Chemical Toxicology of Reactive Intermediates Formed by the

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

10.1021/tx700202w CCC: $40.75  2008 American Chemical Society Published on Web 08/16/2007

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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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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.

PerspectiVe


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


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,

PerspectiVe


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-


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.

PerspectiVe

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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.

References (1) Miller, E. C., and Miller, J. A. (1947) The presence and significance of bound aminoazo dyes in the livers of rats fed p-dimethylaminoazobenzene. Cancer Res. 7, 468–480. (2) Wheeler, G. P., and Skipper, H. E. (1957) Studies with mustards. III. In vivo fixation of C14from nitrogen mustard-C14H3 in nucleic acid fractions of animal tissues. Arch. Biochem. Biophys. 72, 465– 475. (3) Miller, J. A. (1970) Carcinogenesis by chemicals: An overviewG. H. A. Clowes memorial lecture. Cancer Res. 30, 559–576. (4) Miller, E. C., andMiller, J. A. (1985) Some historical perspectives on the metabolism of xenobiotic chemicals to reactive electrophiles. In BioactiVation of Foreign Compounds (Anders, M. W., Ed.) pp 3–28, Academic Press, Orlando. (5) Brodie, B. B., Reid, W. D., Cho, A. K., Sipes, G., Krishna, G., and Gillette, J. R. (1971) Possible mechanism of liver necrosis caused by aromatic organic compounds. Proc. Natl. Acad. Sci. U.S.A. 68, 160–164. (6) Anders, M. W., Ed. (1985) BioactiVation of Foreign Compounds, Academic Press, Orlando. (7) Pumford, N. R., and Halmes, N. C. (1997) Protein targets of xenobiotic reactive intermediates. Annu. ReV. Pharmacol. Toxicol. 37, 91–117. (8) Uetrecht, J. (2003) Bioactivation. In Drug Metabolizing Enzymes. Cytochrome P450 and Other Enzymes in Drug DiscoVery and DeVelopment (Fisher, M. B., Lee, J. S., and Obach, R. S., Eds.) pp 87–146, Marcel Dekker, New York. (9) Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A. (2004) Drug-protein adducts: An industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol. 17, 3–16. (10) Kalgutkar, A. S., Gardner, I., Obach, R. S., Shaffer, C. L., Callegari, E., Henne, K. R., Mutlib, A. E., Dalvie, D. K., Lee, J. S., Nakai, Y., O’Donnell, J. P., Boer, J., and Harriman, S. P. (2005) A comprehensive listing of bioactivation pathways of organic functional groups. Curr. Drug Metab. 6, 161–225. (11) Walgren, J. L., Mitchell, M. D., and Thompson, D. C. (2005) Role of metabolism in drug-induced idiosyncratic hepatotoxicity. Crit. ReV. Toxicol. 35, 325–361. (12) Schanne, F. A., Kane, A. B., Young, E. E., and Farber, J. L. (1979) Calcium dependence of toxic cell death: a final common pathway. Science 206, 700–702. (13) Smith, M. T., Thor, H., and Orrenius, S. (1981) Toxic injury to isolated hepatocytes is not dependent on extracellular calcium. Science 213, 1257–1259. (14) Orrenius, S., and Nicotera, P. (1994) The calcium ion and cell death. J. Neural Transm. 43, 1–11. (15) Degterev, A., Huang, Z., Boyce, M., Li, Y., Jagtap, P., Mizushima, N., Cuny, G. D., Mitchison, T. J., Moskowitz, M. A., and Yuan, J. (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 1, 112–119. (16) Orrenius, S., Gogvadze, V., and Zhivotovsky, B. (2007) Mitochondrial oxidative stress: implications for cell death. Annu. ReV. Pharmacol. Toxicol. 47, 143–183. (17) Anders, M. W., andPohl, L. R. (1985) Halogenated alkanes. In BioactiVation of Foreign Compounds (Anders, M. W., Ed.) pp 283– 315, Academic Press, New York. (18) Anders, M. W., Lash, L., Dekant, W., Elfarra, A. A., and Dohn, D. R. (1988) Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. CRC Crit. ReV. Toxicol. 18, 311–341. (19) Anders, M. W. (1991) Metabolism and toxicity of hydrochlorofluorocarbons: Current knowledge and needs for the future. EnViron. Health Perspect. 96, 185–191. (20) Dekant, W., and Vamvakas, S. (1993) Glutathione-dependent bioactivation of xenobiotics. Xenobiotica 23, 873–887. (21) Anders, M. W., Dekant, W., and Vamvakas, S. (1995) Glutathionedependent bioactivation of xenobiotics. In Biothiols (Packer, L., and Cadenas, E., Eds.) pp 135–163, Marcel Dekker, New York. (22) Anders, M. W., and Dekant, W. (1998) Glutathione-dependent bioactivation of haloalkenes. Annu. ReV. Pharmacol. Toxicol. 38, 501–537. (23) Anders, M. W. (2004) Glutathione-dependent bioactivation of haloalkanes and haloalkenes. Drug Metab. ReV. 36, 583–594. (24) Potter, C. L., Gandolfi, A. J., Nagle, R., and Clayton, J. W. (1981) Effects of inhaled chlorotrifluoroethylene and hexafluoropropene on the rat kidney. Toxicol. Appl. Pharmacol. 59, 431–440. (25) Kennedy, G. L., Jr. (1990) Toxicology of fluorine-containing monomers. Crit. ReV. Toxicol. 21, 149–170. (26) Keller, D. A., Kennedy, G. L., Jr., Ross, P. E., Kelly, D. P., and Elliott, G. S. (2000) Toxicity of tetrafluoroethylene and S-(1,1,2,2tetrafluoroethyl)-L-cysteine in rats and mice. Toxicol. Sci. 56, 414– 423.

156 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 (27) Gonsowski, C. T., Laster, M. J., Eger, E. I., II, Ferrell, L. D., and Kerschmann, R. L. (1994) Toxicity of compound A in rats. Effect of a 3-hour administration. Anesthesiology 80, 556–565. (28) Buckley, L. A., Clayton, J. W., Nagle, R. B., and Gandolfi, A. J. (1982) Chlorotrifluoroethylene nephrotoxicity in rats: A subacute study. Fundam. Appl. Toxicol. 2, 181–186. (29) Parker, V. H. (1965) A biochemical study of the toxicity of S-dichlorovinyl-L-cysteine. Food Cosmet. Toxicol. 3, 75–84. (30) Gandolfi, A. J., Nagle, R. B., Soltis, J. J., and Plescia, F. H. (1981) Nephrotoxicity of halogenated vinyl cysteine compounds. Res. Commun. Chem. Pathol. Pharmacol. 33, 249–261. (31) Bonhaus, D. W., and Gandolfi, A. J. (1981) Conjugation and bioactivation of chlorotrifluoroethylene. Life Sci. 29, 2399–2405. (32) Dohn, D. R., and Anders, M. W. (1982) The enzymatic reaction of chlorotrifluoroethylene with glutathione. Biochem. Biophys. Res. Commun. 109, 1339–1345. (33) Dohn, D. R., Quebbemann, A. J., Borch, R. F., and Anders, M. W. (1985) Enzymatic reaction of chlorotrifluoroethene with glutathione: 19 F NMR evidence for stereochemical control of the reaction. Biochemistry 24, 5137–5143. (34) Dohn, D. R., Leininger, J. R., Lash, L. H., Quebbemann, A. J., and Anders, M. W. (1985) Nephrotoxicity of S-(2 chloro-1,1,2-trifluoroethyl)glutathione and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, the glutathione and cysteine conjugates of chlorotrifluoroethene. J. Pharmacol. Exp. Ther. 235, 851–857. (35) Lash, L. H., and Anders, M. W. (1989) Uptake of nephrotoxic S-conjugates by isolated rat renal proximal tubular cells. J. Pharmacol. Exp. Ther. 248, 531–537. (36) Lock, E. A., and Ishmael, J. (1998) The nephrotoxicity and hepatotoxicity of 1,1,2,2-tetrafluoroethyl-L-cysteine in the rat. Arch. Toxicol. 72, 347–354. (37) Commandeur, J. N. M., Oostendorp, R. A. J., Schoofs, P. R., Xu, B., and Vermeulen, N. P. E. (1987) Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Biochem. Pharmacol. 36, 4229–4237. (38) Boogaard, P. J., Commandeur, J. N. M., Mulder, G. J., Vermeulen, N. P. E., and Nagelkerke, J. F. (1989) Toxicity of the cysteine-Sconjugates and mercapturic acids of four structurally related difluoroethylenes in isolated proximal tubular cells from rat kidney. Uptake of the conjugates and activation to toxic metabolites. Biochem. Pharmacol. 38, 3731–3741. (39) Commandeur, J. N. M., Brakenhoff, J. P. G., De Kanter, F. J. J., and Vermeulen, N. P. E. (1988) Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Biochem. Pharmacol. 37, 4495–4504. (40) Dekant, W., Lash, L. H., and Anders, M. W. (1987) Bioactivation mechanism of the cytotoxic and nephrotoxic S-conjugate S-(2-chloro1,1,2-trifluoroethyl)-L-cysteine. Proc. Natl. Acad. Sci. U.S.A. 84, 7443–7447. (41) Banki, K., Elfarra, A. A., Lash, L. H., and Anders, M. W. (1986) Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine to hydrogen sulfide and the role of hydrogen sulfide in S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine-induced mitochondrial toxicity. Biochem. Biophys. Res. Commun. 138, 707–713. (42) Hayden, P. J., Yang, Y., Ward, A. J. I., Dulik, D. M., McCann, D. J., and Stevens, J. L. (1991) Formation of difluorothionoacetyl-protein adducts by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine metabolites: Nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine. Biochemistry 30, 5935–5943. (43) Harris, J. W., Dekant, W., and Anders, M. W. (1992) In vivo detection and characterization of protein adducts resulting from bioactivation of haloethene cysteine S-conjugates by19F NMR: chlorotrifluoroethene and tetrafluoroethene. Chem. Res. Toxicol. 5, 34–41. (44) Hargus, S. J., and Anders, M. W. (1991) Immunochemical detection of covalently modified kidney proteins in S-(1,1,2,2-tetrafluoroethyl)L-cysteine-treated rats. Biochem. Pharmacol. 42, R17–R20. (45) Fisher, M. B., Hayden, P. J., Bruschi, S. A., Dulik, D. M., Yang, Y., Ward, A. J. I., and Stevens, J. L. (1993) Formation, characterization, and immunoreactivity of lysine thioamide adducts from fluorinated nephrotoxic cysteine conjugates in vitro and in vivo. Chem. Res. Toxicol. 6, 223–230. (46) Cooper, A. J. L., and Anders, M. W. (1990) Glutamine transaminase K and cysteine conjugate β-lyase. Ann. N. Y. Acad. Sci. 585, 118– 127. (47) Cooper, A. J. L. (1994) Enzymology of cysteine S-conjugate β-lyases. AdV. Pharmacol. 27, 73–115. (48) Cooper, A. J., and Pinto, J. T. (2006) Cysteine S-conjugate β-lyases. Amino Acids 30, 1–15. (49) Cooper, A., Bruschi, S., and Anders, M. (2002) Toxic, halogenated cysteine Sconjugates and targeting of mitochondrial enzymes of energy metabolism. Biochem. Pharmacol. 64, 553–564. (50) Abraham, D. G., Thomas, R. J., and Cooper, A. J. L. (1995) Glutamine transaminase K is not a major cysteine Sconjugate β-lyase

Anders

(51) (52)

(53)

(54)

(55) (56)

(57) (58)

(59)

(60)

(61)

(62)

(63)

(64) (65)

(66) (67)

(68)

(69) (70) (71)

of rat kidney mitochondria: Evidence that a high-molecular-weight enzyme fulfills this role. Mol. Pharmacol. 48, 855–860. Cooper, A. J., Bruschi, S. A., Iriarte, A., and Martinez-Carrion, M. (2002) Mitochondrial aspartate aminotransferase catalyses cysteine S-conjugate β-lyase reactions. Biochem. J. 368, 253–261. Cooper, A. J., Bruschi, S. A., Conway, M., and Hutson, S. M. (2003) Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate β-lyases, but turnover leads to inactivation. Biochem. Pharmacol. 65, 181–192. Cooper, A. J., Krasnikov, B. F., Okuno, E., and Jeitner, T. M. (2003) L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate β-lyase activity: A contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes? Biochem. J. 376, 169–178. Bruschi, S. A., West, K. A., Crabb, J. W., Gupta, R. S., and Stevens, J. L. (1993) Mitochondrial HSP60 (P1 protein) and a HSP70-like protein (mortalin) are major targets for modification during S-(1,1,2,2tetrafluoroethyl)-L-cysteine-induced nephrotoxicity. J. Biol. Chem. 268, 23157–23161. Abraham, D. G., and Cooper, A. J. L. (1991) Glutamine transaminase K and cysteine S-conjugate β-lyase activity stains. Anal. Biochem. 197, 421–427. Cooper, A. J., Wang, J., Gartner, C. A., and Bruschi, S. A. (2001) Co-purification of mitochondrial HSP70 and mature protein disulfide isomerase with a functional rat kidney high-Mr cysteine S-conjugate β-lyase. Biochem. Pharmacol. 62, 1345–1353. Bruschi, S. A., and Lindsay, J. G. (1994) Mitochondrial stress protein actions during chemically induced renal proximal tubule cell death. Biochem. Cell Biol. 72, 663–667. Bruschi, S. A., Lindsay, J. G., and Crabb, J. W. (1998) Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death. Proc. Natl. Acad. Sci. U.S.A. 95, 13413– 13418. Ho, H. K., Hu, Z. H., Tzung, S. P., Hockenbery, D. M., Fausto, N., Nelson, S. D., and Bruschi, S. A. (2005) BCL-xL overexpression effectively protects against tetrafluoroethylcysteine-induced intramitochondrial damage and cell death. Biochem. Pharmacol. 69, 147– 157. Wakabayashi, N., Dinkova-Kostova, A. T., Holtzclaw, W. D., Kang, M. I., Kobayashi, A., Yamamoto, M., Kensler, T. W., and Talalay, P. (2004) Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers. Proc. Natl. Acad. Sci. U.S.A. 101, 2040– 2045. Nguyen, T., Sherratt, P. J., Nioi, P., Yang, C. S., and Pickett, C. B. (2005) Nrf2 controls constitutive and inducible expression of AREdriven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. J. Biol. Chem. 280, 32485–32492. Ho, H. K., White, C. C., Fernandez, C., Fausto, N., Kavanagh, T. J., Nelson, S. D., and Bruschi, S. A. (2005) Nrf2 activation involves an oxidative-stress independent pathway in tetrafluoroethylcysteineinduced cytotoxicity. Toxicol. Sci. 86, 354–364. Park, L. C. H., Gibson, G. E., Bunik, V., and Cooper, A. J. L. (1999) Inhibition of select mitochondrial enzymes in PC12 cells exposed to S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. Biochem. Pharmacol. 58, 1557– 1565. Ovadi, J., and Srere, P. A. (2000) Macromolecular compartmentation and channeling. Int. ReV. Cytol. 192, 255–280. Islam, M. M., Wallin, R., Wynn, R. M., Conway, M., Fujii, H., Mobley, J. A., Chuang, D. T., and Hutson, S. M. (2007) A novel branched-chain amino acid metabolon: protein-protein interactions in a supramolecular complex. J. Biol. Chem. 282, 11893–11903. Strum, D. P., Johnson, B. H., and Eger, E. I., II (1987) Stability of sevoflurane in soda lime. Anesthesiology 67, 779–781. Morio, M., Fujii, K., Satoh, N., Imai, M., Kawakami, T., Negishi, A., Kumagai, Y., and Kawai, T. (1992) Reaction of sevoflurane and its degradation products with soda lime. Toxicity of the byproducts. Anesthesiology 77, 1155–1164. Mazze, R. I., Callan, C. M., Galvez, S. T., Delgado-Herrera, L., and Mayer, D. B. (2000) The effects of sevoflurane on serum creatinine and blood urea nitrogen concentrations: a retrospective, twenty-twocenter, comparative evaluation of renal function in adult surgical patients. Anesth. Analg. (Baltimore) 90, 683–688. Anders, M. W. (2005) Formation and toxicity of anesthetic degradation products. Annu. ReV. Pharmacol. Toxicol. 45, 147–176. Lash, L. H., Nelson, R. M., Van Dyke, R. A., and Anders, M. W. (1990) Purification and characterization of human kidney cytosolic conjugate β-lyase activity. Drug Metab. Dispos. 18, 50–54. Iyer, R. A., and Anders, M. W. (1996) Cysteine conjugate β-lyasedependent biotransformation of the cysteine Sconjugates of the sevoflurane degradation product Compound A in human, nonhuman primate, and rat renal tissue. Fundam. Appl. Toxicol. 30 (1), 315.

PerspectiVe (72) Jin, L., Davis, M. R., Kharasch, E. D., Doss, G. A., and Baillie, T. A. (1996) Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevoflurane. Chem. Res. Toxicol. 9, 555–561. (73) Iyer, R. A., and Anders, M. W. (1997) Cysteine conjugate β-lyasedependent biotransformation of the cysteine Sconjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A). Chem. Res. Toxicol. 10, 811–819. (74) Iyer, R. A., Frink, E. J., Jr., Ebert, T. J., and Anders, M. W. (1998) Cysteine conjugate β-lyase-dependent metabolism of Compound A (2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound A. Anesthesiology 88, 611–618. (75) Kharasch, E. D., and Jubert, C. (1999) Compound A uptake and metabolism to mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid during low-flow sevoflurane anesthesia: biomarkers for exposure, risk assessment, and interspecies comparison. Anesthesiology 91, 1267–1278. (76) Martin, J. L., Laster, M. J., Kandel, L., Kerschmann, R. L., Reed, G. F., and Eger, E. I., II (1996) Metabolism of Compound A by renal cysteine-S-conjugate β-lyase is not the mechanism of compound A-induced renal injury in the rat. Anesth. Analg. (Baltimore) 82, 770774. . (77) Kharasch, E. D., Hoffman, G. M., Thorning, D., Hankins, D. C., and Kilty, C. G. (1998) Role of the renal cysteine conjugate β-lyase pathway in inhaled compound A nephrotoxicity in rats. Anesthesiology 88, 1624–1633. (78) Kharasch, E. D., Schroeder, J. L., Bammler, T., Beyer, R., and Srinouanprachanh, S. (2006) Gene expression profiling of nephrotoxicity from the sevoflurane degradation product fluoromethyl-2,2difluoro-1-(trifluoromethyl)vinyl ether (“compound A”) in rats. Toxicol. Sci. 90, 419–431. (79) McKinney, L. L., Picken, J. C., Jr., Weakley, F. B., Eldridge, A. C., Campbell, R. E., Cowan, J. C., and Biester, H. E. (1959) Possible toxic factor of trichloroethylene-extracted soybean oil meal. J. Am. Chem. Soc. 81, 909–915. (80) McKinney, L. L., Weakley, F. B., Eldridge, A. C., Campbell, R. E., Cowan, J. C., Picken, J. C., Jr., and Biester, H. E. (1957) S(Dichlorovinyl)-L-cysteine: An agent causing fatal aplastic anemia in calves. J. Am. Chem. Soc. 79, 3932–3933. (81) Lock, E. A., Sani, Y., Moore, R. B., Finkelstein, M. B., Anders, M. W., and Seawright, A. A. (1996) Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves. Arch. Toxicol. 70, 607–619. (82) Terracini, B., and Parker, V. H. (1965) A pathological study on the toxicity of S-dichlorovinyl-L-cysteine. Food Cosmet. Toxicol. 3, 67– 74. (83) Anderson, P. M., and Schultze, M. O. (1965) Cleavage of S-(1,2dichlorovinyl)-L-cysteine by an enzyme of bovine origin. Arch. Biochem. Biophys. 111, 593–602. (84) Bhattacharya, R. K., and Schultze, M. O. (1972) Properties of DNA treated with S-(1,2-dichlorovinyl)-L-cysteine and a lyase. Arch. Biochem. Biophys. 153, 105–115. (85) Bhattacharya, R. K., and Schultze, M. O. (1974) Action of a metabolite of S-(1,2-dichlorovinyl)-L-cysteine on poly-L-lysine, polyL-arginine, and their soluble DNA complexes. Arch. Biochem. Biophys. 161, 40–45. (86) Derr, R. F., and Schultze, M. O. (1963) The metabolism of35S-(1,2dichlorovinyl)-L-cysteine in the rat. Biochem. Pharmacol. 12, 465– 474. (87) Derr, R. F., Schultze, M. O., Mizuno, N. S., Joel, D. D., and Sautter, J. H. (1963) The metabolism of35S-(1,2-dichlorovinyl)-L-cysteine in the calf. Biochem. Pharmacol. 12, 475–488. (88) Guthrie, J. P. (1979) The enol content of simple carbonyl compounds: An approach based upon pKa estimation. Can. J. Chem. 57, 1177– 1185. (89) Su, C.-C., Lin, C.-K., Wu, C.-C., and Lien, M.-H. (1999) Ab initio study on the keto-enol tautomerism of the R-substituted acetaldehydes XH2CCH:O (X ) H, BH2, CH3, NH2, OH, F, CN, NC, and Cl): Comparison with the tautomerism in R-substituted acetaldimines and acetyl derivatives. J. Phys. Chem. A 103, 3289–3293. (90) Zhang, T.-L., Wang, L., Hashmi, M., Anders, M. W., Thorpe, C., and Ridge, D. P. (1995) Fourier-transform ion cyclotron resonance mass spectrometric evidence for the formation of R-chloroethenethiolates and thioketenes from chloroalkene-derived, cytotoxic 4-thiaalkanoates. Chem. Res. Toxicol. 8, 907–910. (91) Dekant, W., Berthold, K., Vamvakas, S., Henschler, D., and Anders, M. W. (1988) Thioacylating intermediates as metabolites of S-(1,2dichlorovinyl)-L-cysteine and S-(1,2,2-trichlorovinyl)-L-cysteine formed by cysteine conjugate β-lyase. Chem. Res. Toxicol. 1, 175–178. (92) Dekant, W., Berthold, K., Vamvakas, S., and Henschler, D. (1988) Thioacylating agents as ultimate intermediates in the β-lyase catalyzed


(93)

(94)

(95) (96)

(97)

(98) (99)

(100) (101) (102) (103) (104)

(105)

(106) (107) (108)

(109)

(110)

(111)

(112)

(113)

metabolism of S-(pentachlorobutadienyl)-L-cysteine. Chem.-Biol. Interact. 67, 139–148. Vamvakas, S., Dekant, W., Berthold, K., Schmidt, S., Wild, D., and Henschler, D. (1987) Enzymatic transformation of mercapturic acids derived from halogenated alkenes to reactive and mutagenic intermediates. Biochem. Pharmacol. 36, 2741–2748. Vamvakas, S., Müller, D. A., Dekant, W., and Henschler, D. (1988) DNA-binding of sulfur-containing metabolites from35S-(pentachlorobutadienyl)-L-cysteine in bacteria and isolated renal tubular cells. Drug Metab. Drug Interact. 6, 349–358. Schrenk, D., and Dekant, W. (1989) Covalent binding of hexachlorobutadiene metabolites to renal and hepatic mitochondrial DNA. Carcinogenesis 10, 1139–1141. Dekant, W., Urban, G., Görsmann, C., and Anders, M. W. (1991) Thioketene formation from R-haloalkenyl 2-nitrophenyl disulfides: Models for biological reactive intermediates of cytotoxic S-conjugates. J. Am. Chem. Soc. 113, 5120–5122. Völkel, W., and Dekant, W. (1998) Chlorothioketene, the ultimate reactive intermediate formed by cysteine conjugate β-lyase-mediated cleavage of the trichloroethene metabolite S-(1,2-dichlorovinyl)-Lcysteine, forms cytosine adducts in organic solvents, but not in aqueous solution. Chem. Res. Toxicol. 11, 1082–1088. Hayden, P. J., and Stevens, J. L. (1990) Cysteine conjugate toxicity, metabolism, and binding to macromolecules in isolated rat kidney mitochondria. Mol. Pharmacol. 37, 468–476. Chen, Y., Cai, J., Anders, M. W., Stevens, J. L., and Jones, D. P. (2001) Role of mitochondrial dysfunction in S-(1,2-dichlorovinyl)L-cysteine-induced apoptosis. Toxicol. Appl. Pharmacol. 170, 172– 180. Shaik, Z. P., Fifer, E. K., and Nowak, G. (2007) Protein kinase B/Akt modulates nephrotoxicant-induced necrosis in renal cells. Am. J. Physiol. 292, F292–F303. Sharp, J. H., Trudell, J. R., and Cohen, E. N. (1979) Volatile metabolites and decomposition products of halothane in man. Anesthesiology 50, 2–8. Cohen, E. N., Trudell, J. R., Edmunds, H. N., and Watson, E. (1975) Urinary metabolites of halothane in man. Anesthesiology 43, 392– 401. Wark, H., Earl, J., Chau, D. D., Overton, J., and Cheung, H. T. A. (1990) A urinary cysteine-halothane metabolite: validation and measurement in children. Br. J. Anaesth. 64, 469–473. Finkelstein, M. B., Dekant, W., Kende, A. S., and Anders, M. W. (1995) R-Thiolactones as novel intermediates in the cysteine conjugate β-lyase-catalyzed bioactivation of bromine-containing cysteine S-conjugates. J. Am. Chem. Soc. 117, 9590–9591. Finkelstein, M. B., Dekant, W., and Anders, M. W. (1996) Cysteine conjugate β-lyase-catalyzed bioactivation of bromine-containing cysteine S-conjugates: Stoichiometry and formation of 2,2-difluoro3-halothiiranes. Chem. Res. Toxicol. 9, 227–231. Huisgen, R. (1989) From thiiranes to thiocarbonyl S-sulfides: recent results. Phosphorus, Sulfur, Silica 43, 63–94. Middleton, W. J., Howard, E. G., and Sharkey, W. H. (1965) Fluorothiocarbonyl compounds. I. Preparation of thio ketones, thioacyl halides, and thio esters. J. Org. Chem. 30, 1375–1384. Commandeur, J. N. M., King, L. J., Koymans, L., and Vermeulen, N. P. E. (1996) Bioactivation of S-(2,2-dihalo-1,1-difluoroethyl)-Lcysteines and S-(trihalovinyl)-L-cysteines by cysteine S-conjugate β-lyase: Indications for formation of both thionoacylating species and thiiranes as reactive intermediates. Chem. Res. Toxicol. 9, 1092– 1102. Shim, J.-Y., and Richard, A. M. (1997) Theoretical evaluation of two plausible routes for bioactivation of S-(1,1difluoro-2,2-dihaloethyl)-L-cysteine conjugates: Thiirane vs thionoacyl fluoride pathway. Chem. Res. Toxicol. 10, 103–110. Burek, J. D., Nitschke, K. D., Bell, T. J., Wackerle, D. L., Childs, R. C., Beyer, J. E., Dittenber, D. A., Rampy, L. W., and McKenna, M. J. (1984) Methylene chloride: A two-year inhalation toxicity and oncogenicity study in rats and hamsters. Fundam. Appl. Toxicol. 4, 30–47. Nitschke, K. D., Burek, J. D., Bell, T. J., Kociba, R. J., Rampy, L. W., and McKenna, M. J. (1988) Methylene chloride: A 2-year inhalation toxicity and oncogenicity study in rats. Fundam. Appl. Toxicol. 11, 48–59. Kari, F., Foley, J. F., Seilkop, S. K., Maronpot, R. R., and Anderson, M. W. (1993) Effect of varying exposure regimens on methylene chloride-induced lung and liver tumors in female B6C3F1 mice. Carcinogenesis 14, 819–826. Reitz, R. H., Mendrala, A. L., and Guengerich, F. P. (1989) In vitro metabolism of methylene chloride in human and animal tissues: Use in physiologically based pharmacokinetic models. Toxicol. Appl. Pharmacol. 97, 230–246.

158 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 (114) Sweeney, L. M., Kirman, C. R., Morgott, D. A., and Gargas, M. L. (2004) Estimation of interindividual variation in oxidative metabolism of dichloromethane in human volunteers. Toxicol. Lett. 154, 201– 216. (115) Starr, T. B., Matanoski, G., Anders, M. W., and Andersen, M. E. (2006) Workshop overview: Reassessment of the cancer risk of dichloromethane in humans. Toxicol. Sci. 91, 20–28. (116) Ahmed, A. E., and Anders, M. W. (1976) Metabolism of dihalomethanes to formaldehyde and inorganic halide. I. In vitro studies. Drug Metab. Dispos. 4, 357–361. (117) Ahmed, A. E., and Anders, M. W. (1978) Metabolism of dihalomethanes to formaldehyde and inorganic halide. II. Studies on the mechanism of the reaction. Biochem. Pharmacol. 27, 2021–2025. (118) Meyer, D. J., Coles, B., Pemble, S. E., Gilmore, K. S., Fraser, G. M., and Ketterer, B. (1991) Theta, a new class of glutathione transferases purified from rat and man. Biochem. J. 274, 409–414. (119) Thier, R., Taylor, J. B., Pemble, S. E., Humphreys, W. G., Persmark, M., Ketterer, B., and Guengerich, F. P. (1993) Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes. Proc. Natl. Acad. Sci. U.S.A. 90, 8576–8580. (120) Mainwaring, G. W., Nash, J., Davidson, M., and Green, T. (1996) Isolation of a mouse theta glutathione S-transferase active with methylene chloride. Biochem. J. 314, 445–448. (121) Thier, R., Delbanco, E. H., Wiebel, F. A., Hallier, E., and Bolt, H. M. (1998) Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using35S-glutathione. Arch. Toxicol. 72, 811–815. (122) Hallier, E., Langhof, T., Dannappel, D., Leutbecher, M., Schroder, K., Goergens, H. W., Muller, A., and Bolt, H. M. (1993) Polymorphism of glutathione conjugation of methyl bromide, ethylene oxide and dichloromethane in human blood: Influence on the induction of sister chromatid exchanges (SCE) in lymphocytes. Arch. Toxicol. 67, 173–178. (123) Mainwaring, G. W., Williams, S. M., Foster, J. R., Tugwood, J., and Green, T. (1996) The distribution of theta-class glutathione S-transferases in the liver and lung of mouse, rat and human. Biochem. J. 318, 297–303. (124) Goldmacher, V. S., and Thilly, W. G. (1983) Formaldehyde is mutagenic for cultured human cells. Mutat. Res. 116, 417–422. (125) Wheeler, J. B., Stourman, N. V., Thier, R., Dommermuth, A., Vuilleumier, S., Rose, J. A., Armstrong, R. N., and Guengerich, F. P. (2001) Conjugation of haloalkanes by bacterial and mammalian glutathione transferases: Mono- and dihalomethanes. Chem. Res. Toxicol. 14, 1118–1127. (126) Böhme, H., Fischer, H., and Frank, R. (1949) Darstellung und Eigenschaften der R-halogenierten Thioäther. Justus Liebig’s Ann. Chem. 563, 54–72. (127) Marsch, G. A., Mundkowski, R. G., Morris, B. J., Manier, M. L., Hartman, M. K., and Guengerich, F. P. (2001) Characterization of nucleoside and DNA adducts formed by S-(1-acetoxymethyl)glutathione and implications for dihalomethane-glutathione conjugates. Chem. Res. Toxicol. 14, 600–608. (128) Marsch, G. A., Botta, S., Martin, M. V., McCormick, W. A., and Guengerich, F. P. (2004) Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl)glutathione and by glutathione S-transferase-mediated activation of dihalomethanes. Chem. Res. Toxicol. 17, 45–54. (129) Kluwe, W. M., McNish, R., and Hook, J. B. (1981) Acute nephrotoxicities and hepatotoxicities of 1,2-dibromo-3-chloropropane and 1,2-dibromoethane in male and female F344 rats. Toxicol. Lett. 8, 317–321. (130) Wong, L. C., Winston, J. M., Hong, C. B., and Plotnick, H. (1982) Carcinogenicity and toxicity of 1,2-dibromoethane in the rat. Toxicol. Appl. Pharmacol. 63, 155–165. (131) Rannug, U. (1980) Genotoxic effects of 1,2-dibromoethane and 1,2dichloroethane. Mutat. Res. 76, 269–295. (132) Morgan, D. L., Bucher, J. R., Elwell, M. R., Lilja, H. S., and Murthy, A. S. (1990) Comparative toxicity of ethylene dichloride in F344/N, Sprague-Dawley and Osborne-Mendel rats. Food Chem. Toxicol. 28, 839–845. (133) Daniel, F. B., Robinson, M., Olson, G. R., York, R. G., and Condie, L. W. (1994) Ten and ninety-day toxicity studies of 1,2-dichloroethane in Sprague-Dawley rats. Drug Chem. Toxicol. 17, 463–477. (134) Whorton, M. D., and Foliart, D. E. (1983) Mutagenicity, carcinogenicity and reproductive effects of dibromochloropropane (DBCP). Mutat. Res. 123, 13–30. (135) Teitelbaum, D. T. (1999) The toxicology of 1,2-dibromo-3-chloropropane (DBCP): A brief review. Int. J. Occup. EnViron. Health 5, 122–126. (136) Guengerich, F. P. (2003) Activation of dihaloalkanes by thioldependent mechanisms. J. Biochem. Mol. Biol. 36, 20–27.

Anders (137) Shih, T.-W., and Hill, D. L. (1981) Metabolic activation of 1,2dibromoethane by glutathione transferase and by microsomal mixed function oxidase: Further evidence for formation of two reactive metabolites. Res. Commun. Chem. Pathol. Pharmacol. 33, 449–461. (138) Rannug, U., Sundvall, A., and Ramel, C. (1978) The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. I. Activation through conjugation with glutathione in vitro. Chem.-Biol. Interact. 20, 1–16. (139) van Bladeren, P. J., Breimer, D. D., Rotteveel-Smijs, G. M. T., de Jong, R. A. W., Buijs, W., van der Gen, A., and Mohn, G. R. (1980) The role of glutathione conjugation in the mutagenicity of 1,2dibromoethane. Biochem. Pharmacol. 29, 2975–2982. (140) van Bladeren, P. J., Breimer, D. D., Rotteveel-Smijs, G. M. T., and Mohn, G. R. (1980) Mutagenic activation of dibromomethane and diiodomethane by mammalian microsomes and glutathione Stransferases. Mutat. Res. 74, 341–346. (141) Elfarra, A. A., Baggs, R. B., and Anders, M. W. (1985) Structurenephrotoxicity relationships of S-(2-chloroethyl)-DL-cysteine and analogs: Role for an episulfonium ion. J. Pharmacol. Exp. Ther. 233, 512–516. (142) Webb, W. W., Elfarra, A. A., Webster, K. D., Thom, R. E., and Anders, M. W. (1987) Role for an episulfonium ion in S-(2chloroethyl)-DL-cysteine-induced cytotoxicity and its reaction with glutathione. Biochemistry 26, 3017–3023. (143) van Bladeren, P. J., van der Gen, A., Breimer, D. D., and Mohn, G. R. (1979) Stereoselective activation of vicinal dihalogen compounds to mutagens by glutathione conjugation. Biochem. Pharmacol. 28, 2521–2524. (144) van Bladeren, P. J., Breimer, D. D., van Huijgevoort, J. A. T. C. M., Vermeulen, N. P. E., and van der Gen, A. (1981) The metabolic formation of N-acetyl-S-2-hydroxyethyl-L-cysteine from tetradeutero1,2-dibromoethane. Relative importance of oxidation and glutathione conjugation in vivo. Biochem. Pharmacol. 30, 2499–2502. (145) Dohn, D. R., and Casida, J. E. (1987) Thiiranium ion intermediates in the formation and reactions of S-(2-haloethyl)-L-cysteines. Bioorg. Chem. 15, 115–124. (146) Peterson, L. A., Harris, T. M., and Guengerich, F. P. (1988) Evidence for an episulfonium ion intermediate in the formation of S-[2-(N7guanyl)ethyl]glutathione in DNA. J. Am. Chem. Soc. 110, 3284– 3291. (147) Mangold, J. B., and Abdel-Monem, M. M. (1983) Stereochemical aspects of conjugation reactions catalyzed by rat liver glutathione S-transferase isozymes. J. Med. Chem. 26, 66–71. (148) Schasteen, C. S., and Reed, D. J. (1983) The hydrolysis and alkylation activities of S-(2-haloethyl)-L-cysteine analogs-evidence for extended half-life. Toxicol. Appl. Pharmacol. 70, 423–432. (149) Ozawa, N., and Guengerich, F. P. (1983) Evidence for formation of an S-[2-(N7-guanyl)ethyl]glutathione adduct in glutathione-mediated binding of the carcinogen 1,2-dibromoethane to DNA. Proc. Natl. Acad. Sci. U.S.A. 80, 5266–5270. (150) Koga, N., Inskeep, P. B., Harris, T. M., and Guengerich, F. P. (1986) S-[2-(N7-Guanyl)ethyl]glutathione, the major DNA adduct formed from 1,2-dibromoethane. Biochemistry 25, 2192–2198. (151) Inskeep, P. B., Koga, N., Cmarik, J. L., and Guengerich, F. P. (1986) Covalent binding of 1,2-dihloalknes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione. Cancer Res. 46, 2839–2844. (152) Foureman, G. L., and Reed, D. J. (1987) Formation of S-[2-(N7guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane. Biochemistry 26, 2028–2033. (153) Kim, D. H., and Guengerich, F. P. (1990) Formation of the DNA adduct S-[2-(N7-guanyl)ethyl]glutathione from ethylene dibromide: Effects of modulation of glutathione and glutathione S-transferase levels and lack of a role for sulfation. Carcinogenesis 11, 419–424. (154) Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase. J. Biol. Chem. 277, 37920– 37928. (155) Liu, L., Hachey, D. L., Valadez, G., Williams, K. M., Guengerich, F. P., Loktionova, N. A., Kanugula, S., and Pegg, A. E. (2004) Characterization of a mutagenic DNA adduct formed from 1,2dibromoethane by O6-alkylguanine-DNA alkyltransferase. J. Biol. Chem. 279, 4250–4259. (156) Liu, L., Watanabe, K., Fang, Q., Williams, K. M., Guengerich, F. P., and Pegg, A. E. (2007) Effect of alterations of key active site residues in O6-alkylguanine-DNA Alkyltransferase on its ability to modulate the genotoxicity of 1,2-dibromoethane. Chem. Res. Toxicol. 20, 155– 163. (157) Pearson, P. G., Søderlund, E. J., Dybing, E., and Nelson, S. D. (1990) Metabolic activation of 1,2-dibromo-3-chloropropane: Evidence for the formation of reactive episulfonium ion intermediates. Biochemistry 29, 4971–4981.

PerspectiVe (158) Omichinski, J. G., Brunborg, G., Holme, J. A., Søderlund, E. J., Nelson, S. D., and Dybing, E. (1988) The role of oxidative and conjugative pathways in the activation of 1,2-dibromo-3-chloropropane to DNA-damaging products in rat testicular cells. Mol. Pharmacol. 34, 74–79. (159) Humphreys, W. G., Kim, D. H., and Guengerich, F. P. (1991) Isolation and characterization of N7-guanyl adducts derived from 1,2dibromo-3-chloropropane. Chem. Res. Toxicol. 4, 445–453. (160) Dunn, C. D. (1974) The chemical and biological properties of busulphan (“Myleran”). Exp. Hematol. 2, 101–117. (161) Roberts, J. J., and Warwick, G. P. (1961) The mode of action of alkylating agents—III. The formation of 3-hydroxytetrahydrothiophene1:1-dioxide from 1:4-dimethanesulphonyloxybutane (Myleran), S-βalanyltetrahydrothiophenium mesylate, tetrahydrothiophene and tetrahydrothiophene-1:1-dioxide in the rat, rabbit and mouse. Biochem. Pharmacol. 6, 217–227. (162) Buijs, W., van der Gen, A., Mohn, G. R., and Breimer, D. D. (1984) The direct mutagenic activity of R,ω-dihalogenoalkanes in Salmonella typhimurium. Strong correlation between chemical properties and mutagenic activity. Mutat. Res. 141, 11–14. (163) Marchand, D. H., and Abdel-Monem, M. M. (1985) Glutathione S-transferases catalyzed conjugation of 1,4-disubstituted butanes with glutathione in vitro. Biochem. Biophys. Res. Commun. 128, 360– 367. (164) Marchand, D. H., Remmel, R. P., and Abdel-Monem, M. M. (1988) Biliary excretion of a glutathione conjugate of busulfan and 1,4diiodobutane in the rat. Drug Metab. Dispos. 16, 85–92. (165) Onkenhout, W., van Loon, W. M., Buijs, W., van der Gen, A., and Vermeulen, N. P. (1986) Biotransformation and quantitative determination of sulfur-containing metabolites of 1,4-dibromobutane in the rat. Drug Metab. Dispos. 14, 608–612. (166) Park, S. B., Osterloh, J. D., Vamvakas, S., Hashmi, M., Anders, M. W., and Cashman, J. R. (1992) Flavin-containing monooxygenasedependent stereoselective S-oxygenation and cytotoxicity of cysteine S-conjugates and mercapturates. Chem. Res. Toxicol. 5, 193–201. (167) Kahn, S. D., and Hehre, W. J. (1986) Modeling chemical reactivity. 3. Stereochemistry of Michael additions of vinyl sulfoxides. J. Am. Chem. Soc. 108, 7399–7400. (168) Sausen, P. J., and Elfarra, A. A. (1991) Reactivity of cysteine S-conjugate sulfoxides: Formation of S-[1-chloro-2-(S-glutathionyl)-vinyl]-L-cysteine sulfoxide by the reaction of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide with glutathione. Chem. Res. Toxicol. 4, 655–660. (169) Rosner, E., Müller, M., and Dekant, W. (1998) Stereo- and regioselective conjugation of S-halovinyl mercapturic acid sulfoxides by glutathione S-transferases. Chem. Res. Toxicol. 11, 12–18. (170) Rosner, E., and Dekant, W. (1999) Stereoselective formation of glutathione S-conjugates from halovinylmercapturate sulphoxides. Xenobiotica 29, 327–340. (171) Sausen, P. J., and Elfarra, A. A. (1990) Cysteine conjugate S-oxidase: Characterization of a novel enzymatic activity in rat hepatic and renal microsomes. J. Biol. Chem. 265, 6139–6145. (172) Sausen, P. J., Duescher, R. J., and Elfarra, A. A. (1993) Further characterization and purification of the flavin-dependent S-benzylL-cysteine S-oxidase activities of rat liver and kidney microsomes. Mol. Pharmacol. 43, 388–396. (173) Ripp, S. L., Overby, L. H., Philpot, R. M., and Elfarra, A. A. (1997) Oxidation of cysteine S-conjugates by rabbit liver microsomes and cDNA-expressed flavin-containing mono-oxygenases: Studies with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine. Mol. Pharmacol. 51, 507– 515. (174) Werner, M., Birner, G., and Dekant, W. (1995) The role of cytochrome P450A1/2 in the sex-specific sulfoxidation of the hexachlorobutadiene metabolite, N-acetyl-S-(pentachlorobutadienyl)L-cysteine in rats. Drug Metab. Dispos. 23, 861–868. (175) Werner, M., Guo, Z., Birner, G., Dekant, W., and Guengerich, F. P. (1995) The sulfoxidation of the hexachlorobutadiene metabolite N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine is catalyzed by human cytochrome P450 3A enzymes. Chem. Res. Toxicol. 8, 917–923. (176) Werner, M., Birner, G., and Dekant, W. (1996) Sulfoxidation of mercapturic acids derived from tri-and tetrachloroethene by cyto-


(177)

(178)

(179)

(180)

(181)

(182)

(183)

(184) (185) (186)

(187) (188) (189) (190)

(191)

(192) (193) (194)

chromes P450 3A: A bioactivation reaction in addition to deacetylation and cysteine conjugate β-lyase mediated cleavage. Chem. Res. Toxicol. 9, 41–49. Birner, G., Werner, M., Rosner, E., Mehler, C., and Dekant, W. (1998) Biotransformation, excretion, and nephrotoxicity of the hexachlorobutadiene metabolite (E)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide. Chem. Res. Toxicol. 11, 750–757. Elfarra, A. A., and Krause, R. J. (2007) S-(1,2,2-Trichlorovinyl)-Lcysteine sulfoxide, a reactive metabolite of S-(1,2,2-trichlorovinyl)L-cysteine formed in rat liver and kidney microsomes, is a potent nephrotoxicant. J. Pharmacol. Exp. Ther. 321, 1095–1101. Altuntas, T. G., Zager, R. A., and Kharasch, E. D. (2003) Cytotoxicity of S-conjugates of the sevoflurane degradation product fluoromethyl2,2-difluoro-1-(trifluoromethyl) vinyl ether (Compound A) in a human proximal tubular cell line. Toxicol. Appl. Pharmacol. 193, 55–65. Altuntas, T. G., Park, S. B., and Kharasch, E. D. (2004) Sulfoxidation of cysteine and mercapturic acid conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A). Chem. Res. Toxicol. 17, 435–445. Sheffels, P., Schroeder, J. L., Altuntas, T. G., Liggitt, H. D., and Kharasch, E. D. (2004) Role of cytochrome P4503A in cysteine S-conjugates sulfoxidation and the nephrotoxicity of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)viny l ether (compound A) in rats. Chem. Res. Toxicol. 17, 1177–1189. Lash, L. H., Sausen, P. J., Duescher, R. J., Cooley, A. J., and Elfarra, A. A. (1994) Roles of cysteine conjugate β-lyase and S-oxidase in nephrotoxicity: Studies with S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine sulfoxide. J. Pharmacol. Exp. Ther. 269, 374–383. Lash, L. H., Putt, D. A., Hueni, S. E., Krause, R. J., and Elfarra, A. A. (2003) Roles of necrosis, apoptosis, and mitochondrial dysfunction in S-(1,2-dichlorovinyl)-L-cysteine sulfoxide-induced cytotoxicity in primary cultures of human renal proximal tubular cells. J. Pharmacol. Exp. Ther. 305, 1163–1172. Elfarra, A. A., and Anders, M. W. (1984) Renal processing of glutathione conjugates: Role in nephrotoxicity. Biochem. Pharmacol. 33, 3729–3732. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973) Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211–217. National Toxicology Program (1986) Toxicology and Carcinogenesis Studies of Dichloromethane (Methylene Chloride) (CAS No. 75-092) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). U.S. Department of Health and Human Services, Washington, DC. Kubic, V. L., and Anders, M. W. (1975) Metabolism of dihalomethanes to carbon monoxide. II. In vitro studies. Drug Metab. Dispos. 3, 104–112. Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991) Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168–179. Ahmed, A. E., and Anders, M. W. (1977) Studies on the bioorganic mechanism of the metabolism of dihalomethanes to formaldehyde and inorganic halide. Toxicol. Appl. Pharmacol. 41, 160. Blocki, F. A., Logan, M. S., Baoli, C., and Wackett, L. P. (1994) Reaction of rat liver glutathione S-transferases and bacterial dichloromethane dehalogenase with dihalomethanes. J. Biol. Chem. 269, 8826–8830. Gargas, M. L., Clewell, H. J., III, and Andersen, M. E. (1986) Metabolism of inhaled dihalomethanes in vivo: Differentiation of kinetic constants for two independent pathways. Toxicol. Appl. Pharmacol. 82, 211–223. Elfarra, A. A., and Hwang, I. Y. (1993) Targeting of 6-mercaptopurine to the kidneys. Metabolism and kidney-selectivity of S-(6-purinyl)L-cysteine analogs in rats. Drug Metab. Dispos. 21, 841–845. Elfarra, A. A., Duescher, R. J., Hwang, I. Y., Sicuri, A. R., and Nelson, J. A. (1995) Targeting 6-thioguanine to the kidney with S-(guanin-6-yl)-L-cysteine. J. Pharmacol. Exp. Ther. 274, 1298–1304. Hwang, I. Y., and Elfarra, A. A. (1991) Kidney-selective prodrugs of 6-mercaptopurine: Biochemical basis of the kidney selectivity of S-(6-purinyl)-L-cysteine and metabolism of new analogues in rats. J. Pharmacol. Exp. Ther. 258, 171–177.

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