B chromatography under dissociating conditions. Histones which are crosslinked to DNA are released by chemical hydrolysis of the DNA and identified by SDS gel electrophoresis. The results indicate that, of the histones, H1 and H3 become crosslinked to the DNA most readily under our irradiation conditions. (author)

  15. Few constraints limit the design of quinone methide-oligonucleotide self-adducts for directing DNA alkylation†

    OpenAIRE

    Rossiter, Clifford S.; Modica, Emilia; Kumar, Dalip; Rokita, Steven E

    2010-01-01

    Nucleotide sequences minimally containing adenosine, cytosine or guanosine are sufficient to form intrastrand oligonucleotide quinone methide self-adducts reversibly for subsequent alkylation of complementary DNA. The general lack of sequence restrictions should now allow for alkylation of most any target of interest although reaction is most efficient when the self-adducts contain guanine residues and do not form hairpin structures.

  16. DNA adducts in marine mussel Mytilus galloprovincialis living in polluted and unpolluted environments. Chapter 12. Book chapter

    International Nuclear Information System (INIS)

    A generally applicable (32)P-postlabeling assay was used to examine the presence of DNA adducts in mussels experimentally exposed to known carcinogens and in mussels collected from sites impacted by wastewaters. Mussels exposed to seawater artificially polluted with 2-aminofluorene showed exclusively one adduct which was identified to be dG-C8-2-aminofluorene. Under the same experimental conditions, Diesel-2 oil did not induce any detectable adducts. When mussel digestive gland DNA was collected and analyzed from one unpolluted site, two moderately impacted sites, and one site heavily impacted by cannery wastewaters, mussel DNA from the unpolluted and only one moderately polluted site showed the presence of 6 to 10 adducts. This indicates they were not related to the pollution. This was further supported by the absence of dose-related adducts. Clear evidence for the presence of pollution-related DNA adducts was, however, found in juvenile mussels collected from an oil refinery site. One major and three minor adducts were detected in these mussels with no adducts detected in juvenile mussels from an unpolluted site

  17. AlkB recognition of a bulky DNA base adduct stabilized by chemical cross-linking

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    E.coli AlkB is a direct DNA/RNA repair protein that oxidatively reverses N1 alkylated purines and N3 alkylated pyrimidines to regular bases.Previous crystal structures have revealed N1-methyl adenine(1-meA) recognition by AlkB and a unique base flipping mechanism,but how the AlkB active site can accommodate bulky base adducts is largely unknown.Employing a previously developed chemical cross-linking technique,we crystallized AlkB with a duplex DNA containing a caged thymine base(cagedT).The structure revealed a flexible hairpin lid and a reorganized substrate recognition loop used by AlkB to accommodate cagedT.These observations demonstrate,at the molecular level,how bulky DNA adducts may be recognized and processed by AlkB.

  18. DNA adducts in human urinary bladder and other tissues.

    OpenAIRE

    Phillips, D. H.; Hewer, A.

    1993-01-01

    Tobacco smoking is associated with an increased risk of cancer in a number of organs, including bladder and lung. Tobacco smoke contains at least 50 known chemical carcinogens that exert their biological effects through their covalent binding to cellular DNA. Examining human DNA for the presence of altered nucleotides is a means of monitoring exposure to genotoxic chemicals. DNA isolated from 73 human bladder biopsies has been analyzed by 32P-postlabeling for the presence of aromatic/hydropho...

  19. Accumulation of miscoding etheno-DNA adducts and highly expressed DNA repair during liver fluke-induced cholangiocarcinogenesis in hamsters.

    Science.gov (United States)

    Dechakhamphu, Somkid; Pinlaor, Somchai; Sitthithaworn, Paiboon; Bartsch, Helmut; Yongvanit, Puangrat

    2010-09-10

    Infection by Opisthorchis viverrini, a risk factor for cholangiocarcinoma (CCA) may act through chronic inflammation, oxidative stress and lipid peroxidation (LPO)-related damage and growth stimuli. 1,N6-etheno-2'-deoxyadenosine (epsilondA), and 3,N4-etheno-2'-deoxycytidine (epsilondC), markers for LPO-derived DNA damage were highly increased in white blood cell and urine of O. viverrini-infected Thai patients. In order to investigate tissue specificity etheno adducts were measured in a cholangiocarcinogenesis model, in O. viverrini-infected hamsters that had received N-nitrosodimethylamine (NDMA, 12.5 ppm in dw) for 2 months. epsilondA- and epsilondC-levels were analyzed in paraffin-embedded liver sections by a novel immunohistochemical method, from 21 up to 180 days post-O. viverrini-infection. In inflamed areas of the liver, etheno adducts were localized in the nuclei of inflammatory cells and in the epithelial lining of the bile duct. Semi-quantitative image analysis showed higher adduct levels in the liver of O. viverrini-infected hamsters, treated with or w/o NDMA when compared with untreated controls. Levels were found highest in the liver of O. viverrini-infected plus NDMA-treated hamsters. Adducts increased in an age-dependent manner from O. viverrini-infection until CCA development. Increased adduct formation paralleled histopathological changes in plasma alkaline phosphatase (ALP) activity, bile duct hyperplasia, dysplasia, precancerous lesions, and CCA appearance. Also elevated expression of alkyladenine DNA glycosylase (AAG), which excises 1,N6-ethenoadenine (epsilonA) was linked to higher adduct formation, suggesting imbalanced repair. Our results implicate accumulation of inflammation-related, promutagenic DNA damage in target tissue and possibly imbalanced repair in the onset of cholangiocarcinogenesis. PMID:20541562

  20. Use of the /sup 32/P-postlabeling method to detect DNA adducts of 2-amino-3-methylimidazolo(4,5-f)quinoline (IQ) in monkeys fed IQ: identification of the N-(deoxyguanosin-8-yl)-IQ adduct

    Energy Technology Data Exchange (ETDEWEB)

    Snyderwine, E.G.; Yamashita, K.; Adamson, R.H.; Sato, S.; Nagao, M.; Sugimura, T.; Thorgeirsson, S.S.

    1988-10-01

    Eight DNA adducts of 2-amino-3-methylimidazolo(4,5-f)-quinoline (IQ) were found by the standard /sup 32/P-postlabeling method in livers from male Cynomolgus monkeys fed IQ (5 days/week, 3 weeks, 20 mg/kg, nasal-gastric intubation). The IQ-DNA adduct fingerprints observed in monkeys were identical to those observed in rats that received IQ (0.03%) in the diet for 2 weeks. The C8-guanine-IQ adduct was identified by comigration with the synthetic 3',5'-bisphosphate derivative of N(-deoxyguanosin-8-yl)-IQ. DNA modified in vitro with N-hydroxy-IQ showed seven adducts, including the C8-guanine-IQ adduct, that were identical to those found in monkeys and rats. Thus it appears that N-hydroxy-IQ, the reactive metabolite of IQ, was responsible for all adducts found in vivo, except one. In order to detect adducts in other organs that were present at lower levels, the intensification (ATP-deficient) method for /sup 32/P-postlabeling was used. Five of the adducts detected under standard conditions, including the C8-guanine-IQ adduct, were also detected under intensification conditions. The total level of DNA-IQ adducts was highest in the liver, followed by the kidney, colon and stomach, and bladder. The adduct patterns were identical in all organs examined. The results indicate that IQ is potentially genotoxic in primates and therefore a likely human carcinogen.

  1. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Brown, Kyle L.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves

  2. Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.

    OpenAIRE

    Kovács, Katalin; Anna, Lívia; Rudnai, Péter; Schoket, Bernadette

    2011-01-01

    Bulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical...

  3. Mechanism of repair of 5'-topoisomerase II-DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2

    Energy Technology Data Exchange (ETDEWEB)

    Schellenberg, Matthew J; Appel, C Denise; Adhikari, Sanjay; Robertson, Patrick D; Ramsden, Dale A; Williams, R Scott [NIH; (Georgetown); (UNC)

    2012-10-28

    The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl–linked topo II–DNA adducts. Here, X-ray structures of mouse Tdp2–DNA complexes reveal that Tdp2 β–2-helix–β DNA damage–binding 'grasp', helical 'cap' and DNA lesion–binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single–metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.

  4. Lipid peroxidation-derived etheno-DNA adducts in human atherosclerotic lesions

    Energy Technology Data Exchange (ETDEWEB)

    Nair, Jagadeesan [Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); De Flora, Silvio [Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa (Italy); Izzotti, Alberto [Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa (Italy); Bartsch, Helmut [Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany)]. E-mail: h.bartsch@dkfz.de

    2007-08-01

    Atherosclerosis and cancer are characterized by uncontrolled cell proliferation and share common risk factors, such as cigarette smoking, dietary habits and ageing. Growth of smooth muscle cells (SMCs) in atherosclerotic plaques may result from DNA damage, caused either by exogenous mutagens or by agents endogenously generated due to oxidative stress and lipid peroxidation (LPO). Hydroxy-2-nonenal (HNE), a major LPO product, binds covalently to cellular DNA to form the exocyclic etheno-DNA-base adducts, 1,N {sup 6}-ethenodeoxyadenine ({epsilon}dA) and 3,N {sup 4}-ethenodeoxycytosine ({epsilon}dC). By applying an ultrasensitive {sup 32}P-postlabeling-immunoaffinity method, {epsilon}dA and {epsilon}dC were quantified in abdominal aorta SMCs from 13 atherosclerotic patients and 3 non-smoking subjects without atherosclerotic lesions. The levels of etheno-adducts ranged for {epsilon}dA from 2.3 to 39.6/10{sup 8} dA and for {epsilon}dC from 10.7 to 157.7/10{sup 8} dC, with a high correlation between {epsilon}dA and {epsilon}dC (r = 0.84, P = 0.0001). Etheno-adduct levels were higher in atherosclerotic smokers than in ex-smokers for both {epsilon}dA (means 15.2 versus 7.3, P = 0.06) and {epsilon}dC (71.9 versus 51.6, not significant). {epsilon}dC levels were higher in either ex-smokers (P = 0.03) or smokers (P = 0.07) than in non-smokers. There was a poor correlation between either {epsilon}dA or {epsilon}dC and 8-hydroxy-2'-deoxyguanosine, whereas significant positive correlations were detected with the levels of several postlabeled bulky aromatic DNA adducts. In conclusion, two different types of DNA damage may be involved in atherosclerotic plaque formation and progression: (i) bulky aromatic compounds, to which aorta SMCs are chronically exposed in smokers, can either covalently bind to DNA, induce redox-cycling via quinone intermediates and/or activate local chronic inflammatory processes in the arterial wall; ii) this in turn leads to a self perpetuating

  5. TRANSPLATIN-CONJUGATED TRIPLEX-FORMING OLIGONUCLEOTIDES FORM ADDUCTS WITH BOTH STRANDS OF DNA

    OpenAIRE

    Campbell, Meghan A.; Miller, Paul S.

    2009-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to polypurine•polypyrimidine tracts in DNA and as a consequence, perturb normal functioning of a targeted gene. The effectiveness of such anti-gene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here we report that attachment of N-7-platinated guanine nucleosides to the 3′- and/or 5′-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenos...

  6. Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

    Science.gov (United States)

    Adams, S P; Laws, G M; Selden, J R; Nichols, W W

    1994-05-15

    Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling. PMID:8059938

  7. Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

    Science.gov (United States)

    Chen, Fangyi; Tang, Qi; Bian, Ke; Humulock, Zachary T; Yang, Xuedong; Jost, Marco; Drennan, Catherine L; Essigmann, John M; Li, Deyu

    2016-04-18

    The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments. PMID:26919079

  8. 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

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

    International Nuclear Information System (INIS)

    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, 32P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal (β 95%CI; 0.46 (0.08, 0.84)) and cord blood (β 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-01

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

  11. 32P-Postlabelling/HPLC analysis of various styrene-induced DNA adducts in mice

    Czech Academy of Sciences Publication Activity Database

    Koskinen, M.; Vodička, Pavel; Vodičková, L.; Hemminki, K.

    2001-01-01

    Roč. 6, č. 3 (2001), s. 175-189. ISSN 1354-750X R&D Projects: GA ČR GA313/99/1460 Grant ostatní: GA-(EU) QLK4-1999-01368 Institutional research plan: CEZ:AV0Z5039906 Keywords : DNA adducts Subject RIV: EA - Cell Biology Impact factor: 1.118, year: 2001

  12. Kinetics of formation of specific styrene oxide adducts in double-stranded DNA

    Czech Academy of Sciences Publication Activity Database

    Koskinen, M.; Vodičková, L.; Vodička, Pavel; Warner, S. C.; Hemminki, K.

    2001-01-01

    Roč. 138, č. 2 (2001), s. 111-124. ISSN 0009-2797 R&D Projects: GA ČR GA313/99/1460 Grant ostatní: EU(XC) QLK4-1999-01368 Institutional research plan: CEZ:AV0Z5039906 Keywords : biomonitoring * DNA adducts Subject RIV: EA - Cell Biology Impact factor: 1.706, year: 2001

  13. Modulation of the Effect of Prenatal PAH Exposure on PAH-DNA Adducts in Cord Blood by Plasma Antioxidants

    OpenAIRE

    Kelvin, Elizabeth A.; Edwards, Susan; Jedrychowski, Wieslaw; Schleicher, Rosemary L.; Camann, David; Tang, Deliang; Perera, Frederica P.

    2009-01-01

    The fetus is more susceptible than the adult to the effects of certain carcinogens, such as polycyclic aromatic hydrocarbons (PAH). Nutritional factors, including antioxidants, have been shown to have a protective effect on carcinogen-DNA adducts and cancer risk in adults. We investigated whether the effect of prenatal airborne PAH exposure, measured by personal air monitoring during pregnancy, on the level of PAH-DNA adducts in a baby's cord blood is modified by the concentration of micronut...

  14. 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

  15. 32P-postlabelling analysis of DNA adducted with urinary mutagens from smokers of black tobacco.

    Science.gov (United States)

    Peluso, M; Castegnaro, M; Malaveille, C; Talaska, G; Vineis, P; Kadlubar, F; Bartsch, H

    1990-08-01

    In order to characterize the tobacco-derived mutagens excreted in the urine of tobacco smokers, 32P-postlabelling techniques were used to examine DNA adducts formed from these mutagens with calf thymus DNA in the presence of a metabolic activation system (rat liver S9, Aroclor 1254-induced, with or without acetyl coenzyme A). Using either nuclease P1 or butanol extraction procedures, four-six and three spots, respectively, were reproducibly found on the autoradiograms in the case of the urine extract from two smokers of black tobacco. Using the urinary extract from a non-smoker, only three faint spots were detected after nuclease P1 enrichment. DNA adducts produced in smokers' urine were then compared with those formed by four N-hydroxyarylamines, N-hydroxy-2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline, N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoxaline, N-hydroxy-2-naphthylamine and N-hydroxy-4-aminobiphenyl. Visual inspection revealed that none of the reference aromatic amines contributed to the adduct pattern produced by the urinary mutagen(s). However, primary aromatic amines are mainly implicated as urinary mutagens because: (i) they produce frameshift mutations in Salmonella typhimurium strains, (ii) they are easily extractable with blue cotton and (iii) their mutagenicity is abolished by a nitrite treatment procedure for deamination. PMID:2387016

  16. 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

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

    International Nuclear Information System (INIS)

    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-(N2deoxyguanosyl)-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/108 nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/108 nucleotides, with a median of 75/108 nucleotides. PAH-DNA adduct values above 150/108 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 of

  18. DNA adduct formation and oxidative stress in colon and liver of Big Blue rats after dietary exposure to diesel particles

    DEFF Research Database (Denmark)

    Dybdahl, Marianne; Risom, Lotte; Møller, Peter;

    2003-01-01

    in liver accompanied by enhanced vitamin C levels. In plasma, we found no significant effects on oxidative damage to proteins and lipids, antioxidant enzymes or vitamin C levels. Our data indicate that gastrointestinal exposure to DEP induces DNA adducts and oxidative stress resulting in DNA strand breaks...... on oxidative DNA damage (8-oxodG) in colon or liver DNA or in the urine. However, the mRNA expression of OGG1, encoding an enzyme involved in repair of 8-oxodG, was increased by DEP in both liver and colon. DNA adduct levels measured by 32P-post-labelling were elevated in colon and liver, and the expression...

  19. 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.

  20. 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).

  1. 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)

    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 32P-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)

  2. Formation and persistence of sterigmatocystin-DNA adducts in rat liver determined via 32P-postlabeling analysis

    International Nuclear Information System (INIS)

    A 32P-postlabeling method has been employed to detect the in vitro and in vivo modification of DNA by the mycotoxin sterigmatocystin (ST). Dose-dependent ST-DNA adduct formation was detected in the liver of male Fischer 344 rats over a 27-fold range of ST administered. In addition, ST-DNA adducts, formed in rats given a 9 mg/kg dose, were found to persist up to 105 days after treatment at a level of 0.5% of the 2-h value. Loss of these adducts from liver DNA was observed to exhibit a triphasic profile: rapid loss during the first 24 h followed by a slower decline from 1 to 14 days post dosing and an extremely slow decline from day 14 to 105 post treatment. This experimental approach to the study of mycotoxin-DNA interactions permits the quantitative description of DNA modification in ST-treated animals. (Auth.)

  3. Mutagenic properties of the 8-amino-2'-deoxyguanosine DNA adduct in mammalian cells.

    OpenAIRE

    X. Tan; Suzuki, N; Johnson, F; Grollman, A P; Shibutani, S

    1999-01-01

    The DNA adduct 8-amino-2'-deoxyguanosine (8-amino-dG) is found in liver DNA of rats treated with the hepatocarcinogen 2-nitropropane. Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of 8-amino-dG in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'-TCCTCCTX1G2CCTCTC and 5'-TCCTCCTG1X2CCTCTC, X = dG or 8-amino-dG) with the lesion positioned at codon 60 or 61 of the non-coding strand of the human c-Ha- ras1 gene were inserted into single-strand...

  4. 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

  5. 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; Autrup, Herman; Haugen, Aage

    )-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the...... levels in lung tissue and TP53 mutations....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-02-09

    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.

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

    International Nuclear Information System (INIS)

    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

  8. Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Klaene, Joshua J; Flarakos, Caroline; Glick, James; Barret, Jennifer T; Zarbl, Helmut; Vouros, Paul

    2016-03-25

    LC-MS using electrospray ionization is currently the method of choice in bio-organic analysis covering a wide range of applications in a broad spectrum of biological media. The technique is noted for its high sensitivity but one major limitation that hinders achievement of its optimal sensitivity is the signal suppression due to matrix inferences introduced by the presence of co-extracted compounds during the sample preparation procedure. The analysis of DNA adducts of common environmental carcinogens is particularly sensitive to such matrix effects as sample preparation is a multistep process which involves "contamination" of the sample due to the addition of enzymes and other reagents for digestion of the DNA in order to isolate the analyte(s). This problem is further exacerbated by the need to reach low levels of quantitation (LOQ in the ppb level) while also working with limited (2-5μg) quantities of sample. We report here on the systematic investigation of ion signal suppression contributed by each individual step involved in the sample preparation associated with the analysis of DNA adducts of polycyclic aromatic hydrocarbon (PAH) using as model analyte BaP-dG, the deoxyguanosine (dG) adduct of benzo[a]pyrene (BaP). The individual matrix contribution of each one of these sources to analyte signal was systematically addressed as were any interactive effects. The information was used to develop a validated analytical protocol for the target biomarker at levels typically encountered in vivo using as little as 2μg of DNA and applied to a dose response study using a metabolically competent cell line. PMID:26607319

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

    International Nuclear Information System (INIS)

    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 32P-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

  10. Mechanism of Translesion Synthesis Past an Equine Estrogen-DNA Adduct by Y-Family DNA Polymerases

    OpenAIRE

    Yasui, Manabu; Suzuki, Naomi; Liu, Xiaoping; Kim, Yoshinori Okamoto Sung Yeon; Laxmi, Y. R. Santosh; Shibutani, Shinya

    2007-01-01

    4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) η, κ and ι in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol η, κ and ι. Pol η inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and ...

  11. 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...... individuals were included in this study, excluding 125 cases, which developed lung cancer within the first 3 yr after blood sampling. Bulky DNA adduct levels were measured by 32P-postlabeling technique and polymorphisms in carcinogen metabolism and DNA repair genes were determined. Potential predictors of...

  12. Myeloperoxidase - 463A variant reduces benzo(a)pyrene diol epoxide DNA adducts in skin of coal tar treated patients

    Energy Technology Data Exchange (ETDEWEB)

    Rojas, M.; Godschalk, R.; Alexandrov, K.; Cascorbi, I.; Kriek, E.; Ostertag, J.; Van Schooten, F.J.; Bartsch, H. [German Cancer Research Center, Heidelberg (Germany). Div. of Toxicology & Cancer Risk Factors

    2001-07-01

    The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo(a)pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. The authors determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)-DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the -453G into A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. The data show for the first time: (i) the in vivo formation of BPDE-DNA adducts in human skin treated with coal tar; (ii) that the MPO-463AA/AG genotype reduced BPDE-DNA adduct levels in human skin.

  13. Formation of 1,4-dioxo-2-butene-derived adducts of 2′-deoxyadenosine and 2′-deoxycytosine in oxidized DNA

    OpenAIRE

    Chen, Bingzi; Vu, Choua C.; Byrns, Michael C.; Peter C. Dedon; Peterson, Lisa A.

    2006-01-01

    Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M1dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the β-elimination product of the 2-phosphoryl-1,4-dioxobutane ...

  14. Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation.

    Science.gov (United States)

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Chin, Chiu; Huang, William; Lepor, Herbert; Wu, Xue-Ru; Rom, William N; Chen, Lung-Chi; Tang, Moon-shong

    2015-10-20

    Second-hand smoke (SHS) is associated with 20-30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers. PMID:26431382

  15. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair genes polymorphisms

    Czech Academy of Sciences Publication Activity Database

    Binková, Blanka; Chvátalová, Irena; Lněničková, Zdena; Milcová, Alena; Tulupová, Elena; Farmer, P. B.; Šrám, Radim

    2007-01-01

    Roč. 620, - (2007), s. 49-61. ISSN 0027-5107 R&D Projects: GA MŽP SI/340/2/00; GA MŽP SL/740/5/03 Grant ostatní: EU(GB) 2000-00091 Institutional research plan: CEZ:AV0Z50390512 Source of funding: R - rámcový projekt EK Keywords : DNA adducts * genetic polymorphisms * metabolic genes Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 4.159, year: 2007

  16. 2-Aminofluorene metabolism and DNA adduct formation by mononuclear leukocytes from rapid and slow acetylator mouse strains.

    Science.gov (United States)

    Levy, G N; Chung, J G; Weber, W W

    1994-02-01

    Following exposure of mice to the arylamine carcinogen 2-aminofluorene, DNA-carcinogen adducts can be found in the target tissues liver and bladder, and also in circulating leukocytes. Evidence is presented here that mouse mononuclear leukocytes (MNL) are capable of metabolizing 2-aminofluorene to DNA-binding metabolites which give rise to the adducts found in the MNL. Both lymphocytes and monocytes were able to acetylate arylamines during 18 h of culture. The degree of acetylation was determined by the N-acetyltransferase genotype of the mice as shown through use of acetylator congenic strains which differ only in the Nat-2 gene. Cultured MNL from rapid acetylator mice (C57BL/6J and A.B6-Natr) produced about twice as much N-acetylaminofluorene from 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as cells from slow acetylator mice (B6.A-Nat(s) and A/J). Other differences in arylamine metabolism by MNL in culture were observed and shown to be due to genetic factors, currently unidentified, other than N-acetyltransferase. DNA adduct formation following incubation of MNL with the arylamine carcinogen 2-aminofluorene was related to both acetylation capacity and to other genetic metabolic factors in the mouse genome. MNL from rapid acetylator mice with the C57BL/6J background (B6) had 3-fold the DNA adduct levels of cells from the corresponding slow acetylator congenic (B6.A-Nat(s)). Similarly, MNL from rapid acetylator mice with the A/J background (A.B6-Natr) had twice the DNA adduct levels of those from their corresponding slow congenic (A). Adduct levels in MNL from C57BL/6J were nearly the same as those of MNL from A/J, again indicating the involvement of loci other than acetylation in DNA adduct formation. The finding of genetically dependent arylamine carcinogen metabolism and DNA adduct formation in cultured MNL suggests the possibility of using cultured MNL for assessing individual susceptibility to arylamine

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

    International Nuclear Information System (INIS)

    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 32P-postlabeling procedure. The data demonstrate the DNA-damaging potential of long term fresh cigarette smoke exposure and suggest the ability of the tissue to partially recover from such damage following cessation of the exposure

  18. 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

    International Nuclear Information System (INIS)

    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 physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1'-hydroxyestragole. The model was extended by linking the area under the curve for 1'-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1'-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.

  19. Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA).

    Science.gov (United States)

    Schoket, B; Doty, W A; Vincze, I; Strickland, P T; Ferri, G M; Assennato, G; Poirier, M C

    1993-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA), the most frequently used immunoassay for the determination of polycyclic aromatic hydrocarbon-DNA adducts in human tissues, has been modified to achieve approximately a 6-fold increase in sensitivity. The new assay, a competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) has utilized the same rabbit antiserum as the ELISA, antiserum elicited against DNA modified with benzo[a]pyrene. However, the alkaline phosphatase conjugate has been replaced with a biotin-europium-labeled streptavidin signal amplification system, and the release of europium into the solution forms a highly fluorescent chelate complex that is measured by time-resolved fluorometry. The DELFIA has achieved a 5- to 6-fold increase in sensitivity for measurement of DNA samples modified in vitro with benzo[a]pyrene, for cultured cells exposed to radiolabeled benzo[a]pyrene, and for human samples from occupationally exposed workers. The assay has been validated by comparison of adduct levels determined by DELFIA, ELISA, and radioactivity in DNA from mouse keratinocytes exposed to radiolabeled benzo[a]pyrene. Human lymphocyte DNA samples from 104 Hungarian aluminum plant workers were assayed by ELISA and compared to blood cell DNA samples from 69 Italian coke oven workers assayed by DELFIA. The standard curves demonstrated that the limit of detection of 4.0 adducts in 10(8) nucleotides for polycyclic aromatic hydrocarbon-DNA adducts by ELISA, using 35 micrograms of DNA/microtiter plate well, has been decreased to 1.3 adducts in 10(8) nucleotides by DELFIA, using 20 micrograms of DNA/microtiter well. If 35 micrograms of DNA were used in the DELFIA, the calculated detection limit would be 0.7 adducts in 10(8) nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8348058

  20. Fast repair of oxidizing OH radical adducts of dGMP, dAMP and DNA by hydroxycinnamic acid derivatives. A pulse radiolytic study

    International Nuclear Information System (INIS)

    Using a pulse radiolytic technique, it has been demonstrated that the interaction of oxidizing OH adducts of DNA with hydroxycinnamic acid derivatives proceeds via an electron transfer process with rate constant of (5-7) x 108dm3mol-1s-1. Besides, the rate for fast repair of OH adducts of dAMP and DNA (ssDNA and dsDNA) are slower than the corresponding rate for the rest OH adducts of DNA constituents, such as dGMP (2-3*109 dm1mol-1s-1). The slower rate for oxidizing OH adduct of dAMP and DNA has indicated that repair of oxidizing OH adduct of dAMP may be the rate determining process during the interaction of hydroxycinnamic acid derivatives with OH adduct of DNA containing the varieties of OH adducts of DNA components. On other hand, the yields for oxidizing OH radical adduct with dGMP and dAMP were determined to be 50% and 30% respectively

  1. 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.

  2. The mechanism of cytotoxicity and DNA adduct formation by the anticancer drug ellipticine in human neuroblastoma cells

    OpenAIRE

    Poljaková, Jitka; Eckschlager, Tomáš; Hraběta, Jan; Hřebačková, Jana; Smutný, Svatopluk; Frei, Eva; Martínek, Václav; Kizek, René; Stiborová, Marie

    2009-01-01

    Abstract Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the molecular mechanism of DNA-mediated ellipticine action in human neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cancer cell lines was investigated. Treatment of neuroblastoma cells with ellipticine resulted in apoptosis induction, which was verified by the ...

  3. Association between plasma BPDE‐Alb adduct concentrations and DNA damage of peripheral blood lymphocytes among coke oven workers

    Science.gov (United States)

    Wang, Hong; Chen, Weihong; Zheng, Hongyan; Guo, Liang; Liang, Huashan; Yang, Xiaobo; Bai, Yun; Sun, Jianya; Su, Yougong; Chen, Yongwen; Yuan, Jing; Bi, Yongyi; Wei, Qingyi; Wu, Tangchun

    2007-01-01

    Objectives Coke oven emissions (COE) containing polycyclic aromatic hydrocarbons (PAHs) can induce both benzo[a]pyrene‐r‐7, t‐8, t‐9,c‐10‐tetrahydotetrol‐albumin (BPDE‐Alb) adducts and DNA damage. However, the relation between these biomarkers for early biological effects is not well documented in coke oven workers. Methods In this study, the authors recruited 207 male workers exposed to COE and 102 controls not exposed to COE in the same steel plant in northern China. They measured BPDE‐Alb adduct concentrations in plasma with reverse‐phase high performance liquid chromatography and DNA damage in peripheral blood lymphocytes with alkaline comet assay. Results The results showed that the median concentration of BPDE‐Alb adducts in the exposed group (34.36 fmol/mg albumin) was significantly higher than that in the control group (21.90 fmol/mg albumin, p = 0.012). The mean Olive tail moment (Olive TM) of DNA damage in the exposed and control groups were 1.20 and 0.63, respectively (p = 0.000). Multivariate logistic regression analysis revealed that the odds ratio (OR) for BPDE‐Alb adduct and Olive TM associated with the exposure were 1.72 (95% CI 1.06 to 2.81) and 1.96 (95% CI 1.20 to 3.19), respectively. These results show significant correlations between the concentrations of BPDE‐Alb adduct and Olive TM levels in exposed group (r = 0.235, p = 0.001) but not in control group (r = 0.093, p = 0.353). Conclusion The results suggest that occupational exposure to COE may induce both BPDE–Alb adducts and DNA damage in the lymphocytes of coke oven workers and that these two markers are useful for monitoring exposure to COE in the workplace. PMID:17449561

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

    International Nuclear Information System (INIS)

    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-3H]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

  5. The relevance and significance of O6-, N7-Alkylguanines and N3-Alkyladenine DNA adducts from tobacco smoke.

    Czech Academy of Sciences Publication Activity Database

    Chadt, Jiří; Koskinen, M.; Vodička, Pavel

    Vídeň, 2006. [Tobacco Herm Reduction and Perception of Risk. 08.03.2006-11.03.2006, Vídeň] R&D Projects: GA ČR GA310/05/2626 Institutional research plan: CEZ:AV0Z50390512 Keywords : DNA Adduct Subject RIV: EB - Genetics ; Molecular Biology

  6. Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers and prospects in human biomonitoring

    Czech Academy of Sciences Publication Activity Database

    Vodička, Pavel; Koskinen, M.; Arand, M.; Hemminki, K.

    2002-01-01

    Roč. 511, - (2002), s. 239-254. ISSN 1383-5742 R&D Projects: GA ČR GA313/99/1460 Institutional research plan: CEZ:AV0Z5039906 Keywords : styrene * DNA adducts Subject RIV: FM - Hygiene Impact factor: 7.085, year: 2002

  7. Isolation of methylcarbamoyl-adducts of adenine and cytosine following in vitro reaction of methyl isocyanate with calf thymus DNA.

    Science.gov (United States)

    Segal, A; Solomon, J J; Li, F J

    1989-01-01

    Methylisocyanate (MIC) is the direct-acting acylating compound involved in the Bhopal, India disaster which occurred on December 3rd, 1984. The accidental release of MIC resulted in at least 2000 deaths, thousands of injuries and exposure of at least 200,000 people to varying amounts of MIC. We have studied how MIC reacts with 2'-deoxyribonucleosides at pH 7.0 and 37 degrees C for 1 h. MIC acylates exocyclic amino groups resulting in the following methylcarbamoyl (MC) adducts: N6-MC-Ade (0.5% yield) and N4-MC-dCyd (6%). No adducts were detected with dThd and dGuo. UV, NMR and mass spectrometry were employed to spectroscopically characterize these adducts. MIC was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 1 h) and yielded N6-MC-Ade (0.3 nmol/mg DNA) and N4-MC-dCyd (2.0 nmol/mg DNA). The inability of others to observe genetic mutations by MIC in Salmonella and Drosophila is consistent with the exocyclic adducts at N4 of Cyt and N6 of Ade where normal hydrogen bonding can occur after rotation of the methylcarbamoyl group anti to the Watson-Crick side of the molecule assuming that MIC binds to DNA within the intact cell. PMID:2731306

  8. 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.

  9. 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...... 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...

  10. Isolation and identification of the adducts of mitomycin C and porfiromycin with DNA formed in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Chowdary, D.R.

    1989-01-01

    The antitumor antibiotics, mitomycin C (MC) and porfiromycin (PM), are shown to form covalent complexes with DNA in vitro, under reductive activation conditions (both chemical and enzymatic). Three major covalent adducts have been isolated and identified as (1) N{sup 2}-guanine adduct with MC (structure 4a), (2) N{sup 2}-guanine adduct with 10-decarbamoyl mitomycin ((10-DMC); structure 16a), and a bisadduct of MC linked to two Gs at their N{sup 2}-positions (structure 6). The adducts of PM with DNA formed in vitro are analogous (structures 19, 20, and 21). Formation of adducts 6 and 16a in CHO mammalian cells has been shown after exposing them to MC or 10-DMC, whereas formation of crosslink 6 in vivo has been demonstrated after injecting rats with MC. The experiments done in tissue cultures with (1a-{sup 3}H)-polyfiromycin show ({sup 3}H)-label in the unmodified A, G, and T thus suggesting the demethylation of PM to MC in cells. The methyl group containing ({sup 3}H) label was incorporated into nucleosides via de novo purine and thymidylate biosynthesis. A consolidated enzymatic scheme for the hydrolysis of MC-modified DNA has been established and the resistance of such DNA to cleavage by several nucleases has been shown. Thus, only DNase I/SVD/alkaline phosphatase or nuclease P{sub 1}/SVD/alkaline phosphatase combinations can degrade MC-modified DNA into nucleosides. A modified version of {sup 32}P-postlabeling has been developed with in vitro authentic standards and this can be conveniently used in the future to detect MC-modified lesions obtained in vivo. By utilizing the alkaline ethidium bromide fluorescence assay, the crosslinking effect of MC, PM, and 10-DMC has been shown to occur in cells.

  11. Analysis of DNA adducts of some low molecular weight aldehydes : Methods development and application in human biomonitoring

    OpenAIRE

    Fang, Jia-Long

    1997-01-01

    Doctoral Thesis present at the Karolinska Institute, 1997 Center for Nutrition and Toxicology Departrnent of Biosciences at NOVUM Karolinska Institute 141 57 Huddinge Malondialdehyde (MA), acetaldehyde (Aa) and methylglyoxal (MG) are ubiquitously present in the environment and endogenously forrned in animals and humans. They have been shown to be genotoxic and to readily react with DNA to form DNA adducts under physiological conditions. The present studies ...

  12. 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.

  13. 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)

    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, N6-ethenodeoxyadenosine (εdA) and 3, N4-ethenodeoxycytidine (εdC) by immunoaffinity/32P-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

  14. Screening for DNA adducts by data-dependent constant neutral loss-triple stage mass spectrometry with a linear quadrupole ion trap mass spectrometer.

    Science.gov (United States)

    Bessette, Erin E; Goodenough, Angela K; Langouët, Sophie; Yasa, Isil; Kozekov, Ivan D; Spivack, Simon D; Turesky, Robert J

    2009-01-15

    A two-dimensional linear quadrupole ion trap mass spectrometer (LIT/MS) was employed to simultaneously screen for DNA adducts of environmental, dietary, and endogenous genotoxicants, by data-dependent constant neutral loss scanning followed by triple-stage mass spectrometry (CNL-MS3). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M + H - 116]+) in the MS/MS scan mode triggered the acquisition of MS3 product ion spectra of the aglycone adducts [BH2]+. Five DNA adducts of the tobacco carcinogen 4-aminobiphenyl (4-ABP) were detected in human hepatocytes treated with 4-ABP, and three DNA adducts of the cooked-meat carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were identified in the livers of rats exposed to MeIQx, by the CNL-MS3 scan mode. Buccal cell DNA from tobacco smokers was screened for DNA adducts of various classes of carcinogens in tobacco smoke including 4-ABP, 2-amino-9H-pyrido[2,3-b]indole (AalphaC), and benzo[a]pyrene (BaP); the cooked-meat carcinogens MeIQx, AalphaC, and 2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (PhIP); and the lipid peroxidation products acrolein (AC) and trans-4-hydroxynonenal (HNE). The CNL-MS3 scanning technique can be used to simultaneously screen for multiple DNA adducts derived from different classes of carcinogens, at levels of adduct modification approaching 1 adduct per 108 unmodified DNA bases, when 10 microg of DNA is employed for the assay. PMID:19086795

  15. Measurement of DNA repair in Chinese hamster fibroblasts employing flow cytometry and monoclonal antibodies to DNA adducts

    International Nuclear Information System (INIS)

    The authors examined the utility of measuring DNA repair in single cells employing flow cytometric quantitation of fluorescent monoclonal antibodies directed against specific DNA adducts. Two antibodies were employed; the first directed against single strand (ss) bromodeoxyuridine (anti-BrdUrd) and the second against UV light induced ss-thymine dimers (anti-TT). Sensitivity with both monoclonals was highly dependent on DNA denaturation, with the most effective shown to be a 0.5N HCl histone extraction followed by 50% formamide for 30 min at 800C. Unscheduled synthesis following 30 J/m/sup 2/ UV irradiation in Gl/GO plateau phase CHO cells was demonstrated employing the anti-BrdUrd AB method combined with DNA counter-staining with propidium iodide. Data suggest that anti-BrdUrd Ab recognition of newly replicated sequences following UV irradiation may be strongly dependent on chromatin conformation. A linear correlation was observed for mean anti-TT AB fluorescence and UV dose up to a total of 3000 J/m/sup 2/. Also, a rapid reduction in cellular fluorescence, presumably reflecting dimer excision was observed when the cells were returned to 370C before fixation. Finally, data from various repair deficient CHO cells will be compared employing these methods

  16. DIFFERENCES IN DETECTION OF DNA ADDUCTS IN THE 32P-POSTLABELING ASSAY AFTER EITHER 1-BUTANOL EXTRACTION OR NUCLEASE P1 TREATMENT

    Science.gov (United States)

    The use of nuclease Pl treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabe1ling assay for DNA adducts have been compared. lthough similar results were obtained with the two methods for standard adducts formed with benzo(a)pyrene diol epoxide I, nucl...

  17. Structure of cis-[Pt(NH3)(2-picoline)]2+ and DNA adduct and its bonding characteristics

    Institute of Scientific and Technical Information of China (English)

    JIA; Muxin; LIU; Kai; YANG; Zuoyin; CHEN; Guangju

    2004-01-01

    Several methods including molecular mechanics, molecular dynamics, ONIOM that combines quantum chemistry with molecular mechanics and standard quantum chemistry are used to study the configuration and electron structures of an adduct of the DNA segment d(ATACATG*G*TACATA)·d(TATGTACCATGTAT) with cis-[Pt(NH3)(2-Picoline)]2+. The investigation shows that the configuration optimized by ONIOM is similar to that determined by NMR. Strong chemical bonds between Pt of the complex and two N7s of neighboring guanines in the DNA duplex and hydrogen bond between the NH3 of the complex and O6 of a nearby guanine have a large impact on the configuration of the adduct. Chemical bonds, the aforementioned hydrogen bond, and the interaction between a methyl of the complex and a methyl of the base in close proximity are critical for the complex to specifically recognize DNA.

  18. DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy)

    International Nuclear Information System (INIS)

    The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a 'cleaner' reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of β-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r=0.60 in 2001; r=0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization period

  19. Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease Pl treatment

    International Nuclear Information System (INIS)

    The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo(a)pyrene diol epoxide I, nuclease P1 treatment resulted in a significant reduction in detection of major adducts 1-amino-6-nitropyrene, 1-amino-8-nitropyrene, 2-aminofluorene, 2-naphthylamine and 4-aminobiphenyl modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene modified DNA. These results suggest that at least initially, both modications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitro-aromatics and aromatic amines

  20. Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease P1 treatment.

    Science.gov (United States)

    Gallagher, J E; Jackson, M A; George, M H; Lewtas, J; Robertson, I G

    1989-04-01

    The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo[a]pyrene diol epoxide I (BPDE-I), nuclease P1 treatment resulted in a significant reduction in detection of major adducts from 1-amino-6-nitropyrene (1-amino-6-NP), 1-amino-8-nitropyrene (1-amino-8-NP), 2-aminofluorene (2-AF), 2-naphthylamine (2-NA) and 4-aminobiphenyl (4-ABP) modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene (2-AAF) modified DNA. These results suggest that, at least initially, both modifications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitroaromatics and aromatic amines. PMID:2540901

  1. Role of retinoic acid in the modulation of benzo(a)pyrene-DNA adducts in human hepatoma cells: Implications for cancer prevention

    International Nuclear Information System (INIS)

    Carcinogen-DNA adducts could lead to mutations in critical genes, eventually resulting in cancer. Many studies have shown that retinoic acid (RA) plays an important role in inducing cell apoptosis. Here we have tested the hypothesis that levels of carcinogen-DNA adducts can be diminished by DNA repair and/or by eliminating damaged cells through apoptosis. Our results showed that the levels of total DNA adducts in HepG2 cells treated with benzo(a)pyrene (BP, 2 μM) + RA (1 μM) were significantly reduced compared to those treated with BP only (P = 0.038). In order to understand the mechanism of attenuation of DNA adducts, further experiments were performed. Cells were treated with BP (4 μM) for 24 h to initiate DNA adduct formation, following which the medium containing BP was removed, and fresh medium containing 1 μM RA was added. The cells were harvested 24 h after RA treatment. Interestingly, the levels of total DNA adducts were lower in the BP/RA group (390 ± 34) than those in the BP/DMSO group (544 ± 33), P = 0.032. Analysis of cell apoptosis showed an increase in BP + RA group, compared to BP or RA only groups. Our results also indicated that attenuation of BP-DNA adducts by RA was not primarily due to its effects on CYP1A1 expression. In conclusion, our results suggest a mechanistic link between cellular apoptosis and DNA adduct formation, phenomena that play important roles in BP-mediated carcinogenesis. Furthermore, these results help understand the mechanisms of carcinogenesis, especially in relation to the chemopreventive properties of nutritional apoptosis inducers.

  2. 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 t....... 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....... 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...

  3. SEPARATION AND CHARACTERIZATION OF TETROL METABOLITES OF BENZO[A]PYRENE-DNA ADDUCTS USING HPLC AND SOLID-MATRIX ROOM TEMPERATURE LUMINESCENCE. (R824100)

    Science.gov (United States)

    AbstractFour tetrols of benzo[a]pyrene-DNA adducts were separated using reversed-phase high performance liquid chromatography. Chromatographic fractions containing a given tetrol were readily characterized with solid-matrix room temperature luminescence techniques. So...

  4. Proceedings of a workshop on DNA adducts: Biological significance and applications to risk assessment Washington, DC, April 13-14, 2004

    International Nuclear Information System (INIS)

    In April 2004, the Health and Environmental Sciences Institute, a branch of the International Life Sciences Institute, with support from the National Institute of Environmental Health Sciences, organized a workshop to discuss the biological significance of DNA adducts. Workshop speakers and attendees included leading international experts from government, academia, and industry in the field of adduct detection and interpretation. The workshop initially examined the relationship between measured adduct levels in the context of exposure and dose. This was followed by a discussion on the complex response of cells to deal with genotoxic insult in complex, interconnected, and interdependent repair pathways. One of the major objectives of the workshop was to address the recurring question about the mechanistic and toxicological relevance of low-concentration measured adducts and the presentations in the session entitled 'Can low levels of DNA adducts predict adverse outcomes?' served as catalysts for further discussions on this subject during the course of the workshop. Speakers representing the regulatory community and industry reviewed the value, current practices, and limitations of utilizing DNA adduct data in risk assessment and addressed a number of practical questions pertaining to these issues. While no consensus statement emerged on the biological significance of low levels of DNA adducts, the workshop concluded by identifying the need for more experimental data to address this important question. One of the recommendations stemming from this workshop was the need to develop an interim 'decision-logic' or framework to guide the integration of DNA adduct data in the risk assessment process. HESI has recently formed a subcommittee consisting of experts in the field and other key stakeholders to address this recommendation as well as to identify specific research projects that could help advance the understanding of the biological significance of low levels of DNA

  5. 5-Methylchrysene metabolism in mouse epidermis in vivo, diol epoxide--DNA adduct persistence, and diol epoxide reactivity with DNA as potential factors influencing the predominance of 5-methylchrysene-1,2-diol-3,4-epoxide--DNA adducts in mouse epidermis

    International Nuclear Information System (INIS)

    5-Methylchrysene (5-MeC) can form two bay region dihydrodiol epoxides: 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I) which has the methyl group and the epoxide ring in the same bay region, and 7,8-dihydroxy-9,10-epoxy-7,8,10-tetrahydro-5-methylchrysene (DE-II). In a previous study, we observed that the ratio of DE-I:DNA adducts to DE-II:DNA adducts in mouse epidermis, 24 h after application of [3H]5-MeC metabolites was 2.7 to 1. To investigate the basis for this observation we have now studies: (i) the formation of [3H]5-MeC in mouse epidermis in vivo at various time intervals from 0.33 to 24 h; (ii) the persistence of DE-I:DNA adducts and DE-II:DNA adducts in mouse epidermis at 4-48 h after application of [3H]5-MeC; and (iii) the reactions of DE-I and DE-II with calf thymus DNA in vitro. In contrast to results obtained with mouse liver 9000 grams supernatant, the dihydrodiol precursors of DE-I and DE-II were present in equivalent quantities in mouse epidermis in vivo at every time point studied. The ratio of DE-I:DNA adducts to DE-II:DNA adducts in mouse epidermis was constant throughout the time period studied. However, the extent of formation of DE-I:DNA adducts was greater than that of DE-II:DNA adducts upon reaction of DE-I or DE-II with calf thymus DNA in vitro. These results suggest that differences in reactivity with DNA of DE-I and DE-II may bw responsible for the higher levels in mouse epidermis of DE-I:DNA adducts compared with DE-II:DNA adducts and provide a possible basis for the observed enhancing effect of a bay region methyl group on the carcinogenicity of polynuclear aromatic hydrocarbons

  6. DNA adduct formation and induction of apoptosis in rat liver epithelial "stem-like" cells exposed to carcinogenic PAHs

    Czech Academy of Sciences Publication Activity Database

    Topinka, Jan; Sevastyanova, Oksana; Marvanová, S.; Vondráček, Jan; Nováková, Zuzana; Krčmář, P.; Pěnčíková, K.; Machala, M.

    Los Angeles, 2007. s. 1. [Annual Meeting 2007. 14.04.2007-18.04.2007, Los Angeles] R&D Projects: GA AV ČR(CZ) KJB6004407 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : DNA adducts * cytochromes P450 * cytotoxicity Subject RIV: BO - Biophysics

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang-In [Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010 (United States); Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Yoon, Jae-In [Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010 (United States); Cole, Kathleen J. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Pfeifer, Gerd P. [Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010 (United States); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Besaratinia, Ahmad, E-mail: ania@coh.org [Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010 (United States)

    2011-11-01

    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 {sup 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.

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

    International Nuclear Information System (INIS)

    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 32P-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.

  9. Analysis of biomarkers in a Czech population exposed to heavy air pollution. Part I. Bulky DNA adducts

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

    Roč. 28, č. 1 (2013), s. 89-95. ISSN 0267-8357 R&D Projects: GA MŽP(CZ) SP/1B3/8/08; GA MŠk 2B08005; GA ČR GAP503/11/0084 Institutional research plan: CEZ:AV0Z50390703 Institutional support: RVO:68378041 Keywords : Air pollution * biomarkers * DNA adducts Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.497, year: 2013

  10. 32P-post-labelling analysis of DNA adducts formed in the upper gastrointestinal tissue of mice fed bracken extract or bracken spores.

    OpenAIRE

    A. C. Povey; D. Potter; O'Connor, P J

    1996-01-01

    Bracken toxicity to both domestic and laboratory animals is well established and tumours are formed when rodents are treated with either bracken extracts or bracken spores. In this study we have administered bracken spores and extract to mice in order to investigate whether such exposure leads to the formation of DNA adducts. DNA, isolated from the upper gastrointestinal tract and liver, was digested to 3'-nucleotides. Adducts were extracted with butanol, 32P-post-labelled, separated by thin ...

  11. Regional distribution of xenobiotic metabolizing enzymes and DNA adducts in the respiratory tract - relationship to respiratory epithelial populations

    International Nuclear Information System (INIS)

    Specific regions from one-half of the nasal, tracheal, bronchial, and pulmonary airways of dogs were excised and analyzed for the presence of xenobiotic metabolizing enzymes. Substrates for different isozymes of cytochrome P-450, including benzo[a]pyrene (BaP), nitropyrene (NP), ethoxycoumarin, and ethoxyresorufin as well as selected Phase II enzymes were measured. Experiments were also conducted in which rats were exposed nose-only to dilute diesel exhaust (10 mg particles/m3) for up to 12 weeks. DNA adducts were measured in various regions of the respiratory tract after the 12-week exposure, using the highly sensitive 32P-postlabeling assay. The data indicate that there are significant differences in the metabolic capability and DNA adduct distribution along the respiratory tract. Quantitative differences that were noted along the respiratory tract are probably due to both differences in the capability of a given region to metabolize the organic chemicals associated with diesel exhaust and differences in the deposition patterns of the diesel exhaust. Differences in the metabolic capability, and therefore DNA adduct distribution, along the respiratory tracts of different species could explain, in part, why certain portions of the respiratory tract are more susceptible to tumor formation than are other portions

  12. 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.

  13. DNA adduct formation and oxidative stress in colon and liver of Big Blue (R) rats after dietary exposure to diesel particles

    DEFF Research Database (Denmark)

    Dybdahl, M.; Risom, L.; Møller, P.;

    2003-01-01

    in liver accompanied by enhanced vitamin C levels. In plasma, we found no significant effects on oxidative damage to proteins and lipids, antioxidant enzymes or vitamin C levels. Our data indicate that gastrointestinal exposure to DEP induces DNA adducts and oxidative stress resulting in DNA strand breaks...... on oxidative DNA damage (8-oxodG) in colon or liver DNA or in the urine. However, the mRNA expression of OGG1, encoding an enzyme involved in repair of 8-oxodG, was increased by DEP in both liver and colon. DNA adduct levels measured by P-32-post-labelling were elevated in colon and liver, and the expression...

  14. Tissue differences, dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L.) exposed to benzo[a]pyrene

    International Nuclear Information System (INIS)

    Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a) to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b) to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c) to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 μg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 μg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 μg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0±0.1 mg B[a]P/g tissue dry wt at 100 μg/l, mean±SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 μg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 μg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2

  15. 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.

    Science.gov (United States)

    Punt, Ans; Paini, Alicia; Spenkelink, Albertus; Scholz, Gabriele; Schilter, Benoit; van Bladeren, Peter J; Rietjens, Ivonne M C M

    2016-04-18

    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 extending a previously defined PBK model for estragole in humans to include (i) new data on interindividual variation in the kinetics for the major PBK model parameters influencing the formation of 1'-sulfooxyestragole, (ii) an equation describing the relationship between 1'-sulfooxyestragole and DNA adduct formation, (iii) Monte Carlo modeling to simulate interindividual human variation in DNA adduct formation in the population, and (iv) a comparison of the predictions made to human data on DNA adduct formation for the related alkenylbenzene methyleugenol. Adequate model predictions could be made, with the predicted DNA adduct levels at the estimated daily intake of estragole of 0.01 mg/kg bw ranging between 1.6 and 8.8 adducts in 10(8) nucleotides (nts) (50th and 99th percentiles, respectively). This is somewhat lower than values reported in the literature for the related alkenylbenzene methyleugenol in surgical human liver samples. The predicted levels seem to be below DNA adduct levels that are linked with tumor formation by alkenylbenzenes in rodents, which were estimated to amount to 188-500 adducts per 10(8) nts at the BMD10 values of estragole and methyleugenol. Although this does not seem to point to a significant health concern for human dietary exposure, drawing firm conclusions may have to await further validation of the model's predictions. PMID:26952143

  16. 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.

  17. 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)

    (+)-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 [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H 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 [6-15N]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 15N 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

  18. DNA adduct formation by 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) in rat colon

    OpenAIRE

    Lin, Chun-xing; Mondan, Yasumasa; Umemoto, Atsushi

    2001-01-01

    A food-born carcinogen, 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) induces cancer in the rat colon. The mechanism for colonic DNA adduct formation leading to cancer by IQ was studied using a colostomized F344 rat model. In this model, the transverse colon of the rat was colostomized, which produced a fecal stream-positive proximal colon and a negative distal colon were produced. When IQ (50 mg/kg) was administered into the distal colon of the colostomized rats (n=5), the ratio of the DNA ...

  19. Structural Basis for Bulky-Adduct DNA-Lesion Recognition by the Nucleotide Excision Repair Protein Rad14.

    Science.gov (United States)

    Simon, Nina; Ebert, Charlotte; Schneider, Sabine

    2016-07-25

    Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8-guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion-detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures. PMID:27223336

  20. NMR solution structures of adducts derived from the binding of polycyclic aromatic diol epoxides to DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cosman, M.; Patel, D.J. [Memorial Sloan Kettering Cancer Center, New York, NY (United States). Cellular Biochemistry and Biophysics Program; Hingerty, B.E. [Oak Ridge National Lab., TN (United States). Health and Safety Research Div.; Amin, S. [American Health Foundation, Valhalla, NY (United States); Broyde, S.; Geacintov, N.E. [New York Univ., NY (United States)

    1995-12-31

    Site-specifically modified oligonucleotides were derived from the reactions of stereoisomeric polycyclic aromatic diol epoxide metabolite model compounds with oligonucleotides of defined base composition and sequence. The NMR solution structures of ten different adducts studied so far are briefly described, and it is shown that stereochemical factors and the nature of the oligonucleotide context of the complementary strands, exert a powerful influence on the conformational features of these adducts.

  1. 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)

    32P-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/108 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.

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

    International Nuclear Information System (INIS)

    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

  3. 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.

  4. 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)

    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 32P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N2-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

  5. 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.

  6. Tamoxifen Forms DNA Adducts In Human Colon After Administration Of A Single (14C)-Labeled Therapeutic Dose

    International Nuclear Information System (INIS)

    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 (14C)-labeled therapeutic (20 mg) dose, ∼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-N2-tamoxifen adduct was detected in samples from three patients, at levels ranging from 1-7 adducts/109 nucleotides. No (14C)-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 α-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 ∼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.

  7. Punicalagin and Ellagic Acid Demonstrate Antimutagenic Activity and Inhibition of Benzo[a]pyrene Induced DNA Adducts

    Directory of Open Access Journals (Sweden)

    Maryam Zahin

    2014-01-01

    Full Text Available Punicalagin (PC is an ellagitannin found in the fruit peel of Punica granatum. We have demonstrated antioxidant and antigenotoxic properties of Punica granatum and showed that PC and ellagic acid (EA are its major constituents. In this study, we demonstrate the antimutagenic potential, inhibition of BP-induced DNA damage, and antiproliferative activity of PC and EA. Incubation of BP with rat liver microsomes, appropriate cofactors, and DNA in the presence of vehicle or PC and EA showed significant inhibition of the resultant DNA adducts, with essentially complete inhibition (97% at 40 μM by PC and 77% inhibition by EA. Antimutagenicity was tested by Ames test. PC and EA dose-dependently and markedly antagonized the effect of tested mutagens, sodium azide, methyl methanesulfonate, benzo[a]pyrene, and 2-aminoflourine, with maximum inhibition of mutagenicity up to 90 percent. Almost all the doses tested (50–500 μM exhibited significant antimutagenicity. A profound antiproliferative effect on human lung cancer cells was also shown with PC and EA. Together, our data show that PC and EA are pomegranate bioactives responsible for inhibition of BP-induced DNA adducts and strong antimutagenic, antiproliferative activities. However, these compounds are to be evaluated in suitable animal model to assess their therapeutic efficacy against cancer.

  8. 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.

  9. The solid-matrix phosphorescence of (±)-anti-dibenzo[a,l]pyrene diol epoxide-DNA adducts and benzo[e]pyrene

    International Nuclear Information System (INIS)

    New solid-matrix phosphorescence (SMP) methods for (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P were developed. The methods can be used to detect and characterize (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P by employing SMP spectra, intensities, and lifetimes acquired with the heavy-atom salt, TlNO3, on Whatman 1PS paper. With the SMP data, a number of photophysical parameters were calculated such as biexponential SMP decay curves, pre-exponential factors, and fractional contribution to SMP decay curves. The SMP results were compared with earlier SMP data for (±)-anti-BPDE-DNA adducts and tetrol I-1. The SMP results show that small molecular-weight compounds like B[e]P can be readily detected and characterized by SMP. For example, the limit of detection for B[e]P was 0.60 pmol. Comparison of the SMP properties of the (±)-anti-DB[a,l]PDE-DNA adducts with earlier SMP data for the (±)-anti-BPDE-DNA adducts showed major differences in the SMP spectra, intensities, and lifetimes. The methods developed are important for the comparison of the SMP properties of different diol epoxides of PAH bonded to DNA

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

    Directory of Open Access Journals (Sweden)

    Inmaculada Buendia Jimenez

    2015-01-01

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

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

    Science.gov (United States)

    Buendia Jimenez, Inmaculada; Richardot, Pascaline; Picard, Pascaline; Lepicard, Eve M.; De Meo, Michel; Talaska, Glenn

    2015-01-01

    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. PMID:26357419

  12. 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...

  13. 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)

    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

  14. Variation in PAH-related DNA adduct levels among non-smokers: the role of multiple genetic polymorphisms and nucleotide excision repair phenotype.

    Science.gov (United States)

    Etemadi, Arash; Islami, Farhad; Phillips, David H; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T; Taylor, Philip R; Boffetta, Paolo; Abnet, Christian C; Dawsey, Sanford M; Malekzadeh, Reza; van Schooten, Frederik J

    2013-06-15

    Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to xenobiotic metabolism and 12 SNPs in eight DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by32P-postlabeling. Multivariable regression models were compared by Akaike's information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 10(8) nucleotides (mean: 5.8 ± 3.1). DNA adduct level was significantly lower in homozygotes for NAT2 slow alleles and ERCC5 non-risk-allele genotype, and was higher in the MPO homozygote risk-allele genotype. The sum of risk alleles in these genes significantly correlated with the log-adduct level (r = 0.4, p studies, with large inter-individual variation which could best be explained by a combination of Phase I genes and NER capacity. PMID:23175176

  15. The effect of plant phenolics on the formation of 7,12-dimethylbenz[a]anthracene-DNA adducts and TPA-stimulated polymorphonuclear neutrophils chemiluminescence in vitro

    International Nuclear Information System (INIS)

    Phenolics, common plant constituents, form up an important part of human diet and are considered potential chemopreventive agents. In the present study, structurally diverse phenolics, such as tannic acid, protocatechuic acid, chlorogenic acid and resveratrol, were investigated for their inhibitory effects on covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA in vitro and the suppression of oxidative burst in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human polymorphonuclear neutrophils (PMNs). 32P-postlabeling analysis of DNA incubated with DMBA in the presence of 3-methylcholanthrene (3-MC)-induced microsomes produced three major adducts derived from anti-, syn- and anti-dihydrodiol epoxides through reactions with dGuo and dAdo, respectively. Phenolic compounds at the concentration of 150 μM reduced the levels of all DMBA-DNA adducts by 55-98%. The most dramatic effect was observed in case of tannic acid, which completely inhibited the formation of DMBA-dAdo adducts. Chlorogenic acid was the least effective inhibitor of DMBA-DNA adducts formation particularly syn-DMBADE-dAdo (20%). Human neutrophils showed a significant dose-related decrease of TPA-induced chemiluminescence after pretreatment with phenolic compounds. The most effective inhibitors were tannic acid and resveratrol with IC50=5.19 and 5.76 μM, respectively. These results suggest that the suppression of reactive oxygen species (ROS) and carcinogen-DNA adducts formation may be important for anticarcinogenic activity of the examined phenolics

  16. 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

  17. Two food-borne heterocyclic amines: Metabolism and DNA adduct formation of amino-alpha-carbolines

    DEFF Research Database (Denmark)

    Frederiksen, Hanne

    2005-01-01

    -adducts have also been studied. Characteristic for the amino-a-carbolines are that relatively large amounts of these compounds in rat and human hepatic microsomes are activated to potent carcinogenic compounds compared with other heterocyclic amines, but further in vivo studies of the amino-a-carbolines are...

  18. 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.

  19. Reduction of cisplatin-induced nephrotoxicity in vivo by selenomethionine: the effect on cisplatin-DNA adducts.

    Science.gov (United States)

    García Sar, Daniel; Montes-Bayón, Maria; Blanco González, Elisa; Sierra Zapico, Luisa M; Sanz-Medel, Alfredo

    2011-06-20

    Cisplatin is one of the most effective chemotherapeutic agents, although its clinical use is limited by severe renal toxicity. This toxicity seems to be related to the accumulation of the drug in kidney tissues, leading to renal failure. For this reason, several compounds have been evaluated to ameliorate the nephrotoxicity induced by cisplatin. In the present investigation, we report the effect of the oral administration of selenomethionine before intraperitoneal cisplatin treatment. The preadministration of this Se species has been shown to have an important effect in reducing renal damage induced by cisplatin by increasing the excreted urea and improving creatinine clearance. Quantification of the level of DNA--cisplatin adducts in kidney and liver tissues was carried out by postcolumn isotope dilution analysis using liquid chromatography-inductively coupled plasma (LC-ICP-MS) as speciation set up. The level of DNA--cisplatin adducts in rats given Se-methionine in the drinking water before cisplatin administration was considerably lower in kidney tissues with respect to the animals drinking only water. Such effects were not observed in liver tissue. Initial speciation studies of Pt and Se conducted in kidney tissues of exposed animals by HPLC-ICP-MS have revealed the presence of cisplatin as part of a complex with Se-methionine, which can be eventually excreted into urine. This Pt--Se complex could explain the observed reduction of the kidney damage in Se-methionine-treated animals. PMID:21491944

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

    International Nuclear Information System (INIS)

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

  1. Analysis of cigarette-smoke-induced DNA adducts by butanol extraction and nuclease P1-enhanced 32P-postlabeling in human lymphocytes and granulocytes.

    OpenAIRE

    Savela, K; Hemminki, K

    1993-01-01

    In an earlier study, we analyzed the aromatic DNA adducts separated from lymphocytes and granulocytes of smokers and nonsmokers using the nuclease P1-enhanced 32P-postlabeling assay. Here we compare the butanol extraction and nuclease P1-enhanced procedure on the same kind of samples. The DNA adducts of 42 per 10(8) nucleotides from smokers' lymphocytes were statistically higher (p < 0.05) than those of 11 from nonsmokers', when analyzed by the nuclease P1 treatment, but not by the 1-butanol ...

  2. 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

    highest among mother-newborn pairs who lived near medium-traffic-density (> 400-2500 vehicle km/24 h; p <0.01) places. MN frequencies among newborns from women who lived at high-traffic-density homes ( > 2500 vehicle km/24 h) were significantly increased (p = 0.02). This trend remained after adjusting for......Exposure to traffic-related air pollution in urban environment is common and has been associated with adverse human health effects. In utero exposures that result in DNA damage may affect health later in life. Early effects of maternal and in utero exposures to traffic-related air pollution were...... 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...

  3. Mutations Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene in lacI Transgenic B6C3F1 Mouse Lung Result from Stable DNA Adducts

    Science.gov (United States)

    Dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P) are carcinogenic polycyclic aromatic hydrocarbons (PAH) that are each capable of forming a variety of covalent adducts with DNA. Some of the DNA adducts formed by these PAHs have been demonstrated to spontaneously depurina...

  4. 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)

    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

  5. 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

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shuangying, E-mail: shuangying.yu@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Tang, Song, E-mail: song.tang@usask.ca [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Mayer, Gregory D., E-mail: greg.mayer@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Cobb, George P., E-mail: george_cobb@baylor.edu [Department of Environmental Science, Baylor University, One Bear Place #97266, Waco, TX 76798 (United States); Maul, Jonathan D., E-mail: jonathan.maul@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States)

    2015-02-15

    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

  6. Food borne bacterial models for detection of benzo[a]pyrene-DNA adducts formation using RAPD-PCR.

    Science.gov (United States)

    Lanzone, Valentina; Tofalo, Rosanna; Fasoli, Giuseppe; Perpetuini, Giorgia; Suzzi, Giovanna; Sergi, Manuel; Corrado, Federica; Compagnone, Dario

    2016-05-01

    Random amplified polymorphic DNA (RAPD) PCR is a feasible method to evaluate genotoxin-induced DNA damage and mutations. In this study, Lactobacillus plantarum ATCC 14917T, Enterococcus faecium DSMZ 20477T, Escherichia coli PQ37 and Saccharomyces cerevisiae S441 were screened for DNA genetic alterations by DNA fingerprinting using M13 and LA1 primers after treatment with three compounds forming covalent adducts with DNA [benzo[a]pyrenediol epoxide (BPDE), methyl methanesulfonate and 1,2,3,4-diepoxybutane (DEB)]. M13 RAPD fingerprinting revealed that the total number of bands decreased in all treated DNA compared to control samples and generally the lost bands were characterized by high molecular weight. Some extra bands were detected for L. plantarum and E. faecium, while in E. coli and S. cerevisiae DNAs BPDE and DEB treatments did not result in new extra bands. Besides qualitatively analysis, cluster analysis based on Unweighted Pair-Group Method with Average algorithm was performed to compare DNA fingerprints before and after treatments. This analysis confirmed the absence of significant differences between negative controls and treated DNA in S. cerevisiae and E. coli however the disappearance of some bands can be detected. The data indicate that this approach can be used for DNA damage detection and mutations induced by genotoxic compounds and highlighted the possible use of L. plantarum and E. faecium M13 based fingerprinting as reference for hazard identification in risk assessment. PMID:26991971

  7. Effect of increased intake of dietary animal fat and fat energy on oxidative damage, mutation frequency, DNA adduct level and DNA repair in rat colon and liver

    DEFF Research Database (Denmark)

    Vogel, Ulla; Daneshvar, Bahram; Autrup, Herman;

    2003-01-01

    The effect of high dietary intake of animal fat and an increased fat energy intake on colon and liver genotoxicity and on markers of oxidative damage and antioxidative defence in colon, liver and plasma was investigated in Big Blue rats. The rats were fed ad libitum with semi-synthetic feed...... DNA-adduct level measured by 32P-postlabelling decreased in both liver and colon with increased fat intake. In liver, this was accompanied by a 2-fold increase of the mRNA level of nucleotide excision repair (NER) gene ERCC1. In colon, a non-statistically significant increase in the ERCC1 mRNA levels...

  8. MeIQx-induced DNA adduct formation and mutagenesis in DNA repair deficient CHO cells expressing human CYP1A1 and rapid or slow acetylator NAT2

    Science.gov (United States)

    Bendaly, Jean; Zhao, Shuang; Neale, Jason R.; Metry, Kristin J.; Doll, Mark A.; States, J. Christopher; Pierce, William M.; Hein, David W.

    2007-01-01

    2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient chinese hamster ovary (CHO) cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A1 activity did not differ significantly (p > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of sulfamethazine N-acetyltransferase (p = 0.0001) and N-hydroxy-MeIQx O-acetyltransferase (p = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis following MeIQx treatment. dG-C8-MeIQx was the primary DNA adduct formed and levels were dose-dependent in each cell line and in the order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 & NAT2*5B < transfected with CYP1A1 & NAT2*4. MeIQx DNA adduct levels were significantly higher (p < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MeIQx –induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiological studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators. PMID:17627018

  9. In vitro bypass of the major malondialdehyde- and base propenal-derived DNA adduct by human Y-family DNA polymerases κ, ι, and Rev1.

    Science.gov (United States)

    Maddukuri, Leena; Eoff, Robert L; Choi, Jeong-Yun; Rizzo, Carmelo J; Guengerich, F Peter; Marnett, Lawrence J

    2010-09-28

    3-(2'-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M(1)dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M(1)dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M(1)dG → A and M(1)dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M(1)dG in vivo. Previous reports described the bypass of M(1)dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M(1)dG by the human Y-family DNA polymerases κ, ι, and Rev1. M(1)dG was incorporated into template-primers containing either dC or dT residues 5' to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol ι, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M(1)dG:dC > M(1)dG:dG > M(1)dG:dT ∼ M(1)dG:dA, but neither hPol ι nor Rev1 extended M(1)dG-containing template-primers. Liquid chromatography-mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3'-GXC-5' template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M(1)dG in the 3'-GXT-5' template sequence. The results indicate that DNA hPol κ or the combined action of hPol ι or Rev1 and hPol κ bypass M(1)dG residues in DNA and generate products that are consistent with some of the mutations induced by M(1)dG in mammalian cells. PMID:20726503

  10. In Vitro Bypass of the Major Malondialdehyde- and Base Propenal-Derived DNA Adduct by Human Y-family DNA Polymerases κ, ι, and Rev1†

    Science.gov (United States)

    2010-01-01

    3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M1dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M1dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M1dG → A and M1dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M1dG in vivo. Previous reports described the bypass of M1dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M1dG by the human Y-family DNA polymerases κ, ι, and Rev1. M1dG was incorporated into template-primers containing either dC or dT residues 5′ to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol ι, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M1dG:dC > M1dG:dG > M1dG:dT ∼ M1dG:dA, but neither hPol ι nor Rev1 extended M1dG-containing template-primers. Liquid chromatography−mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3′-GXC-5′ template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M1dG in the 3′-GXT-5′ template sequence. The results indicate that DNA hPol κ or the combined action of hPol ι or Rev1 and hPol κ bypass M1dG residues in DNA and generate products that are consistent with some of the mutations induced by M1dG in mammalian cells. PMID:20726503

  11. 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.

  12. Urinary Metabolites of the Dietary Carcinogen PhIP are Predictive of Colon DNA Adducts After a Low Dose Exposure in Humans

    International Nuclear Information System (INIS)

    Epidemiologic evidence indicates that exposure to heterocyclic amines (HAs) in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of HAs which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of HA bioactivation in humans, the most mass abundant HA, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers were administered a dietary relevant dose of [14C]PhIP 48-72 h prior to surgery to remove colon tumors. Urine was collected for 24 h after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All ten subjects were phenotyped for CYP1A2, NAT2, and SULT1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N2-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N2-glucuronide, had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N2-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group

  13. Urinary Metabolites of the Dietary Carcinogen PhIP are Predictive of Colon DNA Adducts After a Low Dose Exposure in Humans

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M; Dingley, K; Nowell, S; Ubick, E; Mulakken, N; Nelson, D; Lang, N; Felton, J; Turteltaub, K

    2006-04-28

    Epidemiologic evidence indicates that exposure to heterocyclic amines (HAs) in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of HAs which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of HA bioactivation in humans, the most mass abundant HA, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers were administered a dietary relevant dose of [{sup 14}C]PhIP 48-72 h prior to surgery to remove colon tumors. Urine was collected for 24 h after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All ten subjects were phenotyped for CYP1A2, NAT2, and SULT1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N{sup 2}-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide, had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.

  14. Effects of dietary fish oil on the depletion of carcinogenic PAH-DNA adduct levels in the liver of B6C3F1 mouse.

    Directory of Open Access Journals (Sweden)

    Guo-Dong Zhou

    Full Text Available Many carcinogenic polycyclic aromatic hydrocarbons (PAHs and their metabolites can bind covalently to DNA. Carcinogen-DNA adducts may lead to mutations in critical genes, eventually leading to cancer. In this study we report that fish oil (FO blocks the formation of DNA adducts by detoxification of PAHs. B6C3F1 male mice were fed a FO or corn oil (CO diet for 30 days. The animals were then treated with seven carcinogenic PAHs including benzo(apyrene (BaP with one of two doses via a single intraperitoneal injection. Animals were terminated at 1, 3, or 7 d after treatment. The levels of DNA adducts were analyzed by the (32P-postlabeling assay. Our results showed that the levels of total hepatic DNA adducts were significantly decreased in FO groups compared to CO groups with an exception of low PAH dose at 3 d (P = 0.067. Total adduct levels in the high dose PAH groups were 41.36±6.48 (Mean±SEM and 78.72±8.03 in 10(9 nucleotides (P = 0.011, respectively, for the FO and CO groups at 7 d. Animals treated with the low dose (2.5 fold lower PAHs displayed similar trends. Total adduct levels were 12.21±2.33 in the FO group and 24.07±1.99 in the CO group, P = 0.008. BPDE-dG adduct values at 7 d after treatment of high dose PAHs were 32.34±1.94 (CO group and 21.82±3.37 (FO group in 10(9 nucleotides with P value being 0.035. Low dose groups showed similar trends for BPDE-dG adduct in the two diet groups. FO significantly enhanced gene expression of Cyp1a1 in both the high and low dose PAH groups. Gstt1 at low dose of PAHs showed high levels in FO compared to CO groups with P values being 0.014. Histological observations indicated that FO played a hepatoprotective role during the early stages. Our results suggest that FO has a potential to be developed as a cancer chemopreventive agent.

  15. 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

  16. 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

  17. ANALYSIS OF BENZO(A)PYRENE-INDUCED DNA ADDUCTS IN MCF-7 BREAST CANCER CELLS WITH DIFFERENT LEVELS OF HSP70 EXPRESSION

    Science.gov (United States)

    Analysis of benzo[a]pyrene-induced DNA adducts in MCF-7 breast cancer cells withdifferent levels of HSP7O expression.L.C. King1, L.D. Adams1, E.Winkfield1, J.A. Barnes2, S.D. Hester1 and J.W. Allen1. 1USEnvironmental Protection Agency, Research Triangle Park, NC 2771...

  18. 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

  19. Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk.

    Science.gov (United States)

    Lewin, Michelle H; Bailey, Nina; Bandaletova, Tanya; Bowman, Richard; Cross, Amanda J; Pollock, Jim; Shuker, David E G; Bingham, Sheila A

    2006-02-01

    Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N-nitrosocompounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, high-fiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O(6)-carboxymethyl guanine (O(6)CMG) was significantly (P colorectal cancer. PMID:16452248

  20. Absence of formation of benzo[a]pyrene/DNA adducts in the cuttlefish (Sepia officinalis, Mollusca: Cephalopoda)

    Energy Technology Data Exchange (ETDEWEB)

    Lee, P.G.; Lu, L.J.W.; Salazar, J.J.; Holoubek, V. (Univ. of Texas Medical Branch, Galveston, TX (United States))

    1994-01-01

    Benzo[a]pyrene (B[a]P) injected intramuscularly into the base of the arms of cuttlefish was released continuously from the injection site and removed from the organism. Only a portion of the compound accumulated in the body. Twenty-four hr after its injection, 75% of B[a]P applied in olive oil was removed from the cuttlefish, and 1.2% was found in the body outside the head, in site of injection. If the carcinogen was dissolved in dimethylformamide, the removal of B[a]P was slower, so that only 18% of the injected B[a]P was removed from the organism and 0.36% accumulated in the body outside the head 24 hr after injection. The high level of B[a]P in gills and hemolymph 4 hr after injection and the kinetics of the decrease of its concentration with time indicate that these two organs could be involved in the excretion of B[a]P from the body. The B[a]P/DNA adducts characteristic for vertebrates could not be demonstrated in gills, skin, brain, hepatopancreas, and lymphocytes of the cuttlefish 24 hr after injection. The dose of the carcinogene injected into the cuttlefish was 2-4 times higher than the dose resulting in the formation of a high level of B[a]P/DNA adducts in vertebrates. A different metabolism of B[a]P in the tissue of cephalopods, compared to vertebrates, could be less favorable to the process leading to malignant transformation and could explain the absence from the literature of reports of tumors in cephalopods. 15 refs., 1 fig., 2 tabs.

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

    DEFF Research Database (Denmark)

    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...... found in these two peaks, and the ratio of radioactivity between the peaks was nearly 1. In colonic DNA, the ratio between Structures 1 and 11 was approximately 2. These observations add aflatoxin B1 to the list of chemical procarcinogens metabolized by cultured human tissues and in which the carcinogen-DNA...... adducts are similar to the adducts formed in animal tissue susceptible to the carcinogenic action of aflatoxin B1....

  2. 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

  3. DNA-adduct formation in lungs, nasal mucosa, and livers of rats exposed to urban roadside air in Kawasaki City, Japan

    International Nuclear Information System (INIS)

    The potency of ambient air for DNA-adduct formation was estimated using Wistar rats. The animals were maintained in a small-animal facility located beside a main highway intersection in Kawasaki City, Japan, for up to 60 weeks and were exposed to roadside air contaminated mainly with automobile emission (exposure group, EG) or to clean air (control group, CG). Compared to CG, the relative adduct levels (RAL) were increased significantly in EG lungs (17.1-fold (P<0.05)), nasal mucosa, and livers after exposure for 4 weeks. However, there were no significant differences in RAL between EG and CG after exposure for 12 weeks, but they were elevated again in EG after exposure for 48 or 60 weeks. These results suggest that roadside air in this region can cause the generation of DNA adducts. This activity of ambient roadside air can be estimated using experimental animals, indicating that biological monitoring of DNA-adduct formation may be a powerful tool to assess the effect of ambient air on human health

  4. In Vitro Bypass of the Major Malondialdehyde- and Base Propenal-Derived DNA Adduct by Human Y-family DNA Polymerases κ, ι, and Rev1†

    OpenAIRE

    Maddukuri, Leena; Robert L Eoff; Choi, Jeong-Yun; Rizzo, Carmelo J.; Guengerich, F. Peter; Marnett, Lawrence J.

    2010-01-01

    3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M1dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M1dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M1dG → A and M1dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M1dG in vivo. Previous reports described the bypass of M1dG...

  5. 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...

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

    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...

  7. 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/mu g 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...

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

    International Nuclear Information System (INIS)

    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/m3). 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/cm2) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. Conclusion: The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely

  9. Analysis of Pyridyloxobutyl and Pyridylhydroxybutyl DNA Adducts in Extra-hepatic Tissues of F344 Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

    OpenAIRE

    Zhang, Siyi; Wang, Mingyao; Villalta, Peter W.; Lindgren, Bruce R.; Upadhyaya, Pramod; Lao, Yanbin; Hecht, Stephen S.

    2009-01-01

    The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rats. NNK and NNAL require metabolic activation to express their carcinogenicity. Cytochrome P450-catalyzed α-hydroxylation at the methyl position of NNK or NNAL generates reactive intermediates, which alkylate DNA to form pyridyloxobutyl (POB)-DNA adducts or pyridylhydroxybutyl (PHB)-DNA adducts. NNK ...

  10. Sequence-specific Hydrolysis of Single-stranded DNA by PNA-Cerium (Ⅳ) Adduct

    Institute of Scientific and Technical Information of China (English)

    He Bai SHEN; Feng WANG; Yong Tao YANG

    2005-01-01

    A novel artificial site specific cleavage reagent, with peptide nucleic acid (PNA) as sequence-recognizing moiety and cerium (Ⅳ) ions as "scissors" for cleaving target DNA, was synthesized. Subsequently, it was employed in the cleavage of target 26-mer single-stranded DNA (ssDNA), which has 10-mer sequence complementary with PNA recognizer in the hybrids,under physiological conditions. Reversed-phase high-performance liquid chromatogram (RPHPLC) experiments indicated that the artificial site specific cleavage reagent could cleave the target DNA specifically.

  11. Cisplatin adducts on a GGG sequence within a DNA duplex studied by NMR spectroscopy and molecular dynamics simulations.

    Science.gov (United States)

    Téletchéa, Stéphane; Skauge, Tormod; Sletten, Einar; Kozelka, Jirí

    2009-11-16

    The antitumor drug cisplatin(cis-[PtCl2(NH3)2]) reacts with cellular DNA to form GG intrastrand adducts between adjacent guanines as predominant lesions. GGG sites have been shown to be hotspots of platination. To study the structural perturbation induced by binding of cisplatin to two adjacent guanines of a GGG trinucleotide,we examined here the decanucleotide duplex d[(G1C2C3G*4 G*5 G6T7-C8G9C10).d(G11C12G13A14C15C16C17G18-G19C20)] (dsCG*G*G) intrastrand cross-linked at the G* guanines by cis-{Pt(NH3)2}2+ using NMR spectroscopy and molecular dynamics (MD) simulations.The NMR spectra of dsCG*G*G were found to be similar to those of previously characterized DNA duplexes cross-linked by cisplatin at apyG*G*X site (py=pyrimidine; X=C,T, A). This similarity of NMR spectra indicates that the base at the 3'-side of the G*G*-Pt cross-link does not affect the structure to a large extent. An unprecedented reversible isomerization between the duplex dsCG*G*G (bearing a G*4 G*5 -Pt chelate) and duplex dsGG*G*T (bearing a G*5 G*6 -Pt chelate)was observed, which yielded a 40:60 equilibrium between the two intrastrand GG-Pt cross-links. No formation of interstrand cross-links was observed.NMR spectroscopic data of dsCG*G*G indicated that the deoxyribose of the 5'-G* adopts an N-type conformation, and the cytidines C3, C15,and C16 have average phase angles intermediate between S and N. The NMR spectroscopic chemical shifts of dsGG*G*T showed some fundamental differences to those of pyG*G*-platinum adducts but were in agreement with the NMR spectra reported previously for the DNA duplexes crosslinked at an AG*G*C sequence by cisplatin or oxaliplatin. The presence of apurine instead of a pyrimidine at the 5'-side of the G*G* cross-link seems therefore to affect the structure of the XG* step significantly. PMID:19813235

  12. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    International Nuclear Information System (INIS)

    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

  13. 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.

  14. 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.

  15. 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.

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

    International Nuclear Information System (INIS)

    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 32P-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

  17. Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne benzo(a)pyrene exposure: implications for biomarker determination.

    Science.gov (United States)

    Lee, Jin Wuk; Kim, Yong Hwa; Yoon, Seokjoo; Lee, Sung Kyu

    2014-09-01

    Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t-test, p platypus is a useful species for assessing the risk of waterborne BaP exposure. PMID:23192953

  18. Analysis of O(6)-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine and Other DNA Adducts in Rats Treated with Enantiomeric or Racemic N'-Nitrosonornicotine.

    Science.gov (United States)

    Yang, Jing; Villalta, Peter W; Upadhyaya, Pramod; Hecht, Stephen S

    2016-01-19

    (S)-N'-Nitrosonornicotine [(S)-NNN] and racemic NNN are powerful oral and esophageal carcinogens in the F344 rat, whereas (R)-NNN has only weak activity. Tumor formation in these tissues of rats treated with racemic NNN was far greater than the sum of the activities of the individual enantiomers. We hypothesized that metabolites of (R)-NNN enhanced levels of DNA adducts produced by (S)-NNN. A test of that hypothesis necessitated the development of a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the analysis of O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo), a highly mutagenic DNA adduct not previously quantified in rats treated with NNN. The new method, with a limit of detection of 6.5 amol for diluted standard and 100 amol for DNA samples, was applied in this study. Groups of nine F344 rats were treated with doses as follows: 7 ppm (R)-NNN, 7 ppm (S)-NNN, and 14 ppm racemic NNN; 14 ppm (R)-NNN, 14 ppm (S)-NNN, and 28 ppm racemic NNN; or 28 ppm (R)-NNN, 28 ppm (S)-NNN, and 56 ppm racemic NNN for 5 weeks, and tissues were analyzed for DNA adducts. We found statistically significant, but modest, synergistic enhancement of levels of O(6)-POB-dGuo in the esophagus but not the oral cavity of rats treated with racemic NNN (low and median doses only) compared to the sum of the amounts formed in these tissues of rats treated with (S)-NNN or (R)-NNN. There was no synergy in the formation of other POB-DNA adducts of NNN in oral cavity and esophagus, nor was there any evidence for synergy in nasal respiratory and olfactory epithelium, lung, or liver. Our results provide the first quantitation of O(6)-POB-dGuo in DNA from tissues of rats treated with NNN and evidence for synergy in DNA adduct formation as one possible mechanism by which (R)-NNN enhances the carcinogenicity of (S)-NNN in rats. PMID:26633576

  19. Variation in PAH-related DNA adduct levels among non-smokers: the role of multiple genetic polymorphisms and nucleotide excision repair phenotype

    OpenAIRE

    Etemadi, Arash; Islami, Farhad; Phillips, David H.; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T.; Taylor, Philip R.; Boffetta, Paolo; Abnet, Christian C.; Dawsey, Sanford M.

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to...

  20. DNA Adduct Formation from Metabolic 5'-Hydroxylation of the Tobacco-Specific Carcinogen N'-Nitrosonornicotine in Human Enzyme Systems and in Rats.

    Science.gov (United States)

    Zarth, Adam T; Upadhyaya, Pramod; Yang, Jing; Hecht, Stephen S

    2016-03-21

    N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2'-hydroxylation and 5'-hydroxylation. While most previous studies have focused on 2'-hydroxylation in target tissues of rats, available evidence suggests that 5'-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5'-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2'-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7-500 ppm), py-py-dI was the major DNA adduct resulting from 5'-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R)-py-py-dI to (S)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5'-hydroxylation of NNN. Rat hepatocytes treated with (S)- or (R)-NNN or (2'S)- or (2'R)-5'-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. In vitro studies with human liver S9 fraction or human hepatocytes incubated with NNN (2-500 μM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2'-hydroxylation of NNN. (S)-NNN formed more total py-py-dI adducts than (R)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5'-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans

  1. Molecular modeling and spectroscopic studies of semustine binding with DNA and its comparison with lomustine-DNA adduct formation.

    Science.gov (United States)

    Agarwal, Shweta; Chadha, Deepti; Mehrotra, Ranjana

    2015-01-01

    Chloroethyl nitrosoureas constitute an important family of cancer chemotherapeutic agents, used in the treatment of various types of cancer. They exert antitumor activity by inducing DNA interstrand cross-links. Semustine, a chloroethyl nitrosourea, is a 4-methyl derivative of lomustine. There exist some interesting reports dealing with DNA-binding properties of chloroethyl nitrosoureas; however, underlying mechanism of cytotoxicity caused by semustine has not been precisely and completely delineated. The present work focuses on understanding semustine-DNA interaction to comprehend its anti-proliferative action at molecular level using various spectroscopic techniques. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is used to determine the binding site of semustine on DNA. Conformational transition in DNA after semustine complexation is investigated using circular dichroism (CD) spectroscopy. Stability of semustine-DNA complexes is determined using absorption spectroscopy. Results of the present study demonstrate that semustine performs major-groove-directed DNA alkylation at guanine residues in an incubation-time-drug-concentration-dependent manner. CD spectral outcomes suggest partial transition of DNA from native B-conformation to C-form. Calculated binding constants (Ka) for semustine and lomustine interactions with DNA are 1.53 × 10(3) M(-1) and 8.12 × 10(3) M(-1), respectively. Moreover, molecular modeling simulation is performed to predict preferential binding orientation of semustine with DNA that corroborates well with spectral outcomes. Results based on comparative study of DNA-binding properties of semustine and lomustine, presented here, may establish a correlation between molecular structure and cytotoxicity of chloroethyl nitrosoureas that may be instrumental in the designing and synthesis of new nitrosourea therapeutics possessing better efficacy and fewer side effects. PMID:25350567

  2. RAPID TECHNIQUE FOR ESTIMATING DNA BINDING, USED TO EVALUATE 1-NITROPYRENE ADDUCT FORMATION

    Science.gov (United States)

    A simple and rapid procedure for estimating binding of radiolabelled material to DNA and protein is described. Protein was extracted from lysed rabbit alveolar macrophages with chloroform: iso-amyl-alcohol:phenol extraction. Nucleic acids were precipitated from the lysate, and hy...

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

    Science.gov (United States)

    The abstract reports the results of studies assessing the relative DNA damage potential of extracts of exhaust particles resulting from the combustion of petroleum diesel, biodiesel, and petroleum diesel-biodiesel blends. Results indicate that the commercially available B20 petr...

  4. 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.

  5. Noni juice reduces lipid peroxidation–derived DNA adducts in heavy smokers

    OpenAIRE

    Wang, Mian-Ying; Peng, Lin; Jensen, Claude J; Deng, Shixin; Brett J. West

    2013-01-01

    Food plants provide important phytochemicals which help improve or maintain health through various biological activities, including antioxidant effects. Cigarette smoke–induced oxidative stress leads to the formation of lipid hydroperoxides (LOOHs) and their decomposition product malondialdehyde (MDA), both of which cause oxidative damage to DNA. Two hundred forty-five heavy cigarette smokers completed a randomized, double-blind, placebo-controlled clinical trial designed to investigate the e...

  6. 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

  7. 1,N(2)-propanodeoxyguanosine adduct formation in aortic DNA following inhalation of acrolein.

    OpenAIRE

    A Penn; Nath, R.; Pan, J; Chen, L; Widmer, K; Henk, W; Chung, F L

    2001-01-01

    Recent reports indicate that many of the cytotoxic and health-threatening components of environmental tobacco smoke (ETS) reside in the vapor phase of the smoke. We have reported previously that inhalation of 1,3-butadiene, a prominent vapor phase component of ETS, accelerates arteriosclerotic plaque development in cockerels. In this study we asked whether inhaled acrolein, a reactive aldehyde that is also a prominent vapor-phase component of ETS, damages artery-wall DNA and accelerates plaqu...

  8. Effects of one 150R x-ray exposure on DMBA-DNA adduct formation in Syrian hamster cheek pouch epithelium in vivo

    International Nuclear Information System (INIS)

    The authors' prior studies showed enhancement of DMBA carcinogenesis in hamster buccal pouch epithelium by repeated 20R head and neck x-ray exposures. Possible mechanisms included radiation-induced increases in covalent binding of DMBA to epithelial cell DNA. This pilot study assessed, at one time period, the effect of a single modest x-ray exposure on in vivo DMBA-DNA adduct formation in the cheek pouch. Hamsters received one 0.05ml topical application of 5.0μg 3H-DMBA (900μCi) in DMSO to the right pouch. Two hours later, 1/2 of the hamsters received a single 150R head and neck x-ray exposure. 24 hours after DMBA treatments, pouches were exercised and epithelium was isolated. Epithelial DNA was extracted and purified using homogenization, phenol-chloroform-isoamyl alcohol extraction, and spermine precipitation. DMBA-DNA adducts were calculated via scintillation spectrometry of radioactivity and fluorometry of DNA, and were expressed as μg DMBA bound / mg DNA. There was no significant difference between irradiated and non-irradiated groups in DMBA bound. This may reflect a true lack of radiation effect on DMBA binding, a temporal displacement of increased binding not detectable at 24 hours, or too small a binding increase from one x-ray exposure to detect. Ongoing studies are addressing these possibilities

  9. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

    International Nuclear Information System (INIS)

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP

  10. Ring-Opening of the γ-OH-PdG Adduct Promotes Error-Free Bypass by the Sulfolobus solfataricus DNA Polymerase Dpo4

    OpenAIRE

    Shanmugam, Ganesh; Minko, Irina G.; Banerjee, Surajit; Christov, Plamen P.; Kozekov, Ivan D.; Rizzo, Carmelo J.; Lloyd, R. Stephen; Egli, Martin; Michael P. Stone

    2013-01-01

    Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N 2-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus ...

  11. Benzo pyrene-induced DNA adducts and gene expression profiles in target and non-target organs for carcinogenesis in mice

    OpenAIRE

    Zuo, Jie; Brewer, Daniel S; Arlt, Volker M.; Colin S. Cooper; Phillips, David H.

    2014-01-01

    Background Gene expression changes induced by carcinogens may identify differences in molecular function between target and non-target organs. Target organs for benzo[a]pyrene (BaP) carcinogenicity in mice (lung, spleen and forestomach) and three non-target organs (liver, colon and glandular stomach) were investigated for DNA adducts by 32P-postlabelling, for gene expression changes by cDNA microarray and for miRNA expression changes by miRNA microarray after exposure of animals to BaP. Resul...

  12. 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

  13. 3,2'-Dimethyl-4-aminobiphenyl-DNA adduct formation in tumor target and nontarget organs of rapid and slow acetylator Syrian hamsters cogenic at the NAT2 locus.

    Science.gov (United States)

    Feng, Y; Jiang, W; Deitz, A C; Hein, D W

    1996-10-01

    DNA adduct formation is an important step in initiation of the carcinogenic process. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is a well-documented multiorgan carcinogenic aromatic amine in rodents. In the present study, DMABP-DNA adduct levels were measured in rapid (Bio. 82.73/H-Pat(r)) and slow (Bio. 82.73/H-Pat(s)) acetylator Syrian hamsters congenic at the NAT2 locus following a single injection of 33 or 100 mg/kg body wt DMABP. Two DNA adducts, N-(deoxyguanosin-8-yl)-DMABP and 5-(deoxyguanosin-N2-yl)-DMABP, were identified and quantitated by 32P-postlabeling assay. After injection of 33 mg/kg, DMABP-DNA adducts were detected in urinary bladder at 6, 18, 24, and 48 hr with adduct levels increasing up to 48 hr postinjection. DMABP-DNA adducts were not detected in liver, colon, and heart. After injection of 100 mg/kg, DMABP-DNA adducts were detected in urinary bladder, liver, prostate, colon, and heart at 48 hr postinjection. DMABP-DNA adduct levels were significantly higher in urinary bladder (primary tumor target organ) than in the other organs of both rapid and slow acetylator congenic hamsters. N-(deoxyguanosin-8-yl)-DMABP levels were significantly higher in liver and prostate than in colon and heart of rapid and slow acetylator congenic hamsters, whereas 5-(deoxyguanosin-N2-yl)-DMABP levels were significantly higher in prostate than in colon and heart of rapid and slow acetylator congenic hamsters. DMABP-DNA adduct levels in each tissue examined did not differ significantly between rapid and slow acetylator hamsters following either 33 or 100 mg/kg injection. The tissue-dependent differences in DMABP-DNA adduct levels observed in the Syrian hamster differ from those reported in the rat and are consistent with previous studies that show DMABP induces primarily urinary bladder tumors in the Syrian hamster. PMID:8887447

  14. Accumulation and persistence of nicotine derived DNA and hemoglobin adducts in mice after multiple administration of 14C-nicotine at low dose level

    International Nuclear Information System (INIS)

    The hypothetic role of nicotine in causing smoking related diseases has not been well established. Based on our early finding of the genotoxicity of nicotine, a sub-chronic study on the accumulation and persistence of nicotine derived DNA and hemoglobin (Hb) adducts in mice following multiple low dose exposures was carried out by accelerator mass spectrometry (AMS). AMS is a sophisticated ultrasensitive nuclear method, which facilitates the detection of adduction of DNA and other bio-macromolecules with xenobiotics at human relevant environmental dose levels. Briefly, in this study [N-14CH3]-nicotine (s.a. 16.2 μCi/μmol) was administered to mice by gavage once daily at 3.0 μg/kg b.w., which is equivalent to an estimated nicotine dose inhaled by a 70 kg person smoking 5 cigarettes, for 14 consecutive days. Lung DNA, liver DNA and Hb were isolated from tissues and blood samples which were collected at 1, 3, 5, 7, 10, 14, 15, 17, 21 and 25 days time point, respectively. (orig.)

  15. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

    Directory of Open Access Journals (Sweden)

    Zhi Liu

    Full Text Available The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue-DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of

  16. In vitro studies of the genotoxic effects of bitumen and coal-tar fume condensates: comparison of data obtained by mutagenicity testing and DNA adduct analysis by 32P-postlabelling.

    Science.gov (United States)

    De Méo, M; Genevois, C; Brandt, H; Laget, M; Bartsch, H; Castegnaro, M

    1996-08-14

    Bitumens contain traces of polycyclic aromatic compounds (PACs), a part of which will end up in the fumes emitted during hot handling of bitumen-containing products, e.g. during roadpaving. Although exposure of workers to these fumes is low, it might lead to health problems. Studies on bitumen fume condensates (BFCs) showed weak to moderate mutagenic activities, but studies on DNA adduct formation have not been reported. Therefore, a study was initiated in which fumes were generated from two road grade bitumens, in such a way that they were representative of the fumes produced in the field. The combined vapour/particulates were tested in vitro for their ability to produce DNA adducts and in modified Ames mutation assays, using a number of different strains. An attempt was made to relate the results to chemical data, such as the content of a number of individual polycyclic aromatic hydrocarbons (PAHs) and with a measure for the total PAC content. As a reference material fume condensate from coal-tar (coal-tar pitch volatiles; CTPV) were subjected to the same tests. All fume condensates tested were mutagenic to all strains and induced the formation of DNA adducts. The patterns of DNA adducts, obtained by 32P-postlabelling, arising from the BFCs were qualitatively different from the patterns of adducts obtained from the CTPVs, implying qualitative differences in the nature of the compounds responsible for the formation of these adducts. This is corroborated by the observation that for BFCs quantitative adduct levels are higher than would be expected based on the PAH content. These data thus indicate that the PAHs analysed are not the sole components responsible for adduct formation from BFCs, but that an important contribution comes from other (hetero- and/or substituted-) PACs. PMID:8760390

  17. DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo

    International Nuclear Information System (INIS)

    The presence of relatively high levels of prostaglandin H synthase (PHS) in the dog urinary bladder and its ability to mediate the activation of carcinogenic arylamines to DNA-bound products in vitro suggests the involvement of this enzyme in arylamine-induced bladder carcinogenesis. Since the PHS-dependent metabolism of 2-naphthylamine (2-NA) had been shown to yield both ring- and N-oxidation products in vitro, the authors compared the reactivity of 3H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene, and 2-amino-1-naphthol (2-AN) toward DNA and protein. In the PHS-incubation system, all three derivatives bound at high levels to protein, but only N-OH-2-NA and 2-AN bound appreciably to DNA. Though ring-oxidation has usually been considered a detoxification pathway, the covalent binding of [3H]2-AN to DNA was found to occur readily under aerobic conditions and was enhanced at acidic pH. At pH 5 in air, the reactivity of [3H]2-AN with nucleic acids and protein was in the order: serum albumin greater than tRNA greater than poly G greater than poly C greater than DNA greater than poly A greater than rRNA greater than poly U. Enzymatic hydrolysis of DNA reacted with [3H]2-AN and subsequent analysis by h.p.l.c. indicated the presence of several carcinogen-nucleoside adducts. The major product was characterized as N4-(deoxyguanosin-N2-yl)-2-amino-1,4-naphthoquinoneimine; and two minor products were tentatively identified as N4-(deoxyadenosin-N6-yl)-2-amino-1,4-naphthoquinoneimine and a deoxyguanosin-N2-yl adduct of a naphthoquinoneimine dimer. These adducts accounted for approximately 60% of the total DNA binding obtained by incubation of [3H]2-NA with PHS in vitro and for approximately 20% of the [3H]2-NA bound to dog urothelial DNA in vivo

  18. 3,N4-ethenocytosine, a highly mutagenic adduct, is a primary substrate for Escherichia coli double-stranded uracil-DNA glycosylase and human mismatch-specific thymine-DNA glycosylase

    OpenAIRE

    Saparbaev, Murat; Laval, Jacques

    1998-01-01

    Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,N4-ethenocytosine (ɛC), 1,N6-ethenoadenine (ɛA), N2,3-ethenoguanine, and 1,N2-ethenoguanine cause mutations. The ɛA residues are excised by the human and the Escherichia coli 3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repa...

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

    OpenAIRE

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

    2014-01-01

    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 mutag...

  20. Detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-treated human fibroblasts by means of immunofluorescence microscopy and quantitative immunoautoradiography

    International Nuclear Information System (INIS)

    An immunohistochemical procedure was developed for the detection of adducts in DNA of cultured cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) with the use of antibodies raised in rabbits against N-(guanosin-8-yl)-2-acetyl-aminofluorene (Guo-8-AAF) conjugated to bovine serum albumin. Binding of these antibodies to the nuclear DNA was visualized by means of immunofluorescence microscopy with fluorescein-conjugated anti-rabbit-Ig antibodies, or by means of immunoautoradiography with 125I-labeled protein A. The dose-response curve obtained when the number of autoradiographic grains developed over the nuclei was plotted as a function of the concentration of N-AcO-AAF used to treat the cells, indicated that the extent of specific antibody-binding is determined by the amount of adducts in the cells. DNA modification levels allowing for 20% survival of the cells could be detected with the immunofluorescence technique, while cells exposed to concentrations of N-AcO-AAF resulting in 60% survival were still positive with the immunoautoradiographic method

  1. In vitro studies of the genotoxic effects of bitumen and coal-tar fume condensates: comparison of data obtained by mutagenicity testing and DNA adduct analysis by {sup 32}P-postlabelling

    Energy Technology Data Exchange (ETDEWEB)

    De Meo, M.; Genevois, C.; Brandt, H.; Laget, M.; Bartsch, H.; Castegnaro, M. [Laboratoire de Biogenotoxicologie et Mutagenese Environnementale, Marseille (France). Faculte de Pharmcie

    1996-08-14

    Bitumens contain traces of polycyclic aromatic compounds (PACs), a part of which will end up in fumes emitted during hot handling of bitumen-containing products, e.g. during road paving. Exposure of workers to these fumes might lead to health problems. Studies on bitumen fume condensates (BFCs) showed mutagenic activities, but studies on DNA adduct formation have not been reported. Thus a study was initiated in which fumes were generated from two road grade bitumens which were representative of the fumes produced in the field. The combined vapour/particulates were tested in vitro for their ability to produce DNA adducts and in modified Ames mutation assays. Results were related to chemical data, such as the content of a number of individual polycyclic aromatic hydrocarbons (PAHs) and with a measure for total PAC content. As a reference material fume condensate from coal tar (coal-tar pitch volatiles; CTPV) were subjected to the same tests. All fume condensates were tested were mutagenic to all strains and induced the formation of DNA adducts. The patterns of DNA adducts arising from the BFCs were qualitatively different from the patterns of adducts obtained from the CTPVs, implying qualitative differences in the nature of the compounds responsible for their formation. This is corroborated by the observation that for BFCs quantitative adduct levels are higher than would be expected based on the PAH content. These data thus indicate that the PAHs analysed are not the sole components responsible for adduct formation from BFCs, but that an important contribution comes from other (hetero- and/or substituted-) PACs. 32 refs., 2 figs., 3 tabs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Kun-Ming [Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Sacks, Peter G. [Department of Basic Sciences, College of Dentistry, New York University, New York, NY 10010 (United States); Spratt, Thomas E.; Lin, Jyh-Ming; Boyiri, Telih [Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Schwartz, Joel [University of Illinois, College of Dentistry, Chicago, IL 60612 (United States); Richie, John P.; Calcagnotto, Ana [Department of Public Health Sciences, Penn State College of Medicine, Hershey, PA 17033 (United States); Das, Arunangshu; Bortner, James [Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Zhao, Zonglin [Department of Basic Sciences, College of Dentistry, New York University, New York, NY 10010 (United States); Department of Environmental Medicine, School of Medicine, New York University, New York, NY 10010 (United States); Amin, Shantu [Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033 (United States); Guttenplan, Joseph [Department of Basic Sciences, College of Dentistry, New York University, New York, NY 10010 (United States); Department of Environmental Medicine, School of Medicine, New York University, New York, NY 10010 (United States); El-Bayoumy, Karam, E-mail: kee2@psu.edu [Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033 (United States)

    2009-05-22

    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.

  3. 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)

    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.

  4. 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.; Autrup, H.; Risom, L.; Hald, M.T.; Daneshvar, Bahram; Dragsted, Lars; Poulsen, H.E.; Loft, S.

    2002-01-01

    . Investigations of oxidative stress biomarkers produced inconclusive results. Oxidative DNA damage detected by the endonuclease III enzyme and 7-hydro-8-oxo-2'-deoxyguanosine in colon, liver and/or urine was unaltered by IQ. However, there was increased level of gamma-glutamyl semialdehyde in liver proteins......The contribution of oxidative stress, different types of DNA damage and expression of DNA repair enzymes in colon and liver mutagenesis induced by 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in four groups of six Big Blue rats fed diets with 0, 20, 70, and 200 mg IQ/kg for 3...... systemic oxidative stress. However, the level of total vitamin C was increased in plasma, with the largest fraction being in the reduced form. In conclusion, our results indicate that DNA adducts rather than oxidative stress are responsible for the initiation of IQ-induced carcinogenesis of the liver and...

  5. Reduced nucleotide excision repair and GSTM1-null genotypes influence anti-B(a)PDE-DNA adduct levels in mononuclear white blood cells of highly PAH-exposed coke oven workers

    Energy Technology Data Exchange (ETDEWEB)

    Sofia Pavanello; Alessandra Pulliero; Ewa Siwinska; Danuta Mielzynska; Erminio Clonfero [University of Padova, Padova (Italy). Occupational Health Section, Department of Environmental Medicine and Public Health

    2005-07-01

    It is important to identify the potential genetic-susceptible factors that are able to modulate individual responses to exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). In the present study we evaluated the influence of four polymorphisms of nucleotide excision repair (NER) genes and that of glutathione S-transferase {mu}1 (GSTM1-active or -null) on benzo(a)pyrene diol epoxide (B(a)PDE)-DNA adduct levels from the lympho-monocyte fraction (LMF) of highly PAH benzo(a)pyrene -exposed Polish coke oven workers with individual urinary post-shift excretion of 1-pyrenol exceeding the proposed biological exposure index. The bulky {+-}-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-B(a)PDE)-DNA adduct levels were detected by high-performance liquid chromatography fluorescence analysis and genotypes by polymerase chain reaction. We found that workers with the low DNA repair capacity of XPC-PAT+/+ and XPA-A23A genotypes had increased anti-B(a)PDE-DNA adduct levels, DNA adducts were also raised in workers without GSTM1 activity. Workers with unfavourable XPC-PAT+/+ and XPA-A23A NER genotypes, alone or combined with GSTM1-null genotype were in the tertile with the highest adduct level. The increase in anti-B(a)PDE-DNA adduct levels was related in a multiple linear regression analysis to PAH exposure lack of GSTM1 activity and to low DNA repair capacity of the XPC-PAT+/+ genotype. The influence of the XPA-A23A genotype was not evident in this statistical analysis, and no associations with XPD polymorphisms, dietary habits or tobacco smoking were found. The modulation of anti-B(a)PDE-DNA adducts in the LMF by GSTM1-null and some low-activity NER genotypes may be considered as a potential genetic susceptibility factor capable of modulating individual responses to PAH genotoxic exposure and the consequent risk of cancer in coke oven workers.

  6. 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

  7. 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 ostatní: 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

  8. Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

    Science.gov (United States)

    Pan, Jishen; Sinclair, Elizabeth; Xuan, Zhuoli; Dyba, Marcin; Fu, Ying; Sen, Supti; Berry, Deborah; Creswell, Karen; Hu, Jiaxi; Roy, Rabindra; Chung, Fung-Lung

    2016-07-01

    The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125μM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA. PMID:27036235

  9. Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N[superscript 2]-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Christov, Plamen P.; Kozekova, Albena; Rizzo, Carmelo J.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2014-10-02

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of {omega}-6 polyunsaturated fatty acids in vivo. Michael addition of the N{sub 2}-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N{sub 2}-dGuo (1,N{sub 2}-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua {yields} Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, the (6S,8R,11S)-1,N

  10. 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

    2015-09-30

    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 <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-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

  11. 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

  12. High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts.

    Science.gov (United States)

    Hemeryck, Lieselot Y; Decloedt, Anneleen I; Vanden Bussche, Julie; Geboes, Karen P; Vanhaecke, Lynn

    2015-09-10

    Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O(6)-methylguanine (O(6)-MeG), O(6)-carboxymethylguanine (O(6)-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation. PMID:26388482

  13. Chromosomal aberrations and oxidative DNA adduct 8-hydroxy-2-deoxyguanosine as biomarkers of radiotoxicity in radiation workers

    Directory of Open Access Journals (Sweden)

    Sanaa A. El-Benhawy

    2016-07-01

    Conclusions: Scoring of chromosome aberrations such as breaks, fragments and dicentrics is a reliable method to detect previous exposure to ionizing radiation. This type of monitoring may be used as a biological dosimeter instead of physical dosimetry.8-OHdG is a useful oxidative DNA marker among radiation workers and those exposed to environmental carcinogens.

  14. 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 ostatní: 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

  15. 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.

  16. Structural and Functional Analysis of Sulfolobus solfataricus Y-Family DNA Polymerase Dpo4-Catalyzed Bypass of the Malondialdehyde−Deoxyguanosine Adduct

    Energy Technology Data Exchange (ETDEWEB)

    Eoff, Robert L.; Stafford, Jennifer B.; Szekely, Jozsef; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter; Marnett, Lawrence J.; (Vanderbilt)

    2010-01-12

    Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-{alpha}]purin-10(3H)-one (M{sub 1}dG). When paired opposite cytosine in duplex DNA at physiological pH, M{sub 1}dG undergoes ring opening to form N{sup 2}-(3-oxo-1-propenyl)-dG (N{sup 2}-OPdG). Previous work has shown that M{sub 1}dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M{sub 1}dG. To probe the mechanism by which translesion polymerases bypass M{sub 1}dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M{sub 1}dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M{sub 1}dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M{sub 1}dG. Two crystal structures were determined, including a 'type II' frameshift deletion complex and another complex with Dpo4 bound to a dC-M{sub 1}dG pair located in the postinsertion context. Importantly, M{sub 1}dG was in the ring-closed state in both structures, and in the structure with dC opposite M{sub 1}dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M{sub 1}dG and illustrate how the lesion may affect replication events.

  17. Binding of mismatch repair protein MutS to mispaired DNA adducts of intercalating ruthenium(II) arene complexes

    Czech Academy of Sciences Publication Activity Database

    Castellano-Castillo, M.; Kostrhunová, Hana; Marini, Victoria; Kašpárková, Jana; Sadler, P.J.; Malinge, J.-M.; Brabec, Viktor

    2008-01-01

    Roč. 13, - (2008), s. 993-999. ISSN 0949-8257 R&D Projects: GA AV ČR(CZ) KAN200200651; GA AV ČR(CZ) 1QS500040581; GA MŠk(CZ) LC06030 Grant ostatní: GA MŠk(CZ) ME08017 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * ruthenium * mismatch repair Subject RIV: BO - Biophysics Impact factor: 3.600, year: 2008

  18. Binding of MMR protein MutS to mispaired DNA adducts of intercalating Ru(II) arene complexes

    Czech Academy of Sciences Publication Activity Database

    Kostrhunová, Hana; Marini, Victoria; Kašpárková, Jana; Sadler, P.J.; Malinge, J.-M.; Brabec, Viktor

    Wroclaw, 2008. s. 219. ISBN 978-83-60043-10-3. [9th European Biological Inorganic Chemistry Conference. 02.09.2008-06.09.2008, Wroclaw] R&D Projects: GA ČR(CZ) GA305/05/2030; GA MŠk(CZ) LC06030; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : Ru(II) arene complexes * DNA * MutS Subject RIV: BO - Biophysics

  19. 32P-postlabeling assay for carcinogen-DNA adducts: description of beta shielding apparatus and semi-automatic spotting and washing devices that facilitate the handling of multiple samples

    International Nuclear Information System (INIS)

    The utilization of the 32P-postlabeling assay in combination with TLC for the sensitive detection and estimation of aromatic DNA adducts has been increasing. The procedure consists of 32P-labeling of carcinogen-adducted 3'-nucleotides in the DNA digests using γ-32P ATP and polynucleotide kinase, separation of 32P-labeled adducts by TLC, and their detection by autoradiography. During both 32P-labeling and initial phases of TLC, a relatively high amount of γ-32P 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 32P beta radiation, but also allow quick and easy handling of a large number of samples. Specifically, the equipment includes: (i) a multi-tube carousel rack having 15 wells to hold capless Eppendorf tubes and a rotatable lid with an aperture to access individual tubes; (ii) a pipette shielder; (iii) two semi-automatic spotting devices to apply radioactive solutions to TLC plates; (iv) a multi-plate holder for TLC plates; and (v) a mechanical device for washing multiple TLC plates. Item (i) is small enough to be held in one-hand, vortexed, and centrifuged to mix the solutions in each tube while beta radiation is shielded. Items (iii) to (iv) aid in the automation of the assay. (author)

  20. Aflatoxin B1-Associated DNA Adducts Stall S Phase and Stimulate Rad51 foci in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Michael Fasullo

    2010-01-01

    Full Text Available AFB1 is a potent recombinagen in budding yeast. AFB1 exposure induces RAD51 expression and triggers Rad53 activation in yeast cells that express human CYP1A2. It was unknown, however, when and if Rad51 foci appear. Herein, we show that Rad53 activation correlates with cell-cycle delay in yeast and the subsequent formation of Rad51 foci. In contrast to cells exposed to X-rays, in which Rad51 foci appear exclusively in G2 cells, Rad51 foci in AFB1-exposed cells can appear as soon as cells enter S phase. Although rad51 and rad4 mutants are mildly sensitive to AFB1, chronic exposure of the NER deficient rad4 cells to AFB1 leads to increased lag times, while rad4 rad51 double mutants exhibit synergistic sensitivity and do not grow when exposed to 50 μM AFB1. We suggest RAD51 functions to facilitate DNA replication after replication fork stalling or collapse in AFB1-exposed cells.

  1. DNA single strand breakage, DNA adducts, and sister chromatid exchange in lymphocytes and phenanthrene and pyrene metabolites in urine of coke oven workers.

    OpenAIRE

    W. Popp; Vahrenholz, C.; Schell, C; Grimmer, G.; Dettbarn, G; Kraus, R.; Brauksiepe, A; Schmeling, B; Gutzeit, T; von Bülow, J; Norpoth, K

    1997-01-01

    OBJECTIVES: To investigate the specificity of biological monitoring variables (excretion of phenanthrene and pyrene metabolites in urine) and the usefulness of some biomarkers of effect (alkaline filter elution, 32P postlabelling assay, measurement of sister chromatid exchange) in workers exposed to polycyclic aromatic hydrocarbons (PAHs). METHODS: 29 coke oven workers and a standardised control group were investigated for frequencies of DNA single strand breakage, DNA protein cross links (al...

  2. Effect of increased intake of dietary animal fat and fat energy on oxidative damage, mutation frequency, DNA adduct level and DNA repair in rat colon and liver

    DEFF Research Database (Denmark)

    Vogel, Ulla Birgitte; Danesvar, B.; Autrup, H.;

    2003-01-01

    liver, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat colon...... was observed. Intake of lard fat resulted in increased ascorbate synthesis and affected markers of oxidative damage to proteins in liver cytosol, but not in plasma. The effect was observed at all lard doses and was not dose-dependent. However, no evidence of increased oxidative DNA damage was found in...

  3. 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

    2016-03-01

    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. Environ. Mol. Mutagen. 57:125-136, 2016. © 2016 Wiley Periodicals, Inc. PMID:26734915

  4. 18. Adduct detection in human monitoring for carcinogen exposure

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Determination of the covalently bound products (adducts) of carcinogens with DNA or proteins may be used for the monitoring of exposure to these compounds. Protein adducts are generally stable and are not enzymatically repaired, and the use of these for cxposure monitoring is normally carried out with globin or albumin, because

  5. 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)

    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 P32 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

  6. 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 ostatní: 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

  7. The Impact of Glucuronidation on the Bioactivation and DNA Adduction of the Cooked-Food Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M A; Ubick, E A; Felton, J S

    2005-03-31

    UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of many different chemicals. Glucuronidation is especially important for detoxifying reactive intermediates from metabolic reactions, which otherwise can be biotransformed into highly reactive cytotoxic or carcinogenic species. Detoxification of certain food-borne carcinogenic heterocyclic amines (HAs) is highly dependent on UGT1A-mediated glucuronidation. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant carcinogenic HA found in well-done cooked meat, is extensively glucuronidated by UGT1A proteins. In humans, CYP1A2 catalyzed N-hydroxylation and subsequent UGT1A-mediated glucuronidation is a dominant pathway in the metabolism of PhIP. Therefore, changes in glucuronidation rates could significantly alter PhIP metabolism. To determine the importance of UGT1A-mediated glucuronidation in the biotransformation of PhIP, UGT1A proficient Wistar and UGT1A deficient Gunn rats were exposed to a single 100 {micro}g/kg oral dose of [{sup 14}C]-PhIP. Urine was collected over 24 h and the PhIP urinary metabolite profiles were compared between the two strains. After the 24 h exposure, livers and colon were removed and analyzed for DNA adduct formation by accelerator mass spectrometry. Wistar rats produced several PhIP and N-hydroxy-PhIP glucuronides that accounted for {approx}25% of the total amount of recovered urinary metabolites. In the Gunn rats, PhIP and N-hydroxy-PhIP glucuronides were reduced by 68-92%, compared to the Wistar rats, and comprised only 4% of the total amount of recovered urinary metabolites. PhIP-DNA adduct analysis from the Gunn rats revealed a correlation between reduced PhIP and N-hydroxy-PhIP glucuronide levels in the urine and increased hepatic DNA adducts, compared to the Wistar rats. These results indicate that UGT1A-mediated glucuronidation of PhIP and N-hydroxy-PhIP is an important pathway for PhIP detoxification. Failure to form glucuronide conjugates

  8. 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)

    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

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

    DEFF Research Database (Denmark)

    Autrup, Herman; Daneshvar, Bahram; Dragsted, Lars Ove; Gamborg, Mikael; Hansen, Åse Marie; Loft, Steffen; Okkels, Henrik; Nielsen, Flemming; Nielsen, Per Sabro; Raffn, Edith; Wallin, Håkan; Knudsen, Lisbeth Ehlert

    1999-01-01

    = 0.001). Significant correlations were also observed between urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and AAS in plasma (p = 0.001) and PAM-albumin adducts (p = 0.002). The influence of the glutatione S-transferase (GST) M1 deletion on the correlation between the biomarkers was studied in the...

  10. 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)

    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 × 108 dm3 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)

  11. Acetaldehyde adducts with hemoglobin.

    OpenAIRE

    Stevens, V.J.; Fantl, W J; Newman, C B; Sims, R V; Cerami, A.; Peterson, C M

    1981-01-01

    Clinical studies on the minor hemoglobins (hemoglobin A1a-c) have suggested that a novel adduct may form in people abusing alcohol. Such patients were found to have an elevated concentration of minor hemoglobins, but normal or subnormal amounts of glycosylated hemoglobin (hemoglobin A1c) as determined by radioimmunoassay, Acetaldehyde, a reactive metabolite of ethanol, was postulated to form adducts with hemoglobin A that change its chromatographic properties. At physiological concentrations,...

  12. In vivo DNA/RNA adduction of 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BaP) in the liver of rainbow trout (Oncorhynchus mykiss)

    Energy Technology Data Exchange (ETDEWEB)

    Schnitz, A.R.; O' Connor, J.M. (New York University Medical Center, NY (United States))

    1992-07-01

    Juvenile rainbow trout were exposed via two intramuscular injections to either 14C-DMBA for 24 hr or 14C-BaP for 48 hr, after which the livers were removed for DNA extraction and analysis. In the fish exposed to 14C-BaP, 0.2 ng was bound to the DNA, representing 0.5% of the total liver PAH-derived radioactivity and 2.38% of the administered dose. Liver DNA and RNA were found to contain 0.5% of the administered dose, respectively. Liver analysis of rainbow trout exposed to 14C-DMBA demonstrated that 0.4 ng and 0.3 ng were bound to the DNA and RNA, respectively. This represents 1.0% and 0.6% of the liver DMBA burden, respectively. The DNA adduct concentrations formed were comparable to both in vitro and in vivo experiments with both mammals and fishes, indicating that relatively small, environmentally realistic' doses of PAH have the ability to bind significantly to critical cellular macromolecules of young fish in vivo.

  13. 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

    International Nuclear Information System (INIS)

    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

  14. 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

  15. Correlation of 32P-postlabelling-detection of DNA adducts in mouse skin in vivo with the polycyclic aromatic compound content and mutagenicity in Salmonella typhimurium of a range of oil products

    International Nuclear Information System (INIS)

    The in vivo genotoxic activities in mouse skin of the dimethyl sulphoxide (DMSO) extracts of a range of oil products [residual aromatic extract; untreated heavy paraffinic distillate aromatic extract; mildly refined light naphthenic base oil; bitumen (vacuum residue); high viscosity index base oil obtained by catalytic hydrogenation] were evaluated by 32P-postlabelling DNA analysis. The results of quantitative 32P-postlabelling analyses of epidermal DNA from mice treated with the DMSO extracts showed linear relationships with the total polycyclic aromatic compound (PAC) contents, determined by the Institute of Petroleum method IP 346 and also the 3-6 ring PAC contents, measured by on-line liquid-liquid extraction using flow injection analysis. The 32P-postlabelling data also showed a linear relationship with the mutagenicity indices of these oil products determined in S. typhimurium TA98 using the modified Ames Salmonella microsome test. The in vivo genotoxicity of the DMSO extracts from the oil products was low, judged by 32P-postlabelling analysis of DNA adducts measured in epidermal DNA of treated mouse skin, and ranging from 2 to 723 attomole/μg DNA per mg oil product. The in vivo 32P-postlabelling data from this study are consistent with these materials expressing low genotoxicity in mouse skin in vivo. The DMSO extraction procedure coupled with 32P-postlabelling DNA analysis is useful for ranking the relative genotoxic potency in vivo of a wide range of oil products. In general the trend observed is similar to rankings based on physicochemical measurements of total PAC contents or 3-6 ring PAC contents of the oil products. (orig.)

  16. Correlation of {sup 32}P-postlabelling-detection of DNA adducts in mouse skin in vivo with the polycyclic aromatic compound content and mutagenicity in Salmonella typhimurium of a range of oil products

    Energy Technology Data Exchange (ETDEWEB)

    Booth, E.D.; Loose, R.W.; Watson, W.P. [Toxicology Department, Shell International Chemicals B.V., Amsterdam (Netherlands); Brandt, H.C.A. [Product Development Department, Shell International Oil Products B.V., Amsterdam (Netherlands)

    1998-07-01

    The in vivo genotoxic activities in mouse skin of the dimethyl sulphoxide (DMSO) extracts of a range of oil products [residual aromatic extract; untreated heavy paraffinic distillate aromatic extract; mildly refined light naphthenic base oil; bitumen (vacuum residue); high viscosity index base oil obtained by catalytic hydrogenation] were evaluated by {sup 32}P-postlabelling DNA analysis. The results of quantitative {sup 32}P-postlabelling analyses of epidermal DNA from mice treated with the DMSO extracts showed linear relationships with the total polycyclic aromatic compound (PAC) contents, determined by the Institute of Petroleum method IP 346 and also the 3-6 ring PAC contents, measured by on-line liquid-liquid extraction using flow injection analysis. The {sup 32}P-postlabelling data also showed a linear relationship with the mutagenicity indices of these oil products determined in S. typhimurium TA98 using the modified Ames Salmonella microsome test. The in vivo genotoxicity of the DMSO extracts from the oil products was low, judged by {sup 32}P-postlabelling analysis of DNA adducts measured in epidermal DNA of treated mouse skin, and ranging from 2 to 723 attomole/{mu}g DNA per mg oil product. The in vivo {sup 32}P-postlabelling data from this study are consistent with these materials expressing low genotoxicity in mouse skin in vivo. The DMSO extraction procedure coupled with {sup 32}P-postlabelling DNA analysis is useful for ranking the relative genotoxic potency in vivo of a wide range of oil products. In general the trend observed is similar to rankings based on physicochemical measurements of total PAC contents or 3-6 ring PAC contents of the oil products. (orig.) With 4 figs., 1 tab., 44 refs.

  17. 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome P4501A1 and rapid or slow acetylator N-acetyltransferase 2.

    Science.gov (United States)

    Bendaly, Jean; Zhao, Shuang; Neale, Jason R; Metry, Kristin J; Doll, Mark A; States, J Christopher; Pierce, William M; Hein, David W

    2007-07-01

    2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism

  18. 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.

  19. 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.

    Science.gov (United States)

    Siddens, Lisbeth K; Bunde, Kristi L; Harper, Tod A; McQuistan, Tammie J; Löhr, Christiane V; Bramer, Lisa M; Waters, Katrina M; Tilton, Susan C; Krueger, Sharon K; Williams, David E; Baird, William M

    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 8h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8h 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 4h 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 8h 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. PMID:26049101

  20. 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

    International Nuclear Information System (INIS)

    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

  1. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5′ Adducts

    OpenAIRE

    Hong Yan; Margaret Tammaro; Shuren Liao

    2016-01-01

    Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5′ end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-prot...

  2. 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)

    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 b5, 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 b5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b5 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 b5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR

  3. DNA Repair by Reversal of DNA Damage

    OpenAIRE

    Yi, Chengqi; He, Chuan

    2013-01-01

    Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-,...

  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. Assessment of three classes of DNA adducts in human placentas from smoking and non-snoking women in the Czech Republic

    Science.gov (United States)

    Three classes of DNA damage were assessed in human placentas collected (2000-2004) 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 ...

  7. A physiologically based in silico model for trans-2-hexenal detoxicifcation and DNA adduct formation in human including interindividual variation indicates efficient detoxification and a negligible genotoxicity risk

    NARCIS (Netherlands)

    Kiwamoto, R.; Spenkelink, A.; Rietjens, I.M.C.M.; Punt, A.

    2013-01-01

    A number of a,ß-unsaturated aldehydes are present in food both as natural constituents and as flavouring agents. Their reaction with DNA due to their electrophilic a,ß-unsaturated aldehyde moiety may result in genotoxicity as observed in some in vitro models, thereby raising a safety concern. A ques

  8. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5′ Adducts

    Directory of Open Access Journals (Sweden)

    Hong Yan

    2016-07-01

    Full Text Available Topoisomerase 2 (Top2 is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5′ end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc. In this configuration, the two sides of the nicked DNA are held together by the strong protein-protein interactions between the two subunits of Top2, allowing the nicks to be faithfully resealed in situ. Top2ccs are normally transient, but can be trapped by cancer drugs, such as etoposide, and subsequently processed into DSBs in cells. If not properly repaired, these DSBs would lead to genome instability and cell death. Here, I review the current understanding of the mechanisms by which DSBs are induced by etoposide, the unique features of such DSBs and how they are repaired. Implications for the improvement of cancer therapy will be discussed.

  9. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5' Adducts.

    Science.gov (United States)

    Yan, Hong; Tammaro, Margaret; Liao, Shuren

    2016-01-01

    Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5' end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-protein interactions between the two subunits of Top2, allowing the nicks to be faithfully resealed in situ. Top2ccs are normally transient, but can be trapped by cancer drugs, such as etoposide, and subsequently processed into DSBs in cells. If not properly repaired, these DSBs would lead to genome instability and cell death. Here, I review the current understanding of the mechanisms by which DSBs are induced by etoposide, the unique features of such DSBs and how they are repaired. Implications for the improvement of cancer therapy will be discussed. PMID:27376333

  10. Quantitative evaluation of cellular uptake, DNA incorporation and adduct formation in cisplatin sensitive and resistant cell lines: Comparison of different Pt-containing drugs.

    Science.gov (United States)

    Corte-Rodríguez, M; Espina, M; Sierra, L M; Blanco, E; Ames, T; Montes-Bayón, M; Sanz-Medel, A

    2015-11-01

    The use of Pt-containing compounds as chemotherapeutic agents facilitates drug monitoring by using highly sensitive elemental techniques like inductively coupled plasma mass spectrometry (ICP-MS). However, methodological problems arise when trying to compare different experiments due to the high variability of biological parameters. In this work we have attempted to identify and correct such variations in order to compare the biological behavior of cisplatin, oxaliplatin and pyrodach-2 (a novel platinum-containing agent). A detailed study to address differential cellular uptake has been conducted in three different cell lines: lung adenocarcinoma (A549); cisplatin-sensitive ovarian carcinoma (A2780); and cisplatin-resistant ovarian carcinoma (A2780cis). The normalization of Pt results to cell mass, after freeze-drying, has been used to minimize the errors associated with cell counting. Similarly, Pt accumulation in DNA has been evaluated by referencing the Pt results to the DNA concentration, as measured by (31)P monitoring using flow-injection and ICP-MS detection. These strategies have permitted to address significantly lower Pt levels in the resistant cells when treated with cisplatin or oxaliplatin as well as an independent behaviour from the cell type (sensitive or resistant) for pyrodach-2. Similarly, different levels of incorporation in DNA have been found for the three drugs depending on the cell model revealing a different behavior regarding cell cisplatin resistance. Further speciation experiments (by using complementary HPLC-ICP-MS and HPLC-ESI-Q-TOF MS) have shown that the main target in DNA is still the N7 of the guanine but with different kinetics of the ligand exchange mechanism for each of the compounds under evaluation. PMID:26352094

  11. Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-05-05

    Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA than that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study.

  12. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal.

    Science.gov (United States)

    Shireman, Laura M; Kripps, Kimberly A; Balogh, Larissa M; Conner, Kip P; Whittington, Dale; Atkins, William M

    2010-12-15

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  13. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal☆

    Science.gov (United States)

    Shireman, Laura M.; Kripps, Kimberly A.; Balogh, Larissa M.; Conner, Kip P.; Whittington, Dale; Atkins, William M.

    2010-01-01

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro- 2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  14. Metabolism of benzo(a)pyrene and identification of the major benzo(a)pyrene-DNA adducts in cultured human colon

    DEFF Research Database (Denmark)

    1978-01-01

    The metabolism of benzo(a)pyrene in cultured human colon has been investigated. Nontumorous colonie tissue was collected at the time of either surgery or "immediate autopsy" from patients with or without colonic cancer. After 24 hr in culture the expiants were exposed to [3H]benzo(a)pyrene for an...... can metabolize benzo(a)pyrene by pathways similar to those found in human bronchus and in cells of experimental animals....... acid increased the binding levels of benzo(a)pyrene to DNA. An increased level of frans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in the culture media was also observed when expiants were coincubated with either taurodeoxycholic acid or lithocholic acid. These results indicate that cultured human colon......The metabolism of benzo(a)pyrene in cultured human colon has been investigated. Nontumorous colonie tissue was collected at the time of either surgery or "immediate autopsy" from patients with or without colonic cancer. After 24 hr in culture the expiants were exposed to [3H]benzo(a)pyrene for...

  15. Effect of the diaminocyclohexane carrier ligand on platinum adduct formation, repair, and lethality

    International Nuclear Information System (INIS)

    Platinum compounds with the diaminocyclohexane (dach) carrier ligand are of particular interest because cell lines that have developed resistance to platinum compounds in general often retain sensitivity to dach-platinum compounds, suggesting that the dach carrier ligand affects the formation, repair, or lethality of platinum-DNA adducts. The effect of the dach ligand on platinum adduct formation was assessed by using the (HaeIII-HindIII)146 fragment of pBR322 treated to give equal amounts of dach- or ethylene-diamine-platinum adducts. The sites of adduct formation were mapped by digestion with Escherichia coli ABC excinuclease. There were no significant effects of the dach carrier ligand on the types or sites of platinum adduct formation. The effect of the dach ligand on platinum adduct repair was determined by using synthetic oligomers designed to have single, specific platinum adducts. These data suggest that if the carrier ligand has any effect on the repair of platinum adducts, it is more likely to exert that effect on the repair of platinum monoadducts or GNG diadducts rather than on the more abundant AG or GG diadducts. [14C]Thiourea incorporation was used to quantitate the rate of monoadduct to diadduct conversion. Finally, the effect of the dach ligand on platinum adduct lethality was assessed by determining the effect of dach- and en-platinum adducts on the transformation efficiency of pBR322 into a repair-deficient (recA- uvrA-) strain of E. coli. These data suggest that the dach carrier ligand can significantly affect the ability of platinum-DNA adducts to block essential processes such as replication and transcription

  16. Marked dependence on carrier-ligand bulk but not on carrier-ligand chirality of the duplex versus single-strand forms of a DNA oligonucleotide with a series of G-Pt(II)-G intrastrand cross-links modeling cisplatin-DNA adducts.

    Science.gov (United States)

    Beljanski, Vladimir; Villanueva, Julie M; Doetsch, Paul W; Natile, Giovanni; Marzilli, Luigi G

    2005-11-16

    The N7-Pt-N7 adjacent G,G intrastrand DNA cross-link responsible for cisplatin anticancer activity is dynamic, promotes local "melting" in long DNA, and converts many oligomer duplexes to single strands. For 5'-d(A1T2G3G4G5T6A7C8C9C10A11T12)-3' (G3), treatment of the (G3)2 duplex with five pairs of [LPt(H2O)2]2+ enantiomers (L = an asymmetric diamine) formed mixtures of LPt-G3 products (1 Pt per strand) cross-linked at G3,G4 or at G4,G5 in all cases. L chirality exerted little influence. For primary diamines L with bulk on chelate ring carbons (e.g., 1,2-diaminocyclohexane), the duplex was converted completely into single strands (G3,G4 coils and G4,G5 hairpins), exactly mirroring results for cisplatin, which lacks bulk. In sharp contrast, for secondary diamines L with bulk on chelate ring nitrogens (e.g., 2,2'-bipiperidine, Bip), unexpectedly stable duplexes having two platinated strands (even a unique G3,G4/G4,G5 heteroduplex) were formed. After enzymatic digestion of BipPt-G3 duplexes, the conformation of the relatively nondynamic G,G units was shown to be head-to-head (HH) by HPLC/mass spectrometric characterization. Because the HH conformation dominates at the G,G lesion in duplex DNA and in the BipPt-G3 duplexes, the stabilization of the duplex form only when the L nitrogen adducts possess bulk suggests that H-bonding interactions of the Pt-NH groups with the flanking DNA lead to local melting and to destabilization of oligomer duplexes. The marked dependence of adduct properties on L bulk and the minimal dependence on L chirality underscore the need for future exploration of the roles of the L periphery in affecting anticancer activity. PMID:16277526

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

    International Nuclear Information System (INIS)

    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

  18. 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)

    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,N6-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

  19. 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

    The contribution of oxidative stress, different types of DNA damage and expression of DNA repair enzymes in colon and liver mutagenesis induced by 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in four groups of six Big Blue rats fed diets with 0, 20, 70, and 200 mg IQ/kg for 3....... Investigations of oxidative stress biomarkers produced inconclusive results. Oxidative DNA damage detected by the endonuclease III enzyme and 7-hydro-8-oxo-2'-deoxyguanosine in colon, liver and/or urine was unaltered by IQ. However, there was increased level of gamma-glutamyl semialdehyde in liver proteins......, 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...

  20. 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

    The contribution of oxidative stress, different types of DNA damage and expression of DNA repair enzymes in colon and liver mutagenesis induced by 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in four groups of six Big Blue rats fed diets with 0, 20, 70, and 200 mg IQ/kg for 3....... Investigations of oxidative stress biomarkers produced inconclusive results. Oxidative DNA damage detected by the endonuclease III enzyme and 7-hydro-8-oxo-2'-deoxyguanosine in colon, liver and/or urine was unaltered by IQ. However, there was increased level of gamma-glutamyl semialdehyde in liver proteins......, 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...

  1. Hafnium tetrachloride adducts with aminophenols

    International Nuclear Information System (INIS)

    Adducts of hafnium tetrachloride with aminophenols of a general composition of HfCl4x4L have been obtained by addition of ethyl acetate solutions of hafnium tetrachloride with solutions of o-aminophenol in dioxan, m-aminophenol in ethyl acetate and n-aminophenol in dioxan at a ratio Hf/L=1/2. In the investigated adducts, aminophenols are connected to hafnium both through an oxygen atom and a nitrogen atom, the latter's coordination being preferable. A thermal investigation of synthesized complexes has determined the quantity of heat evolving on addition of 4 moles of aminophenol to 1 mole of crystalline hafnium tetrachloride

  2. DNA adducts and human atherosclerotic lesions

    Czech Academy of Sciences Publication Activity Database

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

    Buffalo : Lippincott Williams Wilkins, 2000, s. 124. [ISEE/ISEA Conference 2000. Buffalo (US), 19.08.2000-23.08.2000] R&D Projects: GA MŽP SI/340/2/00 Grant ostatní: EU(XE) (PHARE II, AC/HEA/18) Subject RIV: DN - Health Impact of the Environment Quality

  3. Synthesis and mutagenesis of the butadiene-derived N3 2'-deoxyuridine adducts.

    Science.gov (United States)

    Fernandes, Priscilla H; Hackfeld, Linda C; Kozekov, Ivan D; Hodge, Richard P; Lloyd, R Stephen

    2006-07-01

    1,3-Butadiene is a known carcinogen and mutagen that acts through a variety of metabolic intermediates that react with DNA, forming stable and unstable lesions on dG, dA, dC, and dT. The N3 2'-deoxyuridine adducts are a highly stable, stereoisomeric mixture of adducts derived from the reaction of cytosine with the monoepoxide metabolite of butadiene, followed by spontaneous deamination. In this study, the phosphoramidites and subsequent oligodeoxynucleotides containing the N3 2'-deoxyuridine adducts have been constructed and characterized. Using a single-stranded shuttle vector DNA, the mutagenic potential of these adducts has been tested following replication in mammalian cells. Replication past the N3 2'-deoxyuridine adducts was found to be highly mutagenic with an overall mutation yield of approximately 97%. The major mutations that were observed were C to T transitions and C to A transversions. In vitro, these adducts posed a complete block to both the Klenow fragment of Escherichia coli polymerase I and polymerase epsilon, while these lesions significantly blocked polymerase delta. These data suggested a possible involvement of bypass polymerases in the in vivo replication of these lesions. Overall, these findings indicate that the N3 2'-deoxyuridine adducts are highly mutagenic lesions that may contribute to butadiene-mediated carcinogenesis. PMID:16841966

  4. Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation

    OpenAIRE

    Shrivastav, Nidhi; Li, Deyu; Essigmann, John M.

    2009-01-01

    The reaction of DNA-damaging agents with the genome results in a plethora of lesions, commonly referred to as adducts. Adducts may cause DNA to mutate, they may represent the chemical precursors of lethal events and they can disrupt expression of genes. Determination of which adduct is responsible for each of these biological endpoints is difficult, but this task has been accomplished for some carcinogenic DNA-damaging agents. Here, we describe the respective contributions of specific DNA les...

  5. Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay

    OpenAIRE

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-01-01

    Enzymes that form transient DNA–protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA–protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiia...

  6. The boron trifluoride nitromethane adduct

    Science.gov (United States)

    Ownby, P. Darrell

    2004-02-01

    The separation of the boron isotopes using boron trifluoride·organic-donor, Lewis acid·base adducts is an essential first step in preparing 10B enriched and depleted crystalline solids so vital to nuclear studies and reactor applications such as enriched MgB 2, boron carbide, ZrB 2, HfB 2, aluminum boron alloys, and depleted silicon circuits for radiation hardening and neutron diffraction crystal structure studies. The appearance of this new adduct with such superior properties demands attention in the continuing search for more effective and efficient means of separation. An evaluation of the boron trifluoride nitromethane adduct, its thermodynamic and physical properties related to large-scale isotopic separation is presented. Its remarkably high separation factor was confirmed to be higher than the expected theoretical value. However, the reportedly high acid/donor ratio was proven to be an order of magnitude lower. On-going research is determining the crystal structure of deuterated and 11B enriched 11BF 3·CD 3NO 2 by X-ray and neutron diffraction.

  7. Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

    OpenAIRE

    Sproviero, Michael; Verwey, Anne M.R.; Rankin, Katherine M.; Witham, Aaron A.; Soldatov, Dmitriy V.; Richard A. Manderville; Fekry, Mostafa I.; Sturla, Shana J.; Sharma, Purshotam; Wetmore, Stacey D.

    2014-01-01

    Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of auth...

  8. Formation of adduct of cerium (4) thenoyltrifluoroacetonate

    International Nuclear Information System (INIS)

    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

  9. Triple helix-forming oligonucleotides target psoralen adducts to specific chromosomal sequences in human cells.

    OpenAIRE

    Oh, D H; Hanawalt, P C

    1999-01-01

    The ability to target photochemical adducts to specific genomic DNA sequences in cells is useful for studying DNA repair and mutagenesis in intact cells, and also as a potential mode of gene-specific therapy. Triple helix-forming DNA oligonucleotides linked to psoralen (psoTFOs) were designed to deliver UVA-induced psoralen photoadducts to two distinct sequences within the human interstitial collagenase gene. A primer extension assay demonstrated that the appropriate psoTFO selectively damage...

  10. Bilateral failure of adduction following orbital decompression.

    OpenAIRE

    Kinsella, F; Kyle, P.; Stansfield, A

    1990-01-01

    We report a case of bilateral complete failure of adduction following bilateral translid antralethmoidal orbital decompression. We believe the probable mechanism is neuropraxia (temporary dysfunction) of the third cranial nerves' supply to the medial recti, owing to these nerves' occupying an anatomically abnormal position. Partial recovery of adduction occurred over the ensuing six months.

  11. Hydrogen abstraction reactions by amide electron adducts

    International Nuclear Information System (INIS)

    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-)ND2. 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)

  12. Peroxidase-catalyzed formation of (deoxy)guanosine adducts by Sudan I

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Semanská, M.; Cvačka, Josef; Martínek, V.; Stiborová, M.

    Brno : Masaryk University, 2009. C32-C32. ISBN 978-80-86441-40-5. [Central European NMR Meeting. NMR Valtice /24./. 26.04.2009-29.04.2009, Valtice] Institutional research plan: CEZ:AV0Z40550506 Keywords : Sudan I * DNA adducts * NMR Subject RIV: CC - Organic Chemistry

  13. Detection of 1,N2-propanodeoxyguanosine adducts of 2-hexenal in organs of Fischer 344 rats by a 32P-post-labeling technique.

    Science.gov (United States)

    Schuler, D; Eder, E

    1999-07-01

    2-Hexenal is an alpha,beta-unsaturated carbonyl compound which is mutagenic, genotoxic and forms cyclic 1,N2-propanodeoxyguanosine adducts like similar propenals for which carcinogenicity was shown, e.g. acrolein or crotonaldehyde. Since humans have a permanent intake of 2-hexenal via vegetarian food this genotoxic compound is considered to play a role in human carcinogenicity. The data base is, however, presently not sufficient for a cancer risk assessment. To date no long term carcinogenicity study on 2-hexenal has been published. Detection of respective DNA adducts of this substance in animals or humans could allow cancer risk assessment. Therefore, we have developed a 32P-post-labeling technique based on nuclease P1 enrichment and TLC separation of the labeled adducts. The respective adducts are stable over a wide pH range from pH 4 to pH 11 and relatively stable against nuclease P1. The detection limit was 0.03 adducts per 10(6) nucleotides and the recovery was 10%. With this method we have shown in vivo formation of 1,N 2-propanodeoxyguanosine adducts of 2-hexenal for the first time and found the respective DNA adducts in different organs of Fischer 344 rats after gavage of 500, 200 and 50 mg 2-hexenal/kg body wt. No adducts could be detected in the organs of untreated rats. There is a clear dependence of the adduct level and the CBI (covalent binding index) on the dose. The CBI of 2-hexenal calculated on the basis of our adduct levels is extremely low (0.06). Since intake of 2-hexenal via fruit and vegetables is very low the cancer risk from 2-hexenal intake via food must also be considered as very low according to a first raw estimation on the basis of CBI and intake. The situation deserves, however, a more precise risk assessment in the future. PMID:10383910

  14. Hip adduction and abduction strength profiles in elite soccer players

    DEFF Research Database (Denmark)

    Serner, Andreas; Petersen, Jesper; Madsen, Thomas Moller;

    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....

  15. 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-...

  16. New adducts of Lapachol with primary amines

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Mirelly D.F.; Litivack-Junior, Jose T.; Antunes, Roberto V.; Silva, Tania M.S.; Camara, Celso A., E-mail: ccelso@dq.ufrpe.b [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Dept. de Quimica

    2011-07-01

    New adducts of lapachol with neat primary aliphatic amines were obtained in a solvent-free reaction in good to reasonable yields (52 to 88%), at room temperature. The new compounds containing a phenazine moiety were obtained from suitable functionalized aminoalkyl compounds, including ethanolamine, 3-propanolamine, 2-methoxy-ethylamine, 3-methoxy-propylamine, n-butylamine and 2-phenetylamine. (author)

  17. uvrC gene function has no specific role in repair of N-2-aminofluorene adducts.

    OpenAIRE

    Bichara, M; Fuchs, R P

    1987-01-01

    In Escherichia coli, plasmid DNA modified with N-2-aminofluorene adducts survived equally well in wild-type, uvrA, or uvrB strains. Increased sensitivity was found in uvrC and uvrD strains. Moreover, N-2-aminofluorene-mediated toxicity in the uvrC background was reversed when an additional uvrA mutation was introduced into the strain.

  18. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal☆

    OpenAIRE

    Shireman, Laura M.; Kripps, Kimberly A.; Balogh, Larissa M.; Conner, Kip P.; Whittington, Dale; Atkins, William M.

    2010-01-01

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with cat...

  19. Detection of environmental carcinogens-DNA

    International Nuclear Information System (INIS)

    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 32P 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)

  20. Covalent adduction of nitrogen mustards to model protein nucleophiles.

    Science.gov (United States)

    Thompson, Vanessa R; DeCaprio, Anthony P

    2013-08-19

    Protein adducts have the potential to serve as unique biomarkers of exposure to compounds of interest. Many xenobiotics (or their metabolites) are electrophilic and therefore reactive with nucleophilic amino acid residues on proteins. Nitrogen mustards are reactive xenobiotics with potential use as chemical warfare agents (CWA) or agents of terrorist attack, in addition to being employed as chemotherapeutic agents. The present study utilized cysteine-, lysine-, and histidine-containing model peptides to characterize in vitro adduction of the nitrogen mustards mechloroethamine (HN-2) and tris-(2-chlorethyl)amine (HN-3) to these nucleophilic amino acid residues by means of liquid chromatography-tandem mass spectrometry. The study assessed the structure of adducts formed, the time course of adduct formation, concentration-response relationships, and temporal stability of adducts. Adduction was hypothesized to occur on all three model peptides via initial formation of a reactive aziridinium intermediate for both mechloroethamine and tris-(2-chlorethyl)amine, followed by covalent adduction to nucleophilic residues. While adduction was found to occur most readily with cysteine, it was also observed at lysine and histidine, demonstrating that adduction by mechloroethamine and tris-(2-chlorethyl)amine is possible at multiple nucleophilic sites. Following solid phase extraction cleanup, adducts formed with mechloroethamine were stable for up to three weeks. Adducts formed with tris-(2-chlorethyl)amine were less stable; however, hydrolyzed secondary adducts were observed throughout the three week period. This study demonstrates that the nitrogen mustards mechloroethamine and tris-(2-chlorethyl)amine form stable adducts with reactive protein nucleophiles other than cysteine. PMID:23859065

  1. Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells.

    Science.gov (United States)

    Kanuri, Manorama; Minko, Irina G; Nechev, Lubomir V; Harris, Thomas M; Harris, Constance M; Lloyd, R Stephen

    2002-05-24

    8-Hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin- 10(3H)-one,3-(2'-deoxyriboside) (1,N(2)-gamma-hydroxypropano deoxyguanosine, gamma-HOPdG) is a major DNA adduct that forms as a result of exposure to acrolein, an environmental pollutant and a product of endogenous lipid peroxidation. gamma-HOPdG has been shown previously not to be a miscoding lesion when replicated in Escherichia coli. In contrast to those prokaryotic studies, in vivo replication and mutagenesis assays in COS-7 cells using single stranded DNA containing a specific gamma-HOPdG adduct, revealed that the gamma-HOPdG adduct was significantly mutagenic. Analyses revealed both transversion and transition types of mutations at an overall mutagenic frequency of 7.4 x 10(-2)/translesion synthesis. In vitro gamma-HOPdG strongly blocks DNA synthesis by two major polymerases, pol delta and pol epsilon. Replicative blockage of pol delta by gamma-HOPdG could be diminished by the addition of proliferating cell nuclear antigen, leading to highly mutagenic translesion bypass across this adduct. The differential functioning and processing capacities of the mammalian polymerases may be responsible for the higher mutation frequencies observed in this study when compared with the accurate and efficient nonmutagenic bypass observed in the bacterial system. PMID:11889127

  2. ELISA-like Analysis of Cisplatinated DNA Using Magnetic Separation

    OpenAIRE

    Kristyna Smerkova; Marcela Vlcnovska; Simona Dostalova; Vedran Milosavljevic; Pavel Kopel; Tomas Vaculovic; Sona Krizkova; Marketa Vaculovicova; Vojtech Adam; Rene Kizek

    2015-01-01

    Cisplatin belongs to the most widely used cytostatic drugs. The determination of the presence of the DNA-cisplatin adducts may not only signal the guanine-rich regions but also monitor the interaction reaction between DNA and the drug in terms of speed of interaction. In this work, the combined advantages of magnetic particles-based isolation/purification with fluorescent properties of quantum dots (QDs) and antibodies targeted on specific recognition of DNA-cisplatin adducts are demonstra...

  3. Risk assessment of DNA-reactive carcinogens in food

    International Nuclear Information System (INIS)

    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 B1 (AFB1), a limit of 20 ppb or ∼30 μg/day has been set and is considered a tolerable daily intake (TDI). Since AFB1 is considered a potent carcinogen, doses of 32P-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

  4. Association of DNA adducts and genetic polymorphisms with birth weight

    Czech Academy of Sciences Publication Activity Database

    Šrám, Radim; Binková, Blanka; Dejmek, Jan; Chvátalová, Irena; Solanský, I.; Topinka, Jan

    2006-01-01

    Roč. 608, - (2006), s. 121-128. ISSN 1383-5718 R&D Projects: GA MŽP SL/5/160/05; GA MŽP SL/740/5/03; GA MŽP(CZ) SI/340/2/00 Institutional research plan: CEZ:AV0Z50390512 Keywords : pregnancy outcomes * birth weight * genetic polymorphism Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 2.122, year: 2006

  5. 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 ostatní: 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

  6. Properties of ferrocene derivative C60 adduct

    International Nuclear Information System (INIS)

    Properties of ferrocene derivative C60 adduct were investigated by thermal analyses, x-ray diffraction, 57Fe Moessbauer spectroscopy in order to examine interaction of iron with fullerene. Thermal treatment may be applied to remove organic groups to obtain sample containing C60Fe with C60 arranged in the fcc lattice and iron dispersed between fullerenes. We performed calculations based on the semi-empirical quantum chemistry model P M3 for a few exohedral complexes with Fe at various sites relatively to C60

  7. Intramolecular Tetrylene Lewis Adducts: Synthesis and Reactivity.

    Science.gov (United States)

    Schneider, Julia; Krebs, Kilian M; Freitag, Sarah; Eichele, Klaus; Schubert, Hartmut; Wesemann, Lars

    2016-07-01

    A series of benzyl(diphenylphosphino) and o-phenyl(diphenlyphosphino) substituted germylenes and plumbylenes were synthesized by nucleophilic substitution between the respective lithium reagent and tetrylene halide. The Lewis pairs were characterized by X-ray crystallography and NMR spectroscopy. The reactivity of the tetrylenes was investigated with respect to azide addition. In the germylene case, the germaniumimide was formed as the kinetically controlled product, which rearranges upon heating to give the phosphinimide. The stannylene and plumbylene derivatives react with adamantylazide to give the azide adducts. 1-Pentene reacts diastereoselectively with the phosphagermirane to give a cyclic addition product. Trimethysilylacetylene shows an addition with the benzylphosphino-substituted germylene and plumbylene to give the cycloheteropentene molecules. The addition product between phenylacetylene and the four membered Ge-P adduct shows after addition at room temperature a 1,4-phenylmigration to give a cyclic phosphine. Alkylnitrene insertion into a Ge-C bond of the alkyne addition product of the phosphagermirane was found in reaction with adamantylazide. PMID:27273819

  8. Suicide of EMT-6 tumor cells by decays from radioactively-labelled sensitizer adducts

    International Nuclear Information System (INIS)

    Nitroaromatic radiosensitizers become metabolically bound preferentially to hypoxic cells and at least 10/sup 9/ adducts/cell can be tolerated as non-toxic. EMT-6 tumor cells have been incubated in hypoxia in the presence of /sup 3/H-Misonidazole and /sup 125/I-Azomycin Riboside for various times and the amount of /sup 3/H or /sup 125/I bound/cell was determined. Cells were stored as monolayers at 250C for up to 96 hr to accumulate radioactive decays and transferred at various times to 370C for colony-forming assays. No radiation inactivation was measured in cells which had incorporated at least 10/sup 6/ /sup 3/H or 10/sup 5/ /sup 125/I atoms. Previous studies had shown that -- 1% of MISO adducts to EMT-6 cells was associated with cellular DNA. These data indicate that the radiation-induced damage produced by these quantities of bound /sup 3/H or /sup 125/I causes little or not cell inactivation. The results of current studies to measure the colony-forming ability of sensitizer-labelled cells which have been stored in liquid nitrogen to facilitate the accumulation of more decays will be reported. These data suggest that a ''sensitizer-adduct suicide technique'' as a hypoxic cell selective adjunct to other cancer therapies is not feasible. These data are also instructive for those who attempt to develop radiolabelled ''tumor specific'' antibodies for therapeutic purposes

  9. Fast repair of dAMP hydroxyl radical adduct by verbascoside via electron transfer

    Institute of Scientific and Technical Information of China (English)

    石益民; 王文锋; 姚思德; 林维真; 韩镇辉; 师彦平; 贾忠建; 郑荣梁

    1999-01-01

    DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very impotant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells’ inadequate repair capacity. The repair activity and its mechanism of verbaseoside, isolated from Pedicularis species, towards dAMP-OH·was studied with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mmol/L dAMP aqueous solution containing verbascoside, the transient absorption spectrum of the hydroxyl adduct of dAMP decayed with the formation of that of the phenoxyl radical of verbascoside well under 100 microseconds after electron pulse irradiation. The result indicated that dAMP hydroxyl adducts can be repaired by verbascoside. The rate constants of the repair reaction was deduced to be 5.9×108 dm3·mol-1·s-1. A deeper understanding of this new repair mechanism will undo

  10. 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

    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....

  11. Biocatalytic Reductions of Baylis - Hillman Adducts

    Energy Technology Data Exchange (ETDEWEB)

    A Walton; W Conerly; Y Pompeu; B Sullivan; J Stewart

    2011-12-31

    Baylis-Hillman adducts are highly useful synthetic intermediates; to enhance their value further, we sought enantiocomplementary alkene reductases to introduce chirality. Two solutions emerged: (1) a wild-type protein from Pichia stipitis (OYE 2.6), whose performance significantly outstrips that of the standard enzyme (Saccharomyces pastorianus OYE1), and (2) a series of OYE1 mutants at position 116 (Trp in the wild-type enzyme). To understand how mutations could lead to inverted enantioselectivity, we solved the X-ray crystal structure of the Trp116Ile OYE1 variant complexed with a cyclopentenone substrate. This revealed key protein-ligand interactions that control the orientation of substrate binding above the FMN cofactor.

  12. Analysis of heat-labile sites generated by reactions of depleted uranium and ascorbate in plasmid DNA.

    Science.gov (United States)

    Wilson, Janice; Young, Ashley; Civitello, Edgar R; Stearns, Diane M

    2014-01-01

    The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single-strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate, cisplatin, and chromic chloride were compared with those formed by reaction of uranyl acetate and ascorbate. Uranyl ion in the presence of ascorbate produced U-DNA adducts that converted to SSB on heating. Piperidine, which acted on DNA methylated by methyl methanesulfonate to convert methyl-DNA adducts to SSB, served in the opposite fashion as U-DNA adducts by decreasing the level of SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cisplatin and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect the formation of uranium-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from the presence of free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. The results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding the mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposure in humans. PMID:24218036

  13. Structure and function of the translesion DNA polymerases and interactions with damaged DNA

    Directory of Open Access Journals (Sweden)

    F. Peter Guengerich

    2015-03-01

    Pre-steady-state kinetic analysis has been used to develop minimum kinetic models with rate constants of (the eight individual reaction steps in the catalytic cycle. The use of single-tryptophan mutants of Sulfolobus solfataricus Dpo4 and human (h pol κ has led to discernment of the steps for the conformation change (associated with dNTP binding and relocation and nucleotidyl transfer. X-ray crystal structures have been obtained for a number of the DNA adduct/DNA polymerase pairs in both binary and ternary complexes. Two isomeric etheno guanine adducts differ considerably in their interactions with DNA polymerases, explaining the base preferences. Further, even when several DNA polymerases cause the same mispairs with a single DNA adduct, the structural bases for this can differ considerably.

  14. Protein adduct formation as a molecular mechanism in neurotoxicity.

    Science.gov (United States)

    Lopachin, Richard M; Decaprio, Anthony P

    2005-08-01

    Chemicals that cause nerve injury and neurological deficits are a structurally diverse group. For the majority, the corresponding molecular mechanisms of neurotoxicity are poorly understood. Many toxicants (e.g., hepatotoxicants) of other organ systems and/or their oxidative metabolites have been identified as electrophiles and will react with cellular proteins by covalently binding nucleophilic amino acid residues. Cellular toxicity occurs when adduct formation disrupts protein structure and/or function, which secondarily causes damage to submembrane organelles, metabolic pathways, or cytological processes. Since many neurotoxicants are also electrophiles, the corresponding pathophysiological mechanism might involve protein adduction. In this review, we will summarize the principles of covalent bond formation that govern reactions between xenobiotic electrophiles and biological nucleophiles. Because a neurotoxicant can form adducts with multiple nucleophilic residues on proteins, the challenge is to identify the mechanistically important adduct. In this regard, it is now recognized that despite widespread chemical adduction of tissue proteins, neurotoxicity can be mediated through binding of specific target nucleophiles in key neuronal proteins. Acrylamide and 2,5-hexanedione are prototypical neurotoxicants that presumably act through the formation of protein adducts. To illustrate both the promise and the difficulty of adduct research, these electrophilic chemicals will be discussed with respect to covalent bond formation, suspected protein sites of adduction, and proposed mechanisms of neurotoxicity. The goals of future investigations are to identify and quantify specific protein adducts that play a causal role in the generation of neurotoxicity induced by electrophilic neurotoxicants. This is a challenging but critical objective that will be facilitated by recent advances in proteomic methodologies. PMID:15901921

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

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, J.

    1994-12-01

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

  16. Reaction of chromium(VI) with glutathione or with hydrogen peroxide: identification of reactive intermediates and their role in chromium(VI)-induced DNA damage.

    OpenAIRE

    Aiyar, J; Berkovits, H J; Floyd, R A; Wetterhahn, K E

    1991-01-01

    The types of reactive intermediates generated upon reduction of chromium(VI) by glutathione or hydrogen peroxide and the resulting DNA damage have been determined. In vitro, reaction of chromium(VI) with glutathione led to formation of two chromium(V) complexes and the glutathione thiyl radical. When chromium(VI) was reacted with DNA in the presence of glutathione, chromium-DNA adducts were obtained, with no DNA strand breakage. The level of chromium-DNA adduct formation correlated with chrom...

  17. Crystalline guanine adducts of natural and synthetic trioxacarcins suggest a common biological mechanism and reveal a basis for the instability of trioxacarcin A.

    Science.gov (United States)

    Pröpper, Kevin; Dittrich, Birger; Smaltz, Daniel J; Magauer, Thomas; Myers, Andrew G

    2014-09-15

    X-ray crystallographic characterization of products derived from natural and fully synthetic trioxacarcins, molecules with potent antiproliferative effects, illuminates aspects of their reactivity and mechanism of action. Incubation of the fully synthetic trioxacarcin analog 3, which lacks one of the carbohydrate residues present in the natural product trioxacarcin A (1) as well as oxygenation at C2 and C4 yet retains potent antiproliferative effects, with the self-complimentary duplex oligonucleotide d(AACCGGTT) led to production of a crystalline covalent guanine adduct (6). Adduct 6 is closely analogous to gutingimycin (2), the previously reported guanine adduct derived from incubation of natural trioxacarcin A (1) with duplex DNA, suggesting that 3 and 1 likely share a common basis of cytotoxicity. In addition, we isolated a novel, dark-red crystalline guanine adduct (7) from incubation of trioxacarcin A itself with the self-complimentary duplex oligonucleotide d(CGTATACG). Crystallographic analysis suggests that 7 is an anthraquinone derivative, which we propose arises by a sequence of guanosine alkylation within duplex DNA, depurination, base-catalyzed elimination of the trioxacarcinose A carbohydrate residue, and oxidative rearrangement to form an anthraquinone. We believe that this heretofore unrecognized chemical instability of natural trioxacarcins may explain why trioxacarcin analogs lacking C4 oxygenation exhibit superior chemical stabilities yet, as evidenced by structure 3, retain a capacity to form lesions with duplex DNA. PMID:25176186

  18. A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

    Directory of Open Access Journals (Sweden)

    Thu Le Trinh

    Full Text Available AS1411 (previously known as AGRO100 is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox, to AS1411 to form a synthetic Drug-DNA Adduct (DDA, termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

  19. Structural basis for recognition of 5'-phosphotyrosine adducts by TDP2

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Ke; Kurahash, Kayo; Gao, Rui; Tsutakawa, Susan E.; Tainer, John A.; Pommier, Yves; Aihara, Hideki

    2012-12-19

    The DNA repair enzyme TDP2 resolves 5'-phosphotyrosyl-DNA adducts, and is responsible for resistance to anti-cancer drugs that target covalent topoisomerase-DNA complexes. TDP2 also participates in key signaling pathways during development and tumorigenesis, and cleaves a protein-RNA linkage during picornavirus replication. The crystal structure of zebrafish TDP2 bound to DNA reveals a deep and narrow basic groove that selectively accommodates the 5'-end of single-stranded DNA in a stretched conformation. The crystal structure of the full-length C. elegans TDP2 shows that this groove can also accommodate an acidic peptide stretch in vitro, with Glu and Asp sidechains occupying the DNA backbone phosphate binding sites. This extensive molecular mimicry suggests a potential mechanism for auto-regulation and how TDP2 may interact with phosphorylated proteins in signaling. Our study provides a framework to interrogate functions of TDP2 and develop inhibitors for chemotherapeutic and antiviral applications.

  20. The importance of Pyr(6-4)Pyo adducts for survival and mutation induction in mammalian cells

    International Nuclear Information System (INIS)

    The biological consequences of a variety of DNA photoproducts are being studied in Chinese hamster ovary cells. By comparing the rate of induction of resistance to 6-thioguanine following irradiation at 254 nm and 313 nm, the authors find a similar mutation rate at equitoxic doses. Thus, enhanced mutation frequency does not appear to be a consequence of Pyr(6-4)Pyo adducts, which are produced at 254 nm but not at 313 nm. When the level of dimerised photoproducts is measured in a radioimmunoassay, Pyr(6-4)Pyo adducts, which are highly antigenic, are readily detected. By comparing the kinetics of removal of antibody-binding sites following irradiation at 254mn and 313 nm, it is evident that these lesions are repaired at the same rate as cyclobutane dimers

  1. Covalent thiol adducts arising from reactive intermediates of cocaine biotransformation.

    Science.gov (United States)

    Schneider, Kevin J; DeCaprio, Anthony P

    2013-11-18

    Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic

  2. Study on the Interaction between Antitumor Drug Daunomycin and DNA

    Institute of Scientific and Technical Information of China (English)

    CHENG Gui-Fang; ZHAO Jie; TU Yong-Hua; HE Pin-Gang; FANG Yu-Zhi

    2005-01-01

    A detection of anthracycline antitumor drug daunomycin (DNR) reacting with DNA in simulate metabolism in vitro has been made. It was found that DNR could react with DNA to form DNR-DNA adducts. The adduct compositions of DNR with fish sperm DNA and thermally denaturated DNA were determined. The equilibrium association constant K of DNR with fish sperm DNA is 1.98 × 105 L/mol and that of DNR with denaturated DNA is 2.29 × 104 L/mol. Semiquinone free radicals, metabolic products of DNR, can destroy both fish sperm DNA and its thermally denaturated DNA. It is verified by hyperchromic effect increase observed in UV spectrum and AFM experiments. The mechanism of DNA degradation has also been investigated. Results obtained allow one to explain the reason of side effect of anthracycline drug and give the way to depress, which were of clinical significance.

  3. Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation*

    OpenAIRE

    Zhang, Huidong; Beckman, Jeff W.; Guengerich, F. Peter

    2009-01-01

    The human DNA polymerase κ homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces “−1” frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N2-etheno-2′-deoxyguanosine, with a larger stacking surface than canonical purines, showed t...

  4. Application of the adductome approach to assess intertissue DNA damage variations in human lung and esophagus

    International Nuclear Information System (INIS)

    Methods for determining the differential susceptibility of human organs to DNA damage have not yet been explored to any large extent due to technical constraints. The development of comprehensive analytical approaches by which to detect intertissue variations in DNA damage susceptibility may advance our understanding of the roles of DNA adducts in cancer etiology and as exposure biomarkers at least. A strategy designed for the detection and comparison of multiple DNA adducts from different tissue samples was applied to assess esophageal and peripherally- and centrally-located lung tissue DNA obtained from the same person. This adductome approach utilized LC/ESI-MS/MS analysis methods designed to detect the neutral loss of 2'-deoxyribose from positively ionized 2'-deoxynucleoside adducts transmitting the [M+H]+ > [M+H-116]+ transition over 374 transitions. In the final analyses, adductome maps were produced which facilitated the visualization of putative DNA adducts and their relative levels of occurrence and allowed for comprehensive comparisons between samples, including a calf thymus DNA negative control. The largest putative adducts were distributed similarly across the samples, however, differences in the relative amounts of putative adducts in lung and esophagus tissue were also revealed. The largest-occurring lung tissue DNA putative adducts were 90% similar (n = 50), while putative adducts in esophagus tissue DNA were shown to be 80 and 84% similar to central and peripheral lung tissue DNA respectively. Seven DNA adducts, N2-ethyl-2'-deoxyguanosine (N2-ethyl-dG), 1,N6-etheno-2'-deoxyadenosine (εdA), α-S- and α-R-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (1,N2-PdG1, 1,N2-PdG2), 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxy-pyrimido[1,2-a] purine-(3H)-one (8-OH-PdG) and the two stereoisomers of 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a] purine-(3H)-one (6-OH-PdG) were unambiguously detected in all tissue DNA samples by

  5. [sup 129]I Moessbauer spectroscopic study of metallocene-iodine adducts

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Satoru (Dept. of Chemistry, Faculty of Science, Hiroshima Univ. (Japan)); Sakai, Hiroshi (Dept. of Chemistry, Faculty of Science, Hiroshima Univ. (Japan)); Watanabe, Masanobu (Dept. of Chemistry, Coll. of Arts and Sciences, Univ. of Tokyo (Japan)); Maeda, Yutaka (Research Reactor Inst., Kyoto Univ., Osaka (Japan))

    1994-05-01

    A [sup 129]I Moessbauer spectroscopic study of iodine adducts of ferrocenophane, biruthenocene, and osmocene is reported. The spectra show the existence of iodine bonded to the central metals of metallocenes in addition to triiodide anions. The valence state of iron in the ferrocenophane-iodine adduct is the same as those of ruthenium and osmium in their adducts. (orig.)

  6. Synthesis of Borinic Acids and Borinate Adducts Using Diisopropylaminoborane.

    Science.gov (United States)

    Marciasini, Ludovic; Cacciuttolo, Bastien; Vaultier, Michel; Pucheault, Mathieu

    2015-07-17

    In situ formation of aryl Grignard under Barbier condition and diisopropylaminoborane as boron source allows a complete control of the addition onto the boron electrophile. Analytically pure borinic acid derivatives were produced at the gram scale without column chromatography and isolated as borinates adducts, with ethanolamine or 8-hydroxyquinoline, after workup. PMID:26183591

  7. Characterization of trypsin-derived peptides acrylamide-adducted hemoglobin

    International Nuclear Information System (INIS)

    Even though there are a number of sources for human exposure to acrylamide, reliable biomarkers of exposure are not available. In an effort to develop such a biomarker, the authors are characterizing peptides derived from trypsin digests of acrylamide-adducted hemoglobin. For this, radiolabeled acrylamide was incubated with this, radiolabeled acrylamide was incubated with purified human hemoglobin (Ao) and decomposition products removed by dialysis. When the adducted hemoglobin was separated by reverse-phase HPLC, radioactivity eluted with the α and β subunits, suggesting covalent binding. Digestion of individual subunits with trypsin followed by reverse phase HPLC, indicated that most of the radioactivity associated with the α subunit co-eluted with a single peptide. Similar results were observed for the β subunit except that significant amounts of radioactivity eluted with the solvent front, suggesting that radioactivity was released by trypsin digestion. Currently, these preparation are under further characterization by electrospray ionization mass spectrometry. This approach will aid in the identification of the adducted will aid in the identification of the adducted peptide and subsequent preparation of an acrylamide-specific antibody

  8. Stability, accumulation and cytotoxicity of an albumin-cisplatin adduct

    DEFF Research Database (Denmark)

    Møller, Charlotte; Tastesen, Hanne Sørup; Gammelgaard, Bente;

    2010-01-01

    The accumulation and cytotoxicity of a 10 µmol L¿¹ equimolar human serum albumin-cisplatin adduct (HSA-Pt) was investigated in suspension Ehrlich Ascites Tumor Cells (EATC) and adherent Ehrlich Lettré Ascites Cells (Lettré). HSA-Pt did not induce apoptosis nor was it taken up by the cells to any...

  9. Triphosgene mediated chlorination of Baylis-Hillman adducts

    Indian Academy of Sciences (India)

    Narender Reddy Thatikonda; Naga Sesha Sai Pavan Kumar Chebolu; Mahendar Budde; Jayathirtha Rao Vaidya

    2012-03-01

    An efficient method for the preparation of allyl chlorides from Baylis-Hillman adducts has been developed using triphosgene/pyridine system. This method is best illustrated by its advantages like operational simplicity, excellent yields, short reaction time, simple procedure and stereoselectivity.

  10. Cooperative cluster formation, DNA bending and base-flipping by O6-alkylguanine-DNA alkyltransferase

    OpenAIRE

    Tessmer, Ingrid; Melikishvili, Manana; Fried, Michael G.

    2012-01-01

    O6-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 pr...

  11. Detection of protein adduction derived from dauricine by alkaline permethylation.

    Science.gov (United States)

    Xie, Honglei; Liu, Yuyang; Peng, Ying; Zhao, Dongmei; Zheng, Jiang

    2016-06-01

    Dauricine is a bisbenzylisoquinoline alkaloid derivative and has shown multiple pharmacological properties. Despite this, our previous study demonstrated that dauricine induced severe lung toxicity in experimental animals. Metabolic activation of dauricine to the corresponding quinone methide intermediate is suggested to play an important role in dauricine-induced cytotoxicity. Protein adduction derived from the reactive intermediate is considered to initiate the process of the toxicity. In the present study, we developed an alkaline permethylation- and mass spectrometry-based approach to detect dauricine-derived protein adduction. Protein samples were permethylated in the presence of NaOH and CH3I at 80 °C, followed by LC-MS/MS analysis. A thioether product was produced in the reaction. Not only does this technique quantify dauricine-derived protein adduction but also it tells the nature of the interaction between the target proteins and the reactive intermediate of dauricine. The recovery, precision, limit of detection, limit of quantity, and method detection limit were found to be 102.8 %±1.7 %, 1.89 %, 1.32 fmol/mL, 4.93 fmol/mL and 3.37 fmol/mL respectively. The surrogate recovery and surrogate RSD values were 81.5-103.0 % and 2.59 %, respectively. This analytical method has proven sensitive, selective, reliable, and feasible to assess total protein adduction derived from dauricine, and will facilitate the mechanistic investigation of dauricine and other bisbenzylisoquinoline toxicities. Graphical Abstract Alkaline permethylation of dauricine derived protein adduct. PMID:27071763

  12. Quantification of Carnosine-Aldehyde Adducts in Human Urine.

    Science.gov (United States)

    da Silva Bispo, Vanderson; Di Mascio, Paolo; Medeiros, Marisa

    2014-10-01

    Lipid peroxidation generates several reactive carbonyl species, including 4-hydroxy-2-nonenal (HNE), acrolein (ACR), 4-hydroxy-2-hexenal (HHE) and malondialdehyde. One major pathwayof aldehydes detoxification is through conjugation with glutathione catalyzed by glutathione-S-transferases or, alternatively, by conjugation with endogenous histidine containing dipeptides, such as carnosine (CAR). In this study, on-line reverse-phase high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection was utilized for the accurate quantification of CAR- ACR, CAR-HHE and CAR-HNE adducts in human urinary samples from non-smokers young adults. Standard adducts were prepared and isolated by HPLC. The results showed the presence of a new product from the reaction of CAR with ACR. This new adduct was completely characterized by HPLC/MS-MSn, 1H RMN, COSY and HSQC. The new HPLC/MS/MS methodology employing stable isotope-labeled internal standards (CAR-HHEd5 and CAR-HNEd11) was developed for adducts quantification. This methodology permits quantification of 10pmol CAR-HHE and 1pmol of CAR-ACR and CAR-HNE. Accurate determinations in human urine sample were performed and showed 4.65±1.71 to CAR-ACR, 5.13±1.76 to CAR-HHE and 5.99±3.19nmol/mg creatinine to CAR-HNE. Our results indicate that carnosine pathways can be an important detoxification route of a, ß -unsaturated aldehydes. Moreover, carnosine adducts may be useful as redox stress indicator. PMID:26461323

  13. Experimental and computational evidence for hydrogen bonding interaction between 2′-deoxyadenosine conjugate adduct and amino-terminated organic film on Si(001)

    Energy Technology Data Exchange (ETDEWEB)

    Szwajca, A., E-mail: Anna.Szwajca@amu.edu.pl [Faculty of Chemistry, A" . Mickiewicz University, Umultowska 89 b, 61-614 Poznań (Poland); Krzywiecki, M. [Institute of Physics-CSE, Silesian University of Technology, Konarskiego 22B, 44-100 Gliwice (Poland); Pluskota-Karwatka, D. [Faculty of Chemistry, A" . Mickiewicz University, Umultowska 89 b, 61-614 Poznań (Poland)

    2015-08-03

    A simple method for immobilization of malonaldehyde-acetaldehyde conjugate adduct with DNA base onto an amino-terminated surface of silicon from water solution is proposed. The Si(001) surface which contains OH groups was modified with 3-aminopropyltrimethoxysilane (APTMS) to serve as a linker between the silica surface and the organic adduct. The 2′-deoxyadenosine adduct was adsorbed on the APTMS/Si surface from acetonitrile/water solution. This nucleoside derivative is stable under laboratory conditions and emits a natural fluorescence, allowing for its adsorption on the APTMS/Si surface to be easily verified by fluorescence microscopy, non-contact atomic force microscopy and attenuated total reflectance Fourier transform infrared spectroscopy. The degree of surface coverage by the adduct was evaluated by X-ray photoelectron spectroscopy (XPS). Analysis of the XPS spectra revealed bands at 400.2 eV and 533.1 eV which are characteristic of a hydrogen bonded –NH{sub 2} and –OH group. This observation implies that the free electron donating –NH{sub 2} groups from the APTMS layer makes hydrogen bonds with the fluorescent adduct and immobilize it on the surface. The wetting angle of the APTMS/Si surface before and after adsorption of the nucleoside derivative does not differ significantly, which points to the involvement of an – OH group from 2′-deoxyadenosine to be involved in hydrogen bonding. These experimental results were further supported using quantum chemical calculations to demonstrate that the 2′deoxyadenosine adduct makes hydrogen bonds with the APTMS molecule. Furthermore, this hydrogen bond involves the –NH{sub 2} group from APTMS and –OH group at carbon atoms C3 and C6 from the deoxyribose ring of 2′deoxyadenosine. - Highlights: • DNA base adduct was immobilized onto amino-terminated silicon surface. • Hydrogen bonds were observed between aminosilane molecules and deoxyribose ring. • Fluorescent film was characterized by

  14. Experimental and computational evidence for hydrogen bonding interaction between 2′-deoxyadenosine conjugate adduct and amino-terminated organic film on Si(001)

    International Nuclear Information System (INIS)

    A simple method for immobilization of malonaldehyde-acetaldehyde conjugate adduct with DNA base onto an amino-terminated surface of silicon from water solution is proposed. The Si(001) surface which contains OH groups was modified with 3-aminopropyltrimethoxysilane (APTMS) to serve as a linker between the silica surface and the organic adduct. The 2′-deoxyadenosine adduct was adsorbed on the APTMS/Si surface from acetonitrile/water solution. This nucleoside derivative is stable under laboratory conditions and emits a natural fluorescence, allowing for its adsorption on the APTMS/Si surface to be easily verified by fluorescence microscopy, non-contact atomic force microscopy and attenuated total reflectance Fourier transform infrared spectroscopy. The degree of surface coverage by the adduct was evaluated by X-ray photoelectron spectroscopy (XPS). Analysis of the XPS spectra revealed bands at 400.2 eV and 533.1 eV which are characteristic of a hydrogen bonded –NH2 and –OH group. This observation implies that the free electron donating –NH2 groups from the APTMS layer makes hydrogen bonds with the fluorescent adduct and immobilize it on the surface. The wetting angle of the APTMS/Si surface before and after adsorption of the nucleoside derivative does not differ significantly, which points to the involvement of an – OH group from 2′-deoxyadenosine to be involved in hydrogen bonding. These experimental results were further supported using quantum chemical calculations to demonstrate that the 2′deoxyadenosine adduct makes hydrogen bonds with the APTMS molecule. Furthermore, this hydrogen bond involves the –NH2 group from APTMS and –OH group at carbon atoms C3 and C6 from the deoxyribose ring of 2′deoxyadenosine. - Highlights: • DNA base adduct was immobilized onto amino-terminated silicon surface. • Hydrogen bonds were observed between aminosilane molecules and deoxyribose ring. • Fluorescent film was characterized by spectroscopy and

  15. Detection of 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine occurring endogenously in DNA.

    Science.gov (United States)

    Watson, W P; Aston, J P; Barlow, T; Crane, A E; Potter, D; Brown, T

    1999-01-01

    1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the

  16. DNA damage in humans exposed to environmental and dietary polycyclic aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Schoket, Bernadette [National Institute of Environmental Health, Jozsef Fodor National Centre of Public Health, Gyaliut 2-6, Budapest (Hungary)

    1999-03-08

    The paper describes recent research on human DNA damage related to environmental and dietary polycyclic aromatic hydrocarbon (Pah) exposures. The study populations either represent general populations of large geographical regions, or their exposure situation may have relevance to the general population. In Silesia, Poland, and Northern Bohemia, Czech Republic, where coal-based industry and domestic heating are the major sources of PAHs, significant differences have been observed in white blood cell DNA adducts and cytogenetic biomarkers between environmentally exposed and rural control populations, and significant seasonal variations of DNA damage have been detected. Bus drivers, traffic policemen and local residents have been involved in biomarker studies in Copenhagen, Athens, Genoa and Cairo, and differences have been measured in the level of DNA damage of urban and rural populations. Burning of smoky coal in unvented homes in Xuan Weiregion, China, causes high PAH exposure of residents, which has been reflected in DNA adduct levels in different tissues. Indoor wood burning in open fireplaces did not increase human DNA adduct levels. Oil-well fires left burning in Kuwait after the Persian Gulf war created an unprecedented environmental pollution. However, insignificant environmental PAH levels were measured several miles from these fires. Aromatic and PAH-DNA adduct level sin white blood cells of US Army soldiers were lower during their deployment in Kuwait, than in Fulda, Germany, where they were stationed before and after serving in Kuwait. The contribution of dietary PAH exposure to blood cell DNA adduct levels had been demonstrated in studies in which volunteers consumed heavily charbroiled beef. Environmental tobacco smoke did not cause detectable changes, as measured by{sup 32}P-postlabelling, in DNA adduct levels in non-smokers. In the reviewed studies, observed DNA adduct levels were generally in the range of 1 to 10 adducts, and not higher than 40

  17. 8-Oxo-2'-deoxyguanosine adducts in human biomonitoring; Zur Validitaet von 8-Oxo-2'-deoxyguanosin-Addukten in weissen Blutzellen im Rahmen des Human Biomonitoring

    Energy Technology Data Exchange (ETDEWEB)

    Marczynski, B. [Ruhr-Univ., Bochum (DE). Berufsgenossenschaftliches Forschungsinstitut fuer Arbeitsmedizin (BGFA); Wilhelm, M. [Hygiene, Sozial- und Umweltmedizin, Ruhr-Univ. Bochum, Bochum (Germany)

    2001-07-01

    This review summarizes the role of 8-oxo-2'-deoxyguanosine (8-oxodG, 8-OHdG) adducts. Reactive oxygen species are able to cause oxidative stress which can lead to oxidative DNA damage. According to present knowledge the increased production of reactive oxygen species plays an important role in the development of tumors in exposed individuals. Oxidative DNA adducts can be used as indirect markers in the biomonitoring of mutagenic and carcinogenic substances. HPLC combined to an electrochemical detector are appropriate tools to determine the amount of 8-oxodG in peripheral white blood cells. Recent data from different countries reveal that 8-oxodG levels in white blood cells amount to 0.5 8-oxodG/10{sup 5}dG. We found increased 8-oxodG adduct levels in the DNA of white blood cells of PAH-exposed workers. However, several critical factors must be considered with respect to the reliability of 8-oxodG adducts as effect biomarkers. For example, the correlation between the 8-oxodG levels in peripheral white blood cells and in target organs is still unknown. Data from larger populations are lacking. Therefore, the representative reference values for this adduct cannot yet be established. Finally, oxidatve DNA-adducts are non-specific and their adduct level can be affected by many factors (e.g. lifestyle). The significance of this effect biomarker must be further validated in environmental medicine. (orig.) [German] In dieser Uebersicht werden Aspekte der oxidativen DNA-Schaedigung unter besonderer Beruecksichtigung von 8-Oxo-2'-deoxyguanosin-Addukten als Effektmarker dargelegt. Oxidative DNA-Schaedigung wird u.a. durch reaktive Sauerstoffspezies hervorgerufen. Reaktive Sauerstoffspezies werden vor allem endogen durch verschiedene pathophysiologische Prozesse, aber auch bei erhoehter Exposition gegenueber Fremdstoffen vermehrt gebildet. Es wird angenommen, dass die erhoehte Bildung reaktiver Sauerstoffspezies eine Rolle in der Tumorentstehung exponierter Personen

  18. Vitamin C for DNA damage prevention

    Czech Academy of Sciences Publication Activity Database

    Šrám, Radim; Binková, B.; Rössner ml., Pavel

    2012-01-01

    Roč. 733, 1-2 (2012), s. 39-49. ISSN 0027-5107 R&D Projects: GA MŠk 2B08005; GA MŽP(CZ) SP/1B3/50/07 Institutional research plan: CEZ:AV0Z50390703 Keywords : Chromosomal aberrations * DNA adducts * DNA repair Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.902, year: 2012

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

    International Nuclear Information System (INIS)

    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

  20. SEM-EDX STUDIES OF CHITOSAN DERIVATIVES-METAL ADDUCTS

    OpenAIRE

    Galo Cárdenas; Edelio Taboada; Armando Bravo; S. Patricia Miranda

    2003-01-01

    Chitosan was obtained from shrimps shells (pleuroncodes monodon) using chemical methods. A series of chitosan (QS)charged with solution of copper, cobalt, nickel and mercury ions were prepared at room temperature using the batch method. N-3,5-diethylamino benzoyl chitosan (QDAB); N,O-dimercapto succinate chitosan (QNOT) and 4-aminobenzoate chitosan (QAB) derivatives were prepared. The chitosan metal adducts with Cu, Co, Ni and Hg ions and derivatives maximum loading is discussed. Chitosan and...

  1. Tunable degradation of maleimide-thiol adducts in reducing environments

    OpenAIRE

    Baldwin, Aaron D.; Kiick, Kristi L.

    2011-01-01

    Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here we show that the resulting succinimide thioether formed by a Michael type addition of a thiol to N-ethylmaleimide (NEM), generally accepted as stable, can in fact undergo retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies (1H NMR...

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

    OpenAIRE

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

    2009-01-01

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

  3. PHOSPHATO AND PHOSPHONATO ADDUCTS: SYNTHESIS AND SPECTROSCOPIC STUDY

    Directory of Open Access Journals (Sweden)

    Mouhamadou Birame Diop

    2014-05-01

    Full Text Available Two new adducts have been synthesized and studied by infrared and NMR spectroscopy. The suggested structures are discrete or of infinite chain type with a phosphate behaving as a bidentate ligand, a phosphonate acting as a monodentate ligand, the environments around the tin centre being tetrahedral or trigonal bipyramidal. In all the studied compounds, supramolecular architectures are obtained when hydrogen bonds are considered.

  4. 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.

  5. Ion Pairs or Neutral Molecule Adducts? Cooperativity in Hydrogen Bonding

    Science.gov (United States)

    DeKock, Roger L.; Schipper, Laura A.; Dykhouse, Stephanie C.; Heeringa, Lee P.; Brandsen, Benjamin M.

    2009-01-01

    We performed theoretical studies on the systems NH[subscript 3] times HF times mH[subscript 2]O, NH[subscript 3] times HCl times mH[subscript 2]O, with m = 0, 1, 2, and 6. The molecules with m = 0 form hydrogen-bonded adducts with little tendency to form an ion-pair structure. The molecule NH[subscript 3] times HCl times H[subscript 2]O cannot be…

  6. The repair of cyclobutane dimers and PYR(6-4) PYO adducts in normal and repair-deficient mammalian cells

    International Nuclear Information System (INIS)

    Antiserum raised against DNA irradiated with a high dose of far UV light was found to have at least two antibody populations. A competitive assay in which the labelled antigen (/sup 32/P-labelled poly (dA):poly (dT)) was irradiated with far UV light (254nm) was shown to be specific for Pyr (6-4) Pyo adducts. Labelled antigen irradiated with near UV light (320nm) competed specifically for cyclobutane dimers. This assay had the same specificity as one consisting of labelled antigen irradiated with far UV light in competition for antiserum raised against DNA irradiated with near UV light in the presence of acetophenone. The authors conclude from these results that their previous data for removal of antibody-binding sites demonstrated repair of Pyr(6-4)Pyo adducts in normal, but not UV-hypersensitive, mammalian cell lines. Using the cyclobutane dimer-specific assay they have been able to correlate the removal of antibody-binding sites with repair kinetics previously published for T4 endonuclease V sensitive sites in mouse, hamster, normal human and Xeroderma pigmentosum cells

  7. Tamoxifen DNA damage detected in human endometrium using accelerator mass spectrometry.

    Science.gov (United States)

    Martin, Elizabeth A; Brown, Karen; Gaskell, Margaret; Al-Azzawi, Farook; Garner, R Colin; Boocock, David J; Mattock, Elizabeth; Pring, David W; Dingley, Karen; Turteltaub, Kenneth W; Smith, Lewis L; White, Ian N H

    2003-12-01

    This study was aimed to establish whether tamoxifen binds irreversibly to uterine DNA when given to women. Patients were given a single therapeutic dose of [(14)C]tamoxifen citrate orally (20 mg, 0.37 or 1.85 MBq) approximately 18 h prior to hysterectomy or breast surgery. Nonmalignant uterine tissue was separated into myometrium and endometrium. DNA and protein were isolated and bound radiolabel determined by the sensitive technique of accelerator mass spectrometry. Levels of irreversible DNA binding of tamoxifen in the endometrium of treated patients were 237 +/- 77 adducts/10(12) nucleotides (mean +/- SE, n = 10). In myometrial tissues, a similar extent of DNA binding was detected (492 +/- 112 adducts/10(12) nucleotides). Binding of tamoxifen to endometrial and myometrial proteins was 10 +/- 3 and 20 +/- 4 fmol/mg, respectively. In breast tissue, sufficient DNA could not be extracted but protein binding was an order of magnitude higher than that seen with endometrial proteins (358 +/- 81 fmol/mg). These results demonstrate that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers relative to those previously reported in women after long-term tamoxifen treatment where levels, when detected, ranged from 15000 to 130000 adducts/10(12) nucleotides. Our findings support the hypothesis that the low level of DNA adducts in human uterus is unlikely to be involved with endometrial cancer development. PMID:14679010

  8. The effect of tryptophan on UV-induced DNA photodamage.

    Science.gov (United States)

    Oladepo, Sulayman A; Loppnow, Glen R

    2010-01-01

    Trp-DNA adducts resulting from UV irradiation of pyrimidine bases and nucleotides in the presence of tryptophan (Trp) have been the subject of previous research. However, the relative yield of the adducts compared with the UV screening effect of Trp has not been previously considered. To determine whether Trp-DNA adduct formation or absorption "screening" by Trp is the predominant process when DNA solutions are irradiated with UV light in the presence of Trp, we irradiated Trp-containing DNA oligonucleotide solutions with UVC light and incubated aliquots of those solutions with molecular beacons (MBs) to detect the damage. We observed a rapid decay of fluorescence of the MBs for pure DNA solutions, thereby indicating damage. However, in the presence of Trp, the fluorescence decay is prolonged, with time constants that increase exponentially with Trp concentration. The results are discussed in terms of a beneficial in vivo cellular protection rather than harmful adduct formation and suggest a net sacrificial absorption of UV light by Trp which actually protects the DNA from UV damage. PMID:20492563

  9. Analysis of dibenzo[def,p]chrysene-deoxyadenosine adducts in wild-type and cytochrome P450 1b1 knockout mice using stable-isotope dilution UHPLC-MS/MS.

    Science.gov (United States)

    Harper, Tod A; Morré, Jeff; Lauer, Fredine T; McQuistan, Tammie J; Hummel, Jessica M; Burchiel, Scott W; Williams, David E

    2015-04-01

    The polycyclic aromatic hydrocarbon (PAH), dibenzo[def,p]chrysene (DBC; also known as dibenzo[a,l]pyrene), is a potent carcinogen in animal models and a class 2A human carcinogen. Recent investigations into DBC-mediated toxicity identified DBC as a potent immunosuppressive agent similar to the well-studied immunotoxicant 7,12-dimethylbenz[a]anthracene (DMBA). DBC, like DMBA, is bioactivated by cytochrome P450 (CYP) 1B1 and forms the reactive metabolite DBC-11,12-diol-13,14-epoxide (DBCDE). DBCDE is largely responsible for the genotoxicity associated with DBC exposure. The immunosuppressive properties of several PAHs are also linked to genotoxic mechanisms. Therefore, this study was designed to identify DBCDE-DNA adduct formation in the spleen and thymus of wild-type and cytochrome P450 1b1 (Cyp1b1) knockout (KO) mice using a highly sensitive stable-isotope dilution UHPLC-MS/MS method. Stable-isotope dilution UHPLC-MS/MS identified the major DBC adducts (±)-anti-cis-DBCDE-dA and (±)-anti-trans-DBCDE-dA in the lung, liver, and spleen of both WT and Cyp1b1 KO mice. However, adduct formation in the thymus was below the level of quantitation for our method. Additionally, adduct formation in Cyp1b1 KO mice was significantly reduced compared to wild-type (WT) mice receiving DBC via oral gavage. In conclusion, the current study identifies for the first time DBCDE-dA adducts in the spleen of mice supporting the link between genotoxicity and immunosuppression, in addition to supporting previous studies identifying Cyp1b1 as the primary CYP involved in DBC bioactivation to DBCDE. The high levels of DBC-DNA adducts identified in the spleen, along with the known high levels of Cyp1b1 expression in this organ, supports further investigation into DBC-mediated immunotoxicity. PMID:25868132

  10. DNA repair in spermatocytes and spermatids of the mouse

    International Nuclear Information System (INIS)

    When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced

  11. Calf thymus DNA binding/bonding properties of CC-1065 and analogs as related to their biological activities and toxicities.

    Science.gov (United States)

    Krueger, W C; Prairie, M D

    1992-03-01

    CC-1065 is a potent natural antitumor antibiotic that binds non-covalently and covalently (N-3 adenine adduct) in the minor groove of B-form DNA. Synthetic analogs of CC-1065 do not exhibit the delayed death toxicity of CC-1065 and are efficacious anticancer agents, some of them curative in murine tumor models. In an attempt to understand the different biological properties of CC-1065 and analogs, we have determined the following quantities for CC-1065, enantiomeric CC-1065, and three biologically active analogs and their enantiomers: the calf thymus DNA (CT-DNA) induced molar ellipticity of the adduct (or how rigidly the adduct is held in the right-hand conformation of the minor groove); the stability of the adduct with respect to long incubation times and to digestion by snake venom phosphodiesterase I (SVPD); the stabilizing effect on the CT-DNA helix of the covalently and non-covalently bound species with respect to thermal melting; and the CT-DNA binding/bonding (non-covalent/covalent) profiles at a low molar ratio of nucleotide to drug. The major observations from these studies are as follows: (i) molecules which show large DNA interaction parameters, stable adducts, and significant non-covalent binding exhibit delayed death toxicity; (ii) molecules which show intermediate DNA interaction parameters and stable adducts, but do not show significant non-covalent binding, do not exhibit delayed death toxicity and are biologically active; (iii) molecules which show small DNA interaction parameters and unstable DNA adducts are biologically inactive. The results suggest that a window exists in the affinity for the minor groove of DNA wherein an analog may possess the correct balance of toxicity and activity to make a useful anticancer agent. Outside of this window, the analog causes delayed deaths or has no significant biological activity. PMID:1312395

  12. 32P-Postlabeling test for covalent DNA binding of chemicals in vivo: Application to a variety of aromatic carcinogens and methylating agents

    International Nuclear Information System (INIS)

    Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity

  13. Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences.

    Science.gov (United States)

    Paul, Moumita; Murray, Vincent

    2012-03-01

    The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences. PMID:21678458

  14. Structure of adducts of isoindolo[2,1-a]benzimidazole derivatives with maleimides

    Science.gov (United States)

    Korolev, Oleksandr; Yegorova, Tatyana; Levkov, Igor; Malytskyy, Volodymyr; Shishkin, Oleg; Zubatyuk, Roman; Palamarchuk, Genadiy; Vedrenne, Marc; Baltas, Michel; Voitenko, Zoia

    2015-03-01

    The selectivity of formation and some mechanistic insights during the synthesis of substituted isoindolo[2,1-a]benzimidazoles are discussed. Furthermore, the reactions of the obtained products with maleimides were carried out. Two types rearrangement adducts together with intermediate Michael type adducts were isolated. The influence of the reaction conditions and reagents ratio is discussed. Specific spectral criteria for the identification of the Michael type adducts are indicated.

  15. Knee adduction moment and medial contact force - facts about their correlation during gait

    OpenAIRE

    Kutzner, Ines; Trepczynski, Adam; Heller, Markus O.; Bergmann, Georg

    2013-01-01

    The external knee adduction moment is considered a surrogate measure for the medial tibiofemoral contact force and is commonly used to quantify the load reducing effect of orthopedic interventions. However, only limited and controversial data exist about the correlation between adduction moment and medial force. The objective of this study was to examine whether the adduction moment is indeed a strong predictor for the medial force by determining their correlation during gait. Instrumented kn...

  16. Structural aspects of adducts of N-phthaloylglycine and its derivatives

    Science.gov (United States)

    Barooah, Nilotpal; Sarma, Rupam J.; Batsanov, Andrei S.; Baruah, Jubaraj B.

    2006-06-01

    N-phthaloylglycine forms 2:1 adduct with 1,3-dihydroxybenzene and 1:2 adduct with 2-aminopyrimidine. Whereas N-phthaloylglycine form salts with 2,6-diaminopyridine and with 8-hydroxyquinoline. The 1:1 adduct of N, N'-bis(glycinyl)pyromellitic diimide with dimethylsulphoxide, 2-aminopyrimidine and 4,4'-dihydroxybiphenyl are prepared and characterised. The reaction of N, N'-bis(glycinyl)pyromellitic diimide with 2,6-diaminopyridine gives corresponding salt.

  17. Metabolism of the Antibacterial Triclocarban by Human Epidermal Keratinocytes to Yield Protein Adducts

    OpenAIRE

    Schebb, Nils Helge; Buchholz, Bruce A.; Hammock, Bruce D.; Rice, Robert H.

    2012-01-01

    Previous studies of triclocarban suggest that its biotransformation could yield reactive metabolites that form protein adducts. Since the skin is the major route of triclocarban exposure, present work examined this possibility in cultured human keratinocytes. The results provide evidence for considerable biotransformation and protein adduct formation when cytochrome P450 activity is induced in the cells by TCDD, a model Ah receptor ligand. Since detecting low adduct levels in cells and tissue...

  18. Benzene hemoglobin adducts in mice and rats: Characterization of formation and physiological modeling

    International Nuclear Information System (INIS)

    Benzene is a myelotoxin and a human leukemogen. Humans are exposed to this compound, both occupationally and environmentally. This study was conducted to determine whether formation of benzene-derived adducts with blood hemoglobin (Hb) can be used as a biomarker of exposure to benzene. B6C3F1 mice and F344/N rats were given 0.1 to 10,000 mumol [14C]benzene/kg body wt, orally. Twenty-four hours later, animals were euthanized, and globin was isolated from blood samples. The globin was analyzed by liquid scintillation spectrometry for the presence of [14C]benzene-derived adducts. Hb adduct formation was linear with respect to dose for amounts of up to 500 mumol [14C]benzene/kg body wt, for both rodent species. Within this linear dose-response range, mice formed adducts from [14C]benzene approximately 3.5 times less efficiently [0.022 +/- 0.010 (pmol adducts/mg globin)/(mumol/kg body wt dose)] than did rats [0.076 +/- 0.014 (pmol adducts)/(mumol/kg body wt dose)]. Benzene-derived Hb adducts also accumulated linearly when mice and rats were given up to three daily doses of 500 mumol [14C]benzene/kg body wt. These data were used to develop a physiological model for benzene-derived Hb adduct formation. Both first-order and saturable pathways for adduct formation were incorporated. The results showed that the model simulated the levels of Hb adducts in both mice and rats after oral exposures to benzene and predicted the levels of Hb adducts present after inhalation exposure. These studies suggest that Hb adducts might be useful biomarkers for human exposures to benzene

  19. Synthesis and Characterization of the Adducts of Bis(O-ethyldithiocarbonatocopper(II with Substituted Pyridines

    Directory of Open Access Journals (Sweden)

    Gurpreet Kour

    2013-01-01

    Full Text Available Monomeric five coordinated adducts of bis(O-ethyldithiocarbonatocopper(II of general formula [Cu(C2H5OCS22(L], [L = 2-, 3-, 4-methylpyridines and 2-, 3-, 4-ethylpyridines] have been synthesized and characterized by elemental analysis, i.r. and electronic spectroscopy, magnetic and conductivity measurements. Analytical results show that the adducts have 1 : 1 stoichiometry. The adducts were found to be paramagnetic and their magnetic moments at room temperature lie within the 1.81–1.94 B.M. range and this indicates the presence of one unpaired electron. All the adducts have distorted square pyramidal geometry.

  20. Phosphoryl chloride-methanol adducts: Matrix isolation infrared and DFT studies

    Science.gov (United States)

    Ramanathan, N.; Sankaran, K.

    2013-12-01

    The adducts of phosphoryl chloride (POCl3) and methanol (CH3OH) were studied using matrix isolation infrared spectroscopy and DFT calculations. The 1:1 POCl3:CH3OH binary adduct was generated in a nitrogen matrix at low temperatures and studied using infrared spectroscopy. Formation of the adduct was evidenced by the shifts in the vibrational frequencies of the modes involving POCl3 and CH3OH sub-molecules. The structures, vibrational frequencies and stabilization energies of the adducts were computed at B3LYP/aug-cc-pVDZ level of theory. Our computations located two minima for POCl3:CH3OH adducts on the potential energy surface. However, only one adduct was experimentally identified in the matrix at low temperatures, which was the structure corresponding to the global minimum. The computed vibrational frequencies of the adduct agreed well with the observed experimental frequencies. Atoms In Molecules (AIM) analysis was performed to understand the nature of the interactions in these adducts. Natural Bond Orbital (NBO) analysis was performed to understand the effect of charge-transfer interactions on the stability of adducts.

  1. Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity

    OpenAIRE

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R Scott

    2013-01-01

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases 1,2 . Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5′-adenylated (5′-AMP) DNA lesions 3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for dead...

  2. Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells

    OpenAIRE

    Frank Y. Chen; Harris, Linda C.; Joanna S Remack; Brent, Thomas P.

    1997-01-01

    O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found ...

  3. 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.

  4. Group 13 Superacid Adducts of [PCl2N]3.

    Science.gov (United States)

    Tun, Zin-Min; Heston, Amy J; Panzner, Matthew J; Scionti, Vincenzo; Medvetz, Doug A; Wright, Brian D; Johnson, Nicholas A; Li, Linlin; Wesdemiotis, Chrys; Rinaldi, Peter L; Youngs, Wiley J; Tessier, Claire A

    2016-04-01

    Irrespective of the order of the addition of reagents, the reactions of [PCl2N]3 with MX3 (MX3 = AlCl3, AlBr3, GaCl3) in the presence of water or gaseous HX give the air- and light-sensitive superacid adducts [PCl2N]3·HMX4. The reactions are quantitative when HX is used. These reactions illustrate a Lewis acid/Brønsted acid dichotomy in which Lewis acid chemistry can become Brønsted acid chemistry in the presence of adventitious water or HX. The crystal structures of all three [PCl2N]3·HMX4 adducts show that protonation weakens the two P-N bonds that flank the protonated nitrogen atom. Variable-temperature NMR studies indicate that exchange in solution occurs in [PCl2N]3·HMX4, even at lower temperatures than those for [PCl2N]3·MX3. The fragility of [PCl2N]3·HMX4 at or near room temperature and in the presence of light suggests that such adducts are not involved directly as intermediates in the high-temperature ring-opening polymerization (ROP) of [PCl2N]3 to give [PCl2N]n. Attempts to catalyze or initiate the ROP of [PCl2N]3 with the addition of [PCl2N]3·HMX4 at room temperature or at 70 °C were not successful. PMID:26974866

  5. EMG evaluation of hip adduction exercises for soccer players

    DEFF Research Database (Denmark)

    Serner, Andreas; Jakobsen, Markus Due; Andersen, Lars Louis;

    2014-01-01

    INTRODUCTION: Exercise programmes are used in the prevention and treatment of adductor-related groin injuries in soccer; however, there is a lack of knowledge concerning the intensity of frequently used exercises. OBJECTIVE: Primarily to investigate muscle activity of adductor longus during six...... traditional and two new hip adduction exercises. Additionally, to analyse muscle activation of gluteals and abdominals. MATERIALS AND METHODS: 40 healthy male elite soccer players, training >5 h a week, participated in the study. Muscle activity using surface electromyography (sEMG) was measured bilaterally...

  6. Acetaminophen-cysteine adducts during therapeutic dosing and following overdose

    OpenAIRE

    Judge Bryan S; James Laura P; Green Jody L; Heard Kennon J; Zolot Liza; Rhyee Sean; Dart Richard C

    2011-01-01

    Abstract Background Acetaminophen-cysteine adducts (APAP-CYS) are a specific biomarker of acetaminophen exposure. APAP-CYS concentrations have been described in the setting of acute overdose, and a concentration >1.1 nmol/ml has been suggested as a marker of hepatic injury from acetaminophen overdose in patients with an ALT >1000 IU/L. However, the concentrations of APAP-CYS during therapeutic dosing, in cases of acetaminophen toxicity from repeated dosing and in cases of hepatic injury from ...

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

    International Nuclear Information System (INIS)

    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

  8. 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.

  9. Effect of pH on DNA alkylation by enzyme-activated mitomycin C and porfiromycin.

    Science.gov (United States)

    Yu, F; Pan, S S

    1993-06-01

    DNA adduct formation by enzyme-activated antibiotics, mitomycin C (MMC) or porfiromycin (PFM), at pH 7.6 or pH 6.0 under anaerobic conditions was analyzed by a 32P-postlabeling method. Antibiotic activation by rat liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and bovine milk xanthine oxidase (EC 1.2.3.2) produced similar results. Five 32P-labeled MMC adducts were separated by thin layer chromatography and high performance liquid chromatography from DNA alkylated at either pH. Four of the radioactive spots separated by thin layer chromatography were identified as two monofunctional monoadducts [1" alpha and 1" beta forms of N2-(2" beta,7"-diaminomitosen-1"-yl)-2'-deoxyguanylic acid], one bifunctional monoadduct [N2-(10"-decarbamoyl-2",7"-diaminomitosen-1" alpha-yl)-2'-deoxyguanylic acid], and one cross-linked adduct [N2-(2" beta,7"-diamino-10"-deoxyguanyl-N2-yl-mitosen- 1" alpha-yl)-2'-deoxyguanylic acid]. One minor radioactive spot was not identified. By comparing DNA alkylated at the two pH values, based on equal amounts of 32P radioactivity, similar amounts of cross-links were detected. However, the DNA showed different ratios of the alpha and beta isomers of the monofunctional monoadduct. Furthermore, the DNA alkylated at pH 6.0 showed more bifunctional monoadducts than did the DNA alkylated at pH 7.6. Analysis of alkylated DNA by enzyme-activated PFM showed a similar spectrum of DNA adduct formation. The effect of pH on the distribution of the five PFM-DNA adducts was similar to that observed for the five MMC-DNA adducts. The distribution of adducts in DNA alkylated at the same pH was similar irrespective of which enzyme activated MMC or PFM. The pH of the reaction during DNA and MMC interaction was the determining factor for the quantitative distribution of the adducts. This pH effect may be important for the cytotoxicity of MMC and PFM in tumor cells that have high levels of reductive enzymes with low optimal pH values. PMID:8391116

  10. Yield of photo-adduct formation of LOV domains from Chlamydomonas reinhardtii by picosecond laser excitation

    International Nuclear Information System (INIS)

    The photo-excitation of flavin mononucleotide (FMN) in the wild-type light, oxygen and voltage sensitive (LOV) domains of the blue-light photoreceptors phototropin causes the formation of an intermediate flavin-C(4a)-cysteinyl adduct. The adduct formation due to picosecond laser pulse exaction (wavelength 400nm) is studied on wild-type LOV1 and LOV2 domains of the phototropin phot from Chlamydomonas reinhardtii. The efficiency of adduct formation is probed by detection of the fluorescence signals caused by time-separated picosecond excitation pulses since FMN is fluorescent and the formed adduct is non-fluorescent. Quantum yields of adduct formation of φAd∼0.06 are determined for excitation with intense single-pulses of energy densities, wL, comparable to or larger than the saturation energy density, wsat,S0, of ground-state population depletion. Under repetitive picosecond pulse excitation conditions the efficiency of adduct formation rises with decreasing picosecond pulse energy density and approaches for wLsat,S0 the continuous blue-light exposure quantum yields of adduct formation in the range from φAd=0.5-0.9. Picosecond laser pulse induced energy deposition in the LOV domains causing protein conformational changes is discussed as main origin of the intensity dependent reduction of the efficiency of adduct-formation

  11. Fullerene–Carbene Lewis Acid–Base Adducts

    KAUST Repository

    Li, Huaping

    2011-08-17

    The reaction between a bulky N-heterocylic carbene (NHC) and C60 leads to the formation of a thermally stable zwitterionic Lewis acid-base adduct that is connected via a C-C single bond. Low-energy absorption bands with weak oscillator strengths similar to those of n-doped fullerenes were observed for the product, consistent with a net transfer of electron density to the C60 core. Corroborating information was obtained using UV photoelectron spectroscopy, which revealed that the adduct has an ionization potential ∼1.5 eV lower than that of C60. Density functional theory calculations showed that the C-C bond is polarized, with a total charge of +0.84e located on the NHC framework and -0.84e delocalized on the C 60 cage. The combination of reactivity, characterization, and theoretical studies demonstrates that fullerenes can behave as Lewis acids that react with C-based Lewis bases and that the overall process describes n-doping via C-C bond formation. © 2011 American Chemical Society.

  12. The R46Q, R131Q and R154H Polymorphs of Human DNA Glycosylase/β-Lyase hOgg1 Severely Distort the Active Site and DNA Recognition Site but do not Cause Unfolding†

    OpenAIRE

    Anderson, Peter C.; Daggett, Valerie

    2009-01-01

    Reactive oxygen species can cause widespread cellular damage, including base alterations and strand breaks in DNA. An array of DNA-repair enzymes constitutes an essential part of the line of defense that cells use against oxidative damage to the genome. A DNA glycosylase/β-lyase enzyme, Ogg1, scavenges the genome for 8-oxoguanine, a major mutagenic DNA adduct induced by reactive oxygen species, and catalyzes its excision and subsequent cleavage of the DNA phosphate backbone. Several polymorph...

  13. Repair of DNA treated with γ-irradiation and chemical carcinogens. Progress report, 1980-1983

    International Nuclear Information System (INIS)

    We have studied in vitro DNA repair with the isolation and characterization of DNA glycosylases active in the removable of 3-methyladenine and the problem of repair of DNA in chromatin. The second area of focus has been on transposable elements and carcinogen action. The work on DNA adducts with β-propiolactone was done to define potential new substrates useful in a search for new glycosylases

  14. Photoelectrochemical Sensors for the Rapid Detection of DNA Damage Induced by Some Nanoparticles

    OpenAIRE

    M. Jamaluddin Ahmed; Bin-Tian Zhang; Liang-Hong Guo

    2010-01-01

    Photoelectrochemcal sensors were developed for the rapid detection of oxidative DNA damage induced by titanium dioxide and polystyrene nanoparticles. Each sensor is a multilayer film prepared on a tin oxide nanoparticle electrode using layer- by-layer self assembly and is composed of separate layer of a photoelectrochemical indicator, DNA. The organic compound and heavy metals represent genotoxic chemicals leading two major damaging mechanisms, DNA adduct formation and DNA oxidation. The DN...

  15. The formation of covalent adducts between benzo[a]pyrenediol epoxide and RNA: Structural analysis by mass spectrometry

    International Nuclear Information System (INIS)

    Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention time with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribionucleotides were poly(G) > yeast RNA > poly(A) > poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Major fragmentation pathways resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions

  16. Theoretical Investigation of Detailed Thermodynamic Character of Possible Difunctional Adducts Model

    Institute of Scientific and Technical Information of China (English)

    CHANG Guan-Ru; ZHOU Li-Xin; CHEN Dong

    2006-01-01

    The B3LYP/6-31G* level of theory was used to optimize trans-[Pt(NH3)(Am)G-L], where Am = quinoline or thiazole and L is chosen as the model for functional groups of peptide side chains, and for adenine and guanine sites of DNA as the ultimate target of platinum anticancer drugs. Bond dissociating energy and stability energy of complexes are chosen to study detailedly ther- modynamic character of possible difunctional adducts model. In order to investigate the influence of a polarizable environment on the energy of the Pt-L bond formation, we adopt a new bonding energy formula brought forward by Lippard and his coworkers: ΔH(Sol) = ΔH(SCF) + ΔG(Solv), which is quite appropriate to compare with what is found in experimental studies. Our calculated results demonstrate that N-containing ligands are more favored in view of thermodynamics both in gas phrase and in solution. However, it is worthly to be noted that addition of solvation free energies result in moderate correction of bonding energy in relative ordering, and the largest ones both present in imidazole ligand, not in guanine ligand. Finally, the nature of bond is analyzed in terms of partial charges distribution based on NBO population.

  17. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  18. Structural phase transitions and adduct release in calcium borohydride

    Energy Technology Data Exchange (ETDEWEB)

    Paolone, A.; Palumbo, O.; Rispoli, P.; Miriametro, A.; Cantelli, R.; Luedtke, A.; Rönnebro, E.; Chandra, D.

    2011-09-01

    Ca(BH4)2 compounds were investigated above room temperature by anelastic spectroscopy (AS) and concomitant measurements of thermogravimetry and mass spectrometry (TGA/MS). Both AS and TGA/MS indicate that even after a thermal treatment at 125 °C for 20 h, a non-negligible residual of THF adduct is still present in the sample, which can be removed on a subsequent thermal treatment at temperatures lower than 250 °C. Above 250 °C dehydrogenation takes place. Moreover, AS sensitively detects the occurrence of the α → α’ structural phase transition around 180 °C, and the α’ → β transformation, which is completed around 330 °C. Finally, we also show that both transitions are irreversible and are not accompanied by a latent heat.

  19. SEM-EDX STUDIES OF CHITOSAN DERIVATIVES-METAL ADDUCTS

    Directory of Open Access Journals (Sweden)

    Galo Cárdenas

    2003-12-01

    Full Text Available Chitosan was obtained from shrimps shells (pleuroncodes monodon using chemical methods. A series of chitosan (QScharged with solution of copper, cobalt, nickel and mercury ions were prepared at room temperature using the batch method. N-3,5-diethylamino benzoyl chitosan (QDAB; N,O-dimercapto succinate chitosan (QNOT and 4-aminobenzoate chitosan (QAB derivatives were prepared. The chitosan metal adducts with Cu, Co, Ni and Hg ions and derivatives maximum loading is discussed. Chitosan and derivatives containing copper were analyzed by SEM to find out the morphology of the polymers. Chitosan-Cu and derivatives charged, QDAB-Ni and QDAB-Hg were analyzed by EDX to verify the presence of the metal in the polymeric chain

  20. Vitamin A-aldehyde adducts: AMD risk and targeted therapeutics.

    Science.gov (United States)

    Sparrow, Janet R

    2016-04-26

    Although currently available treatment options for age-related macular degeneration (AMD) are limited, particularly for atrophic AMD, the identification of predisposing genetic variations has informed clinical studies addressing therapeutic options such as complement inhibitors and anti-inflammatory agents. To lower risk of early AMD, recommended lifestyle interventions such as the avoidance of smoking and the intake of low glycemic antioxidant-rich diets have largely followed from the identification of nongenetic modifiable factors. On the other hand, the challenge of understanding the complex relationship between aging and cumulative damage leading to AMD has fueled investigations of the visual cycle adducts that accumulate in retinal pigment epithelial (RPE) cells and are a hallmark of aging retina. These studies have revealed properties of these compounds that provide insights into processes that may compromise RPE and could contribute to disease mechanisms in AMD. This work has also led to the design of targeted therapeutics that are currently under investigation. PMID:27071115

  1. Effects of pesticides on DNA and protein of shrimp larvae Litopenaeus stylirostris of the California Gulf.

    Science.gov (United States)

    Galindo Reyes, J Guillermo; Leyva, Nancy R; Millan, Olivia A; Lazcano, Guadalupe A

    2002-10-01

    Recently, diverse pathologies and massive mortalities have been presented in shrimp hatcheries located along the California Gulf; therefore, toxic responses of shrimp larvae were used as biomarkers of pesticide pollution, because in this region intensive agriculture is practiced. Shrimp larvae were exposed to DDT, azinphosmethyl, permethrine, parathion, chlorpyrifos, malathion, endosulfan, and carbaryl, in order to determine LC50, DNA adducts and/or breaks, and total protein in larvae. The results indicate reductions in protein and DNA in larvae exposed to these pesticides, and in those exposed to DDT, breaks and/or adducts were registered. It is possible that pesticide pollution is a cause of these problems, because reduction in protein indicates a decrease in larvae growth rate and DNA breaks or adducts have been related to pathologies and carcinogenesis in many aquatic organisms. PMID:12568452

  2. Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53

    Science.gov (United States)

    Denissenko, Mikhail F.; Pao, Annie; Tang, Moon-Shong; Pfeifer, Gerd P.

    1996-10-01

    Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

  3. Moessbauer studies of mercury(II) salt adducts of (2)ferrocenophane derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masanobu; Ichikawa, Hajime; Motoyama, Izumi; Sano, Hirotoshi (Tokyo Metropolitan Univ. (Japan). Faculty of Science)

    1983-11-01

    Various adducts of mercuric salts with (2)ferrocenophane and 1,1,2,2-tetramethyl(2)ferrocenophane, were prepared by treating HgX/sub 2/ (X=Cl/sup -/, I/sup -/, CN/sup -/) with the (2)ferrocenophane derivatives in ether. The adducts were studied by Moessbauer spectroscopy and other physicochemical measurements. Anomalously large quadrupole splitting values of the adducts (about 3.10-3.30 mms/sup -1/ at 78 K) suggest the presence of a strong direct interaction between the Fe and Hg atoms.

  4. 32P Postlabelling analysis of urinary mutagens from smokers of black tobacco implicates 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as a major DNA-damaging agent.

    Science.gov (United States)

    Peluso, M; Castegnaro, M; Malaveille, C; Friesen, M; Garren, L; Hautefeuille, A; Vineis, P; Kadlubar, F; Bartsch, H

    1991-04-01

    When mutagens extracted from the urine of two smokers of black tobacco were reacted with DNA in vitro in the presence of a metabolic activation system, several DNA adducts were detected by 32P-postlabelling analysis. Some of these adducts were also visible, but only faintly, on the autoradiogram for a non-smoker's urine. DNA adducts produced in vitro by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or 2-amino-1-methyl-6-phenylimidazo[3,5-b]pyridine could not account for the adduct pattern produced by the urinary mutagens. However, three or four 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-related DNA adducts were present among the five or six adducts observed for smokers in the autoradiograms of urinary mutagen-adducted nucleotides. Mutagenicity testing combined with HPLC fractionation of urinary extracts also supported the postlabelling data which implicates PhIP as a mutagen in the urine of smokers of black tobacco. PMID:2013135

  5. 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.)

  6. NEW THIO S2- ADDUCTS WITH ANTIMONY (III AND V HALIDE: SYNTHESIS AND INFRARED STUDY

    Directory of Open Access Journals (Sweden)

    HASSAN ALLOUCH

    2013-12-01

    Full Text Available Five new S2- adducts with SbIII and SbV halides have been synthesized and studied by infrared. Discrete structures have been suggested, the environment around the antimony being tetrahedral, trigonal bipyramidal or octahedral.

  7. Methods for synthesizing alane without the formation of adducts and free of halides

    Science.gov (United States)

    Zidan, Ragaiy; Knight, Douglas A; Dinh, Long V

    2013-02-19

    A process is provided to synthesize an alane without the formation of alane adducts as a precursor. The resulting product is a crystallized .alpha.-alane and is a highly stable product and is free of halides.

  8. Fast repair activities of quercetin and rutin toward dGMP hydroxyl radical adducts

    International Nuclear Information System (INIS)

    The repair activities and mechanisms of both quercetin and rutin towards the oxidizing deoxyguanosine monophosphate (d GMP) hydroxyl radical adduct were investigated with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mm d GMP aqueous solution containing 0.1 mm quercetin, the transient absorption spectrum of the d GMP hydroxyl radical adduct decays with the formation of phenoxyl radical of quercetin within tens of microseconds. It indicates that there is a repair reaction between d GMP hydroxyl radical adduct and quercetin. The repair activity of rutin towards hydroxyl radical adducts of d GMP was also observed. The rate constants of the repair reactions were calculated to be 3.05x108 and 1.31x108 M-1 s-1for quercetin and rutin, respectively. This result together with our previous studies demonstrated that non-enzymatic, fast repair is a universal repair mechanism of phenolic antioxidants.

  9. Development of methods to measure hemoglobin adducts by gel electrophoresis - Preliminary results

    International Nuclear Information System (INIS)

    Chemical adducts formed on blood hemoglobin may be a useful biomarker for assessing human exposures to these compounds. This paper reports preliminary results in the development of methods to measure such adducts that may be generally applicable for a wide variety of chemicals. Male F344/N rats were intraperitoneally injected with 14C-BaP dissolved in corn oil. Twenty-four hours later, the rats were sacrificed. Blood samples were collected and globin was isolated. Globin protein was then cleaved into peptide fragments using cyanogen bromide and the fragments separated using 2-dimensional gel electrophoresis. The results showed that the adducted 14C-globin fragments migrated to different areas of the gel than did unadducted fragments. Further research is being conducted to develop methods that will allow quantitation of separated adducted globin fragments from human blood samples without the use of a radiolabel. (author)

  10. DNA binding and stimulation of cell division in the carcinogenicity of styrene 7,8-oxide

    OpenAIRE

    Lutz, Werner K; Cantoreggi, S.; Velic, I.

    2013-01-01

    [7-3H)Styrene 7,8-oxide was administered by oral gavage to male CD rats at a dose of 1.3 mg/kg. After 4 h, the forestomach was excised, DNA was isolated, purified to constant specific radioactivity and degraded nzymatically to the 3 '-nucleotides. Highperformance liquid chromatography fractions with the normal nucleotides contained most of the radiolabel, but a minute level of adduct label was also detccted. Using the units of the covalent binding index (micromoles adduct per mole DNA nucleo...

  11. Albumin Adducts of Electrophilic Benzene Metabolites in Benzene-Exposed and Control Workers

    OpenAIRE

    Lin, Yu-Sheng; Vermeulen, Roel; Tsai, Chin H.; Waidyanatha, Suramya; Lan, Qing; Rothman, Nathaniel; Smith, Martyn T.; Zhang, Luoping; Shen, Min; Li, Guilan; Yin, Songnian; Kim, Sungkyoon; Rappaport, Stephen M.

    2006-01-01

    Background Metabolism of benzene produces reactive electrophiles, including benzene oxide (BO), 1,4-benzoquinone (1,4-BQ), and 1,2-benzoquinone (1,2-BQ), that are capable of reacting with blood proteins to produce adducts. Objectives The main purpose of this study was to characterize relationships between levels of albumin adducts of these electrophiles in blood and the corresponding benzene exposures in benzene-exposed and control workers, after adjusting for important covariates. Because se...

  12. Adducts of Amine with Dimeric Rhodium(II) Tetracarboxylates - Formation of Nitrogenous Chiral Center

    International Nuclear Information System (INIS)

    Rhodium(II) tetracarboksylates Rh2(RCO2)4 (1a) are able to produce various complexes with organic ligands, like the 1:1 and 2:1 adducts containing axially bonded ligands (1b), or the compounds with rearranged dirhodium core (1c). The solution of dirhodium(II) tetracarboxylate and a ligand usually contains a mixture of adducts, due to ligand exchange and different equilibriums between species. However, application of nuclear magnetic resonance measurements at reduced temperature allows often observing the signals of all species in the solution. Ligands containing nitrogen atom were the subject of our previous investigation. It was found that dirhodium salts are able to form both 1:1 and 2:1 adducts with an amine, depending on reagents ratio. The present investigations are devoted to the amines with general formula NRR'R', is the amines having potential nitrogenous chiral center. An amine with three various substituents NRR'R' is formally a chiral molecule, but in the solution the molecule forms racemic mixture due to fast pyramidal inversion at the nitrogen atom. However, dirhodium(II) tetracarboxylates bonding the amine acts as an agent freezing pyramidal inversion at nitrogen atom, and results in formation of a new, nitrogenous chiral center in the adduct. Thus, the chiral dirhodium salt (4R)-Rh2[(CF3)(OCH3)(Ph)CO2]4 forms with benzyl-ethyl-methylamine five adducts: (4R)/(S), (4R)/(R) (1:1 adducts) and (R)/(4R)/(R), (R)/(4R)/(S), (S)/(4R)/(S) (2:1 adducts). All these diastereoisomers were detected by means of low temperature NMR (253 K). Similar effect was observed for the adducts with methyl-(1-phenylethyl)- amine, PhCH(NHCH3)CH3, having both nitrogenous and carbon chiral centers. (author)

  13. Formation and characterization of covalent guanosine adducts with electrochemistry—liquid chromatography–mass spectrometry ☆

    OpenAIRE

    Plattner, Sabine; Erb, Robert; Pitterl, Florian; Brouwer, Hendrik-Jan; Oberacher, Herbert

    2012-01-01

    Chemicals can interact with the genetic material giving rise to the formation of covalent adducts. These alterations can lead to adverse consequences, including cancer, reproductive impairment, development anomalies, or genetic diseases. In search for an assay allowing identification of hazardous compounds that might form covalent adducts with nucleic acids, electrochemistry (EC)/liquid chromatography (LC)/mass spectrometry (MS) is presented. EC/LC/MS is a purely instrumental approach. EC is ...

  14. Supercritical fluid extraction (SFE) of actinide complexes. Communication 1. SFE of uranyl trifluoroacetylacetonate adduct with pyridine

    International Nuclear Information System (INIS)

    Comparison of some methods of ascertaining the solubility of metal complexes in supercritical carbon dioxide (SC-CO2) was made. It is shown that solubility of uranyl trifluoroacetylacetonate adduct with pyridine exceeds 100 g/l (36 mg of uranium per ml) of SC-CO2 under the pressure of 300 atm and temperature of 60 deg C. Partial degrading of the uranyl trifluoroacetylacetonate adduct with pyridine is pointed out

  15. Surgical treatment of thumb adduction contracture in children with infantile cerebral palsy

    OpenAIRE

    Vladimir Alexandrovich Novikov; Valery Vladimirovich Umnov

    2015-01-01

    The purpose of the work is to evaluate the effectiveness of different methods of surgical treatment of thumb adduction contracture in children with infantile cerebral palsy.Materials and methods.The present study is based on diagnostic results of children with infantile cerebral palsy with affected upper limb. The main criterion for selection of patients was the presence of thumb adduction contracture, the absence of significant positive outcome in a patient after conservative treatment, the ...

  16. Haemoglobin adducts formed by aromatic amines in smokers: sources of inter-individual variability.

    OpenAIRE

    Ronco, G.; Vineis, P; Bryant, M S; Skipper, P. L.; Tannenbaum, S R

    1990-01-01

    In a previous study we found that aromatic amines, particularly 4-aminobiphenyl, formed haemoglobin adducts at higher concentrations in the blood of smokers compared to non-smokers. We re-analyse here data on haemoglobin adducts of 14 aromatic amines in order to ascertain if the inter-individual variability left unexplained by tobacco smoking could be attributed to differences in individual metabolic patterns. For this purpose we computed residuals from analysis of variance in order to adjust...

  17. Hemoglobin adducts of aromatic amines: associations with smoking status and type of tobacco.

    OpenAIRE

    Bryant, M S; Vineis, P; Skipper, P. L.; Tannenbaum, S R

    1988-01-01

    Hemoglobin adducts of 15 aromatic amines were determined in nonsmokers and smokers of blond- or black-tobacco cigarettes living in Turin, Italy. The subjects were all males age 55 or less and were representative of the population previously examined in a case/control study of bladder cancer. 4-Aminobiphenyl adduct levels were found to be significantly different in the three groups, and the differences were approximately proportional to the relative risk of each group. Adjustment for age and c...

  18. Effects of Metal Ion Adduction on the Gas-Phase Conformations of Protein Ions

    OpenAIRE

    Flick, Tawnya G.; Merenbloom, Samuel I.; Williams, Evan R.

    2013-01-01

    Changes in protein ion conformation as a result of nonspecific adduction of metal ions to the protein during electrospray ionization (ESI) from aqueous solutions were investigated using traveling wave ion mobility spectrometry (TWIMS). For all proteins examined, protein cations (and in most cases anions) with nonspecific metal ion adducts are more compact than the fully protonated (or deprotonated) ions with the same charge state. Compaction of protein cations upon nonspecific metal ion bindi...

  19. Structural Elucidation of a Carnosine-Acrolein Adduct and its Quantification in Human Urine Samples.

    Science.gov (United States)

    Bispo, Vanderson S; de Arruda Campos, Ivan P; Di Mascio, Paolo; Medeiros, Marisa H G

    2016-01-01

    Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic strategies for carnosine. PMID:26783107

  20. Structural Elucidation of a Carnosine-Acrolein Adduct and its Quantification in Human Urine Samples

    OpenAIRE

    Vanderson S. Bispo; Ivan P. de Arruda Campos; Paolo Di Mascio; Medeiros, Marisa H. G.

    2016-01-01

    Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic s...

  1. Scavenging of Toxic Acrolein by Resveratrol and Hesperetin and Identification of Adducts.

    Science.gov (United States)

    Wang, Weixin; Qi, Yajing; Rocca, James R; Sarnoski, Paul J; Jia, Aiqun; Gu, Liwei

    2015-11-01

    The objective of this study was to investigate the ability of resveratrol and hesperetin to scavenge acrolein at pH 7.4 and 37 °C. About 6.4 or 5.2% of acrolein remained after reaction with resveratrol or hesperetin for 12 h at equimolar concentrations. An acrolein-resveratrol adduct and two acrolein-hesperetin adducts were isolated. Their structures were elucidated using mass and NMR spectroscopy. Acrolein reacted with resveratrol at the C-2 and C-3 positions through nucleophilic addition and formed an additional heterocyclic ring. Two similar monoacrolein-conjugated adducts were identified for hesperetin. Spectroscopic data suggested each acrolein-hesperetin adduct was a mixture of four stereoisomers due to the existence of two chiral carbon atoms. Yield of adducts was low at pH 5.4 but increased at pH 7.4 and 8.4. Higher pH also promoted the formation of diacrolein adducts. Results suggest that resveratrol and hesperetin exert health benefits in part through neutralizing toxic acrolein in vivo. PMID:26457480

  2. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    Science.gov (United States)

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin

    2014-04-21

    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC. PMID:24571427

  3. A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates

    International Nuclear Information System (INIS)

    A new sensitive 32P-postlabelling assay for DNA adducts has been developed. When DNA containing bulky adducts, X1, X2, .....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pXipN, because inter-nucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, XipN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides,[32P]pXipN, by T4 polynucleotide kinase-catalyzed [32P]posphate transfer from [γ-32P]ATP. Upon mapping on polyethyleneimine-cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, [32P]pXi and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new 32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3',5'-bisphosphates, [32P]pXip. (author)

  4. Combined chemoassay and mass spectrometric approach to study the reactive potential of electrophiles towards deoxynucleosides as model for DNA.

    Science.gov (United States)

    Schmied-Tobies, Maria I H; Paschke, Heidrun; Reemtsma, Thorsten

    2016-05-01

    The modification of DNA by adduct formation is a potential molecular initiating event of genotoxicity. A chemoassay was established to study adduct formation of electrophiles with deoxynucleosides. Liquid chromatography-mass spectrometry was used to determine the reactivity of the model electrophiles para-benzoquinone, hydroquinone, and 1,4-naphthoquinone with deoxynucleoside (deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and thymidine (dT)) to detect formation of adducts via constant neutral loss scan of deoxyribose (116 Da), and to elucidate adduct structures using high resolution mass spectrometry. Of the four deoxynucleosides dG was most susceptible, followed by dC and para-benzoquinone was the most reactive electrophile. With this approach five dG and four dC adducts were detected, formed by Michael addition and subsequent condensation. Also oxidation occurred with reactive oxygen species (ROS). Three of the adducts formed by benzoquinone have not been reported before. This chemoassay combined with mass spectrometry offers a way (a) to screen a large number of chemicals for their genotoxic potential, (b) to determine novel adducts that may be searched for in in vitro and in vivo studies and thus (c) to better understand the reaction of electrophiles with nucleobases. PMID:26945242

  5. Nitroglycerin induces DNA damage and vascular cell death in the setting of nitrate tolerance.

    Science.gov (United States)

    Mikhed, Yuliya; Fahrer, Jörg; Oelze, Matthias; Kröller-Schön, Swenja; Steven, Sebastian; Welschof, Philipp; Zinßius, Elena; Stamm, Paul; Kashani, Fatemeh; Roohani, Siyer; Kress, Joana Melanie; Ullmann, Elisabeth; Tran, Lan P; Schulz, Eberhard; Epe, Bernd; Kaina, Bernd; Münzel, Thomas; Daiber, Andreas

    2016-07-01

    Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10-1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O (6)-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O (6)-methylguanine-DNA methyltransferase displayed more vascular O (6)-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O (6)-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction. PMID:27357950

  6. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  7. MUTATIONS THAT PROBE THE COOPERATIVE ASSEMBLY OF O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE (AGT) COMPLEXES†

    OpenAIRE

    Adams, Claire A.; Fried, Michael G.

    2011-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively and models of cooperative complexes predict that residues 1–7 of one protein molecule and 163–169 of a neighboring protein are closely juxtaposed. To test these models we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight d...

  8. Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I.

    OpenAIRE

    O'Connor, D; Stöhrer, G

    1985-01-01

    Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and "nick-mers", conditions of single-strand-directed DNA synthesis and nick-translation could be created. Our results show that the polymerase can bypass bot...

  9. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ.

    Science.gov (United States)

    Copeland, William C; Kasiviswanathan, Rajesh; Longley, Matthew J

    2016-01-01

    Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis. PMID:26530671

  10. Novel pyrano and vinylphenol adducts of deoxyanthocyanidins in sorghum sourdough.

    Science.gov (United States)

    Bai, Yunpeng; Findlay, Brandon; Maldonado, Alma Fernanda Sanchez; Schieber, Andreas; Vederas, John C; Gänzle, Michael G

    2014-11-26

    This study determined the fate of deoxyanthocyanidins in sorghum sourdoughs. Sourdoughs prepared from the red sorghum variety Town were fermented with the caffeic acid-decarboxylating strains Lactobacillus plantarum FUA3171 and the decarboxylase negative L. casei FUA3166. Deoxyanthocyanidins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Apigeninidin and methoxyapigeninidin were the major deoxyanthocyanidins prior to fermentation. During fermentation, novel deoxyanthocyanidins were formed. Purification by preparative LC, followed by NMR analysis and high-resolution MS identified two of the compounds as 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin. To identify pathways for their formation, sorghum was fermented with single strains, L. plantarum or L. casei. 6-Deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin were formed only during fermentation with L. plantarum FUA3171, indicating a role of vinylphenol in their formation. Chemical synthesis confirmed that 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin are formed from apigeninidin with vinylphenol but not with p-coumaric acid as reactants. In conclusion, the products of microbial decarboxylation of hydroxycinnamic acids convert apigeninidin and methoxyapigeninidin to pyrano-3-deoxyanthocyanidins and vinylphenol adducts. PMID:25370078

  11. Preferential binding of benzo[a]pyrene diol epoxide to the linker DNA of human foreskin fibroblasts in S phase in the presence of benzamide.

    OpenAIRE

    Kurian, P.; Jeffrey, A M; Milo, G E

    1985-01-01

    Addition of benzamide (BZ) at the onset of S phase inhibited expression of the neoplastic phenotype in human foreskin fibroblasts treated in vitro with (+/-)-7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P diol epoxide) in early S phase. Analysis of the specific B[a]P diol epoxide-DNA adducts revealed that ca. 65% of the total adducts in BZ and non-BZ carcinogen-treated cells was the B[a]P diol epoxide-deoxyguanine adduct. Limited micrococcal nuclease di...

  12. Chromatin structure and DNA damage

    International Nuclear Information System (INIS)

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' → 5' exonuclease activity of T4 DNA polymerase

  13. Specificity and affinity of phenosafranine protein adduct: Insights from biophysical aspects

    Energy Technology Data Exchange (ETDEWEB)

    Ding Fei [Department of Chemistry, China Agricultural University, Beijing 100193 (China); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Zhang Li; Sun Ye [Department of Chemistry, China Agricultural University, Beijing 100193 (China); Diao Jianxiong [College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193 (China); Yang Xinling [Department of Chemistry, China Agricultural University, Beijing 100193 (China); Sun Ying, E-mail: sunying@cau.edu.cn [College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193 (China); Zhang Li, E-mail: zhli.work@gmail.com [Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193 (China)

    2012-03-15

    Phenosafranine is a toxic and recalcitrant compound, whose capacity to intercalate with double stranded DNA has been shown. In this contribution, a biophysical discuss on the conjugation of phenosafranine with two model proteins human serum albumin (HSA) and lysozyme (Lys) has been identified utilizing a combination of molecular modeling, steady state and time-resolved fluorescence and circular dichroism (CD) approaches. The accurate binding domain of phenosafranine in protein has been characterized from molecular modeling, subdomain IIIA of HSA and Trp-62, Trp-63 and Trp-108 residues of Lys was designated to possess high-affinity for this compound, the dominant forces in the protein-phenosafranine adduct are hydrogen bonds and {pi}-{pi} interactions, but hydrophobic interactions between dye and Lys are also not exclude. The data of fluorescence displayed that the complex of phenosafranine with protein produces quenching through static property, this corroborates the time-resolved fluorescence results that the ground state complex formation with a moderate affinity of 10{sup 4} M{sup -1}. Moreover, via synchronous fluorescence, CD and three-dimensional fluorescence we indicated some extent of polypeptide chain of protein partially unfolding upon conjugation with phenosafranine. Through this work, we anticipate it can supply salient clues on the toxicological action of phenosafranine and other azines, which have analogous configuration with phenosafranine. - Highlights: Black-Right-Pointing-Pointer Our study supply salient clues on the toxicological action of toxic phenosafranine. Black-Right-Pointing-Pointer Phenosafranine is situated within subdomain IIIA, Sudlow's site II on HSA. Black-Right-Pointing-Pointer Trp-62, Trp-63 and Trp-108 residues on the Lys molecule are all close to dye. Black-Right-Pointing-Pointer Static property of the phenosafranine induced quenching of protein Trp fluorescence. Black-Right-Pointing-Pointer Protein spatial structure proved

  14. Diazoalkane addition reaction on the fullerene dimer C120O and characterization of the resulting mono-adduct.

    Directory of Open Access Journals (Sweden)

    Uwe Ritter

    2008-06-01

    Full Text Available A mono-adduct of the fullerene dimer C120O was prepared via a diazoalkane addition reaction to obtain rod-like analogue of[60]PCBM opening the possibility to make photovoltaic and photosensitive layers of supra-molecular and anisotropic order.The mono-adduct was obtained as a mixture containing five isomers. The structure of the mono-adduct was verified bymass-, IR-, and 1H-NMR-spectroscopies. The mono-adduct is readily soluble in common fullerene solvents and shows abroader and stronger optical absorption than [60]PCBM. The mono-adduct features a similar acceptor strength as [60]PCBMand [70]PCBM, is stable in air below 150 °C and in nitrogen below 500 °C. The mono-adduct is expected to be a valuablematerial for photovoltaic and photosensitive applications.

  15. DNA adducts formation, induction of oxidative stress and apoptosis in model of rat liver progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Dostálová, L.; Marvanová, S.; Krčmář, P.; Andrysík, Zdeněk; Topinka, J.; Vondráček, Jan; Machala, M.

    Liptovský Ján, 2007. s. 62. ISBN 978-80-969688-5-5. [XXIV. Xenobiochemické sympózium. 22.05.2007-24.05.2007, Liptovský Ján] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : genotoxicity * apoptosis * oval cells Subject RIV: BO - Biophysics

  16. Redox and catalytic reactions of adducts of DNA with osmium tetroxide complexes and carbon electrodes

    Czech Academy of Sciences Publication Activity Database

    Havran, Luděk; Fojta, Miroslav; Kubičárová, Tatiana; Paleček, Emil

    Praha, 2000. s. 39. [J. Heyrovský Memorial Symposium on Advances in Polarography and Related Methods. 30.08.2000-01.09.2000, Praha] Institutional research plan: CEZ:A17/98:Z5-004-9-ii Subject RIV: BO - Biophysics

  17. Disturbance of DNA conformation by the binding of testosterone-based platinum drugs via groove-face and intercalative interactions: a molecular dynamics simulation study

    OpenAIRE

    Cui, Shanshan; Wang, Yan; Chen, Guangju

    2013-01-01

    Background To explore novel platinum-based anticancer agents that are distinct from the structure and interaction mode of the traditional cisplatin by forming the bifunctional intrastrand 1,2 GpG adduct, the monofunctional platinum + DNA adducts with extensive non-covalent interactions had been studied. It was reported that the monofunctional testosterone-based platinum(II) agents present the high anticancer activity. Moreover, it was also found that the testosterone-based platinum agents cou...

  18. Silver adducts of four-branched histidine rich peptides exhibit synergistic antifungal activity.

    Science.gov (United States)

    Leng, Qixin; Woodle, Martin C; Liu, Yijia; Mixson, A James

    2016-09-01

    Previously, a four branched histidine-lysine rich peptide, H3K4b, was shown to demonstrate selective antifungal activity with minimal antibacterial activity. Due to the potential breakdown from proteases, H3K4b was further evaluated in the current study by varying the D- and l-amino acid content in its branches. Whereas analogues of H3K4b that selectively replaced l-amino acids (H3k4b, h3K4b) had improved antifungal activity, the all d-amino acid analogue, h3k4b, had reduced activity, suggesting that partial breakdown of the peptide may be necessary. Moreover, because histidines form coordination bonds with the silver ion, we examined whether silver adducts can be formed with these branched histidine-lysine peptides, which may improve antifungal activity. For Candida albicans, the silver adduct of h3K4b or H3k4b reduced the MIC compared to peptide and silver ions alone by 4- and 5-fold, respectively. For Aspergillus fumigatus, the silver adducts showed even greater enhancement of activity. Although the silver adducts of H3k4b or h3K4b showed synergistic activity, the silver adduct with the all l-amino acid H3K4b surprisingly showed the greatest synergistic and growth inhibition of A. fumigatus: the silver adduct of H3K4b reduced the MIC compared to the peptide and silver ions alone by 30- and 26-fold, respectively. Consistent with these antifungal efficacy results, marked increases in free oxygen radicals were produced with the H3K4b and silver combination. These studies suggest that there is a balance between stability and breakdown for optimal antifungal activity of the peptide alone and for the peptide-silver adduct. PMID:27387239

  19. Repair of DNA treated with γ-irradiation and chemical carcinogens. Final report, June 1, 1981-May 31, 1984

    International Nuclear Information System (INIS)

    Work done in the past three years has been on DNA repair, on genetic transposition and on the effect of carcinogens on alu sequence transcription. DNA repair work was completed on β-propiolactone DNA adducts, on procaryotic and eucaryotic enzymes capable of removal of 3-methyladenine from DNA, and on in vitro repair of neucleosomal core particle DNA and chromatin DNA. Attempts were made to isolate a human transposable element through the isolation of double stranded RNA and probing of a human library. Experiments were also done to determine whether carcinogens altered the expression of alu sequences in human DNA

  20. Formation of metal-ion adducts and evidence for surface-catalyzed ionization in electrospray analysis of pharmaceuticals and pesticides

    Science.gov (United States)

    Thurman, E.M.; Ferrer, I.

    2002-01-01

    The formation of metal ion adducts in liquid chromatography/mass spectrometry positive-ion electrospray analysis of pharmaceuticals and pesticides was investigated. The evidence of surface-catalyzed ionization in the electrospray analysis was also studied. Both positive and negative ion mass spectrometry were used for the analysis of the products. It was found that the sodium adducts formed in the analysis included single, double, and triple sodium adducts. Adduction was found to occur by attachment of the metal ion to carboxyl, carbonyl and aromatic pi electrons of the molecule.

  1. Synthesis of a duplex oligonucleotide containing a nitrogen mustard interstrand DNA-DNA cross-link.

    Science.gov (United States)

    Ojwang, J O; Grueneberg, D A; Loechler, E L

    1989-12-01

    Many cancer chemotherapeutic agents react with DNA and give adducts that block DNA replication, which is thought to result in cytotoxicity, especially in rapidly proliferating cells such as cancer cells. One class of these agents is bifunctionally reactive (e.g., the nitrogen mustards) and forms DNA-DNA cross-links. It is unknown whether inter- or intrastrand cross-links are more effective at blocking DNA replication. To evaluate this, a DNA shuttle vector is being constructed with an interstrand cross-link at a unique site. In the first step of this project, a duplex oligonucleotide containing an interstrand cross-link is isolated by denaturing polyacrylamide gel electrophoresis from the reaction of nitrogen mustard with two partially complementary oligodeoxynucleotides. The purified oligonucleotide product is characterized and shown to be cross-linked in a 5'-GAC-3' 3'-CTG-5' sequence by a nitrogen mustard moiety that is bound at the N(7)-position of the guanines in the opposing strands; the glycosylic bonds of these guanine adducts are stabilized in their corresponding imidazole ring-opened form. Nitrogen mustard is shown to react with a variety of oligonucleotides and, based upon these results, its preferred targets for interstrand cross-linking are 5'-GXC-3' sequences, where X can be any of the four deoxyribonucleotide bases. PMID:2819709

  2. Characterization of model peptide adducts with reactive metabolites of naphthalene by mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Nathalie T Pham

    Full Text Available Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.

  3. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    Science.gov (United States)

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-01

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties. PMID:27387593

  4. Mechanisms of mutagenesis and DNA repair

    International Nuclear Information System (INIS)

    The research deals with mechanisms of excision repair in Escherichia coli exposed to chemicals or ultraviolet (uv) radiation. During the past year attention has been focused on the incision proteins that initiate removal of pyrimidine dimers, benzo[a]pyrene adducts, and other bulky lesions from DNA. The product of the E. Coli uvrD gene was isolated and shown to be important in mediating closure of single strand DNA breaks promoted by the incision complex coded for by the uvrA, uvrB, and uvrC gene products. This suggests that the uvrD gene product (now known to be a helicase) is necessary either for dislodging the incision complex from the nicked DNA or for preparing a DNA primer-template configuration suitable for proper repair resynthesis

  5. Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Valovičová, Z.; Marvanová, S.; Mészárosová, M.; Srančíková, A.; Trilecová, L.; Milcová, A.; Líbalová, H.; Vondráček, Jan; Machala, M.; Topinka, J.; Gábelová, A.

    2009-01-01

    Roč. 665, 1-2 (2009), s. 51-60. ISSN 0027-5107 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA adducts * DNA strand breaks * oxidative stress Subject RIV: BO - Biophysics Impact factor: 3.556, year: 2009

  6. Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts.

    Science.gov (United States)

    Lemons, Angela R; Bledsoe, Toni A; Siegel, Paul D; Beezhold, Donald H; Green, Brett J

    2013-11-29

    Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures. PMID:24012971

  7. Atomic-Resolution Structure of an N(5) Flavin Adduct in D-Arginine Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Guoxing; Yuan, Hongling; Wang, Siming; Gadda, Giovanni; Weber, Irene T. (GSU)

    2011-09-06

    D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other D-amino acids to the corresponding imino acids. The 1.07 {angstrom} atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct.

  8. Atomic-resolution structure of an N5 flavin adduct in D-arginine dehydrogenase.

    Science.gov (United States)

    Fu, Guoxing; Yuan, Hongling; Wang, Siming; Gadda, Giovanni; Weber, Irene T

    2011-07-26

    D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other D-amino acids to the corresponding imino acids. The 1.07 Å atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct. PMID:21707047

  9. Acetaminophen-cysteine adducts during therapeutic dosing and following overdose

    Directory of Open Access Journals (Sweden)

    Judge Bryan S

    2011-03-01

    Full Text Available Abstract Background Acetaminophen-cysteine adducts (APAP-CYS are a specific biomarker of acetaminophen exposure. APAP-CYS concentrations have been described in the setting of acute overdose, and a concentration >1.1 nmol/ml has been suggested as a marker of hepatic injury from acetaminophen overdose in patients with an ALT >1000 IU/L. However, the concentrations of APAP-CYS during therapeutic dosing, in cases of acetaminophen toxicity from repeated dosing and in cases of hepatic injury from non-acetaminophen hepatotoxins have not been well characterized. The objective of this study is to describe APAP-CYS concentrations in these clinical settings as well as to further characterize the concentrations observed following acetaminophen overdose. Methods Samples were collected during three clinical trials in which subjects received 4 g/day of acetaminophen and during an observational study of acetaminophen overdose patients. Trial 1 consisted of non-drinkers who received APAP for 10 days, Trial 2 consisted of moderate drinkers dosed for 10 days and Trial 3 included subjects who chronically abuse alcohol dosed for 5 days. Patients in the observational study were categorized by type of acetaminophen exposure (single or repeated. Serum APAP-CYS was measured using high pressure liquid chromatography with electrochemical detection. Results Trial 1 included 144 samples from 24 subjects; Trial 2 included 182 samples from 91 subjects and Trial 3 included 200 samples from 40 subjects. In addition, we collected samples from 19 subjects with acute acetaminophen ingestion, 7 subjects with repeated acetaminophen exposure and 4 subjects who ingested another hepatotoxin. The mean (SD peak APAP-CYS concentrations for the Trials were: Trial 1- 0.4 (0.20 nmol/ml, Trial 2- 0.1 (0.09 nmol/ml and Trial 3- 0.3 (0.12 nmol/ml. APAP-CYS concentrations varied substantially among the patients with acetaminophen toxicity (0.10 to 27.3 nmol/ml. No subject had detectable APAP

  10. Quantitative analysis of radiation-induced DNA deoxyribose oxidation products

    International Nuclear Information System (INIS)

    Deoxyribose oxidation plays an important role in the chemistry and biology of radical-mediated DNA damage beyond the simple interruption of the DNA backbone, including involvement in complex DNA lesions, cross-linking with DNA repair proteins and the formation of endogenous DNA adducts. This is illustrated by our discovery that 3'-phosphoglycolaldehyde residues, arising from 3'-oxidation of deoxyribose in DNA, form glyoxal and the glyoxal adduct of dG. Our research is driven by the lack of information about the spectrum and quantity of deoxyribose lesions in isolated DNA, human cells and tissues. This problem is compounded by the fact that oxidation of each of the five possible positions in deoxyribose can generate several unique damage products, most of which are toxic to cells. To this end, we have developed a sensitive GC/MS method to identify and quantify virtually all deoxyribose oxidation products in isolated DNA and in cells exposed to oxidizing agents under biological conditions. This method was applied to quantify 3'-phosphoglycolaldehyde residues in DNA oxidized by Fe-EDTA, gamma-radiation and alpha-particles with a detection limit of 30 femtomoles/sample corresponding to two phosphoglycolaldehyde molecules in 10?6 nucleotides for a 170 μg DNA sample. A 13C2 - labeled phosphoglycolaldehyde was used as internal standard. The method was verified by analysis of a synthetic, phosphoglycolaldehyde-containing oligonucleotide. It is widely believed that Fe-EDTA and gamma-radiation induce DNA damage by the formation of hydroxyl radicals and therefore we expected to see similar efficiencies in phosphoglycolaldehyde formation. However, the results reveal large differences in the efficiency of phosphoglycolaldehyde formation by these oxidants and suggest weaknesses in models relating DNA structure to chemical reactivity of DNA. An understanding of the relative quantities of various deoxyribose oxidation products will provide important insights into the basic

  11. Formation of melamium adducts by pyrolysis of thiourea or melamine/NH4 Cl mixtures.

    Science.gov (United States)

    Braml, Nicole E; Sattler, Andreas; Schnick, Wolfgang

    2012-02-01

    Pyrolysis of prominent precursor compounds for the synthesis of carbon nitride type materials (e.g., melamine, thiourea) have been studied in detail. Molecular adducts containing monoprotonated melamium C(6)N(11)H(10)(+) and melaminium HC(3)N(3)(NH(2))(3)(+) ions, respectively, have been identified as intermediates. The adduct C(6)N(11)H(10)Cl·0.5NH(4)Cl was obtained by the reaction of melamine C(3)N(3)(NH(2))(3) with NH(4)Cl at 450 °C. During the pyrolysis of thiourea, guanidinium thiocyanate was initially formed and subsequently the melamium thiocyanate melamine adduct C(6)N(11)H(10)SCN·2C(3)N(3)(NH(2))(3) was isolated at 300 °C. A second melaminium thiocyanate melamine adduct with the formula HC(3)N(3)(NH(2))(3)SCN·2C(3)N(3)(NH(2))(3) represents an intermediary reaction product that is best accessible at low pressures. The crystal structures of the compounds were solved by single-crystal XRD. Unequivocal proton localization at the C(6)N(11)H(10)(+) ion was established. A typical intramolecular and interannular hydrogen bridge and other characteristic hydrogen-bonding motifs were identified. Additionally, the adducts were investigated by solid-state NMR spectroscopy. Our study provides detailed insight into the thermal condensation of thiourea by identifying and characterizing key intermediates involved in the condensation process leading to carbon nitride type materials. Furthermore, factors promoting the formation of melamium adduct phases over melem are discussed. PMID:22223531

  12. Comparison of DNA-Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

    Science.gov (United States)

    Krishnan, Sadagopan; Bajrami, Besnik; Mani, Vigneshwaran; Pan, Shenmin; Rusling, James F.

    2012-01-01

    Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy)32+ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response. PMID:23100998

  13. Effect of external electric field on Cyclodextrin-Alcohol adducts: A DFT study

    Indian Academy of Sciences (India)

    Kundan Baruah; Pradip Kr Bhattacharyya

    2015-06-01

    Effect of external electric fields on the interaction energy between cyclodextrin and alcohol was analyzed in the light of density functional theory (DFT) and density functional reactivity theory (DFRT). Stability of the cyclodextrin-alcohol adducts was measured in terms of DFT based reactivity descriptor, global hardness, electrophilicity, and energy of the HOMO. Stability of adducts was observed to be sensitive towards the strength as well as direction of the applied external electric field. In addition, reactivity pattern follows the maximum hardness and minimum electrophilicity principles.

  14. Adduct formation in LC-ESI-MS of nonylphenol ethoxylates: mass spectrometrical, theoretical and quantitative analytical aspects

    International Nuclear Information System (INIS)

    The analysis of nonylphenol ethoxylate (A9PEOn) surfactants with LC-ESI-MS was investigated in a detailed study of the formation of different types of adducts. Part of the observations was explained by calculating their relative stabilities using molecular dynamics techniques. Strong differences in adduct formation behaviour were found for different oligomers. Beside the common sodium adducts, surfactant dimer adducts [2 x A9PEO1,2 + Na]+, adducts including a solvent molecule [A9PEO1,2 + MeOH + Na]+ and doubly charged adducts [A9PEO>11 + 2 x Na]2+ were found. Molecular dynamics calculations showed that the A9PEOn molecule wraps itself around the complexing sodium ion in a way that negative electronic charges on oxygen have optimum electrostatic interaction with this ion. van der Waals interactions between alkyl chains are of less importance for the stability of these adducts. Both [2 x A9PEO2,5 + Na]+ dimer and [A9PEO2,5 + Na]+ monomer adducts turned out to be stable from an energetic point of view with adducts of A9PEO5 being more stable than adducts of A9PEO2. Only for the monomer adduct the latter is in accordance with experimental observations. Consequences of the formation of several adducts per A9PEOn oligomer for the quantitative analysis of environmental samples were evaluated. In clean samples, it was found that the presence of short-chain A9PEO1,2 can cause an overestimation of long-chain A9PEO>2. In real environmental extracts, other processes like matrix effects have a stronger influence on the quantitative result, and therefore no significant influence of adduct formation processes could be observed. However, inclusion of [A9PEO1,2 + MeOH + Na]+ adduct signals does improve the detection limits of the two short-chain oligomers. Correct quantitative results are obtained when A9PEO1 and A9PEO2 are quantified separately, and longer oligomers with a molar calibration followed by correction of the average molar weight of the A9PEO>2 in the sample

  15. Trabectedin – the DNA minor groove binder

    Directory of Open Access Journals (Sweden)

    G. A. Belitsky

    2015-01-01

    Full Text Available Trabectedin (ET-743, Yondelis is an alkaloid that was originally isolated from the Caribbean Sea squirt, Ecteinascidia turbinata and is now produced synthetically. Its chemical structure consists in three fused tetrahydroisoquinoline rings. Two of them, A and B, binds covalently to guanine residues in the minor groove of the DNA double helix to bend the molecule toward the major groove and the third ring C protrudes from the DNA duplex, apparently allowing interactions with several nuclear proteins. Binding to the minor groove of DNA, trabectedin trigger a cascade of events that interfere with several transcription factors, DNA binding proteins, and DNA repair pathways in particular nucleotide excision repair. It acts both as a DNA-alkylating drug and topoisomerase poison. Trabectedin-DNA adduct traps the nucleotide excision repair proteins repairing the DNA damage in transcribing genes and induces DNA strand breaks. Cells deficient in homologous recombination pathway which repairs these double-strand breaks show increased sensitivity to trabectedin. The most sensitive of them were myxoid liposarcomas. Trabectedin is also effective in chemotherapy-experienced patients with advanced, recurrent liposarcoma or leiomyosarcoma as well as in women with ovarian cancer and breast cancer with BRCAness phenotype. Besides of tumor cells Trabectedin inhibits inflammatory cells by affecting directly monocytes and tumorassociated macrophages and indirectly by inhibiting production of inflammatory mediators, the cytokines and chemokines. It inhibits also the MDR-1 gene, which is responsible for the resistance of cancer cells to chemotherapeutic agents and strikes tumor angiogenesis.

  16. Apoptosis and DNA damage in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    R John Aitken; Adam J Koppers

    2011-01-01

    DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis,resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

  17. Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage

    International Nuclear Information System (INIS)

    DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solid-phase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dual-electrode high-efficient electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates

  18. Close encounters for the first time: Helicase interactions with DNA damage.

    Science.gov (United States)

    Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

    2015-09-01

    DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. PMID:26160335

  19. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-12-29

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

  1. Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases

    OpenAIRE

    Khan, Irfan; Suhasini, Avvaru N.; Banerjee, Taraswi; Sommers, Joshua A.; Kaplan, Daniel L.; Kuper, Jochen; Kisker, Caroline; Brosh, Jr, Robert M

    2014-01-01

    8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ ...

  2. Replication of N[superscript 2],3-Ethenoguanine by DNA Polymerases

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Linlin; Christov, Plamen P.; Kozekov, Ivan D.; Pence, Matthew G.; Pallan, Pradeep S.; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter (Vanderbilt)

    2014-10-02

    The unstable DNA adduct N2,3-ethenoguanine, a product of both exposure to the carcinogen vinyl chloride and of oxidative stress, was built into an oligonucleotide, using an isostere strategy to stabilize the glycosidic bond. This modification was then used to examine the cause of mutations by DNA polymerases, in terms of both the biochemistry of the lesion and a structure of the lesion within a polymerase.

  3. O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism?

    OpenAIRE

    Gouws, Chrisna; Pretorius, Petrus Jacobus

    2011-01-01

    Why does O6-methylguanine-DNA methyltransferase (MGMT), an indispensable DNA repair enzyme, have a mechanism which seems to run counter to its importance? This enzyme is key to the removal of detrimental alkyl adducts from guanine bases. Although the mechanism is well known, an unusual feature surrounds its mode of action, which is its so-called suicidal endpoint. In addition, induction of MGMT is highly variable and its kinetics is atypical. These features raise some questions on the seeming...

  4. Role of DNA lesions and DNA repair in mutagenesis by carcinogens in diploid human fibroblasts

    International Nuclear Information System (INIS)

    The authors investigated the cytotoxicity, mutagenicity, and transforming activity of carcinogens and radiation in diploid human fibroblasts, using cells which differ in their DNA repair capacity. The results indicate that cell killing and induction of mutations are correlated with the number of specific lesions remaining unrepaired in the cells at a particular time posttreatment. DNA excision repair acts to eliminate potentially cytotoxic and mutagenic (and transforming) damage from DNA before these can be converted into permanent cellular effects. Normal human fibroblasts were derived from skin biopsies or circumcision material. Skin fibroblasts from xeroderma pigmentosum (XP) patients provided cells deficient in nucleotide excision repair of pyrimidine dimers or DNA adducts formed by bulky ring structures. Cytotoxicity was determined from loss of ability to form a colony. The genetic marker used was resistance to 6-thioguanine (TG). Transformation was measured by determining the frequency of anchorage-independent cells

  5. Conformational, IR spectroscopic and electronic properties of conium alkaloids and their adducts with C60 fullerene

    Science.gov (United States)

    Zabolotnyi, M. A.; Prylutskyy, Yu I.; Poluyan, N. A.; Evstigneev, M. P.; Dovbeshko, G. I.

    2016-08-01

    Conformational, IR spectroscopic and electronic properties of the components of Conium alkaloids (Conium maculatum) in aqueous environment were determined by model calculations and experiment. With the help of FT-IR spectroscopy the possibility of formation of an adduct between γ-coniceine alkaloid and C60 fullerene was demonstrated, which is important for further application of conium analogues in biomedical purposes.

  6. Situational restriction of elevation in adduction relieved by faden on the medial rectus

    Directory of Open Access Journals (Sweden)

    R Muralidhar

    2016-01-01

    Full Text Available We describe a patient with situational restriction of elevation in adduction in his left eye. Clinical examination pointed to instability of the left medial rectus pulley. This was corrected by Faden on the medial rectus. The importance of this relatively new concept in identifying and treating orbital pulley instability is discussed.

  7. The knee adduction moment measured with an instrumented force shoe in patients with knee osteoarthritis

    NARCIS (Netherlands)

    Noort, van den Josien C.; Esch, van der Martin; Steultjens, Martijn P.M.; Dekker, Joost; Schepers, H. Martin; Veltink, Peter H.; Harlaar, Jaap

    2012-01-01

    The external knee adduction moment (KAdM) during gait is an important parameter in patients with knee osteoarthritis (OA). KAdM measurement is currently restricted to instruments only available in gait laboratories. However, ambulatory movement analysis technology, including instrumented force shoes

  8. Eccentric hip adduction and abduction strength in elite soccer players and matched controls

    DEFF Research Database (Denmark)

    Thorborg, Kristian; Couppé, C; Petersen, J;

    2011-01-01

    Eccentric hip adduction and abduction strength plays an important role in the treatment and prevention of groin injuries in soccer players. Lower extremity strength deficits of less than 10% on the injured side, compared to the uninjured side, have been suggested as the clinical milestone before ...

  9. Adducts compounds of lanthanides (III) trifluoreacetates and yttrium and the N,N - dimenthylformamide

    International Nuclear Information System (INIS)

    Some studies on lanthanides, f transition elements, and yttrium are presented. Adducts of lanthanides trifluoroacetates and N,N -dimethylformamide are described. The characterization of complexes from elementar analysis, conductance measurements, X-ray patterns, vibrational, electronics and fluorescence spectra are analysed. (M.J.C.)

  10. Simulation of Solid-Liquid Equilibrium in a Ternary System with Adduct Compounds

    Czech Academy of Sciences Publication Activity Database

    Malijevská, I.; Sedláková, Zuzana

    Kuala Lumpur : -, 2007, s. 150-151. [Asian Chemical Congress (12ACC) 2007 /12./. Kuala Lumpur (MY), 23.08.2007-25.08.2007] Institutional research plan: CEZ:AV0Z40720504 Keywords : solid-liquid equilibrium * adduct * ternary system Subject RIV: CF - Physical ; Theoretical Chemistry

  11. Structural analysis and aggregation propensity of reduced and nonreduced glycated insulin adducts.

    Science.gov (United States)

    Alavi, Parnian; Yousefi, Reza; Amirghofran, Sara; Karbalaei-Heidari, Hamid Reza; Moosavi-Movahedi, Ali Akbar

    2013-06-01

    The milieu within pancreatic β cells represents a favorable environment for glycation of insulin. Therefore, in this study, insulin samples were individually subjected to glycation under reducing and nonreducing conditions. As monitored by ortho-phthalaldehyde and fluorescamine assays, the reduced glycated insulin adduct demonstrates extensively higher level of glycation than the nonreduced glycated counterpart. Also, gel electrophoresis experiments suggest a significant impact of glycation under a reducing system on the level of insulin oligomerization. Furthermore, reduced and nonreduced glycated insulin adducts respectively exhibit full and partial resistance against dithiothreitol-induced aggregation. The results of thioflavin T and Congo red assays suggest the existence of a significant quantity of amyloid-like entities in the sample of reduced glycated insulin adduct. Both fluorescence and far-ultraviolet circular dichroism studies respectively suggest that the extents of unfolding and secondary structural alteration were closely correlated to the level of insulin glycation. Moreover, the surface tension of two glycated insulin adducts was inversely correlated to their glycation extents and to the quantity of exposed hydrophobic patches. Overall, the glucose-modified insulin molecules under reducing and nonreducing systems display different structural features having significant consequences on aggregation behaviors and surface tension properties. The particular structural constraints of glycated insulin may reduce the binding interaction of this hormone to its receptor which is important for both insulin function and clearance. PMID:23584594

  12. Hemoglobin adducts of epoxybutene in workers occupationally exposed to 1,3-butadiene

    Czech Academy of Sciences Publication Activity Database

    Begemann, P.; Šrám, Radim; Neumann, H. G.

    2001-01-01

    Roč. 74, - (2001), s. 680-687. ISSN 0340-5761 Grant ostatní: EU(XC) CIPA-CT93-0228 Institutional research plan: CEZ:AV0Z5039906 Keywords : biomonitoring * hemoglobin adducts Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 1.558, year: 2001

  13. New adduct of abietane-type diterpene from Salvia leriifolia Benth.

    Science.gov (United States)

    Hussain, Amjad; Adhikari, Achyut; Iqbal Choudhary, M; Ayatollahi, Syed Abdulmajid; Atta-Ur-Rahman

    2016-07-01

    A new adduct of abietane-type diterpene, salvialeriicone (1), was isolated from Salvia leriifolia Benth., along with a new chemical entity nor-abietane diterpene, 2-isopropyl-8,8-dimethyl-7,8-dihydrophenanthrene-1,4,5(6H)-trione (2). Their structures were determined using mass spectrometry, and 1D- and 2D-NMR spectroscopy. PMID:27266891

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-15

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

  15. 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)

    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

  16. 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

  17. Relationship between ambient air pollution and DNA damage in Polish mothers and newborns

    International Nuclear Information System (INIS)

    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

  18. Quantitative analysis of positional isomers of triacylglycerols via electrospray ionization tandem mass spectrometry of sodiated adducts.

    Science.gov (United States)

    Herrera, Lisandra Cubero; Potvin, Michael A; Melanson, Jeremy E

    2010-09-01

    Herein we report a reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC/MS/MS) method for the analysis of positional isomers of triacylglycerols (TAGs) in vegetable oils. The fragmentation behavior of [M + X](+) ions (X = NH(4), Li, Na or Ag) was studied on a quadrupole-time-of-flight (Q-TOF) mass spectrometer under low-energy collision-induced dissociation (CID) conditions. Mass spectra that were dependent on the X(+) ion and the nature and position of the acyl substituents were observed for four pairs of 'AAB/ABA'-type TAGs, namely PPO/POP, OOP/OPO, LLO/LOL and OOL/OLO (where P is 16:0, palmitic acid; O is 18:1, oleic acid; and L is 18:2, linoleic acid). For the majority of [M + X](+) adducts, the loss of the fatty acid in the outer positions (sn-1 or sn-3) was favored over the loss in the central position (sn-2), which enabled the determination of the fractional abundance of the isomers. Ratios of the intensity of fragment ions at various AAB/ABA compositions produced linear calibration curves with positive slopes, comparable to those obtained traditionally by ESI-MS/MS of [M + NH(4)](+) adducts. The only exceptions were the [M + Ag](+) adducts of the PPO/POP system, which produced calibration curves with negative slopes. Sodium adducts provided the most consistent level of isomeric discrimination for the TAGs studied and also offered the most convenience in that they required no additive to the mobile phase. Therefore, calibration curve data derived from [M + Na](+) adducts were applied to the quantification of TAG regioisomers in sunflower and olive oils. The regiospecific analysis showed that palmitic acid was typically located at positions sn-1 or sn-3, whereas unsaturated fatty acids, oleic and linoleic acids were mostly found at the sn-2 position. PMID:20814981

  19. A pulse radiolysis study of the electron reaction with DNA in aqueous solution and ice

    International Nuclear Information System (INIS)

    Research is described into some aspects of the interaction of ionizing radiation with DNA, the main emphasis being on the indirect effect where the radiation causes the formation of intermediates in the surrounding medium which then interact with the DNA. Experiments are described which have been carried out to examine the dependence of the reaction rate constant on ionic strength and counterion charge. They show that for double stranded DNA (dsDNA) the reaction rate constant increases with ionic strength. To see whether quantitative agreement can be obtained between the observations and a physical interpretation the author combines a model for reaction kinetics and a refined model for the electrostatic potential into one to obtain a theoretical rate constant for reaction with a cylindrical polyelectrolyte. Optical absorption spectra of the electron adducts of ss- and dsDNA are shown to exist and they differ significantly from the spectra of the H and OH adducts. The spectra of the electron adducts of the nucleotides have been measured. Experiments are also described devoted to the precursor of the hydrated electron: the thermalized, dry electron. Although from a radiation chemical point of view the experiments yielded a lot of information (a direct observation of the dry electron, the kinetics of electron localization, the role of ice defects in the solvation process, etc.), they proved also that the dry electron does not react with DNA in an ice matrix. (Auth.)

  20. Preparation and Evaluation of Aromatic Amine-Epoxidized Sunflower Free Fatty Acid Adducts As Corrosion Inhibitors in Curable Varnishes

    International Nuclear Information System (INIS)

    Five aromatic amines [o-, m- and p- Toluidine (o-T, m-T and p-T), p- anizidine (p-A) and p- chloroaniline (p-ClA)] were reacted with epoxidized sunflower free fatty acid (ESFA) under severe conditions of inert atmosphere and high temperature. The produced adducts were characterized physically, chemically and by IR spectroscopic analysis. Acid value and oxiran content of the prepared adducts were determined to confirm the participation of carboxylic groups and epoxy groups respectively in the reaction of ESFA with aromatic amines. The prepared adducts of (o-T-ESFA, m-T-ESFA, p-T-ESFA, p-A-ESFA and p-ClA-ESFA) were evaluated as corrosion inhibitors of mild steel in epoxy acrylate oligomer formulations curable by electron beam irradiation. Different concentrations of the prepared aromatic adducts were added in varnish formulations. Physical and mechanical measurements were carried out, in addition to corrosion resistance tests and weight loss of coated steel panels. The efficiency of adducts in varnishes formulation were determined, in which it was found that, the varnish formulations containing the prepared aromatic amine adducts could protect steel from corrosion. Superior corrosion inhibition efficiency was found for the varnish formula containing 0.6% p-A-ESFA adduct. The corrosion inhibition efficiency of the prepared aromatic amine in epoxy acrylate oligomer varnishes follows the order: p-A-ESFA > p-T-ESFA > m-T-ESFA > o-T-ESFA > p-ClA-ESFA