SYNTHESIS AND CHARACTERIZATION OF N-ACETYL-L-CYSTEINE S-CONJUGATES OF BUTADIENE MONOXIDE AND THEIR DETECTION AND QUANTITATION IN URINE OF RATS AND MICE GIVEN BUTADIENE MONOXIDE

Butadiene monoxide (BM), a mutagen and carcinogen, is the major metabolite of 1,3-butadiene in rats and mice. Because mercapturic acids (N-acetyl-L-cysteine S-conjugates) were expected in vivo metabolites of BM, reference BM-mercapturic acids were prepared by the reaction of racemic BM with N-acetyl-L-cysteine. Four isomers were purified and characterized as diastereomeric pairs of S-(2-hydroxy-3-buten-1-yl)-N-acetyl-L-cysteine (I) and S-(1-hydroxy-3-buten-2-yl)-N-acetyl-L-cysteine (II) based on analyses by H-1 NMR, fast atom bombardment mass spectrometry, and high resolution electron impact mass spectrometry. Regioisomers I and II were identified in the urine of rats and mice administered (ip) BM based on GC/MS analyses performed after HPLC fractionation followed by esterification and silylation of the carboxyl and hydroxyl groups, respectively, and comparison of GC retention times with synthetic standards. S-(4-Hydroxy-2-buten-1-yl)-N-acetyl-L-cysteine, a rearrangement pro duct formed during chemical synthesis or storage of both I and II under acidic conditions, was not detected; no other BM metabolites were evident in urine samples using this method. When rats were given BM at a dose of 71.5 to 285 mu mol/kg, their urinary excretion of I and II within 8 h of BM administration exhibited linear relationships with the administered BM dose; the total amount of the BM dose excreted as combined I and II averaged 17 +/- 4% (mean +/- SD, n = 15). No metabolites were detected in urine samples collected between 8 and 24 h after BM dosing. Mice, which are known to be more sensitive to 1,3-butadiene carcinogenicity than rats, excreted similar amounts of mercapturic acids (26 +/- 13%) at the 285 mu mol/kg BM dose within 24 h of BM administration, however, at the 143 and 71.5 mu mol/kg BM doses, they excreted only 7 +/- 3% and 9 +/- 3% of the BM dose as mercapturic acids, respectively. Thus, the ability of the rat to excrete higher levels of BM-mercapturic acids compared to the mouse at low BM doses may partially explain the lower sensitivity of the rat to 1,3-butadiene-induced carcinogenicity. Furthermore, the BM-mercapturic acid analysis method, which has limits of detection of 25 and 40 mu M in rat and mouse urine, respectively, may be used to assess human exposure to 1,3-butadiene.