Cysteine conjugate beta-lyase-catalyzed bioactivation of bromine-containing cysteine S-conjugates: stoichiometry and formation of 2,2-difluoro-3-halothiiranes

Abstract

1,1-Dichloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not 1,1-difluoroalkene-derived S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates, are mutagenic in the Ames test. Recent studies have showed, however, that bromine-containing, 1,1-difluoroalkene-derived S-(2-bromo-2-halo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic [Finkelstein, M. B., et al. (1994) Chem. Res. Toxicol. 7, 157-163] and that alpha-thiolactones are formed as reactive intermediates and glyoxylate as a terminal product [Finkelstein, M. B., et al. (1995) J. Am. Chem. Soc. 117, 9590-9591]. The present studies were undertaken to examine the stoichiometry of cysteine conjugate beta-lyase-catalyzed product formation from a panel of bromine-containing and bromine-lacking cysteine S-conjugates and to search for additional metabolites. The cysteine S-conjugates were incubated with rat renal homogenates, and pyruvate:product (glyoxylate, bromide, fluoride, dihaloacetate, trihaloethene) ratios were measured. Pyruvate:glyoxylate ratios for S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine ranged from 1:0.13 to 1:0.16. With S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine and S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, pyruvate:bromide ratios were 1:1, but with the dibrominated conjugate S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine, the pyruvate:bromide ratio was 1:1.2. All bromine-containing cysteine S-conjugates gave less than complete conversion to fluoride. A search for additional metabolites led to the consideration of 2,2-difluoro-3-halothiiranes as putative intermediates. 2,2-Difluoro-3-halothiiranes may arise by internal displacement of bromide and cyclization of 2-bromo-2-halo-1,1-difluoroethanethiolates, which are beta-elimination products of cysteine S-conjugates. Such halogenated thiiranes may eliminate sulfur to give 1,1-difluoro-2-haloethenes. GC/MS analysis showed that trifluoroethene, 2-chloro-1,1-difluoroethene, and 2-bromo-1,1-difluoroethene were terminal products of S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine, respectively. The bromine-lacking conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine did not yield glyoxylate or trifluoroethene as products, but the formation of chlorofluoroacetate was confirmed. The pyruvate:chlorofluoroacetate ratio was 1:0.38, indicating that other products are formed. This is the first report of the stoichiometry of the beta-lyase-catalyzed biotransformation of haloalkene-derived cysteine S-conjugates and of the formation of 2,2-difluoro-3-halothiiranes as reactive intermediates in the biotransformation of bromine-containing cysteine S-conjugates.