3-Nitrobenzanthrone (3-NBA), a potent environmental mutagen and carcinogen, is known to be activated in vivo to 3-benzanthronylnitrenium ion which forms both NH and C2-bound adducts with DNA and also reacts with glutathione giving rise to urinary 3-aminobenzanthron-2-ylmercapturic acid. In this study, acid hydrolysate of globin from rats dosed intraperitoneally with 3-NBA was analysed by HPLC/MS to identify a novel type of cysteine adduct, 3-aminobenzanthron-2-ylcysteine (3-ABA-Cys), confirmed using a synthesised standard. The 3-ABA-Cys levels in globin peaked after single 3-NBA doses of 1 and 2 mg/kg on day 2 to attain 0.25 and 0.49 nmol/g globin, respectively, thereafter declining slowly to 70-80% of their maximum values during 15 days. After dosing rats for three consecutive days with 1 mg 3-NBA/kg a significant cumulation of 3-ABA-Cys in globin was observed. 3-ABA-Cys was also found in the plasma hydrolysate. Herein, after dosing with 1 and 2 mg 3-NBA/kg the adduct levels peaked on day 1 at 0.15 and 0.51 nmol/ml plasma, respectively, thereafter declining rapidly to undetectable levels on day 15. In addition, sulphinamide adducts were also found in the exposed rats, measured indirectly as 3-aminobenzanthrone (3-ABA) split off from globin by mild acid hydrolysis. Levels of both types of adducts in the globin samples parallelled very well with 3-ABA/3-ABA-Cys ratio being around 1:8. In conclusion, 3-ABA-Cys is the first example of arylnitrenium-cysteine adduct in globin representing a new promising class of biomarkers to assess cumulative exposures to aromatic amines, nitroaromatics and heteroaromatic amines.
- MeSH
- Benz(a)Anthracenes metabolism pharmacokinetics MeSH
- Environmental Biomarkers MeSH
- Cysteine chemistry metabolism MeSH
- Globins chemistry MeSH
- Hydrolysis MeSH
- Carcinogens metabolism pharmacokinetics MeSH
- Plasma metabolism MeSH
- Magnetic Resonance Spectroscopy MeSH
- Rats, Wistar MeSH
- Chromatography, High Pressure Liquid MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspect human carcinogen found in diesel exhaust. Its isomer 2-nitrobenzanthrone (2-NBA) has also been found in ambient air. These isomers differ in mutagenicity in Salmonella by 2-3 orders of magnitude. To identify their urinary metabolites and also to assess the assumed differences in their excretion, rats were dosed orally with 2mg/kg b.w. of either 2-NBA or 3-NBA. Their urine was collected for two consecutive days after dosage. Both LC-ESI-MS and GC-MS confirmed formation of the corresponding aminobenzanthrones (ABA). Excretion of these metabolites within the first day after dosing with 2- and 3-ABA amounted to 0.32±0.06 and 0.83±0.40% of the doses, respectively, while the excretion within the second day was by one order of magnitude lower. A novel mercapturic acid metabolite of 3-NBA was identified in urine by LC-ESI-MS as N-acetyl-S-(3-aminobenzanthron-2-yl)cysteine (3-ABA-MA) by comparison with the authentic standard. Its excretion amounted to 0.49±0.15 and 0.02±0.01% of dose within the first and second day after dosing, respectively. In contrast, no mercapturic acid was detected in the urine of rats dosed with 2-NBA. Observed difference in the mercapturic acid formation between 2- and 3-NBA is a new distinctive feature reflecting differences in the critical step of their metabolism, i.e., benzanthronylnitrenium ion formation that is intrinsically associated with biological activities of these two isomers. Moreover, 3-ABA-MA is a promising candidate biomarker of exposure to the carcinogenic 3-NBA.
- MeSH
- Acetylcysteine urine MeSH
- Benz(a)Anthracenes chemistry pharmacokinetics urine MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Carcinogens chemistry metabolism MeSH
- Rats MeSH
- Air Pollutants chemistry pharmacokinetics urine MeSH
- Magnetic Resonance Spectroscopy MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Rats, Wistar MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: 2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. Understanding which enzymes are involved in metabolism of these toxicants is important in the assessment of individual susceptibility. Here, metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes containing cytochromes P450 (CYPs), their reductase (NADPH:CYP reductase), and NADH:cytochrome b5 reductase was investigated under anaerobic and aerobic conditions. In addition, using the same microsomal systems, 2-NBA and 3-NBA were evaluated to be enzymatically activated under anaerobic conditions to species generating 2-NBA- and 3-NBA-derived DNA adducts. METHODS: High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation and characterization of 2-NBA and 3-NBA metabolites formed by hepatic microsomes of rats and mice under the anaerobic and aerobic conditions. Microsomal systems isolated from the liver of the control (untreated) rats and rats pretreated with Sudan I, β-naphthoflavone (β-NF), phenobarbital (PB), ethanol and pregnenolon 16α-carbonitrile (PCN), the inducers of cytochromes P450 (CYP) 1A1, 1A1/2, 2B, 2E1 and 3A, respectively, were used in this study. Microsomes of mouse models, a control mouse line (wild-type, WT) and Hepatic Cytochrome P450 Reductase Null (HRN) mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in their livers, were also employed. To detect and quantify the 2-NBA- and 3-NBA-derived DNA adducts, the 32P postlabeling technique was used. RESULTS: Both reductive metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), found to be formed predominantly under the anaerobic conditions, and two 3-NBA oxidative metabolites, whose structures have not yet been investigated, were formed by several microsomal systems used in the study. Whereas a 3-NBA reductive metabolite, 3-ABA, was found only in the microsomal systems of control rats, the rats treated with β-NF and PB, and microsomes of WT and HRN mice, all hepatic microsomes tested in the study were capable of activating this carcinogen under the reductive conditions to form DNA adducts. A stability of a reactive intermediate of 3-NBA, N-hydroxy-3-aminobenzanthrone that is formed during 3-NBA reduction to 3-ABA, to form nitrenium (and/or carbenium) ions binding to DNA in individual microsomes as well as binding of these ions to proteins of these microsomes, might be the reasons explaining this phenomenon. In contrast to 3-NBA, its isomer 2-NBA was not metabolized by any of the used enzymatic systems both under the anaerobic and aerobic conditions. Likewise, no DNA adducts were detectable after reaction of 2-NBA in these systems with DNA. CONCLUSIONS: The results found in this study, the first report on the metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes demonstrate that 3-NBA, in contrast to 2-NBA, is reductively activated to form 3-NBA-derived DNA adducts by these enzymatic systems. NADPH:CYP reductase can be responsible for formation of these DNA adducts in rat livers, while NADH:cytochrome b5 reductase can contribute to this process in livers of HRN mice.
- MeSH
- DNA Adducts metabolism MeSH
- Aerobiosis physiology MeSH
- Anaerobiosis physiology MeSH
- Benz(a)Anthracenes pharmacokinetics toxicity MeSH
- Cytochrome-B(5) Reductase genetics metabolism MeSH
- Mice, Inbred Strains MeSH
- Microsomes, Liver enzymology MeSH
- Carcinogens pharmacokinetics toxicity MeSH
- Rats MeSH
- Air Pollutants pharmacokinetics toxicity MeSH
- Inactivation, Metabolic physiology MeSH
- Models, Animal MeSH
- Mice, Knockout MeSH
- Mice MeSH
- NADPH-Ferrihemoprotein Reductase metabolism MeSH
- Rats, Wistar MeSH
- Substrate Specificity physiology MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- Vehicle Emissions toxicity MeSH
- Urban Health MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- DNA Adducts MeSH
- Benz(a)Anthracenes metabolism pharmacology MeSH
- Cytochrome P-450 CYP1A1 metabolism MeSH
- Cytochrome P-450 CYP1A2 metabolism MeSH
- Cytosol drug effects MeSH
- Enzyme Induction drug effects MeSH
- Liver enzymology MeSH
- Carcinogens pharmacology MeSH
- Rats MeSH
- Air Pollutants pharmacology MeSH
- Kidney enzymology MeSH
- Microsomes drug effects MeSH
- NAD(P)H Dehydrogenase (Quinone) metabolism MeSH
- Lung enzymology MeSH
- Rats, Sprague-Dawley MeSH
- Trachea MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
- MeSH
- Benz(a)Anthracenes metabolism MeSH
- Species Specificity MeSH
- Inhibitory Concentration 50 MeSH
- Cytochrome P-450 Enzyme Inhibitors MeSH
- Microsomes, Liver enzymology metabolism MeSH
- Kinetics MeSH
- Rats MeSH
- Humans MeSH
- Oxidation-Reduction MeSH
- Rats, Wistar MeSH
- Recombinant Proteins metabolism MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- Ultraviolet Rays MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- DNA Adducts MeSH
- Benz(a)Anthracenes pharmacology metabolism MeSH
- Biotransformation MeSH
- Cytochrome P-450 CYP1A1 metabolism MeSH
- Carcinogens, Environmental metabolism MeSH
- Rats MeSH
- Kidney metabolism drug effects MeSH
- Humans MeSH
- Microsomes enzymology metabolism drug effects MeSH
- NAD(P)H Dehydrogenase (Quinone) metabolism MeSH
- Lung metabolism drug effects MeSH
- Rats, Wistar MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- DNA Adducts metabolism MeSH
- Benz(a)Anthracenes pharmacology toxicity MeSH
- Cytochrome P-450 CYP1A1 metabolism drug effects MeSH
- Cytosol metabolism drug effects MeSH
- Enzyme Induction MeSH
- Financing, Organized MeSH
- Microsomes, Liver metabolism drug effects MeSH
- Carcinogens pharmacology toxicity MeSH
- Rats MeSH
- Environmental Pollutants pharmacology toxicity MeSH
- Kidney metabolism drug effects MeSH
- Mutagens pharmacology toxicity MeSH
- NAD(P)H Dehydrogenase (Quinone) metabolism drug effects MeSH
- Lung metabolism drug effects MeSH
- Rats, Wistar MeSH
- Phosphorus Radioisotopes MeSH
- Mutagenicity Tests MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
