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)anthraceny metabolismus farmakokinetika MeSH
- bioindikátory MeSH
- cystein chemie metabolismus MeSH
- globiny chemie MeSH
- hydrolýza MeSH
- karcinogeny metabolismus farmakokinetika MeSH
- krevní plazma metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- potkani Wistar MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
- acetylcystein moč MeSH
- benz(a)anthraceny chemie farmakokinetika moč MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- karcinogeny chemie metabolismus MeSH
- krysa rodu rattus MeSH
- látky znečišťující vzduch chemie farmakokinetika moč MeSH
- magnetická rezonanční spektroskopie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- adukty DNA metabolismus MeSH
- aerobióza fyziologie MeSH
- anaerobióza fyziologie MeSH
- benz(a)anthraceny farmakokinetika toxicita MeSH
- cytochrom-B(5)-reduktasa genetika metabolismus MeSH
- inbrední kmeny myší MeSH
- jaterní mikrozomy enzymologie MeSH
- karcinogeny farmakokinetika toxicita MeSH
- krysa rodu rattus MeSH
- látky znečišťující vzduch farmakokinetika toxicita MeSH
- metabolická inaktivace fyziologie MeSH
- modely u zvířat MeSH
- myši knockoutované MeSH
- myši MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- potkani Wistar MeSH
- substrátová specifita fyziologie MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- výfukové emise vozidel toxicita MeSH
- zdraví ve městech MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
