Chem. Res. Toxicol. 1996, 9, 227-231
227
Cysteine Conjugate β-Lyase-Catalyzed Bioactivation of Bromine-Containing Cysteine S-Conjugates: Stoichiometry and Formation of 2,2-Difluoro-3-halothiiranes Martin B. Finkelstein,† Wolfgang Dekant,‡ and M. W. Anders*,† Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, and Institut fu¨ r Toxikologie, Universita¨ t Wu¨ rzburg, Versbacher Strasse 9, D-97078 Wu¨ rzburg, Federal Republic of Germany Received August 14, 1995X
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 R-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 β-lyase-catalyzed product formation from a panel of brominecontaining 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-(2,2dibromo-1,1-difluoroethyl)-L-cysteine, the pyruvate:bromide ratio was 1:1.2. All brominecontaining 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 β-elimination products of cysteine S-conjugates. Such halogenated thiiranes may eliminate sulfur to give 1,1-difluoro2-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 β-lyase-catalyzed biotransformation of haloalkene-derived cysteine S-conjugates and of the formation of 2,2difluoro-3-halothiiranes as reactive intermediates in the biotransformation of brominecontaining cysteine S-conjugates.
Introduction Nephrotoxic haloalkenes undergo glutathione transferase- and cysteine conjugate β-lyase-dependent1 bioactivation to toxic metabolites (1-3). The bioactivation pathway involves hepatic glutathione S-conjugate formation, which is followed by enzymatic hydrolysis to the corresponding cysteine S-conjugates and translocation to the kidney, where β-lyase-dependent bioactivation affords pyruvate, ammonia, and unstable thiols as products. The unstable thiols give rise to electrophilic species whose †
University of Rochester. Universita¨t Wu¨rzburg. Abstract published in Advance ACS Abstracts, December 1, 1995. 1 Abbreviations: β-lyase, cysteine conjugate β-lyase; BCDFC, S-(2bromo-2-chloro-1,1-difluoroethyl)-L-cysteine; BTFC, S-(2-bromo-1,1,2trifluoroethyl)-L-cysteine; CTFC, S-(2-chloro-1,1,2-trifluoroethyl)-Lcysteine; DBDFC, S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine; DCDFC, S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine; TFC, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. ‡
X
0893-228x/96/2709-0227$12.00/0
formation is associated with cell damage and death and with mutagenicity. Although all haloalkene-derived cysteine S-conjugates studied thus far are nephrotoxic and cytotoxic, 1,1dichloroalkene-derived cysteine S-conjugates are mutagenic in the Ames test, whereas 1,1-difluoroalkenederived cysteine S-conjugates are not mutagenic (4-6). Bioactivation of 1,1-dichloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates affords thioketenes as intermediates (7), whereas 1,1-difluoroalkene-derived cysteine S-conjugates form thioacetyl fluorides as reactive intermediates (8). Recent studies have showed, however, that brominecontaining, 1,1-difluoroalkene-derived cysteine S-conjugates are mutagenic in the Ames test (9, 10), which challenges the generalization that 1,1-difluoroalkenederived conjugates are not mutagenic. The observation that bromine-containing, 1,1-difluoroalkene-derived cys© 1996 American Chemical Society
228 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Finkelstein et al. Scheme 1
teine S-conjugates are unexpectedly mutagenic stimulated a search for alternative reactive intermediates and terminal products. Previous studies showed that dihaloacetates are terminal metabolites of bromine-lacking, 1,1-difluoroalkene-derived cysteine S-conjugates; for example, S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteines give dihaloacetates as terminal metabolites (8, 11). Similarly, chloroalkanoates are terminal products of 1,1-dichloroalkene-derived cysteine S-conjugates; S-(pentachlorobutadienyl)-L-cysteine gives 2,3,4,4-tetrachloro-3-butenoate as a terminal metabolite (12). With bromine-containing, 1,1-difluoroalkene-derived cysteine S-conjugates, haloacetates are not detected as terminal metabolites (13); rather, glyoxylate, which arises by hydrolysis of an R-thiolactone intermediate, and both inorganic fluoride and bromide are formed as products. The objective of the present experiments was to study the stoichiometry of β-lyase-catalyzed, β-elimination reactions of a panel of bromine-containing and brominelacking cysteine S-conjugates to determine whether glyoxylate and haloacetates are major or minor metabolites. These experiments were facilitated by the knowledge that β-lyase-catalyzed β-elimination reactions of cysteine S-conjugates (Scheme 1, 1) give pyruvate and a stoichiometric amount of an R-haloalkenylthiolate or R-haloalkylthiolate 2 as initial products (2). Accordingly, pyruvate:product (glyoxylate, dihaloacetate, fluoride, and bromide) ratios were determined for several brominecontaining and bromine-lacking, 1,1-difluoroalkenederived cysteine S-conjugates. Measurement of pyruvate: glyoxylate ratios showed, however, that glyoxylate was a relatively minor (
