S-(3-Aminobenzanthron-2-yl)cysteine in the globin of rats as a novel type of adduct and possible biomarker of exposure to 3-nitrobenzanthrone, a potent environmental carcinogen

Abstract

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.

Hemoglobin Adducts and Urinary Metabolites of Arylamines and Nitroarenes

“…In rat experiments with 3-nitrobenzanthrone, sulfinamide adducts of 3-aminobenzanthrone with Hb and a novel type of Hb-adduct were found. 283 The metabolite 3-benzanthronyl nitrenium ion formed an adduct with cysteine of Hb. After acid hydrolysis, 3-aminobenzanthron-2-ylcysteine was quantified by LC-MS/MS.…”

“…Plotting the Hb-adduct data measured after 24 h against the metHb maximum levels measured after 0.5–2.5 h yields a correlation of r = 1 (ranks). In rat experiments with 3-nitrobenzanthrone, sulfinamide adducts of 3-aminobenzanthrone with Hb and a novel type of Hb-adduct were found . The metabolite 3-benzanthronyl nitrenium ion formed an adduct with cysteine of Hb.…”

New Aminobiphenylcysteine Derivatives in Globin and Urine of Rats Dosed with 4-Aminobiphenyl, a Tobacco Smoke Carcinogen

“…They have been widely used as biomarkers of cumulative exposure to various aromatic amines and nitroarenes. 35−38 It should be noted that 3-nitrobenzanthrone, 1-and 2-naphthylamine, and 1-and 2-nitronaphthalene which were studied previously 25,26 uniformly showed much higher levels of aminoarylcysteine adducts in globin compared to corresponding sulfinamide adducts and appeared to be related to mutagenic potential and carcinogenicity of the parent compounds. However, the example of 4-ABP shows that the predominance of aminoarylcysteine over sulfinamide adducts is not a general trend applying to all arylamines and nitroarenes.…”

“…These can dissociate to release the 4-biphenylnitrenium ion (BPN) as the ultimate electrophilic metabolite, which forms DNA adducts and thereby causes mutations. ,− In addition, N- acetylation of 4-ABP catalyzed by NAT gives N- acetyl-4-aminobiphenyl (AcABP) − which can also be metabolically activated via N -oxidation to N- acetyl -N- hydroxy-4-aminobiphenyl (OHAcABP) and further to active acetate and sulfate esters and N- acetyl-4-biphenylnitrenium (AcBPN). − Like other arylnitrenium ions, both BPN and AcBPN can bind to nucleophilic sites in DNA through their positively charged nitrogen and/or aromatic ring carbon. − However, as the most nucleophilic sites in living organisms are those at cysteine sulfanyl (SH) groups, it can be inferred that arylnitrenium ions can be covalently bound to cysteine SH groups in proteins as well as in glutathione (GSH). Indeed, aminoarylcysteine adducts were recently identified in globin of rats dosed with 3-nitrobenzanthrone, 1- and 2-naphthylamine, and aristolochic acid . Additionally, aminoarylmercapturic acids formed both via glutathione conjugation with arylnitrenium ions and via in vivo cleavage of the above-mentioned aminoarylcysteine globin adducts were identified in rat urine. , Some of these adducts and/or their in vivo cleavage products have been proposed as possible new biomarkers of cumulative exposure and effective dose of arylamines and nitroarenes .…”

“…20−24 However, as the most nucleophilic sites in living organisms are those at cysteine sulfanyl (SH) groups, it can be inferred that arylnitrenium ions can be covalently bound to cysteine SH groups in proteins as well as in glutathione (GSH). Indeed, aminoarylcysteine adducts were recently identified in globin of rats dosed with 3-nitrobenzanthrone, 25 1-and 2-naphthylamine, 26 and aristolochic acid. 27 Additionally, aminoarylmercapturic acids formed both via glutathione conjugation with arylnitrenium ions and via in vivo cleavage of the abovementioned aminoarylcysteine globin adducts were identified in rat urine.…”