THE TOXICITY OF BENZO[a]PYRENE ON SOLE (SOLEA SOLEA) HEPATOCYTES: ASSESSMENT OF GENOTOXIC AND ENZYMATIC EFFECTS

The benzo[a]pyrene is a polycyclic aromatic hydrocarbon known to be genotoxic, mutagenic and carcinogenic in higher vertebrates. The aim of this study was to evaluate in vitro the enzymatic and genotoxic effects of BaP in a benthic fish species, Solea solea. Sole hepatocytes were exposed to BaP in order to measure the modulation of ethoxyresorufin-o-deethylase (EROD) activity and the DNA strand breaks induced by BaP metabolism. Exposures were performed in both culture flasks and microplate wells in order to check for the possible miniaturization of the exposure system. Moreover, sole liver microsomes were exposed to BaP in the presence of standard DNA in order to assess the potential formation of DNA adducts in sole. The results demonstrated the ability of sole hepatic enzymes to metabolize BaP into reactive species responsible for bulky DNA adducts and DNA strand breakage, whatever the tested exposure concentration and the mode of exposure.     

Effect of the Route of Administration on the Induction of Cytogenetic Damage and DNA Adducts in Peripheral Blood Lymphocytes of Rats and Mice by Polycyclic Aromatic Hydrocarbons

Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic end points and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[ a ]anthracene (B[ a ]A), benzo[ b ]fluoranthene (B[ b ]F), benzo[ a ]pyrene (B[ a ]P), or chrysene (Chr) by gavage or by intraperitoneal injection (i.p.). Peripheral blood was removed by cardiac puncture 7 days after PAH administration. Blood samples were analyzed in parallel for sister chromatid exchange (SCE) frequency, the frequency of micronuclei in cytochalasin B-induced binucleate cells (MN bn ), and DNA adduction using 32P-postlabeling. The i.p. route of exposure produced both the highest levels of cytogenetic damage and DNA adducts for each PAH. The mouse was more sensitive than the rat to PAH exposure as measured by SCE induction and the total amount of DNA adducts/ w g DNA.     

Monofunctional and Interstrand DNA Adducts of Platinum(II) Complexes

The effects produced in DNA by monofunctional or interstrand adducts of platinum(II) complexes have been summarized. The monofunctional adducts destabilize DNA in a sequence-dependent manner via conformational distortions, which may have a denaturational character. It has been suggested that this conformational alteration facilitates in DNA the formation of the bidentate DNA adducts, whose formation is associated with a permanent or transient local denaturation. In addition, DNA interstrand cross-linking by cis-diamminedichloroplatinum(II) and its trans isomer produce in DNA lesions that have quite different characteristics. It has been suggested that these differences may have relevance to the distinct antitumor efficacy of the two platinum isomers.     

POLYCYCLIC AROMATIC HYDROCARBONS OF DIESEL AND GASOLINE EXHAUST AND DNA ADDUCT DETECTION IN CALF THYMUS DNA AND LYMPHOCYTE DNA OF WORKERS EXPOSED TO DIESEL EXHAUST

Particles present in urban air pollution are mainly derived from diesel- and gasoline-fueled vehicles. Exhaust emission is able to cause several health effects in humans including mutagenicity and carcinogenicity. Polycyclic aromatic hydrocarbons (PAHs) were measured in diesel and gasoline particulate extracts and DNA binding to CT DNA (±S9 and ±XO) was investigated. A large difference in content of 14 PAHs in diesel and gasoline extracts was observed, showing higher concentration of 14 PAHs, 6 carcinogenic PAHs, benzo[a]pyrene (B[a]P) in diesel than in gasoline extracts. Selected PAH standards of B[a]P, benzo[c]fluoranthene (B[c]F), and 3-nitrobenzanthrone (NBA) were used in ³²P-postlabeling/polyacrylamide gel electrophoresis (PAGE) technique to identify CT DNA adducts formed by diesel particulate extracts. CT DNA adduct formation was higher for diesel extracts in comparison with gasoline extracts; however, no clear origin of DNA adducts derived from B[c]F-, 3-NBA-, B[a]P was detected. ³²P-postlabeling/PAGE was a useful assay for analyzing and identifying PAH-DNA adducts. Occupational exposure to particulate and volatile PAH concentrations were evaluated using personal air samples and lymphocyte DNA adducts as markers of exposure. Overall air PAH concentrations were low in all eight workplaces, consisting of 97% of vapor phase compounds. DNA adducts analyzed by ³²P-postlabeling assay were compared between the butanol and nuclease P1 enrichment procedures. Only in winter samples of exposed workers, butanol extraction revealed significantly higher adduct levels in comparison with those of control persons. No differences in adduct levels between exposed and control persons in summer were detected by using either butanol extraction or nuclease P1 treatment. Total concentrations of particulate and volatile PAHs measured in eight workplaces in winter showed a significant correlation with total DNA adducts analyzed in workers' lymphocytes (r = 0.852N = 8, p = .007).     

Comparison of Animal and Plant DNA-Adducts after Exposure to Benzo(a)pyrene

The occurrence of abnormal hypermodified nucleotides on the DNA upon xenobiotic exposure has long been considered as a characteristic of carcinogenesis and mutagenesis in animal cells. We have previously shown that DNA adducts could also be formed in plants exposed to xenobiotics in natural and controlled conditions. In this study we have compared the DNA adducts formed in different animal species and in different plants after benzo(a)pyrene (B(a)P) exposure.

The main DNA adduct in mice stomach and skin correspond to the 7,8-diol 9,10-epoxide B(a)P-guanine. In liver from rat, fish and Xenopus, this adduct is detected, but is not the major one. In plants analyzed, this adduct is never formed. Ten different adducts are detected in plants. This result indicates that the metabolic pathway leading to genotoxic metabolites is different with species. In conclusion, the result suggest that risk assessment for human and environment due to genotoxic compounds should be realized using multiple species assays.     

Complex Mechanisms of Antimony Genotoxicity in Budding Yeast Involves Replication and Topoisomerase I-Associated DNA Lesions,Telomere Dysfunction and Inhibition of DNA Repair

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.     

Evaluation of PAH: DNA Adduct Formation in Rats Fed Coal Tar-Contaminated Diets

Previous studies have demonstrated that mouse lung is a target organ for the tumorigenic and genotoxic effects of coal tar. The present study evaluated PAH:DNA adduct formation in lung, liver, forestomach, and mammary gland of female CD rats fed various types of coal tar-contaminated diets. Coal tar-contaminated soil, an organic extract of contaminated soil, neat coal tar, and diets containing only B[ a ]P were evaluated. Ingestion of coal tar diets resulted in detectable levels of DNA adducts in lung and forestomach tissue. These adducts were primarily derived from benzo[ c ]fluorene and B[ a ]P. The adduct derived from benzo[ c ]fluorene was the most predominant. No adducts were detected in liver and mammary gland under the conditions employed in this study. The formation of a benzo[ c ]fluorene-derived DNA adduct in rat lung following coal tar exposure is consistent with previous studies performed with mice.     

Genotoxic Anti-Cancer Agents and Their Relationship to DNA Damage,Mitosis, and Checkpoint Adaptation in Proliferating Cancer Cells

When a human cell detects damaged DNA, it initiates the DNA damage response (DDR) that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies.     

DNA Adducts Derived from Diesel Emissions - Assessment of Genotoxic Potency in Vitro and Human Exposure Monitoring

Diesel particle extracts, which originated from three different diesel fuels, were used to study the activation of polycyclic aromatic hydrocarbons (PAHs). DNA adducts were analyzed in vitro calf thymus, human skin tissue culture and in lymphocytes isolated from diesel exposed workers. Direct-acting mutagens (e.g. nitro-PAHs) measured by Ames test were compared with DNA adducts formed in vitro by nitroreductive xanthine oxide enzyme. PAH-DNA adducts were analyzed by ³²P-postlabeling, and when characterizing adducts from skin DNA, a solid-phase micro extraction (SPME) method was developed for sample preparation before HPLC analysis. A good accordance between mutagenicity and DNA adducts showed that the three extracts contain higher amounts of direct-acting PAHs than the PAHs needing S9 activation. Skin DNA adducts demonstrated two-fold differences between the tissue cultures. ³²P-postlabeling and HPLC analysis did not confirm the identity of skin DNA adducts with the BPDE-DNA standard. The pilot study on 13 diesd exposed bus garage and waste collection workers showed low levels of PAH exposure (<50 ng/m³) and lymphocyte PAH-DNA adducts less than 2 adducts/10⁸ nucleotides.     

Dose and Radioadaptive Response Analysis of Micronucleus Induction in Mouse Bone Marrow

Enhanced cellular DNA repair efficiency and suppression of genomic instability have been proposed as mechanisms underlying radio-adaptive responses following low-dose radiation exposures. We previously showed that low-dose γ irradiation does not generate radio-adaptation by lowering radiation-induced cytogenetic damage in mouse spleen. Since radiation may exert tissue-specific effects, we extended these results here by examining the effects of γ radiation on cytogenetic damage and proliferative index in bone marrow erythrocytes of C57BL/6 and BALB/c mice. In C57BL/6 mice, the induction of micronuclei in polychromatic erythrocytes (MN-PCE) was observed at radiation doses of 100 mGy and greater, and suppression of erythroblast maturation occurred at doses of >500 mGy. A linear dose–response relationship for MN-PCE frequencies in C57BL/6 mice was established for radiation doses between 100 mGy and 1 Gy, with departure from linearity at doses of >1 Gy. BALB/c mice exhibited increased MN-PCE frequencies above baseline following a 20 mGy radiation exposure but did not exhibit radio-sensitivity relative to C57BL/6 mice following 2 Gy exposure. Radio-adaptation of bone marrow erythrocytes was not observed in either strain of mice exposed to low-dose priming γ irradiation (single doses of 20 mGy or 100 mGy or multiple 20 mGy doses) administered at various times prior to acute 2 Gy irradiation, confirming the lack of radio-adaptive response for induction of cytogenetic damage or suppression or erythrocyte proliferation/maturation in bone marrow of these mouse strains.     

Stem Cells: The Pursuit of Genomic Stability

Stem cells harbor significant potential for regenerative medicine as well as basic and clinical translational research. Prior to harnessing their reparative nature for degenerative diseases, concerns regarding their genetic integrity and mutation acquisition need to be addressed. Here we review pluripotent and multipotent stem cell response to DNA damage including differences in DNA repair kinetics, specific repair pathways (homologous recombination vs. non-homologous end joining), and apoptotic sensitivity. We also describe DNA damage and repair strategies during reprogramming and discuss potential genotoxic agents that can reduce the inherent risk for teratoma formation and mutation accumulation. Ensuring genomic stability in stem cell lines is required to achieve the quality control standards for safe clinical application.     

Genotoxic and epigenotoxic effects in mice exposed to concentrated ambient fine particulate matter (PM<Subscript>2.5</Subscript>) from São Paulo city,Brazil

Background

The Metropolitan Area of São Paulo has a unique composition of atmospheric pollutants, and positive correlations between exposure and the risk of diseases and mortality have been observed. Here we assessed the effects of ambient fine particulate matter (PM_2.5) on genotoxic and global DNA methylation and hydroxymethylation changes, as well as the activities of antioxidant enzymes, in tissues of AJ mice exposed whole body to ambient air enriched in PM_2.5, which was concentrated in a chamber near an avenue of intense traffic in São Paulo City, Brazil.

Results

Mice exposed to concentrated ambient PM_2.5 (1 h daily, 3 months) were compared to in situ ambient air exposed mice as the study control. The concentrated PM_2.5 exposed group presented increased levels of the oxidized nucleoside 8-oxo-7,8-dihydro-2′-deoxyguanosine in lung and kidney DNA and increased levels of the etheno adducts 1,N⁶-etheno-2′-deoxyadenosine and 1,N²-etheno-2′-deoxyguanosine in kidney and liver DNA, respectively. Apart from the genotoxic effects, the exposure to PM_2.5 led to decreased levels of the epigenetic mark 5-hydroxymethylcytosine (5-hmC) in lung and liver DNA. Changes in lung, liver, and erythrocyte antioxidant enzyme activities were also observed. Decreased glutathione reductase and increased superoxide dismutase (SOD) activities were observed in the lungs, while the liver presented increased glutathione S-transferase and decreased SOD activities. An increase in SOD activity was also observed in erythrocytes. These changes are consistent with the induction of local and systemic oxidative stress.

Conclusions

Mice exposed daily to PM_2.5 at a concentration that mimics 24-h exposure to the mean concentration found in ambient air presented, after 3 months, increased levels of DNA lesions related to the occurrence of oxidative stress in the lungs, liver, and kidney, in parallel to decreased global levels of 5-hmC in lung and liver DNA. Genetic and epigenetic alterations induced by pollutants may affect the genes committed to cell cycle control, apoptosis, and cell differentiation, increasing the chance of cancer development, which merits further investigation.

     

Metabolic and DNA Repair Variations in Susceptibility to Genotoxins

An overview on the influence of metabolic and DNA repair polymorphisms on biological indicators of genotoxic risk in occupational, environmental, or lifestyle exposure is presented in this article. Indicators of genotoxic risk include biomarkers of internal dose (urinary concentration of the substance or its metabolites and urinary mutagenicity), biological effective dose (protein and DNA adducts), and indicators of early biological effects (chromosome aberrations, sister chromatid exchanges, micronuclei, COMET assay, HPRT mutants). Genetic polymorphisms include those involved in the activation and detoxification (i.e., P-450 cytochrome, acetyltransferase, glutathione transferase) of various classes of carcinogens and the newly discovered polymorphisms affecting DNA repair enzymes. Genetic polymorphisms are assessed for their importance in detecting the impact of genotype on biological indicators of genotoxic risk.     

DNA ADDUCTS AS A BIOMARKER OF POLLUTION: FIELD STUDY ON THE GENOTOXIC IMPACT EVOLUTION OF THE ERIKA OIL SPILL ON MUSSELS (MYTILUS EDULIS) OVER A PERIOD OF ELEVEN MONTHS

After the Erika oil spill in December 1999, several thousand tons of heavy fuel were released in marine environment and polluted the Atlantic French coast. DNA adducts were used as a biomarker of the Erika genotoxic impact on DNA of mussel digestive gland sampled at 15 locations from Finistère to Vendée county during a period of 11 months after the accident. Mussels collected in the impacted sites in December 1999 and January 2000 showed the highest total DNA adduct level (165 adducts/10⁸ nucleotides). The level decreased dramatically in February 2000 to a stable level through the rest of the year. The site considered as reference zone was also impacted just after the Erika oil spill. The DNA adduct patterns are similar at all locations, indicating a common genotoxic impact along the coast. Two adducts persisted and were used to characterize and follow the Erika genotoxic impact. The analysis revealed that mussels from three sites (respectively Finistére, Loire Atlantique, and Vendée counties) have been highly impacted by the Erika oil spill.     

METHODS FOR AROMATIC AND HETEROCYCLIC AMINE CARCINOGEN-DNA ADDUCT ANALYSIS BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Xenobiotic-DNA adducts are used as biomarkers to assess the genotoxic effects of carcinogens. Rats were dosed with 4-aminobiphenyl (4-ABP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA was isolated from the colons of vehicle and carcinogen-treated rats and digested using different nucleases and alkaline phosphatase. Deoxyribonucleoside adducts were quantified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. 3′- and 5′- Exonucleases were the most efficient nucleases at isolating dG-C8-ABP adducts. However, bulky adducts such as dG-C8-MeIQx and dG-C8-PhIP were better isolated using nuclease P1 rather than a combination of micrococcal nuclease and spleen phosphodiesterase. The use of DNase I enhanced the detection of all three adducts. We describe LC-MS/MS methods for DNA adduct detection and support the testing of different nucleases that increase DNA digestion efficiency and make available more DNA adducts for detection.     

Genome Maintenance Mechanisms at the Chromatin Level

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress.     

Metabolism and DNA-Binding of 3-Nitrobenzanthrone in Primary Rat Alveolar Type II Cells,in Human Fetal Bronchial,Rat Epithelial and Mesenchymal Cell Lines

3-Nitrobenzanthrone (NBA) is a suspected human carcinogen and has been identified in diesel exhaust and in airborne particulates. Human exposure to NBA is thought to occur primarily via the respiratory tract and bronchial as well as alveolar epithelial cells are believed to be primary targets for lung carcinogenesis. Nitroarenes require metabolic activation to DNA binding metabolites to become genotoxic carcinogens. In this study the metabolism of NBA as well as its metabolic intermediate 3-nitrosobenzanthrone was investigated in cultures of rat lung alveolar type II cells, in human fetal bronchial (HFBE) and rat bronchial epithelial (R3/1) as well as mesenchymal Rwd009 cells. 3-Aminobenzanthrone (ABA) was identified as the major metabolite from both substrates, but also small amounts of 3-acetyl-ABA were observed during short term incubations (6 to 24 h) with NBA. Inhibition studies with allopurinol in alveolar type II cells indicate that the cytosolic enzyme xanthine oxidase contributes substantially to the biotransformation of NBA. ³²?Postlabeling analysis of DNA adducts in these cells demonstrates the formation of 5 and 6 different adducts after exposure of the cells with NBA and 3-nitrosobenzanthrone, respectively. Different oligonucleotides were modified with N?acetoxy-N?acetyl-3-ABA and used as reference materials for postlabeling analysis. Based on co-chromatography experiments, the presence of N?acetoxy-ABA-dA adducts in alveolar type II epithelial cells could be excluded. In conclusion, it was shown that metabolic conversion of NBA is associated with DNA adduct formation in rat alveolar type II epithelial cells. The formation and covalent DNA binding of reactive NBA metabolites may provide the rational for a mechanism of lung carcinogenesis based on direct genotoxicity.     

Mechanisms of Enzyme-Catalyzed Reduction of Two Carcinogenic Nitro-Aromatics, 3-Nitrobenzanthrone and Aristolochic Acid I: Experimental and Theoretical Approaches

This review summarizes the results found in studies investigating the enzymatic activation of two genotoxic nitro-aromatics, an environmental pollutant and carcinogen 3-nitrobenzanthrone (3-NBA) and a natural plant nephrotoxin and carcinogen aristolochic acid I (AAI), to reactive species forming covalent DNA adducts. Experimental and theoretical approaches determined the reasons why human NAD(P)H:quinone oxidoreductase (NQO1) and cytochromes P450 (CYP) 1A1 and 1A2 have the potential to reductively activate both nitro-aromatics. The results also contributed to the elucidation of the molecular mechanisms of these reactions. The contribution of conjugation enzymes such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) to the activation of 3-NBA and AAI was also examined. The results indicated differences in the abilities of 3-NBA and AAI metabolites to be further activated by these conjugation enzymes. The formation of DNA adducts generated by both carcinogens during their reductive activation by the NOQ1 and CYP1A1/2 enzymes was investigated with pure enzymes, enzymes present in subcellular cytosolic and microsomal fractions, selective inhibitors, and animal models (including knock-out and humanized animals). For the theoretical approaches, flexible in silico docking methods as well as ab initio calculations were employed. The results summarized in this review demonstrate that a combination of experimental and theoretical approaches is a useful tool to study the enzyme-mediated reaction mechanisms of 3-NBA and AAI reduction.     

Nucleotide Excision Repair Pathway Activity Is Inhibited by Airborne Particulate Matter (PM10) through XPA Deregulation in Lung Epithelial Cells

Airborne particulate matter with a diameter size of ≤10 µm (PM₁₀) is a carcinogen that contains polycyclic aromatic hydrocarbons (PAH), which form PAH–DNA adducts. However, the way in which these adducts are managed by DNA repair pathways in cells exposed to PM₁₀ has been partially described. We evaluated the effect of PM₁₀ on nucleotide excision repair (NER) activity and on the levels of different proteins of this pathway that eliminate bulky DNA adducts. Our results showed that human lung epithelial cells (A549) exposed to 10 µg/cm² of PM₁₀ exhibited PAH–DNA adducts as well as an increase in RAD23 and XPD protein levels (first responders in NER). In addition, PM₁₀ increased the levels of H4K20me2, a recruitment signal for XPA. However, we observed a decrease in total and phosphorylated XPA (Ser196) and an increase in phosphatase WIP1, aside from the absence of XPA–RPA complex, which participates in DNA-damage removal. Additionally, an NER activity assay demonstrated inhibition of the NER functionality in cells exposed to PM₁₀, indicating that XPA alterations led to deficiencies in DNA repair. These results demonstrate that PM₁₀ exposure induces an accumulation of DNA damage that is associated with NER inhibition, highlighting the role of PM₁₀ as an important contributor to lung cancer.     

DNA Adducts Formed in vitro from Fractionated Extract of Urban Air Particles

This study is a part of an ongoing interdisciplinary project on the health effects of air pollution (Teplice Program) in the highly polluted district of Teplice, Northern Bohemia. In our previous studies we found the relationship between DNA adduct levels detected in white blood cells of selected Teplice population and personal exposure to PAH associated with respirable particles. In this pilot study we used ³²P-postlabeling assay for DNA adduct detection in an in vitro model system for evaluation of genotoxic activity of fractionated urban air extractable organic matter (EOM). To identify some of the specific DNA adducts formed we coupled TLC with HPLC analysis of labeled adducts. The urban air particles were collected by high-volume sampler during January-March 1992 in Teplice. EOM was extracted by dichlormethane (DCM) and crude extract was fractionated into five fractions to obtain a gross partition of different chemical classes. Fractions were incubated with calf thymus DNA (dose 100 μg/ml incubate) under oxidative and reductive conditions using two metabolic activation system: 1) an oxidative rat liver S9 system (S9) and 2) a reductive xanthine oxidase catalyzed system (XO). The different DNA adduct patterns and levels were determined using S9 and XO-mediated metabolism followed by postlabeling with both nuclease P1 and butanol extraction enrichment procedures for all fractions examined. The moderately polar fraction (DCM) contained over 50% of the total DNA adduct forming activity both without and with S9 activation. The highly polar fraction (methanol) contained about 60 % of the DNA adduct forming activity under reductive conditions. Some of the main distinct DNA adducts obtained with S9 and XO mediated metabolism were tentatively identified by HPLC comparing with standards of PAH- and nitro-PAH DNA adducts.