Identification of N-Acetylcysteine Conjugates of 1,2-Dibromo-3-chloropropane: Evidence for Cytochrome P450 and Glutathione Mediated Bioactivation Pathways

Abstract

The haloalkane 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen, mutagen, nephrotoxin, and testicular toxin. The identification of N-acetylcysteine conjugates of DBCP provides information on GSH mediated and cytochrome P450 mediated bioactivation pathways in the expression of DBCP-induced toxicities. N-Acetylcysteine conjugates excreted in the urine of male Sprague-Dawley rats administered DBCP, C1D2-DBCP, C2D1-DBCP, C3D2-DBCP, or D5-DBCP (80 mg/kg) were purified by reverse-phase HPLC as their methyl ester derivatives and characterized by fast atom bombardment tandem mass spectrometry. These metabolites were also converted to tert-butyldimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS) to facilitate the identification of N-acetyl-S-(2,3-dihydroxypropyl)cysteine (Ia), an apparent regioisomer of Ia, 2-(S-(N-acetylcysteinyl))-1,3-propanediol (Ib), N-acetyl-S-(3-hydroxypropyl)cysteine (IIa), and N-acetyl-S-(3-chloro-2-hydroxypropyl)-cysteine (III). Metabolites Ia, Ib, and III displayed quantitative retention of deuterium, an observation consistent with the formation of episulfonium ion intermediate(s) in their biogenesis. Mercapturate IIa retained three atoms of deuterium from D5-DBCP, and two atoms of deuterium from the dideuterio analogs (C1D2-DBCP and C3D2-DBCP), thus invoking P450 mediated formation of 2-bromoacrolein (2-BA) as an intermediate in the biogenesis of IIa. A mechanism is proposed in which conjugate addition of GSH to 2-BA, subsequent episulfonium ion formation, and addition of GSH afford 1,2-(diglutathion-S-yl)propanal. Glutathione mediated reduction is invoked to afford S-(3-hydroxypropyl)GSH which would be excreted in the urine as IIa. The quantitative retention of deuterium from C1D2-DBCP or C3D2-DBCP was indicative of isotopically sensitive branching of P450 metabolism at either C1 or C3 to afford 2-BA. C2D1-DBCP showed a 30% retention of 1 deuterium atom in IIa; the loss of the deuterium is consistent with 2-BA formation, whereas the retention of one deuterium atom is indicative of the formation of metabolite IIa through GSH conjugation of either 2,3-dibromopropanal or 2-bromo-3-chloropropanal. These data indicate that IIa is a marker metabolite for the potent direct-acting mutagen, 2-BA, or its metabolic precursors 2,3-dibromopropanal or 2-bromo-3-chloropropanal. Therefore, evidence has been presented for bioactivation of DBCP by glutathione and cytochrome P450 mediated mechanisms.