Chemical Toxicology of Reactive Intermediates Formed by the

particularly mercapturic acid formation. Abundant evidence now shows that glutathione-dependent biotransformation constitutes an important bioactivati...
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Chem. Res. Toxicol. 2008, 21, 145–159

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Chemical Toxicology of Reactive Intermediates Formed by the Glutathione-Dependent Bioactivation of Halogen-Containing Compounds M. W. Anders* Department of Pharmacology and Physiology, UniVersity of Rochester Medical Center, 601 Elmwood AVenue, Box 711, Rochester, New York 214642 ReceiVed June 4, 2007

The concept that reactive intermediate formation during the biotransformation of drugs and chemicals is an important bioactivation mechanism was proposed in the 1970s and is now accepted as a major mechanism for xenobiotic-induced toxicity. The enzymology of reactive intermediate formation as well as the characterization of the formation and fate of reactive intermediates are now well-established. The mechanism by which reactive intermediates cause cell damage and death is, however, still poorly understood. Although most xenobiotic-metabolizing enzymes catalyze the bioactivation of chemicals, glutathione-dependent biotransformation has been largely associated with detoxication processes, particularly mercapturic acid formation. Abundant evidence now shows that glutathione-dependent biotransformation constitutes an important bioactivation mechanism for halogen-containing drugs and chemicals and has for many compounds been implicated in their organ-selective toxicity and in their mutagenic and carcinogenic potential. The glutathione-dependent biotransformation of haloalkenes is the first step in the cysteine S-conjugate β-lyase pathway for the bioactivation of nephrotoxic haloalkenes. This pathway has been a rich source of reactive intermediates, including thioacyl halides, R-chloroalkenethiolates, 3-halo-R-thiolactones, 2,2,3-trihalothiiranes, halothioketenes, and vinylic sulfoxides. Glutathione-dependent bioactivation of gem-dihalomethanes and 1,2-, 1,3-, and 1,4-dihaloalkanes leads to the formation of R-chlorosulfides, thiiranium ions, sulfenate esters, and tetrahydrothiophenium ions, respectively, and these reactions lead to reactive intermediate formation. Contents 1. Introduction 2. Dimensions of the Chemical Toxicology of RIs Formed from Halogen-Containing Compounds and of the Enzymology of RI Formation 3. Thioacyl Halides 4. R-Chloroalkenethiolates 5. 3-Halo-R-thiolactones 6. 2,2,3-Trihalothiiranes 7. R-Chlorosulfides 8. Thiiranium (Episulfonium) Ions 9. Tetrahydrothiophenium Ions 10. Sulfenate Esters 11. Vinylic Sulfoxides 12. Human Health Implications of the Glutathione-Dependent Bioactivation of Halogen-Containing Compounds 13. Conclusion

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1. Introduction The concept that drug- and chemical-derived reactive intermediates (RIs)1 play a key role in chemical-induced carcinogenesis dates from the 1940s. Metabolites of carcinogenic aminoazo dyes become bound to liver proteins (1), and * To whom correspondence should be addressed. 1 Abbreviations: RIs, reactive intermediates, FMO, flavin-containing monoxygenase; β-lyase, cysteine conjugate β-lyase; GST, glutathione transferase.

metabolites of nitrogen mustard become bound to nucleic acid fractions of experimental animals (2) (for reviews, see refs 3, 4). Later studies implicated the covalent modification of proteins by reactive metabolites of drugs and chemicals in liver damage. The hepatotoxicity of bromobenzene (and other halobenzenes) is associated with its metabolism to a RI: Metabolites of [14C]bromobenzene are covalently bound to liver proteins at the site of necrosis, a metabolite of bromobenzene formed a glutathione conjugate, and bromobenzene epoxide was the putative RI formed (5). These findings were extended to acetaminophen. Since these early studies, the concept that the toxicity of a range of drugs and chemicals is associated with their biotransformation (more commonly referred to as bioactivation) to RIs that covalently modify cellular macromolecules has become well-established (for reviews, see refs 6–11). There are three key questions asked about the bioactivation of drugs and chemicals: One, what is the chemical nature of the metabolites formed? Two, what enzymes catalyze the formation of toxic metabolites? Three, how is the formation and fate of toxic metabolites linked to the production of cell damage and death? Significant progress has been made in identifying and characterizing RIs and about the enzymology of their formation. Although progress has been made into understanding the linkage between RI formation and cell damage and death, much more work is needed to elucidate how RI formation is associated with cell damage and death. Studies on the bioactivation of xenobiotics have made significant strides over the past three decades. Initially, nearly all investigations into the relationship between toxicity and covalent modification of cellular macromolecules were based

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on studies with radiolabeled compounds; the usually uncharacterized radioactivity did provide some insight about the magnitude of covalent binding and its association with enzyme activity but no structural information. Developments in protein chemistry, such as gel electrophoresis, and immunochemical techniques allowed resolution of proteins and, in some cases, pointed to the identity of the modified proteins. This was paralleled by the identification and characterization of the enzymes that catalyze bioactivation reactions. Molecular biological techniques have had a significant effect on studies of bioactivation mechanisms. Techniques to express most xenobiotic-metabolizing enzymes in bacteria, yeast, or insect cells have provided a ready source of pure enzymes and allowed assignment of an enzyme to a specific bioactivation reaction. Methodologies to delete or overexpress enzymes have been of much value in investigating in vivo pathways of bioactivation. The cell biological techniques needed to investigate bioactivation mechanisms will undoubtedly continue to be improved and expanded. As is typically the case, developments in instrumentation paralleled the developments of cell biological techniques and have been of great value in investigating bioactivation mechanisms. The sensitivity and capabilities of contemporary mass and NMR spectrometers are truly impressive and have made the identification of metabolites and adducted proteins accessible. GC/MS was a welcome and most useful development, but LC/MS had a dramatic effect on bioactivation and metabolism studies. For the first time, one could analyze polar metabolites, glutathione and glucuronide conjugates, and peptides without prior derivatization. The availability of LC/MS also opened the door to proteomic studies, which have allowed exploration of the covalently modified proteins present in cells. Hypotheses about the mechanism of toxic cell death have been advanced over the past decades. For example, disturbances in cellular calcium homeostasis were proposed as the final, common pathway in toxic cell death (12), but contrary evidence was soon presented (13). There is ample evidence of perturbations of calcium homeostasis in cell death but not as the final, common pathway (14). Bioactivation of xenobiotics is sometimes associated with apoptotic cell death, but necrosis is more common; necroptosis, a nonapoptotic pathway to cell death, has recently been proposed (15). Also, oxidative stress is also associated with cell damage and death and implicates mitochondrial dysfunction in these phenomena (16). Will the developments in cell biology and analytical chemistry help to answer the question of the relationship between the formation and the fate of RIs and cell damage and death? Probably, but the answer to this question will be the result of a hard slog to windward. One (I’m not) may be tempted to be disappointed in the progress made thus far until one thinks about the complexity of the problem. This is a nontrivial question, but progress to an answer will be a remarkable achievement and will have significant impact in understanding drug and chemical toxicity. This perspective is focused on the reaction mechanisms that lead to the formation of RIs derived from halogen-containing compounds and about the role of glutathione-dependent biotransformation in bioactivation of xenobiotics. A few comments will be made about RI formation and cell death. Neither bioactivation as related to chemical carcinogenesis nor the formation of reactive oxygen metabolites will be addressed. Reviews about the glutathione-dependent bioactivation of halogen-containing compounds have been published (17–23).

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2. Dimensions of the Chemical Toxicology of RIs Formed from Halogen-Containing Compounds and of the Enzymology of RI Formation A diverse and chemically interesting range of RIs is formed from halogen-containing compounds. Indeed, the bioactivation of halogen-containing compounds has revealed some RIs that are unique in chemical experience and others for which there are few precedents. Moreover, a range of xenobiotic- and endobiotic-metabolizing enzymes catalyzes the bioactivation of the compounds of interest. As with most compounds that undergo bioactivation, it is clear that bioactivation is required to elicit a toxic response, but the linkage between bioactivation and cell death and damage remains unclear.

3. Thioacyl Halides 1,1-Difluoroalkenes are monomers used in the synthesis of fluorinated polymers, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene is formed by the degradation of the anesthetic sevoflurane. Tetrafluoroethene, chlorotrifluoroethene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene are selective nephrotoxins in experimental animals (24–27). Although rats exposed to chlorotrifluoroethene excrete fluoride, a known nephrotoxin, the amount of fluoride formed was considered to be too low to account for the observed nephrotoxicity (24, 28). By analogy with studies that showed that the trichloroethene-derived cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine is nephrotoxic (29), it was proposed that 1,1-difluoroethenes may also form nephrotoxic S-(halovinyl)-L-cysteines (30, 31). Further studies on the glutathione-dependent conjugation of chlorotrifluoroethene showed that rat liver cytosolic fractions catalyzed an addition reaction of chlorotrifluoroethene with glutathione to give S-(2-chloro-1,1,2-trifluoroethyl)glutathione (Scheme 1) (32, 33). S-(2-Chloro-1,1,2-trifluoroethyl)glutathione is converted to S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, which undergoes β-lyase-catalyzed bioactivation to nephrotoxic metabolites in rats (34); S-(2-chloro-1,1,2-trifluoroethyl)-DL-Rmethylcysteine, which cannot undergo β-lyase-catalyzed bioactivation, is not nephrotoxic; haloalkene-derived DL-R-methyl cysteine S-conjugates are, however, transported into rat renal tubular cells (35). Similarly, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene also undergoes glutathione-dependent bioactivation (vide infra). S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine is also nephrotoxic in rats (36); the renal anion transport inhibitor probenecid blocks the nephrotoxicity of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, indicating the importance of renal uptake in the bioactivation of cysteine S-conjugates. 1,1-Dichloro-2,2-difluoroethene is also nephrotoxic in rats and apparently undergoes glutathione and cysteine S-conjugate formation and bioactivation by β-lyase (37); N-acetyl-S-(1,1difluoro-2,2-dichloroethyl)-L-cysteine was identified as a metabolite of the parent alkene. The cysteine S-conjugates of tetrafluoroethene, chlorotrifluoroethene, 1,1-dichloro-2,2-difluoroethene, and 1,1-difluoro-2,2-dibromoethene and the corresponding mercapturic acids are nephrotoxic in rats (38); as with S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (vide infra), haloacetic acids, which are identified metabolites of 1,1dichloroalkene-derived cysteine S-conjugates, were not detected as metabolites of N-acetyl-S-(1,1-difluoro-2,2-dichloroethyl)L-cysteine and N-acetyl-S-(1,1-difluoro-2,2-dibromoethyl)-Lcysteine (39), indicating a different mode of bioactivation. [For example, chloroacetic acid and chlorothionoacetic acid are metabolites of S-(1,2-dichlorovinyl)-L-cysteine, as discussed below.]

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Chem. Res. Toxicol., Vol. 21, No. 1, 2008 147 Scheme 1

Thioacyl halides are formed by the cysteine conjugate β-lyase (β-lyase)-catalyzed biotransformation of 1,1-difluoroalkenederived cysteine S-conjugates (Schemes 1 and 2). The cysteine S-conjugates of chlorotrifluoroethene and tetrafluoroethene have been most extensively studied. With S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, the 2-chloro-1,1,2-trifluoroethanethiolate formed has been trapped by reaction with benzyl bromide to give benzyl 2-chloro-1,1,2-trifluoroethyl sulfide (40). The intermediate 2-chloro-1,1,2-trifluoroethanethiolate loses fluoride to give chlorofluorothionoacetyl fluoride; the reaction of the thioacetyl fluoride with diethylamine affords N,N-diethylchlorofluorothioacetamide. Thioacyl halides undergo hydrolysis to give the corresponding carboxylic acids, HF, and H2S (Scheme 1); H2S is highly toxic to mitochondria but is not involved in cysteine S-conjugate-induced mitochondrial dysfunction (41). The thioacyl halides formed from S-(1,1,2,2-tetrafluoroethyl)L-cysteine covalently modify rat renal tubular proteins (42): Incubation of [35S]S-(1,1,2,2-tetrafluoroethyl)-L-cysteine with renal tubular cells and analysis by 19F NMR spectroscopy demonstrated the formation of N-(difluorothionoacetyl)lysine adducts (Scheme 1). Similarly, in rats given S-(2-chloro-1,1,2trifluoroethyl)-L-cysteine or S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, covalently modified renal proteins were identified by 19F NMR spectroscopy as N-(chlorofluorothionoacetyl)lysine or N(difluorothionoacetyl)lysine adducts (43); renal proteins incubated with 2-chloro-1,1,2-trifluoroethyl 2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiolate, also showed the formation of N-(chlorofluorothionoacetyl)lysine adducts. Antibodies raised against trifluoroacetylated proteins have also been used to demonstrate the covalent modification of renal proteins in rats given S-(2-chloro1,1,2-trifluoroethyl)-L-cysteine or S-(1,1,2,2-tetrafluoroethyl)L-cysteine (44, 45). As discussed below, HSP60, mortalin, and the R-ketoglutarate dehydrogenase complex are covalently modified by metabolites of S-(2-chloro-1,1,2-trifluoroethyl)-Lcysteine or S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. The enzymology of the β-lyases involved in the bioactivation of 1,1-difluoroalkene-derived cysteine S-conjugates is complex. Previous studies demonstrated that the cytosolic β-lyase is

identical with glutamine transaminase K (for reviews, see refs 46–48). The nephrotoxicity of cysteine S-conjugates is, however, characterized by mitochondrial dysfunction (49), which lead to work to identify mitochondrial β-lyases. Although glutamine transaminase K is present in mitochondria, the purified enzyme shows little β-lyase activity (50). Eleven pyridoxal phosphatedependent enzymes with β-lyase activity have been identified in mammalian tissues (48), and it is likely that several contribute to the bioactivation of cysteine S-conjugates. Four mitochondrial enzymes with β-lyase activity have been identified: mitochondrial aspartate aminotransferase (51), mitochondrial branched chain aminotransferase also catalyzes β-lyase reactions but is rapidly inactivated during catalysis (52), mitochondrial Lalanine-glyoxylate aminotransferase II also possesses β-lyase activity (53), and GABA amino transferase shows modest β-lyase activity (48). The relative contribution of these enzymes to the bioactivation of nephrotoxic cysteine S-conjugates has not been fully elaborated, but mitochondrial aspartate aminotransferase accounts for 15–20% of the mitochondrial β-lyase activity. Mitochondrial aspartate aminotransferase is syncatalytically inactivated during turnover; this protein also undergoes thioacylation when incubated with S-(1,1,2,2-tetrafluoroethyl)L-cysteine, which may account for the observed inactivation (54). A high molecular weight β-lyase (Mr approximately 330000) is also found in rat kidney mitochondria (55). Subsequent studies showed that mitochondrial HSP70 and mature protein disulfide isomerase copurify and that mitochondrial aspartate aminotransferase accounts for the observed β-lyase activity of this complex (48, 56). The cytotoxicity of 1,1-difluoroalkene-derived cysteine Sconjugates is associated with mitochondrial dysfunction (49). The thioacyl fluoride formed from S-(1,1,2,2-tetrafluoroethyl)L-cysteine, a selective nephrotoxin, reacts with protein-bound lysine residues to give difluorothioamidyl-L-lysine adducts (54). Analysis of the modified proteins shows that mitochondrial P1 protein (HSP60, a chaperonin) and the HSP70 family member mortalin are targets. Subsequent studies show that the modified proteins are subunits of the mitochondrial dehydrogenase multienzyme complex, which plays a key role in regulating

148 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 Scheme 2

cellular respiration (57). Further analysis identified difluorothioamidyl-L-lysine adducts with lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the R-ketoglutarate dehydrogenase complex (58). The adducted R-ketoglutarate dehydrogenase complex was markedly inhibited, but the related pyruvate dehydrogenase complex was not adducted, and no decrease in activity was observed. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine-induced cell death in TAMH cells (a mouse hepatocytes cell line) is dependent on BAX and is blocked by BCL-xL (59); indeed, TAMH cells that overexpress BCL-xL are resistant to S-(1,1,2,2-tetrafluoroethyl)L-cysteine-induced cell death. The activation of Keap1-Nrf2ARE signaling pathway elicits a cytoprotective response and is associated with modification of cysteine residues in Keap1 by electrophiles and oxidative stress (60, 61). With S-(1,1,2,2tetrafluoroethyl)-L-cysteine-induced cytotoxicity, however, Nrf2 activation was independent of oxidative stress (62); this indicates that Nrf2 activation may involve Nrf2 phosphorylation by ERmediated protein kinases [PKR-like endoplasmic reticular kinase (PERK)]. As indicated above, the covalent modification of R-ketoglutarate dehydrogenase complex by metabolites of S-(1,1,2,2tetrafluoroethyl)-L-cysteine is accompanied by inactivation,

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whereas the similar pyruvate dehydrogenase complex is not modified and no decrease in activity is observed (54, 63). These observations lead to the concept that the close association of the R-ketoglutarate dehydrogenase complex and mitochondrial aspartate aminotransferase in a metabolon or supramolecular complex serves to channel RIs to members of the complex and may account for this selectivity (64, 65). The anesthetic sevoflurane undergoes degradation to 2(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene in the anesthetic circuit (66). Hence, human subjects anesthetized with sevoflurane are exposed to 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene, which is nephrotoxic in rats (67). This raises the question of whether 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene may also produce nephrotoxicity in human subjects anesthetized with sevoflurane. Although more than 120 million human subjects worldwide have been anesthetized with sevoflurane, no confirmed cases of 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene-associated nephrotoxicity have been reported (68, 69). This may be attributed to the relatively low β-lyase activity in human kidney tissue as compared with rat kidney tissue (70, 71). 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene is metabolized to the glutathione conjugates S-[2-(fluoromethoxy)1,1,3,3,3-pentafluoropropyl]glutathione and (E)- and (Z)-S-[2(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]glutathione in rats (Scheme 2) (72); in contrast to other 1,1-difluoroalkenes, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene undergoes both addition and addition–elimination reactions. The glutathione conjugates are hydrolyzed by γ-glutamyltransferase and dipeptidases to give S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine and (E)- and (Z)-S-[2-(fluoromethoxy)1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine; β-lyase-catalyzed biotransformation of the cysteine S-conjugates yields 2-(fluoromethoxy)-1,1,3,3,3-propanethiolate and 2-(fluoromethoxy)1,3,3,3-tetrafluoro-1-propenethiolate, which lose HF to give 2-(fluoromethoxy)-3,3,3-trifluorothionopropanoyl fluoride; hydrolysis of the thioacyl fluoride gives 2-(fluoromethoxy)-3,3,3trifluoropropanoic acid; finally, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid is degraded to trifluorolactic acid (Scheme 2) (73). The fate of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene in human subjects mirrors the pathway seen in rats. Analysis of urine samples from human subjects anesthetized with sevoflurane shows the formation of N-acetyl-S-[2-(fluoromethoxy)1,1,3,3,3-pentafluoropropyl]-L-cysteine, (E)- and (Z)-N-acetylS-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine,2-(fluoromethoxy)-3,3,3-trifluoropropanoicacid,trifluorolactic acid, and inorganic fluoride (74, 75). Although these results are consistent with the β-lyase-dependent bioactivation of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene in human subjects and in rats, this conclusion has been challenged based on studies with inhibitors of the β-lyase pathway (76); the β-lyase inhibitor (aminooxy)acetic acid and the γ-glutamyltransferase inhibitor acivicin increase, rather than decrease, as expected, the nephrotoxicity of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene. These observations have been confirmed (77). No alternative pathway for the bioactivation of 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene has, however, been proposed. Although 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene is nephrotoxic and may, by analogy to other nephrotoxic cysteine S-conjugates, induce mitochondrial dysfunction, this has apparently not been investigated. Recent gene expression profiling studies show that a range of genes are up-regulated or downregulated (78); the genes most highly up-regulated include

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Chem. Res. Toxicol., Vol. 21, No. 1, 2008 149 Scheme 3

kidney injury protein, osteopontin, clusterin, tissue inhibitor of metalloproteinase 1, and TNF receptor 2, all of which have been associated with nephrotoxicity.

4. r-Chloroalkenethiolates Calves fed trichloroethene-extracted soybean meal develop a fatal aplastic anemia (79, 80); the toxic agent present in trichloroethene-extracted soybean meal was identified as S-(1,2dichlorovinyl)-L-cysteine, which is formed by the reaction of trichloroethene with cysteine residues on soybean proteins. The hematotoxicity of S-(1,2-dichlorovinyl)-L-cysteine and other cysteine S-conjugates in calves was reinvestigated almost 40 years after the original observation that S-(1,2-dichlorovinyl)L-cysteine produced aplastic anemia in calves, and it was found that the observed hematotoxicity is apparently unique to S-(1,2dichlorovinyl)-L-cysteine (81). S-(1,2-Dichlorovinyl)-L-cysteineinduced hematotoxicity is not observed in rats, mice, guinea pigs, rabbits, cats, or dogs, but nephrotoxicity was observed (82). These observations opened the door to investigations into the selective nephrotoxicity of a range of 1,2-dichloroalkenes and, particularly, to the bioactivation mechanisms involved (for a review, see ref 22). The synthesis of glutathione conjugates of 1,1-dichloroalkenes is catalyzed by cytosolic and microsomal glutathione transferases (GSTs) (Scheme 3) (22). The resulting glutathione S-conjugates are hydrolyzed by γ-glutamyltransferase and dipeptidases to give the corresponding cysteine S-conjugates. The cysteine Sconjugates are bioactivated by mitochondrial β-lyases (vide supra). In vivo studies on the metabolism of S-(1,2-dichlorovinyl)L-cysteine showed the formation of pyruvate, ammonia, and a reactive thiol species (83), which reacts with proteins and DNA (84, 85). [35S]Sulfate is the major metabolite of [35S]-S-(1,2dichlorovinyl)-L-cysteine in the rat (86); the authors speculated that a “S-dichlorovinyl moiety” was also formed. In the calf, [35S]S-(1,2-dichlorovinyl)-L-cysteine is metabolized to [35S]sulfate and eight other unidentified 35S-containing metabolites (87); the highest concentration of radioactivity was found in the kidney, but little was present in bone marrow.

The β-lyase-catalyzed biotransformation of 1,1-dichloroethene-derived cysteine S-conjugates may be expected to give an R-chloroalkenethiolate as the putative initial metabolite (Scheme 3). R-Chloroalkenethiolates have not, however, been observed or trapped in biological systems. R-Chloroethenethiolates are the enol tautomers of chloroacetaldehyde, but the keto–enol tautomerization favors the keto form (88, 89). The formation of 1,2-dichloroethenethiolate from S-propyl 5,6dichloro-4-thia-5-hexenethiolate, an analogue of S-(1,2-dichlorovinyl)-L-cysteine that lacks the R-amino group, has been demonstrated by Fourier transform ion cyclotron resonance mass spectrometry (90). 1,2-Dichloroethenethiolate was directly observed in the gas phase; furthermore, collision-induced decomposition of 1,2-dichloroethenethiolate resulted in the loss of chloride and formation of chlorothioketene (vide infra). In addition, the formation of 1,2-ethenethiolate from N-acetyl-S(1,2-dichlorovinyl)-L-cysteine methyl ester was also observed. Although, as indicated above, intermediates formed by the bioactivation of S-(1,1-dichloroalkenyl)-L-cysteine S-conjugates have not been trapped, thioacylating intermediates, which may be formed from R-chloroalkenethiolates, have been identified (Scheme 3). Incubation of S-(1,2-dichlorovinyl)-L-cysteine with β-lyase or N-dodecylpyridoxal bromide yields chloroacetic acid and chlorothionoacetic acid as metabolites (91); with S-(1,2,3,4,4pentachlorobuta-1,3-dienyl)-L-cysteine as the substrate, 2,3,4,4tetrachlorobutenoic acid and 2,3,4,4-tetrachlorothiobut-3-enoic acid are the major products (92). The thioacylating intermediates formed may be responsible for the covalent binding of metabolites of S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine (93–95). R-Chloroalkenethiolates may, however, lose chloride to give a thioketene (Scheme 3). Hence, R-haloalkenyl 2-nitrophenyl disulfides were prepared as precursors of R-chloroalkenethiolates (96). The reaction of 1,2-dichloro-3,3,3-trifluoro-1-propenyl 2-nitrophenyl disulfide with 1,4-diazabicyclo[2.2.2]octane in THF that contained cyclopentadiene resulted in the formation of (E)- and (Z)-3-(2,2,2-trifluoro-1-chloroethylidene)-2thiabicyclo[2.2.1]hept-5-ene. Moreover, thioketene may dimerize to give 1,3-dithietanes: syn- and anti-2,4-bis(2,2,2-trifluoro-1chloroethylidene)-1,3-dithietanes were also identified. An al-

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ternative route to precursors of thioketenes has also been reported; S-(1,2-dichlorovinyl)thioacetate, which is prepared by the reaction of dichloroethyne with thioacetic acid, is cleaved in presence of base to give chlorothioketene (97). Cleavage of S-(1,2-dichlorovinyl)thioacetate in organic solvents in the presence of cytosine leads to the formation of several identified adducts; when the reaction was carried out in aqueous solution, no thioketene-derived adducts were observed. These findings indicate that thioketenes may not be associated with adduct formation in cells. As with other nephrotoxic cysteine S-conjugates, 1,1-dichloroalkene-derived cysteine S-conjugates induce mitochondrial dysfunction. Metabolites of [35S]S-(1,2-dichlorovinyl)-L-cysteine covalently modify mitochondrial proteins and induce mitochondrial dysfunction (98). Moreover, S-(1,2-dichlorovinyl)-L-cysteine induces cytochrome c release from mitochondria and activates caspase 3, indicating a role for apoptosis in S-(1,2dichlorovinyl)-L-cysteine-induced cytotoxicity (99). Recent studies show that S-(1,2-dichlorovinyl)-L-cysteine induces both apoptosis and necrosis in renal proximal tubular cells and that activation of protein kinase B (Akt) is cytoprotective (100).

5. 3-Halo-r-thiolactones Bromine-containing 1,1-difluoroalkene-derived cysteine Sconjugates undergo reactions that are significantly different from their bromine-lacking congeners, although the initial product of the β-lyase-catalyzed reaction is an ethanethiolate, specifically 2-bromo-2-halo-1,1-difluoroethanethiolate, which may lose HF to give 2-bromo-2-halothioacetyl fluoride. As indicated above, the terminal products of the biotransformation of brominelacking 1,1-difluoroalkene-derived cysteine S-conjugates are dihaloacetic acids. 1-Bromo-1-chloro-2,2-difluoroethene, a metabolite or decomposition product, or both, of the anesthetic halothane (101), is biotransformed to N-acetyl-S-(2-bromo-2chloro-1,1-difluoroethyl)-L-cysteine in human subjects anesthetized with halothane (102, 103), indicating glutathione S-conjugate formation and processing. An investigation of the fate of S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine in rat kidney homogenates and in a pyridoxal model system showed that glyoxylic acid rather than the expected bromochloroacetic acid is the terminal metabolite (104). This observation leads to the proposal that intermediate 2-bromo-2-chloro-1,1-difluoroethanethiolate may undergo a series of reactions that involve the formation of 3-chloro-R-thiolactone; hydrolysis of the R-thiolactone would give mercaptoacetic acid, which may lose H2S to give the observed product glyoxylic acid (Scheme 4). Base hydrolysis of ethyl bromofluorothionoacetate also leads to the formation of glyoxylic acid, which supports the proposed reaction sequence.

6. 2,2,3-Trihalothiiranes Further studies on the fate of bromine-containing 1,1difluoroalkene-derived cysteine S-conjugates showed that glyoxylic acid is a minor (∼20%) metabolite of S-(2-bromo-2chloro-1,1-difluoroethyl)-L-cysteine (105). Stoichiometry studies with S-(2-bromo-2-chloro-1,1- difluoroethyl)-L-cysteine and S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine gave a pyruvate: bromide ratio of 1:1, but less than stoichiometric amounts of fluoride were formed. Hence, a search for additional metabolites was undertaken. The possibility that 2,2-difluoro-3-chlorothiirane may be formed was considered. The formation of 2,2-difluoro3-halothiiranes had been proposed earlier (33, 39), but no experimental data were presented. As shown in Scheme 5, 2-bromo-2-chloro-1,1-difluoroethanethiolate may undergo an internal displacement of bromide to give 2,2-difluoro-3-chlorothiirane. 2,2,3-Trihalothiiranes are apparently not known in chemical experience but would be expected to be highly unstable. Some substituted thiiranes undergo a facile thermal elimination of sulfur to yield alkenes (106, 107). Indeed, analysis of the headspace gas demonstrated the formation of 1,1-difluoro2-chloroethene but only as a minor metabolite (∼5%). Additional evidence for thiirane formation has been presented (108): When S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine is incubated in a pyridoxal model system and in the presence of o-phenylenediamine, 2-fluoroquinoxaline is formed, which can be rationalized by the reaction of 1,1-difluoro-2-chlorothiirane with o-phenylenediamine (Scheme 5). The issue of whether 2,2-dihalothioacetyl fluorides formed from 2,2-dihalo-1,1-difluoroethanethiolates would yield 2,2difluoro-3-halothiiranes or 3-halo-R-thiolactones, or both, has been investigated by computation. Commandeur et al. (108) determined the free enthalpies of formation (∆fG) for the tetrahaloethanethiolates formed from S-(1,1,2,2-tetrahaloethyl)-L-cysteines. With 1,1,2,2-tetrafluoroethanethiolate, the formation of 2,2-difluorothioacetyl fluoride was energetically favored over 2,2,3-trihalothiirane formation. It was also found that the thiirane pathway was favored over the thioacyl fluoride pathway for 2-chloro-1,1,2-trifluoroethanethiolate and 2,2-dichloro-1,1-difluoroethanethiolates. Shim and Richard (109) employed computational methods to identify preferences among the thioacetyl fluoride, thiirane, and R-thiolactone pathways. For 1,1,2,2-tetrafluoroethanethiolate, for example, a preference was shown for 2,2-difluorothioacetyl fluoride formation. In contrast, for 2-bromo-2-halo-1,1difluoroethanethiolate, a preference for trihalothiirane formation was found. The data do not, however, support a pathway leading to 3-halo-R-thiolactone formation.

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Chem. Res. Toxicol., Vol. 21, No. 1, 2008 151 Scheme 5

Scheme 6

7. r-Chlorosulfides R-Chlorosulfides are relatively uncommon RIs. The moststudied example is the GST-catalyzed biotransformation of dichloromethane, which yields S-(chloromethyl)glutathione as an intermediate. Dichloromethane is used in the manufacture of cellulose acetate film and as a solvent, paint remover, blowing agent, extractant, and propellant. Although the acute toxicity of dichloromethane is low as compared with many other solvents, it is carcinogenic in the lung and liver of B6C3F1 mice and increases the incidence of mammary tumors in rats (110–112). Because of human exposure to dichloromethane, it became important to determine the mechanism by which dichloromethane induces tumors in rodents and to apply that knowledge to the likelihood of its carcinogenicity to humans. An investigation of the biotransformation of dichloromethane in rodent and human tissues demonstrated that two enzyme systems catalyze the biotransformation of dichloromethane (113): a high-affinity, saturable pathway catalyzed by P450s and a low-affinity, first-order pathway catalyzed by GSTs. The metabolism of dichloromethane by the GST-dependent pathway was linked to the development of tumors in mice. The U.S. Environmental Protection Agency has, however, classified dichloromethane as a “probable human carcinogen” (http:// www.epa.gov/iris/subst/0070.htm). A recent reassessment of the cancer risk posed by human exposure to dichloromethane that used a refined physiologically based pharmacokinetic model and updated epidemiological findings indicated a significant reduction in the estimated risks over those determined by the U.S. Environmental Protection Agency (114, 115).

The pathway for the glutathione-dependent bioactivation of dichloromethane has been elucidated (116, 117) (Scheme 6): A GST-catalyzed attack of glutathione on dichloromethane yields S-(chloromethyl)glutathione. The reaction of glutathione with dichloromethane is catalyzed by rat, mouse, and human GSTT1-1 (118–120); negligible activity is seen with R- and µ-class GSTs. GSTT1-1 activity is found in human, rat, and mouse liver, kidney, and lung tissue and in human, but not rodent, erythrocytes. GSTT1-1 activity in liver is greater than activity in kidneys (121, 122), and activity is particularly high in mouse lung tissue as compared with human lung tissue, which correlates with the site of tumor induction in mice by dichloromethane (123). S-(Chloromethyl)glutathione may react with water to give S-(hydroxymethyl)glutathione, which is the hemimercaptal of formaldehyde and glutathione. Hence, glutathione is required for the biotransformation of dichloromethane but is not consumed in the reaction. Dichloromethane is mutagenic in some test systems. For some time, the question centered about whether formaldehyde, which is a known mutagen (124), or S-(chloromethyl)glutathione is responsible for the DNA damage required for mutagenesis. Dichloromethane is weakly mutagenic in Salmonella typhimurium TA1535 but is clearly mutagenic in S. typhimurium TA1535 that expresses GSTT1-1 (119, 125); significantly, formaldehyde was not mutagenic in S. typhimurium TA1535 expressing GSTT1-1. Although R-halosulfides have been prepared and shown to undergo rapid hydrolysis (126), S(chloromethyl)glutathione has apparently not been obtained by synthesis; hence, S-(1-acetoxymethyl)glutathione was prepared

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Anders Scheme 7

as a model to study the modification of DNA (119); S-(1acetoxymethyl)glutathione reacts with 2′-deoxyguanosine residues in DNA to give S-[1-(N2-deoxyguanosinyl)methyl]glutathione. Further studies on the reaction of S-(1-acetoxymethyl)glutathione with 2′-deoxyadenosine, 2′-deoxycytosine, and 2′deoxythymidine demonstrated the formation of S-[1(N7-deoxyadenosinyl)methyl]glutathione, S-[1-(N4-deoxycytidyl)methyl]glutathione, and S-[1-(N3-thymidinyl)methyl]glutathione, respectively (127, 128).

8. Thiiranium (Episulfonium) Ions A range of vicinal dihaloalkanes exhibits organ-selective toxicity (129). Three vicinal dihaloalkanes are of historical or contemporary toxicological interest. 1,2-Dibromoethane has been used as a soil and grain fumigant, as a nematocide, as a gasoline additive, and as a solvent; 1,2-dibromoethane is, however, toxic to several organ systems and is carcinogenic in experimental animals (130) and genotoxic in a range of test systems (131). 1,2-Dichloroethane is an intermediate in the synthesis of vinyl chloride and has been used as a gasoline additive and fumigant. 1,2-Dichloroethane exhibits multiorgan toxicity in experimental animals and is genotoxic in several test systems (131–133). 1,2-Dibromo-3-chloropropane has been used as a nematocide. Reproductive toxicity in humans was associated with the manufacture and use of 1,2-dibromo-3-chloropropane (134, 135). Although 1,2-dibromo-3-chloropropane is no longer used in the United States, it is still in use in some countries. Investigations into the mechanisms by which these compounds exert their toxic effects have implicated glutathione-dependent metabolism and thiiranium ion formation as an important bioactivation mechanism associated with the observed mutagenicity of many vicinal dihaloalkanes (136). The key steps in the glutathione-dependent bioactivation of vicinal dihaloalkanes include initial glutathione conjugate formation to give a S-(2-haloethyl)glutathione conjugate, which may undergo thiiranium ion formation (Scheme 7). Enzymatic hydrolysis of the glutathione conjugates yields the corresponding S-(2-haloethyl)-L-cysteine conjugates, which may also form thiiranium ions. Finally, the S-(2-haloethyl)-L-cysteine conjugates may be biotransformed to mercapturic acids, which may also form thiiranium ions. Alternatively, 1,2-dihaloethanes may undergo P450-dependent metabolism to the gem-halohydrin 1,2dihaloethanol, which may lose HX to give a reactive 2-haloacetaldehyde (137). Several lines of evidence implicate thiiranium ion formation in the toxicity and mutagenicity of 1,2-dihaloalkanes. Early studies showed that 1,2-dichloroethane undergoes glutathionedependent conversion to a mutagenic metabolite (138). Moreover, a role for glutathione and GSTs in the bioactivation of 1,2-dibromoethane was demonstrated (139, 140). Other studies

showed that S-(2-chloroethyl)-DL-cysteine, but not S-(3-chloropropyl)-DL-cysteine, S-ethyl-L-cysteine, S-(2-hydroxyethyl)-Nacetyl-DL-cysteine, or S-(2-hydroxyethyl)-DL-cysteine, is nephrotoxic in rats and cytotoxic in isolated rat hepatocytes (141, 142). Several lines of evidence indicate the formation of a thiiranium ion during the glutathione-dependent biotransformation of 1,2-dihaloethanes. Stereochemical experiments provided early evidence for thiiranium ion formation from vicinal-dihaloethanes (143). (Z)-1,2-Dichlorocyclohexane is mutagenic in the Ames test in the presence of rat liver cytosol and glutathione, whereas (E)-1,2-dichlorocyclohexane is only weakly mutagenic; displacement of chloride from (Z)-2-chlorocyclohexane by glutathione would afford a conjugate with an (E)-configuration that is poised for the anti-elimination of chloride, which is required for thiiranium ion formation. Moreover, both N-acetyl-S-[(E)2-chlorocyclohexyl]-L-cysteine methyl ester and N-acetyl-S-[(E)2-bromocyclohexyl]-L-cysteine methyl ester are directly mutagenic in the Ames test. When [1,2-2H4]1,2-dibromoethane is given to rats, [2-hydroxyethyl-2H4]-S-(2-hydroxyethyl)-N-acetylL-cysteine is found in the urine (144); the retention of four deuterium ions indicates conjugation with glutathione and the formation of an intermediate thiiranium ion. (The majority of the excreted mercapturic acids retained only one deuterium atom; these data indicate that the ratio of oxidative to conjugative metabolism is 4:1.) Dohn and Casida (145) showed that S-(1,12 H2-2-hydroxyethyl)-L-cysteine affords an equal mixture of the corresponding S-(1,1-2H2-2-haloethyl)- and S-(2,2-2H2-2-haloethyl)-L-cysteines when treated with concentrated hydrochloric, hydrobromic, or hydroiodic acids. Finally, when [threo-1,22 H2]ethylene dibromide and [erythro-1,2-2H2]ethylene dibromide were incubated with glutathione, rat liver cytosol, and DNA, NMR analysis of the resulting N7-guanyl adducts indicated that the reaction involves three SN2 steps (146): The first is the reaction of glutathione with 1,2-dibromoethane, which proceeds with inversion of configuration (147); the second thiiranium ionforming step also involves an inversion of configuration; finally, the reaction of the thiiranium ion with guanyl residues in DNA also proceeds with inversion of configuration. These data provide strong evidence for the intermediate formation of a thiiranium ion in the reaction of glutathione with vicinal dihaloethanes. A mechanism to explain the pH-dependent hydrolysis of S-(2chloroethyl)-L-cysteine that does not involve an intermediate thiiranium ion has been described (148); the authors propose the formation of thiomorpholine-3-carboxylic acid by displacement of chloride by the amine group of S-(2-chloroethyl)-Lcysteine. Because of the observed mutagenicity of 1,2-dihaloethanes, the reaction of metabolites of 1,2-dihaloethanes with DNA has been investigated. When 1,2-dibromoethane, calf thymus DNA,

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Chem. Res. Toxicol., Vol. 21, No. 1, 2008 153 Scheme 8

Scheme 9

and glutathione were incubated with GSTs, the adduct S-[2(N7-guanyl)ethyl]glutathione was formed; this is the major DNA adduct formed (146, 149–153). Unexpectedly, the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity and cytotoxicity of 1,2-dibromoethane in Escherichia coli (154–156). An investigation of the mechanism showed that 1,2-dibromoethane reacts directly with Cys-145 of the alkyltransferase to give a S-(2bromoethyl) intermediate; the DNA binding function of the alkyltransferase then directs the RI to DNA. Thiiranium ion formation has also been demonstrated for the glutathione-dependent bioactivation of 1,2-dibromo-3-chloropropane. The GST-catalyzed reaction of glutathione with 1,2dibromo-3-chloropropane gives 1-(glutathion-S-yl)-2-(chloromethyl)thiiranium (Scheme 8) (157). Hydrolysis of the thiiranium ion gives 3-chloro-2-(glutathion-S-yl)propan-1-ol, which may also form a thiiranium ion or undergo hydrolysis to S-(2,3dihydroxypropyl)glutathione. Thiiranium ion formation is also implicated in the testicular toxicity of 1,2-dibromo-3-chloropropane (158): [1,1,2,3,3-2H5]1,2-Dibromo-3-chloropropane is as cytotoxic to rat testicular cells as 1,2-dibromo-3-chloropropane. Reaction of synthetic S-(2-bromo-3-chloropropyl)glutathione with DNA gave S-[1-(hydroxymethyl)-2-(N7guanyl)ethyl]glutathione and several other N7-guanyl adducts (Scheme 8) (159); incubation of 1,2-dibromo-3-chloropropane with rat liver cytosol, glutathione, and DNA also resulted in the formation of N7-guanyl adducts but in low yield.

9. Tetrahydrothiophenium Ions 1,4-Bis(methanesulfonoxy)butane (Busulfan, Myleran) is used in the treatment of chronic myelogenous leukemia. It is considered to be a direct-acting agent that does not require enzymatic activation (160) and is metabolized to 3-hydroxysulfolane in rodent species (161). Studies on 1,4-dihalobutanes have been undertaken as models for the fate of 1,4-bis(methanesulfonoxy)butane and to explain the mutagenicity of R,ω-dihaloalkanes (162). 1,4-Disubstituted butanes may undergo metabolism to tetrahydrothiophenium ions: 1,4-Diiodobutane and 1,4-dibromobutane undergo GST-dependent metabolism to γ-glutamyl-β-(S-tetrahydrothiophenium)alanylglycine, which is converted to the cysteine S-conjugate and thence to the mercapturic acid (Scheme 9) (163–165). The mercapturic acid may be metabolized to tetrahydrothiophene, which undergoes flavin-containing monoxygenase (FMO)- and P450 (presumably)-catalyzed oxidation to 3-hydroxysulfolane; alternatively, S-(β-alanyl)tetrahydrothiophenium may undergo a β-lyase-catayzed reaction to give tetrahydrothiophene (164, 165). It is unclear whether this reaction pathway constitutes a detoxication or bioactivation pathway. Some 1,4-disubstituted butanes are mutagenic, but their mutagenicity is not altered in the presence of rat liver 9000g supernatant or cytosol (162, 164).

10. Sulfenate Esters There are few examples of sulfenate ester formation as a bioactivation mechanism. The cysteine S-conjugates and cor-

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Anders Scheme 10

Scheme 11

responding mercapturic acids of both the cis- and the transisomers of the soil fumigant 1,3-dichloropropene are cytotoxic in isolated renal proximal tubular cells and in LLC-PK1 cells (166); moreover, the cytotoxicity is inhibited by methimazole but not by the β-lyase inhibitor (aminooxy)acetic acid, indicating a role for FMO. FMO-catalyzed sulfoxidation of S-(3-chloro1-propenyl)-L-cysteine yields S-(3-chloro-1-propenyl)-L-cysteine sulfoxide that undergoes a [2,3]-sigmatropic rearrangement to the sulfenate ester 2-amino-3-(1-chloroallyloxythio)propanoic acid, which may eliminate acrolein as a RI and cysteine sulfenyl chloride (Scheme 10).

11. Vinylic Sulfoxides Although a role for β-lyase in the bioactivation of nephrotoxic cysteine S-conjugates is well-established, the sulfoxidation of vinylic cysteine S-conjugates to give vinylic sulfoxides has been proposed as another bioactivation mechanism. Vinylic sulfoxides are good Michael acceptors (167), and the reactivity of vinylic cysteine S-conjugate sulfoxides with sulfur nucleophiles, including glutathione, has been demonstrated (168–170) (Scheme 11). Both FMO and P450 catalyze sulfoxidation of cysteine Sconjugates and their mercapturic acids. A FMO that catalyzes the sulfoxidation of S-benzyl-L-cysteine to S-benzyl-L-cysteine sulfoxide has been identified in microsomal fractions of rat liver and kidney (171); S-(1,2-dichlorovinyl)-L-cysteine inhibits Sbenzyl-L-cysteine S-oxidase, indicating that other cysteine S-conjugates are alternative substrates. Further studies showed that the S-benzyl-L-cysteine S-oxidase is identical with pig liver FMO 1A1 (172). An investigation of the substrate selectivities of cDNA-expressed cysteine S-conjugate S-oxidases demonstrated that FMO1 catalyzes the sulfoxidation of S-benzyl-Lcysteine and S-allyl-L-cysteine, but not that of S-(1,2-dichlorovinyl)-L-cysteine and S-(trichlorovinyl)-L-cysteine, whereas FMO3 catalyzed the sulfoxidation of S-benzyl-L-cysteine, Sallyl-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(trichlorovinyl)-L-cysteine (173). The sulfoxidation of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, N-acetyl-S-(trichlorovinyl)-L-cysteine, and N-acetyl-S(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine is catalyzed by P450 3A but not by FMOs (174–177). The mercapturic acids are cytotoxic in rat renal epithelial cells, and their cytotoxicity is reduced by the β-lyase inhibitor (aminooxy)acetic acid, indicating hydrolysis of the mercapturic acids and β-lyasecatalyzed bioactivation of the formed cysteine S-conjugates (176); (aminooxy)acetic acid failed to reduce the cytotoxicity of the corresponding mercapturic acid sulfoxides, indicating that

they are direct-acting electrophiles. Recent studies show, however, that both FMO and P450 are involved in the sulfoxidation of S-(trichlorovinyl)-L-cysteine (178). S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine and their sulfoxides are cytotoxic in human proximal tubular HK-2 cells but only at relatively high concentrations (179). Other studies showed that P450 3A, but not FMO, catalyzes the sulfoxidation of N-acetyl-S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine and N-acetyl(Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (180). In rats given 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene, N-acetyl-S-[2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl]-L-cysteine sulfoxide, but not N-acetyl-(Z)S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]- L cysteine sulfoxide, is excreted in the urine, and a role for P450 3A in the sulfoxidation of the mercapturic acids was established (181); N-acetyl-S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine sulfoxide lacks a vinylic sulfoxide moiety, and it is not apparent how it might contribute to the observed nephrotoxicity of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1propene. S-(1,2-Dichlorovinyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, S-(trichlorovinyl)-L-cysteine, and S-(trichlorovinyl)-L-cysteine sulfoxide are nephrotoxic in rats and cytotoxic in isolated rat renal proximal and distal tubular cells (178, 182); indeed, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide is a more potent nephrotoxin in vivo than S-(1,2-dichlorovinyl)-L-cysteine. The β-lyase inhibitor (aminooxy)acetic acid blocked the nephrotoxicity of S-(1,2-dichlorovinyl)-L-cysteine and S-(trichlorovinyl)-L-cysteine but not that of the sulfoxides. Further studies showed that the FMO inhibitor methimazole reduces the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine in human proximal tubular cells (183). Moreover, N-acetyl S-(1,2,3,4,4pentachlorobutadienyl)-DL-R-methylcysteine sulfoxide is also nephrotoxic in mice (177). The role of vinylic cysteine S-conjugate-derived sulfoxides in the nephrotoxicity and cytotoxicity of cysteine S-conjugates merits further investigation to establish the role of cysteine S-conjugate S-oxidation by FMO or P450 in the nephrotoxicity of vinylic cysteine S-conjugates. Although the exogenously administered cysteine S-conjugate sulfoxides are nephrotoxic, more data are needed to establish that endogenously formed vinylic cysteine S-conjugate sulfoxides contribute to the nephrotoxicity of cysteine S-conjugates. For example, S-(1,2dichlorovinyl)-DL-R-methylcysteine and S-(2-chloro-1,1,2trifluoroethyl)-Dl-R-methylcysteine are not nephrotoxic (34, 184). Studies on the in vivo and in vitro sulfoxidation of these R-methylcysteine S-conjugates, which cannot undergo β-lyasedependent bioactivation, may be instructive.

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12. Human Health Implications of the Glutathione-Dependent Bioactivation of Halogen-Containing Compounds The elucidation of reaction mechanisms that led to the toxic effects of xenobiotics has provided valuable information about issues of human risk assessment and about the extrapolation of experimental–animal data to human health issues. This is no less true for halogen-containing compounds than for the range of xenobiotics that are converted to toxic RIs. Furthermore, the elucidation of reaction mechanisms has led to the development of therapeutic interventions; the use of N-acetyl-L-cysteine in the clinical management of acetaminophen intoxication affords an example (185). The evolution of information about the bioactivation of dichloromethane in human and mouse tissues provides an example of how information about the glutathione-dependent bioactivation of halogenated chemicals can be applied to human risk assessment. The observed carcinogenicity of dichloromethane in female B6C3F1 mice raised concerns about the risk of human exposure to dichloromethane (186). Further studies showed that dichloromethane is metabolized by P450 to carbon monoxide (187, 188) and by θ-class GSTs to formaldehyde (189, 190). The P450-dependent pathway is a high-affinity, lowcapacity pathway, whereas the GST pathway is a low-affinity, high-capacity pathway (191); hence, the P450-dependent pathway is saturated at high substrate concentrations (i.e., those used in the rodent cancer bioassay, g2000 ppm), and the GSTdependent pathway is the dominant route of metabolism. Moreover, the rate constants for the pulmonary metabolism of dichloromethane are higher in the mouse than in the human (113). These and other data were used develop a model for assessing potential human cancer risks from dichloromethane exposure that should lead to substantial reductions in potential human cancer risk (115). The knowledge about the β-lyase pathway has been exploited to target drugs to the kidney. S-(Guanin-6-yl)-L-cysteine and S-(purin-6-yl)-L-cysteine were developed as β-lyase-activated prodrugs of the immunosuppressive and chemotherapeutic agents 6-thioguanine and 6-mercaptopurine (192, 193). In rats given S-(guanin-6-yl)-L-cysteine, renal 6-thioguanine concentrations were four-fold higher than hepatic 6-thioguanine concentrations. Moreover, probenecid, a renal organic ion transport inhibitor, and (aminooxy)acetic acid, a β-lyase inhibitor, reduced the renal concentrations of 6-mercaptopurine in rats given S-(purin-6-yl)-L-cysteine (194), thereby establishing the role of β-lyase in the bioactivation of these prodrugs. These data provide a good example of applying information about chemical bioactivation to develop targeted drug delivery strategies.

13. Conclusion The role of GST-catalyzed reactions in the bioactivation of halogen-containing compounds is well-established. Glutathione S-conjugate formation is often the first step in a bioactivation pathway and may be the only required step, as with gemdihaloalkanes. In the β-lyase pathway, a series of reactions precede the β-lyase-catalyzed bioactivation step that leads to RI formation. The formation of sulfenate esters requires sulfoxidation of an intermediate to lead to RI formation. With some compounds, such as 1,2- and 1,4-dihaloalkanes, the glutathione S-conjugate may undergo a nonenzymatic reaction to give a RI. The commonality in all of these bioactivation reactions is the initial formation of a glutathione S-conjugate.

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