nitrobenzanthrone and 3-aminobenzanthrone

The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/-) and TP53(-/-) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/-) and TP53(-/-) cells compared to TP53(+/+) cells, and p53's impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/-) cells but not in TP53(R248W/+) cells. Our results show that p53's influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.

Topics: Activation, Metabolic; Air Pollutants; Air Pollution; Anthracenes; Benz(a)Anthracenes; Carcinogenesis; Carcinogens, Environmental; Cell Line, Tumor; Cytochrome P-450 CYP1A1; DNA Adducts; DNA Damage; HCT116 Cells; Humans; Inactivation, Metabolic; Schiff Bases; Tumor Suppressor Protein p53; Vehicle Emissions

3-Nitrobenzanthrone (3-NBA), a carcinogenic air pollutant, was investigated for its ability to induce cytochrome P450 (CYP) 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1) in liver, kidney and lung of rats treated by intra-tracheal instillation. The organs used were from a previous study performed to determine the persistence of 3-NBA-derived DNA adducts in target and non-target tissues (Bieler et al., Carcinogenesis 28 (2007) 1117-1121, [22]). NQO1 is the enzyme reducing 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize a human metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), to yield the same reactive intermediate. 3-NBA and 3-ABA are both activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and from liver and kidney. Each compound generated the same five DNA adducts detectable by (32)P-postlabelling. When hepatic cytosols from rats treated with 0.2 or 2mg/kg body weight of 3-NBA were incubated with 3-NBA, DNA adduct formation was 3.2- and 8.6-fold higher, respectively, than in incubations with cytosols from control animals. Likewise, cytosols isolated from lungs and kidneys of rats exposed to 3-NBA more efficiently activated 3-NBA than those of control rats. This increase corresponded to an increase in protein levels and enzymatic activities of NQO1. Incubations of hepatic, pulmonary or renal microsomes of 3-NBA-treated rats with 3-ABA led to an 9.6-fold increase in DNA-adduct formation relative to controls. The highest induction in DNA-adduct levels was found in lung. The stimulation of DNA-adduct formation correlated with expression of CYP1A1/2 induced by the intra-tracheal instillation of 3-NBA. The results demonstrate that 3-NBA induces NQO1 and CYP1A1/2 in livers, lungs and kidneys of rats after intra-tracheal instillation, thereby enhancing its own genotoxic and carcinogenic potential.

Topics: Air Pollutants; Animals; Benz(a)Anthracenes; Carcinogens; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytosol; DNA Adducts; Enzyme Induction; Female; Kidney; Liver; Lung; Microsomes; NAD(P)H Dehydrogenase (Quinone); Rats; Rats, Sprague-Dawley; Trachea

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

Topics: Animals; Benz(a)Anthracenes; Cell Cycle; Cell Death; Cell Line, Tumor; Chemokine CXCL2; DNA Damage; Environmental Pollutants; Interleukin-6; Liver Neoplasms, Experimental; Mice; Mutagens; Receptors, Aryl Hydrocarbon; Signal Transduction

3-aminobenzanthrone (3-ABA) is the metabolite of the carcinogenic air pollutant 3-nitrobenzanthrone (3-NBA). 3-ABA was investigated for its ability to induce cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) in kidney and lung of rats, and for the influence of such induction on DNA adduct formation by 3-ABA and 3-NBA. NQO1 is the enzyme that reduces 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize 3-ABA to the same intermediate. When activated by cytosolic and and/or microsomal fractions isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney, both compounds generated the same DNA-adduct pattern, consisting of five adducts. When pulmonary cytosols isolated from rats that had been treated i.p. with 40 mg/kg bw of 3-ABA were incubated with 3-NBA, DNA adduct formation was up to 1.7-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. In contrast, no induction of NQO1 expression by 3-ABA treatment was found in the kidney. Incubations of 3-ABA with renal and pulmonary microsomes of 3-ABA-treated rats led to an increase of up to a 4.5-fold in DNA-adduct formation relative to controls. The stimulation of DNA-adduct formation correlated with a higher protein expression and activity of CYP1A1 induced by 3-ABA. These results show that by inducing lung and kidney CYP1A1 and NQO1, 3-ABA increases its own enzymatic activation as well as that of the environmental pollutant, 3-NBA, thereby enhancing the genotoxic and carcinogenic potential of both compounds.

Topics: Animals; Benz(a)Anthracenes; Biotransformation; Carcinogens, Environmental; Cytochrome P-450 CYP1A1; DNA Adducts; Humans; Kidney; Lung; Male; Microsomes; NAD(P)H Dehydrogenase (Quinone); Rats; Rats, Wistar

3-Aminobenzanthrone (3-ABA) is the main human metabolite of carcinogenic environmental pollutant 3-nitrobenzanthrone (3-NBA). Understanding which cytochrome P450 (CYP) enzymes are involved in metabolism of this toxicant is important in the assessment of individual susceptibility. Characterization of 3-ABA metabolites formed by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major rat and human CYPs participating in this process are aims of this study.. HPLC with UV detection was employed for the separation and characterization of 3-ABA metabolites. Inducers and inhibitors of CYPs and rat and human recombinant CYPs were used to characterize the enzymes participating in 3-ABA oxidation.. Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize rat liver CYPs metabolizing 3-ABA (measured as consumption of 3-ABA). Kinetics of these reactions catalyzed by rat hepatic microsomes was also evaluated. Based on these studies, we attribute most of 3-ABA metabolism in rat liver to CYP1A and 3A. Among recombinant rat and human CYP enzymes tested in this study, rat CYP3A2 and human CYP3A4/5, followed by CYP1A1 of both organisms were the most effective enzymes converting 3-ABA. Rat hepatic CYP enzymes oxidize 3-ABA up to three metabolites. Two of them were identified to be the products formed by oxidation of 3-ABA on its amino group back to the parent compound from which 3-ABA is generated in organisms, 3-NBA. Namely, N-hydroxylation metabolite, N-hydroxy-3-ABA and 3-NBA were identified to be these 3-ABA oxidation products. These metabolites are formed by CYPs of a 1A subfamily. Another 3-ABA metabolite, whose structure remains to be characterized, is generated not only by CYP1A but also by other CYP enzymes, predominantly by CYPs of a 3A subfamily.. The results found in this study, the first report on the metabolism of 3-ABA by human and rat CYPs, clearly demonstrate that CYPs of 3A and 1A subfamilies are the major enzymes metabolizing 3-ABA.

Topics: Animals; Benz(a)Anthracenes; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Inhibitory Concentration 50; Kinetics; Male; Microsomes, Liver; Oxidation-Reduction; Rats; Rats, Wistar; Recombinant Proteins; Species Specificity; Ultraviolet Rays

FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

Topics: Animals; Benz(a)Anthracenes; Cells, Cultured; DNA Adducts; Epithelial Cells; Lac Operon; Lung; Mice; Mice, Transgenic; Mutagenicity Tests; Mutagens

3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the (32)P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential.

Topics: Animals; Benz(a)Anthracenes; Carcinogens; Cytochrome P-450 CYP1A1; Cytosol; DNA Adducts; Environmental Pollutants; Enzyme Induction; Kidney; Lung; Male; Microsomes, Liver; Mutagenicity Tests; Mutagens; NAD(P)H Dehydrogenase (Quinone); Phosphorus Radioisotopes; Rats; Rats, Wistar

The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca(2+) and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

Topics: Apoptosis; Benz(a)Anthracenes; Carcinogens; Cell Cycle; Cell Line; Cell Proliferation; Cytochrome P-450 CYP1A1; DNA Damage; Enzyme Induction; Enzyme Inhibitors; Epithelial Cells; Humans; Lung; Reactive Oxygen Species

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.

Topics: Animals; Benz(a)Anthracenes; Cytochrome P-450 Enzyme System; DNA Adducts; Enzyme Activation; Humans; Male; Mice; Microsomes, Liver; Peroxidases

3-Nitrobenzanthrone (3-NBA), a suspected human carcinogen occurring in diesel exhaust and air pollution, and its human metabolite 3-aminobenzanthrone (3-ABA) were investigated for their ability to induce biotransformation enzymes in rat liver and the influence of such induction on DNA adduct formation by the compounds. Rats were treated (i.p.) with 0.4, 4, or 40 mg/kg body weight 3-NBA or 3-ABA. When hepatic cytosolic fractions from rats treated with 40 mg/kg body weight 3-NBA or 3-ABA were incubated with 3-NBA, DNA adduct formation, measured by 32P-postlabeling analysis, was 10-fold higher in incubations with cytosols from pretreated rats than with controls. The increase in 3-NBA-derived DNA adduct formation corresponded to a dose-dependent increase in protein levels and enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is the major enzyme reducing 3-NBA in human and rat livers. Incubations of 3-ABA with hepatic microsomes of rats treated with 3-NBA or 3-ABA (40 mg/kg body weight) led to as much as a 12-fold increase in 3-ABA-derived DNA adduct formation compared with controls. The observed stimulation of DNA adduct formation by both compounds was attributed to their potential to induce protein expression and enzymatic activity of cytochromes P450 1A1 and/or -1A2 (CYP1A1/2), the major enzymes responsible for 3-ABA activation in human and rat livers. Collectively, these results demonstrate for the first time, to our knowledge, that by inducing hepatic NQO1 and CYP1A1/2, both 3-NBA and 3-ABA increase the enzymatic activation of these two compounds to reactive DNA adduct-forming species, thereby enhancing their own genotoxic potential.

Topics: Animals; Benz(a)Anthracenes; Carcinogens; Cytochrome P-450 Enzyme System; DNA Adducts; Environmental Pollutants; Enzyme Induction; Humans; Liver; Male; Microsomes, Liver; NAD(P)H Dehydrogenase (Quinone); Rabbits; Rats; Rats, Wistar; RNA, Messenger

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and possible human carcinogen present in diesel exhaust and airborne particulate matter. Nitroreduction is believed to play a crucial role in nitroarene activation and mutagenicity; however, quantification of nitroreduction rate in mammalian samples has proved difficult. In this study, we present a sensitive method to quantify 3-nitrobenzanthrone reductase activity in murine tissues via normal-phase HPLC with fluorescence detection of the reduced product 3-aminobenzanthrone (3-ABA). Calibration linearity was obtained for pure 3-ABA concentrations of 1-500 ng/ml (r2>0.99), with a detection limit of 0.25 ng/ml (S/N=3). Incubation time, substrate concentration, and protein concentration in the reaction mixture were optimized, and the detection limit of the enzyme assay is 0.97 pmol/min/mg protein. The apparent K(m) and V(max) for post-mitochondrial supernatant from Mutatrade markMouse liver (i.e., liver S9) were 23.9 microM and 70.2 pmol/min/mg protein, respectively. Analysis of replicate samples of Mutatrade markMouse liver and lung S9 yielded mean activity values of 39.0+/-3.0 and 61.1+/-4.3 pmol/min/mg, respectively. ANOVA revealed significant effects of tissue type and incubation condition (i.e., with or without N2). The results show significantly higher activity in lung, and, in contrast to that observed for 1-nitropyrene, incubation in open air (i.e., without N2 bubbling) causes only a marginal decrease in activity. Quantification of 3-NBA nitroreductase activity in murine tissues will provide insight into the published tissue-specific mutagenic activity of 3-NBA.

Topics: Animals; Benz(a)Anthracenes; Calibration; Chromatography, High Pressure Liquid; Female; Fluorescence; Kinetics; Liver; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Transgenic; Oxidoreductases; Reproducibility of Results

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 recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were 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. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-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-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a gen

Topics: Air Pollutants; Animals; Benz(a)Anthracenes; Cell Line, Transformed; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; DNA Adducts; Humans; Microsomes, Liver; Rats; Recombinant Proteins

3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3-aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. Recently we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA and its metabolites, we developed a panel of Chinese hamster V79MZ-h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 microM 3-NBA, or its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA). Using both enrichment versions of the (32)P-postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N-acetyl group. Human CYP1A2 was required for the metabolic activation of 3-ABA and 3-Ac-ABA (probably via N-oxidation) and enhanced the activity of 3-NBA (probably via nitroreduction). The lack of acetylated adducts suggests N-deacetylation of 3-Ac-ABA and N-Ac-N-OH-ABA. Thus, N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3-NBA and its metabolites. Moreover, N,O-acetyltransfer reactions catalyzed by human NATs leading to the corresponding N-acetoxyester may be important in the bioactivation of N-Ac-N-OH-ABA. As human exposure to 3-NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3-NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3-NBA.

Topics: Acetylation; Animals; Arylamine N-Acetyltransferase; Arylsulfotransferase; Benz(a)Anthracenes; Biotransformation; Carcinogens; Cell Line; Cricetinae; Cricetulus; Cytochrome P-450 CYP1A2; DNA Adducts; DNA Damage; Fibroblasts; Humans; Isoenzymes; Lung; Molecular Structure; Mutagens; Polymorphism, Genetic; Recombinant Fusion Proteins; Structure-Activity Relationship; Sulfotransferases