DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats

3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it.     

Formation and persistence of DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in target and non-target organs after intratracheal instillation in rats

Sprague-Dawley rats were treated by intratracheal instillation with a single dose of 0.2 mg/kg body wt of 3-nitrobenzanthrone (3-NBA), and whole blood, lungs, pancreases, kidneys, urinary bladders, hearts, small intestines and livers were removed at various times after administration. At five posttreatment times (2 days, 2, 10, 20 and 36 weeks), DNA adducts were analysed in each tissue by (32)P-postlabelling to study their long-term persistence. 3-NBA-derived DNA adducts consisting of the same adduct pattern were observed in all tissues from animals killed between 2 days and 36 weeks and between 2 days and 20 weeks in blood. DNA isolated from whole blood contained the same 3-NBA-specific adduct pattern as that found in tissues. Although total adduct levels in the blood were much lower than those found in the lung, the target organ of 3-NBA tumourigenicity, they were related (20-25%, R(2) = 0.98) to the levels found in lung. In all organs, total adduct levels decreased over time to 20-30% of the initial levels till the latest time point (36 weeks) and showed a biphasic profile, with a rapid loss during the first 2 weeks followed by a much slower decline that reached a stable plateau at 20 weeks after treatment. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in target and non-target organs of the rat. The correlation between DNA adducts in lung and blood suggests that persistent 3-NBA-DNA adducts in the blood may be useful biomarkers for human respiratory exposure to 3-NBA.     

The genotoxic air pollutant 3-nitrobenzanthrone and its reactive metabolite N-hydroxy-3-aminobenzanthrone lack initiating and complete carcinogenic activity in NMRI mouse skin

3-Nitrobenzanthrone (3-NBA), a genotoxic mutagen found in diesel exhaust and ambient air pollution and its active metabolite N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) were tested for initiating and complete carcinogenic activity in the NMRI mouse skin carcinogenesis model. Both compounds were found to be inactive as either tumour initiators or complete carcinogens in mouse skin over a dose range of 25–400 nmol. Topical application of 3-NBA and N-OH-3-ABA produced DNA adduct patterns in epidermis, detected by ³²P-postlabelling, similar to those found previously in other organs of rats and mice. 24 h after a single treatment of 100 nmol DNA adduct levels produced by 3-NBA (18 ± 4 adducts/10⁸ nucleotides) were 6 times lower than those by 7,12-dimethylbenz[a]anthracene (DMBA; 114 ± 37 adducts/10⁸ nucleotides). In contrast, identical treatment with N-OH-3-ABA resulted in adduct levels in the same range as with DMBA (136 ± 25 adducts/10⁸ nucleotides), indicating that initial DNA adduct levels do not parallel tumour initiating activity. When compounds were tested for tumour initiating activity by a single treatment followed by twice-weekly applications of TPA, DNA adducts formed by DMBA, but not by 3-NBA or N-OH-3-ABA, were still detectable 40 weeks after treatment. When tested for activity as complete carcinogens by twice-weekly topical application, 3-NBA and N-OH-3-ABA produced identical DNA adduct profiles in mouse skin, with adducts still detectable after 40 weeks. Only 3-NBA produced detectable adducts in other organs.     

DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by (32)P-postlabeling.

Diesel exhaust is known to induce tumors in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter, 3-nitrobenzanthrone (3-NBA), is a particularly powerful mutagen. We investigated the capacity of 3-NBA to form DNA adducts in vivo that could be used as agent-specific biomarkers of exposure. Female Sprague-Dawley rats were treated orally with 2 mg/kg body weight of 3-NBA, and DNA from various organs was analyzed by (32)P-postlabeling. High levels of 3-NBA-specific adducts were detectable in all organs. Both enrichment versions nuclease P1 digestion and n-butanol extraction resulted in patterns consisting of either 3 or 4 adducts remarkably similar in all tissues examined. The highest level of DNA adducts was found in the small intestine (38 adducts per 10(8) nucleotides) followed by forestomach, glandular stomach, kidney, liver, lung and bladder. To provide information on the nature of the adducts formed in vivo in rats, DNA adducts were cochromatographed in 2 independent systems with standardized deoxyguanosine adducts and deoxyadenosine adducts produced by reaction of 3-NBA in the presence of xanthine oxidase with deoxyribonucleoside 3'-monophosphates in vitro. In both systems, each of the rat adducts comigrated either with a deoxyguanosine or a deoxyadenosine-derived 3-NBA adduct. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vivo, all of which are products derived from reductive metabolites bound to the purine bases (deoxyguanosine 60% and deoxyadenosine 40%).     

Early formation of DNA adducts compared with tumor formation in a long-term tumor study in rats after administration of 2-nitrofluorene

2-Nitrofluorene (NF) is a model compound for nitroarenes whichhas been identified in diesel exhaust and in urban air. Thecurrent study was carried out to observe the carcinogenicityof different doses of NF to rats and DNA adduct formation indifferent organs at an early stage of NF administration. Onegroup of rats was fed basal diet as a control, whereas the otherthree groups of rats were fed basal diet supplemented with differentamounts of NF (0.24, 0.95 and 2.37 mmol NF/kg diet, referredto as low, medium and high dose, respectively). The rats wereexposed to NF continuously for 11 months, after which all groupsof rats were fed basal diet without NF for another 13 months.In the high dose group hepatocellular carcinomas were foundin all rats (20/20), forestomach squamous carcinomas in 11 andcortical kidney carcinomas in 10 rats. Fifteen out of 19 ratsfed the medium dose of NF had hepatocellular carcinomas, 16had forestomach squamous carcinomas and 15 had cortical kidneycarcinomas. The major tumors of the rats fed the low dose ofNF were forestomach squamous carcinomas (10/18). DNA adductsformed in tumor target organs after 1, 2, 6 and 10 days NF administrationwere dose- and time-dependent. Ten days after the start of NFadministration DNA adduct levels were found to be 54, 11 and6 DNA adducts/10⁸ normal nucleotides in forestomach, liver andkidney respectively. In the non-tumor target organs levels inthe range 1.7–4.8 DNA adducts/10⁸ normal nucleotides werefound. DNA adduct formation in this study showed a good correlationwith the localization of tumors, although there is a need foradditional factors for tumor formation. The results indicatethat DNA adduct formation is an important factor for tumor formationand suggest that DNA adducts could be used as biomarkers forgenotoxic risk.     

DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes

Diesel exhaust is known to induce tumours in animals. Of the compounds found in diesel exhaust 3-nitrobenzanthrone (3-NBA) is particularly a powerful mutagen. Recently we showed that 3-NBA is genotoxic in vivo in rats by forming specific DNA adducts derived from nitroreduction. In this study a panel of genetically engineered V79 Chinese hamster cell lines expressing various human cytochrome P450 (CYP) enzymes (CYP1A1, CYP3A4) and/or human NADPH:CYP oxidoreductase (CYPOR) was used to identify CYP enzymes involved in the metabolic activation of 3-NBA. We analyzed the formation of specific DNA adducts by 32P-postlabelling after exposing cells to 1 microM 3-NBA. A similar pattern with a total of four distinct 3-NBA-DNA adducts was found in all cells, identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Total adduct levels ranged from 75 to 132 using nuclease P1 and from 103 to 220 adducts per 10(8) nucleotides, using butanol enrichment. Comparison of DNA binding between different V79MZ derived cells revealed that human CYPOR and CYP3A4 were involved in the metabolic activation of 3-NBA. Furthermore, dose-dependent high adduct levels were detected after exposure to 0.01, 0.1 or 1 microM 3-NBA in the subclone V79NH which exhibits high activities of nitroreductase and N,O-acetyltransferase. Our results suggest that nitroreduction is the major pathway in the human bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA.     

Inhibition of cigarette smoke-related lipophilic DNA adducts in rat tissues by dietary oltipraz

The present study investigated the effects of dietary oltipraz on cigarette smoke-related lipophilic DNA adduct formation. Female Sprague- Dawley rats were exposed daily to sidestream cigarette smoke in a whole- body exposure chamber 6 h/day for 4 consecutive weeks. One group of rats was maintained on control diet while another group received the same diet supplemented with either a low (167 p.p.m.) or high (500 p.p.m.) dose of oltipraz, starting 1 week prior to initiation of smoke exposure until the end of the experiment. Analysis of lipophilic DNA adducts by the nuclease P1-mediated 32P-post-labeling showed up to five smoke-related adducts. Adduct no. 5 predominated in both the lung and the heart while adduct nos 3 and 2 predominated in the trachea and bladder, respectively. Quantitative analysis revealed that the total adduct level was the highest in lungs (270+/-68 adducts/10(10) nucleotides), followed by trachea (196+/-48 adducts/10(10) nucleotides), heart (141+/-22 adducts/10(10) nucleotides) and bladder (85+/-16 adducts/10(10) nucleotides). High dose oltipraz treatment reduced the adduct levels in lungs and bladder by >60%, while the reduction in lungs in the low-dose group was approximately 35%. In trachea, the effect of low and high dietary oltipraz on smoke DNA adduction was equivocal, while smoke-related DNA adducts in the heart were minimally inhibited by high-dose oltipraz. In a repeat experiment that employed a 3-fold lower dose of cigarette smoke, oltipraz (500 p.p.m.) was found to inhibit the formation of DNA adducts in rat lungs and trachea by 80 and 65%, respectively. These data clearly demonstrate a high efficacy of oltipraz in inhibiting the formation of cigarette smoke-induced DNA adducts in the target tissues.      

Identification of three major DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in rat lung at the C8 and N2 position of guanine and at the N6 position of adenine

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3-NBA-derived DNA adducts in rodent tissues by 32P-postlabeling, all of which are derived from reductive metabolites of 3-NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3-NBA-derived DNA adduct standards for 32P-postlabeling by reacting N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised as N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-N-ABA), 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N2-ABA) and 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-C2-ABA), and a deoxyadenosine (dA) adduct was characterised as 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N6-ABA). 3-NBA-derived DNA adducts formed experimentally in vivo and in vitro were compared with the chemically synthesised adducts. The major 3-NBA-derived DNA adduct formed in rat lung cochromatographed with dG3'p-N2-ABA in two independent systems (thin layer and high-performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3-aminobenzanthrone (3-ABA), the major human metabolite of 3-NBA. Similarly, dG3'p-C8-N-ABA and dA3'p-N6-ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3-NBA or 3-ABA, whereas dG3'p-C8-C2-ABA did not cochromatograph with any of the adducts found in vivo. Utilizing different enzymatic systems in vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2, N,O-acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone in DNA, after incubation with 3-NBA and/or 3-ABA.     

Environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosols

3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the (32)P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,O-acetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10- to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs.     

Dose-response relationships for the binding of benzo(a)pyrene metabolites to DNA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated mice

In this study, the formation of benzo(a)pyrene (BP) metabolite:DNA adducts in lung, liver, and forestomach of control and butylated hydroxyanisole (BHA)-treated (5 mg/g diet) female A/HeJ mice was examined as a function of BP dose (p.o.), ranging from 2 to 1351 mumol/kg. The major identified adduct in each tissue at each dose was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI):deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a(-)-BPDEI:deoxyguanosine adduct, and an unidentified adduct were also observed. In lung and liver of untreated animals, the dose-response curves for BPDEI:DNA adduct levels were sigmoidal. In forestomach, there was no indication of saturation of DNA binding over the BP dose range examined. The dose-response curves became linear as BP dose approached zero and thus, no threshold dose existed below which binding of BPDEI to DNA did not occur, at least in lung, liver, and forestomach of these mice. In forestomach, the dose-response curve for BPDEI:DNA adducts in BHA-treated mice, 0.5% of diet for 2 weeks, was parallel to the curve for control animals and thus, the inhibition (45%) of adduct formation is independent of BP dose. In contrast, BHA treatment diminished the curvilinear nature of the dose-response curves for BPDE adducts in lung and liver. The inhibition of BPDEI:DNA adduct formation by BHA in lung and liver was dose dependent. The inhibition of lung (68%) and liver (82%) adduct formation was highest at a BP dose of 270 mumol/kg. As the BP dose approached zero, the inhibition of BPDEI:DNA adduct formation by BHA decreased with BP dose and approached values of approximately 40% (lung) and 55% (liver). The dose dependency of the binding of BP metabolites to protein was also examined. BPDEI:DNA adduct concentrations ranged from 2 to 10% of protein binding concentrations in liver of untreated animals, from 3 to 7% in forestomach, and from 5 to 7% in lung. The dose-response curves for protein binding of BP metabolites in lung and liver from BHA-treated animals were essentially parallel to those in control animals and thus, the inhibition of protein binding by BHA treatment had no dose dependency in these organs. No consistent BHA effect was observed on the amount of binding of BP metabolites to forestomach protein.     

Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:cytochrome p450 reductase

Determining the capability of humans to metabolize the suspected carcinogen 3-nitrobenzanthrone (3-NBA) and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility to this environmental contaminant found in diesel exhaust and ambient air pollution. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-NBA. Using two enrichment procedures of the (32)P-postlabeling method, nuclease P1 digestion and butanol extraction, we found that all hepatic microsomes were competent to activate 3-NBA. DNA adduct patterns with multiple adducts, qualitatively similar to those found recently in vivo in rats, were observed. Additionally one major DNA adduct generated by human microsomes was detected. The role of specific cytochromes p450 (p450) and NADPH:p450 reductase in the human hepatic microsomal samples in 3-NBA activation was investigated by correlating the p450- and NADPH: p450 reductase-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-NBA was attributed to NADPH: p450 reductase. Inhibition of DNA adduct formation in human liver microsomes by alpha-lipoic acid, an inhibitor of NADPH: p450 reductase, supported this finding. Using the purified rabbit enzyme and recombinant human NADPH: p450 reductase expressed in Chinese hamster V79 cells, we confirmed the participation of this enzyme in the formation of 3-NBA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells. The role of individual human recombinant p450s 1A1, 1A2, 1B1, 2A6, 2B6, 2D6, 2C9, 2E1, and 3A4 and of NADPH: p450 reductase in the metabolic activation of 3-NBA, catalyzing DNA adduct formation, was also examined using microsomes of baculovirus-transfected insect cells containing the recombinant enzymes (Supersomes). DNA adducts were observed in all Supersomes preparations, essentially similar to those found with human hepatic microsomes and in human cells. Of all of the recombinant human p450s, p450 2B6 and -2D6 were the most efficient to activate 3-NBA, followed by p450 1A1 and -1A2. These results demonstrate for the first time the potential of human NADPH: p450 reductase and recombinant p450s to contribute to the metabolic activation of 3-NBA by nitroreduction.     

Persistence of benzo(a)pyrene metabolite:DNA adducts in lung and liver of mice

The persistence of benzo(a)pyrene (BP) metabolite:DNA adducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP is not a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10 hr to 28 days, and BP metabolite:DNA adducts were analyzed by high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta,epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a (-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, and an unidentified adduct were also observed. The disappearance of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP adduct in A/HeJ mice followed first-order kinetics over the time period examined, with a half-life of 18 and 9 days in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnover suggest that there is active removal of adducts in liver, but that normal DNA turnover can account for the partial or possibly total observed disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ mice may be related to the relative persistence of adducts and high cell turnover rates in lung. In contrast, the results on formation and persistence of adducts and cell turnover do not provide an explanation for the strain difference in susceptibility to BP-induced pulmonary adenomas. It was also shown that the rates of removal of BP metabolite:DNA adducts in A/HeJ mice are not significantly different at a 500-fold lower BP dose.     

Bioactivation of 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone: evidence for DNA adduct formation mediated by cytochrome P450 enzymes and peroxidases

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in the urine of salt mining workers occupationally exposed to diesel emissions. We evaluated the role of hepatic cytochrome P450 (CYP) enzymes in the activation of 3-ABA in vivo by treating hepatic cytochrome P450 oxidoreductase (POR)-null mice and wild-type littermates intraperitoneally with 0.2 and 2mg/kg body weight of 3-ABA. Hepatic POR-null mice lack POR-mediated CYP enzyme activity in the liver. Using the (32)P-postlabelling method, multiple 3-ABA-derived DNA adducts were observed in liver DNA from wild-type mice, qualitatively similar to those formed in incubations using human hepatic microsomes. The adduct pattern was also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. DNA binding by 3-ABA in the livers of the null mice was undetectable at the lower dose and substantially reduced (by up to 80%), relative to wild-type mice, at the higher dose. These data indicate that POR-mediated CYP enzyme activities are important for the oxidative activation of 3-ABA in livers, confirming recent results indicating that CYP1A1 and -1A2 are mainly responsible for the metabolic activation of 3-ABA in human hepatic microsomes. No difference in DNA binding was found in kidney and bladder between null and wild-type mice, suggesting that cells in these extrahepatic organs have the metabolic capacity to oxidize 3-ABA to species forming the same 3-ABA-derived DNA adducts, independently from the CYP-mediated oxidation in the liver. We determined that different model peroxidases are able to catalyse DNA adduct formation by 3-ABA in vitro. Horseradish peroxidase (HRP), lactoperoxidase (LPO), myeloperoxidase (MPO), and prostaglandin H synthase (PHS) were all effective in activating 3-ABA in vitro, forming DNA adducts qualitatively similar to those formed in vivo in mice treated with 3-ABA and to those found in DNA reacted with N-hydroxy-3-aminobenzanthrone (N-OH-ABA). Collectively, these results suggest that both CYPs and peroxidases may play an important role in metabolizing 3-ABA to reactive DNA adduct forming species.     

Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase

3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity. The formation of 3-NBA-derived DNA adducts was analysed by (32)P-postlabelling after exposing V79 cells to 0.01 micro M 3-NBA or 0.1 micro M N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2. This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.     

Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons

Understanding the kinetics of aromatic-DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched (32)P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P-DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P-DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic-DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.     

Cyclopenta[cd]pyrene-induced tumorigenicity, Ki-ras codon 12 mutations and DNA adducts in strain A/J mouse lung

Cyclopenta[cd]pyrene (CPP) is a ubiquitous cyclopenta-fusedpolycyclic aromatic hydrocarbon. CPP is highly genotoxic inbacterial and mammalian systems inducing gene mutations, sisterchromatid exchanges and morphological transformation. CPP isa mouse skin carcinogen, a mouse skin tumor initiator and inducespulmonary tumors in newborn mice. We have examined the tumorigenicactivity of CPP in strain A/J mice, have determined the formationand persistence of CPP-induced DNA adducts in lung tissue, andanalyzed the mutational spectrum in the Ki-ras oncogene fromCPP induced tumors. CPP dissolved in tricaprylin was administeredby i.p. injection to male A/J mice (20 mice/dose) at 0,10,50,100and 200 mg/kg. Animals were killed 8 months later and the lungsremoved, fixed, and surface adenomas enumerated. CPP provedto be highly tumorigenic in A/J mice in terms of inducing lungadenomas. The observed tumor multiplicities (lung adenomas/mouse)were: 97.7 ± 28.7 at 200 mg/kg, 32.8 ± 15.4 at100 mg/kg, 4.63 ± 2.11 at 50 mg/kg and 0.58 ±0.82 at 10 mg/kg. Tricaprylin-treated controls produced 0.60± 0.58 lung adenomas/mouse. Groups of mice treated underthe same dosing conditions as those in the tumor studies werekilled 1, 3, 7, 14 and 21 days after treatment. The lungs wereremoved, and the DNA was subjected to DNA adduct analysis bythe 32P-postlabeling method. Total CPP-DNA adducts in mouselung peaked at day 3 with 5870 amol CPP adducts/µg DNAafter a single dose of 200 mg/kg. DNA adduct levels decreasedto 1800 amol CPP adducts/µg DNA at day 21. QualitativeDNA adduct analysis revealed four major adducts and one minoradduct. Co-chromatography of the lung DNA from CPP-treated micewith calf thymus DNA treated with CPP-3,4-oxide indicated thatall DNA adducts were oxide derived and comparison with CPP-3,4-oxlde-treatedpolydeoxyguanylic acid suggests that almost all of these adductsare CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12mutation analysis of the DNA from twnors taken from the 100and 200 mg/kg CPP dose groups demonstrated the following patterns:GGT     

Seasonal variation of DNA adduct pattern in human lymphocytes analyzed by 32P-HPLC

³²P-HPLC is a recently published method to generate DNA adductprofiles after exposure to a complex of genotoxic substances.The low detection limit enables characterization of individualDNA adducts in the general population. The ³²P-HPLC method wasapplied to human lymphocytes and granulocytes from Silesia,apolluted industrial region in the south of Poland. Human sampleswere collected at the end of winter and summer to investigatethe seasonal influence on DNA adduct formation. In lymphocytesa strong seasonal variation was seen in total DNA adducts, withwinter values exceeding the summer values by 7.33±3.56times. Granulocytes did not show any seasonal variation. Inwinter-collected lymphocytes the DNA adduct levels were 21.4±16.6/10⁸normal nucleotides (NN) while the summer values were 2.96±2.46±10⁸NN.Granulocytes had 8.06± 7.76 and 9.59±6.19 DNAadducts/10⁸ NN during winter and summer respectively. The lymphocyteDNA adduct profile consisted of at least 16 individual or clustersof DNA adducts. All 16 had a clear winter influence, with awinter: summer ratio of 1.6–15.3, indicating exposureto a complex mixture of genotoxic substances. The DNA adductsanalyzed in human lymphocytes had retention times similar toDNA adducts generated by poycyclic aromatic hydrocarbons. Thesuggested candidates for DNA adducts displayed a similar seasonalvariation in airborne particles to that found in DNA adductsin lymphocytes of humans living in the area. This is the firstapplication of the ³²P-HPLC method to analysis of DNA adductsin human tissues.     

32P-postlabeling analysis of DNA adducts persisting for up to 42 weeks in the skin, epidermis and dermis of mice treated topically with 7,12-dimethylbenz[a]anthracene

The initial and persistent levels of 7,12-dimethylbenz[a]-anthracene (DMBA)-DNA adducts in mouse skin, epidermis and dermis after topical carcinogen application were studied by 32P-postlabeling assay. In the major experiment, a single dose of 1.2 mumol of the carcinogen was applied to the shaved backs of adult female BALB/cANN mice, and DNA was isolated from epidermis and dermis, respectively, 24 h and 1, 2, 3, 4, 8, 16, 24, 36 and 42 weeks later. Total binding at 24 h was approximately 34 and approximately 28 adducts in 10(7) normal nucleotides for epidermal and dermal DNA, respectively. (One adduct in 10(7) nucleotides equals 0.3 fmol adduct/microgram DNA.) While initial binding was higher in epidermal DNA, the adducts were approximately 10 times more persistent in dermal DNA: at 42 weeks, total binding levels were approximately 0.17 and approximately 1.7 adducts in 10(7) nucleotides for epidermis and dermis, respectively. To quantitate low levels of DMBA-DNA adducts, 32P-postlabeling assays were run in the presence of a limiting amount of carrier-free [gamma-32P]ATP; this was found to favor labeling of the adducts, thereby leading to a 20- to 100-fold enhancement of the method's sensitivity for individual adducts. One of the three major DMBA-DNA adducts was more persistent than were the others; the level of this adduct remained constant at approximately 60% of the total in epidermal and dermal DNA during the last 18 weeks of the 42-week observation period. Since a [3H]thymidine-labeling experiment showed a normal epidermal DNA turnover 40 weeks after DMBA treatment, it was concluded that the bulk of the persistent adducts was present in subpopulations of dormant cells. We have hypothesized that such cells, in the absence of a promoting stimulus, are incapable of division because of the adduction and/or mutation of genes critical for growth (proto-oncogenes), and may thus correspond to the 'latent tumor cells', as defined by Berenblum and Shubik in their classical analysis of the attributes of tumor initiation and promotion.     

One-electron oxidation is not a major route of metabolic activation and DNA binding for the carcinogen 7H-dibenzo[c,g]carbazole in vitro and in mouse liver and lung

7H-Dibenzo[c,g]carbazole (DBC) is a potent multi-site, multi-species carcinogen present in a variety of complex mixtures derived from the incomplete combustion of organic matter. Like many carcinogens, DBC requires metabolic activation to an electrophilic species to exert its mutagenic and carcinogenic effects. One-electron oxidation, leading to the formation of radical cation intermediates, has been proposed as a mechanism of metabolic activation for DBC in vitro resulting in unstable DNA adducts. The purpose of this research was to determine whether one-electron oxidation is a mechanism of activation and DNA binding for DBC in vivo. Specific depurinating DBC-DNA adducts formed by one-electron oxidation were analyzed in mouse liver at 4 h following a single i.p. dose of 40 mg/kg of 11 microCi [(14)C]DBC. In addition to five previously published adduct standards, two newly identified adduct standards were characterized by mass spectrometry and NMR, namely DBC-6-N7-Ade and DBC-6-N1-Ade; however, neither was observed in mouse liver. Only the DBC-5-N7-Gua adduct was observed in mouse liver extracts at a level of 6.5 +/- 1. 8 adducts/10(6) nucleotides. In addition, the formation of AP sites and stable DBC-DNA adducts was analyzed in mouse liver and lung at 4, 12 and 24 h following a single i.p. dose of 0.4, 4 or 40 mg/kg DBC (n = 3/group). There was a distinct time- and dose-response of stable DBC-DNA adducts detected by (32)P-post-labeling. There was not a clear dose-response for formation of AP sites; however, a significant increase over control levels was observed at the 4 and 40 mg/kg dose groups at 4 and 12 h post dosing, respectively. Quantitative comparisons indicate that the depurinating DBC-5-N7-Gua adduct constitutes approximately 0.4% of total adducts measured whereas the stable adducts detected by (32)P-post-labeling constitute 99.6% of total adducts measured following a 40 mg/kg dose and a 4 h time-point. The data indicate that one-electron oxidation does occur in mouse liver in vivo. However, one-electron oxidation is a minor mechanism of activation in that the percentage of total adducts formed through this route constitutes a minor percentage of the total adducts formed.     

Effects of dietary supplementation of N-acetylcysteine on cigarette smoke-related DNA adducts in rat tissues

Cigarette smoking plays a major role in the etiology of several human cancers. It is believed that formation of DNA adducts is an initial step in the carcinogenic process. In this study, we have examined the ability of dietary N-acetylcysteine (NAG) to inhibit the formation of cigarette smoke-related DNA adducts in various tissues of rats. Female Sprague-Dawley rats were exposed to cigarette smoke (10 mg TPM/m(3)) in a whole-body exposure chamber for 6 h per day, seven days a week for four weeks. The smoke-exposed groups were provided either an unrefined diet or diets supplemented with low (5,000 ppm) or high (20,000 ppm) dose of NAG. A sham group was given control diet and maintained on filtered ambient air. Tissue DNA analysis of smoke-exposed rats by nuclease P1-version of the P-32-postlabeling assay showed up to 6 adducts in the following descending order expressed as total adducts/10(10) nucleotides: 1 predominant (no. 5) and 4 (no. 1-no. 4) minor adducts in the (219 +/- 36), 6 minor adducts in the heart (93 +/- 11), 5 adducts in the trachea (50 +/- 16), and 4 adducts in the bladder (50 +/- 3.5); sham-treated animals showed 2 or 3 adducts in each tissue but at 4-20-fold lower levels. Dietary intervention with either high or low dose of NAC did not affect the levels of most adducts, except for the following: a 30-40% increase (P<0.05) for adducts 3 and 4 in the lung; a 40-50% decrease (P<0.05) for adduct 2 in the trachea; and a 30% increase (P<0.05) for adduct 2 in the bladder. In a second experiment conducted under identical conditions, most major and minor adducts remained unaffected with NAC intervention, except for adduct 2 in the trachea which was somewhat diminished. These results suggest that dietary NAC intervention does not significantly influence the levels of most major and minor adducts. However, some minor adducts in the lung, trachea and bladder were modulated differentially.