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Sulfoxidation of Cysteine and Mercapturic Acid Conjugates of the Sevoflurane Degradation Product Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl Et...
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Chem. Res. Toxicol. 2004, 17, 435-445

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Sulfoxidation of Cysteine and Mercapturic Acid Conjugates of the Sevoflurane Degradation Product Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl Ether (Compound A) T. Gul Altuntas,†,‡ Sang B. Park,‡ and Evan D. Kharasch*,‡,§ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey, and Departments of Anesthesiology and Medicinal Chemistry, University of Washington, Seattle, Washington Received December 6, 2003

The volatile anesthetic sevoflurane is degraded in anesthesia machines to the haloalkene fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE), which can cause renal and hepatic toxicity in rats. FDVE is metabolized to S-[1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl]-L-cysteine (DFEC) and (E) and (Z)-S-[1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl]-L-cysteine [(E,Z)-FFVC], which are N-acetylated to N-Ac-DFEC and (E,Z)-N-AcFFVC S-conjugates. Some haloalkene S-conjugates undergo sulfoxidation. This investigation tested the hypothesis that FDVE S-conjugates can also undergo sulfoxidation, by evaluating sulfoxide formation by human and rat liver and kidney microsomes and expressed P450s and flavin monooxygenases. Rat, and at lower rates human, liver microsomes oxidized (Z)-N-AcFFVC and N-Ac-DFEC to the corresponding sulfoxides. Much lower rates of (Z)-N-Ac-FFVC, but not N-Ac-DFEC, sulfoxidation occurred with rat and human kidney microsomes. In human liver microsomes, the P450 inhibitor 1-aminobenzotriazole completely inhibited S-oxidation, while heating to inactivate FMO decreased (Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation only 0 and 30%, respectively. Of the various cytochrome P450s examined, P450s 3A4 and 3A5 had the highest S-oxidase activity toward (Z)-N-Ac-FFVC; P450 3A4 was the predominant enzyme forming N-Ac-DFEC-SO. The P450 3A inhibitors troleandomycin and ketoconazole inhibited >95% of (Z)-N-Ac-FFVC sulfoxidation by P450 3A4 and 3A5 and 40-100% of (Z)-N-Ac-FFVC sulfoxidation by human liver microsomes and 15-85% of N-Ac-DFEC sulfoxidation by human liver microsomes. Sulfoxidation of DFEC was also examined in human liver microsomes. Substantial amounts of sulfoxide were observed, even in the absence of NADPH or protein, while enzymatic formation was comparatively minimal. These results show that FDVE S-conjugates undergo P450-catalyzed and nonenzymatic sulfoxidation and that enzymatic sulfoxidation of (Z)-N-Ac-FFVC and N-Ac-DFEC is catalyzed predominantly by P450 3A. The extent of FDVE sulfoxidation in vivo and the toxicologic significance of FDVE sulfoxides remain unknown and merit further investigation.

Introduction FDVE1 (referred to as “compound A” in the sevoflurane labeling) (Figure 1; 1) is the major degradation product * To whom correspondence should be addressed. Tel: 206-543-4070. Fax: 206-685-3079. E-mail: [email protected]. † Ankara University. ‡ Department of Anesthesiology, University of Washington. § Department of Medicinal Chemistry, University of Washington. 1 Abbreviations: FDVE, fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether; GSH, glutathione; GST, glutathione-S-transferase; GGT/ DP, γ-glutamyltransferase/dipeptidase; FMO, flavin-containing monooxygenase; P450, cytochrome P450; DFEG, S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl)glutathione; (E,Z)-FFVG, (E,Z)-S(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)glutathione; DFEC, S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl)-L-cysteine; (E,Z)FFVC, (E,Z)-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)-Lcysteine; N-Ac-DFEC, N-acetyl-S-(1,1-difluoro-2-fluoromethoxy-2(trifluoromethyl)ethyl)-L-cysteine; (E,Z)-N-Ac-FFVC, (E,Z)-N-acetyl-S(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)-L-cysteine; DFECSO, S-[1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl]-L-cysteine sulfoxide; (Z)-N-Ac-FFVC-SO, (Z)-N-acetyl-S-[1-fluoro-2-fluoromethoxy2-(trifluoromethyl)vinyl]-L-cysteine sulfoxide; N-Ac-DFEC-SO, N-acetylS-[1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl]-L-cysteine sulfoxide.

of sevoflurane formed via base-catalyzed dehydrofluorination by the carbon dioxide absorbents in anesthesia machines (1, 2). FDVE is nephrotoxic when administered to rats by inhalation or intraperitoneal injection (3-8). Several other chlorinated and fluorinated alkenes are nephrotoxic, and their nephrotoxocity is associated with a multistep pathway that includes hepatic glutathione S-conjugate formation, enzymatic hydrolysis of the glutathione S-conjugates to cysteine S-conjugates, renal uptake of cysteine S-conjugates, and bioactivation by renal cysteine S-conjugate β-lyase to reactive species, whose reaction with cellular proteins is associated with cell damage and death (9-11). FDVE undergoes enzymatic and nonenzymatic conjugation with GSH to form several FDVE-GSH conjugates, subsequent conversion to the corresponding FDVEcysteine and -mercapturic acid conjugates, and bioactivation of the cysteine conjugates by renal β-lyase. Such conjugation and metabolism have been established in both rats and humans. In rats, in vivo, FDVE undergoes

10.1021/tx034254k CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004

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Figure 1. Pathways of FDVE metabolism in rats and humans. (1) FDVE; (2) DFEG; (3) (E) and (Z)-FFVG; (4) DFEC; (5) (E) and (Z)-FFVC; (6) N-Ac-DFEC; (7) (E) and (Z)-N-Ac-FFVC; (8) 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid; and (9) 3,3,3-trifluorolactic acid.

reaction with GSH to form (R)- and (S)-DFEG (Figure 1; 2) and (E)- and (Z)-FFVG (Figure 1; 3), which undergo cleavage to the corresponding cysteine S-conjugates (Figure 1; 4, 5) (12-14). In rats, N-acetylation forms the mercapturates, (R)- and (S)-N-Ac-DFEC (Figure 1; 6) and (E)- and (Z)-N-Ac-FFVC (Figure 1; 7), which are excreted in urine, as identified by ionspray LC-MS/MS, 19F NMR, and selected ion mode GC-MS (12, 14, 15). The cysteine S-conjugates are also metabolized by rat renal β-lyase in vitro and in vivo to reactive intermediates, which may bind to cellular macromolecules or undergo hydrolysis to 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid (Figure 1; 8) (14-19). The latter has been identified in rat urine by 19F NMR and GC-MS, establishing β-lyase-catalyzed metabolism of FDVE-cysteine conjugates in rats in vivo (14, 16). In human subcellular fractions in vitro, including hepatic and renal microsomes and cytosol and blood, FDVE also undergoes conjugation to form four GSH conjugates (20). FDVE-cysteine S-conjugates are metabolized in vitro to their corresponding mercapturates (6, 7) by human kidney cytosol, microsomes, and mitochondria (17, 21), and mercapturates are also deacetylated to the corresponding cyteine S-conjugates by human renal cytosol in vitro (21). FDVE mercapturates and 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid have been identified by 19F NMR and GC-MS in the urine of patients exposed to FDVE while undergoing sevoflurane anesthesia (15, 22). These results demonstrated that FDVE undergoes GSH conjugation, conversion to corresponding cysteine S-conjugates, and renal β-lyase-catalyzed metabolism in humans in vivo.

A novel pathway of haloalkene S-conjugates bioactivation and toxification, involving rat, rabbit, and/or human hepatic microsomal sulfoxidation of cysteine and mercapturic acid conjugates of dichloropropene, hexachlorobutadiene, trichloroethene, and tetrachloroethene has been identified (23-28). In general, S-conjugates sulfoxidation may be mediated by P450 or flavin monooxygenases. For example, sulfoxidation of S-allyl-L-cysteine and S-benzyl-L-cysteine, and to a lesser extent S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2,2-trichlorovinyl)-L-cysteine, was catalyzed by flavin monooxygenases (29, 30). In contrast, N-acetyl-S-(pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine, N-acetyl-S(1,2-dichlorovinyl)-L-cysteine, and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxidation were catalyzed mainly by P450 (25-27). Sulfoxidation of haloalkyl cysteine S-conjugates can constitute a toxification pathway, which is independent of β-lyase-mediated bioactivation (24-28). It is unknown, however, whether FDVE-cysteine and FDVE-mercapturic acid conjugates can undergo sulfoxidation. Therefore, the objective of this investigation was to test the hypothesis that FDVE-cysteine and -mercapturic acid S-conjugates undergo metabolism in human and rat liver and kidney microsomes to novel sulfoxide metabolites, to determine whether species and tissue differences exist in this oxidative process and to elucidate the enzyme(s) involved.

Experimental Procedures Materials. NADPH, troleandomycin, ketoconazole, methimazole, and 1-aminobenzotriazole were obtained from Sigma-

Sulfoxidation of Haloalkene S-Conjugates Aldrich Co. (St. Louis, MO). DFEC, (Z)-N-Ac-FFVC, and N-AcDFEC were synthesized as previously described (16). Human liver and kidney tissues medically unsuitable for transplant were obtained from the University of Washington Human Liver Bank and the National Disease Research Interchange, respectively. Microsomes were prepared from thawed specimens as described previously (31) and stored at -80 °C until required. Microsomal protein concentrations were measured by the method of Lowry et al. (32) with bovine serum albumin as the standard. Animal experiments were approved by the University of Washington Animal Use Committee in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines. Male Fisher 344 rats (220-240 g, Madison, WI) were treated with 0.1% phenobarbitol in the drinking water for 10 days. Liver and kidney microsomes were prepared the following day. Microsomes from baculovirus-transfected insect cells (Supersomes) expressing human P450 1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5, rat P450 3A1 and 3A2, and human FMO1, FMO3, and FMO5 were obtained from BD Gentest Co. (Woburn, MA). P450 3A4 and P450 3A5 were also purchased from PanVera Co. (Madison, WI). All other reagents were obtained from commercial suppliers and used without further purification. Synthesis of DFEC-SO. DFEC (100 mg) was stirred with hydrogen peroxide (30%, 0.037 mL) in 3 mL of trifluoroacetic acid at 4 °C for 1 h and then at 25 °C. The progress of the reaction was monitored by HPLC. After the starting material was completely consumed, the solvent was removed in vacuo and the product was precipitated by the addition of diethyl ether to give 82 mg of white solid. 1H NMR spectra were recorded on a Varian XL-400 spectrometer, and chemical shifts were referenced to methanol (δ 3.3 for -CH3). 1H NMR (CD3OD): δ5.55 (d of d, 4/3H, J ) 53 Hz, -FCH2O), 5.52 (d of d, 2/3H, J ) 53 Hz, -FCH2O), 5.47 (m, 1H, -F2C-CH-CF3), 4.32 (m, 1H, RH), 3.75 (m, 1H, OS-CH-C), 3.50 (m, 1H, OS-CH-C). LC-ESI: 318 [M + H]+, 340 [M + Na]+. Synthesis of (Z)-N-Ac-FFVC-SO. (Z)-N-Ac-FFVC (20 mg) was dissolved in 1 mL of trifluoroacetic acid, and then, hydrogen peroxide (30%, 0.008 mL) was added at 4 °C. The mixture was stirred for 1 h at 4 °C and then at 25 °C. The progress of the reaction was monitored by HPLC. After the starting material was completely consumed, the solvent was removed in vacuo. The residue was precipitated in diethyl ether and purified by HPLC to give 11 mg of product. 1H NMR (CD3OD): δ 5.61 (d of d, 1H, J ) 52 Hz, FCH2O), 5.50 (d of d, 1H, J ) 39 Hz, FCH2O), 4.81 (m, 1H, RH), 3.79 (m, 1H, OS-CH-C), 3.34-3.62 (m, 1H, OS-CH-C), 1.99 and 2.01 (2 singlets, 3Hs, -CH3). LC-ESI: 340 [M + H]+, 362 [M + Na]+, 130 (-CH2CHNH(Ac)COOH). Synthesis of N-Ac-DFEC-SO. N-Ac-DFEC (15 mg) was dissolved in 1 mL of trifluoroacetic acid, hydrogen peroxide (30%, 0.001 mL) was added at 4 °C, and the mixture was stirred. The progress of the reaction was monitored by HPLC. After the starting material was completely consumed, the solvent was removed in vacuo. The residue was precipitated in diethyl ether and purified by HPLC to give 12 mg of product. 1H NMR (CD3OD): δ 5.35-5.62 (m, 3Hs, J ) 52 Hz, FCH2O and -F2CCH-CF3), 4.82-4.95 (m, 1H, RH), 3.57-3.75 (m, 1H, OS-CHC), 3.32-3.49 (m, 1H, OS-CH-C), 2.01-2.03 (m, 3Hs, -CH3). LC-ESI: 360 [M + H]+, 382 [M + Na]+, 130 (-CH2CHNH(Ac)COOH). Enzyme Systems Involved in FDVE-Mercapturic Acid and -Cysteine Sulfoxidation. Incubation Conditions. Preliminary experiments showed that liver microsomal sulfoxide formation was linear for up to 60 min, 0.1-2 mM (Z)-N-AcFFVC, and 1-4 mg/mL protein. Routine incubations (0.25 mL) contained microsomes (4.0 mg/mL protein) and NADPH (2 mM) in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C, and reactions were started by the addition of 2 mM substrate. Control reactions lacking NADPH, protein, or substrate were run in parallel. Reactions were terminated after 30 min with 20% perchloric acid and placed on ice, vortexed, and centrifuged for 15 min at 3000 rpm to remove precipitated proteins.

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 437 Supernatants (5 µL) were analyzed directly by LC-MS. Assays with expressed human P450s (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), rat P450 3A1 and 3A2 and FMOs (FMO1, FMO3, and FMO5), and (Z)-N-Ac-FFVC and N-AcDFEC were done in the same manner as with microsomes, except 10 pmol/mL P450 or 200 µg/mL FMO was used instead of microsomes. To discriminate between P450 enzymes and FMOs, microsomes were heated for 5 min at 45 °C in the absence of NADPH to inactivate FMO and placed on ice. After NADPH was added and there was a brief preincubation period (5 min, 37 °C), substrates were added and incubations were performed as described above. The mechanism-based P450 3A inhibitor troleandomycin (10 and 100 µM) and the nonselective P450 inhibitor 1-aminobenzotriazole (0.5 mM) (33) were preincubated with human liver microsomes in potassium phosphate buffer (pH 7.4) and NADPH (2 mM) for 15 min at 37 °C, and then, substrates (2 mM) were added to start the 30 min reaction. Competitive inhibitors ketoconazole (1 and 5 µM) and methimazole (1 mM) were coincubated with substrate and human liver microsomes in potassium phosphate buffer (pH 7.4) at 37 °C for 5 min, and the reaction was initiated by the addition of NADPH. After 30 min, the reaction was terminated as above. The effect of troleandomycin (30 and 100 µM) and ketoconazole (1 and 5 µM) on the formation of (Z)-N-Ac-FFVC-SO was also determined with cDNA expressed P450 3A4 and P450 3A5 isoforms (10 pmol/mL). All inhibitors were diluted in methanol (final methanol concentration 1%). To generate samples for LC-MS/MS analysis, mixtures (5 mL) containing 0.1 M potassium phosphate buffer (pH 7.4), phenobarbital-induced rat liver microsomes (4 mg/mL), and (Z)-NAc-FFVC or N-Ac-DFEC (2 mM) were preincubated at 37 °C for 5 min prior to adding 2 mM NADPH. After 60 min, incubations were quenched with the addition of 878 µL of 20% perchloric acid and placed on ice. The incubations were then centrifuged for 15 min and extracted with diethyl ether (3 × 5 mL, samples were centrifuged each time prior to removal of the organic layer). The combined organic layers were evaporated to dryness at 40 °C using a TurboVap LV evaporator (Zymark, Hopkinton, MA). The resulting residue was kept at -20 °C until analysis. At the time of analysis, the samples were reconstituted with 20% acetonitrile in water. Analytical Methods. 1. LC-MS. The LC-MS system (1100 Series MSD, Agilent Technologies, Palo Alto, CA) consisted of a binary solvent delivery system, autosampler, Supelcosil LC18-DB C18 reverse phase HPLC column (150 mm × 3 mm × 3 µm) (Supelco Co., Bellefonte, PA), and quadrupole mass spectrometer equipped with an electrospray interface and operated in the positive ionization mode. The mass spectrometer interface was maintained at 325 °C, with a nitrogen nebulization pressure of 25 psi and a flow of 10 L/min. The gradient mobile phase (0.5 mL/min) was water (0.05% TFA):acetonitrile (0.05% TFA) (90:10) for 1 min, increased to 33% acetonitrile over 6 min and held for 2 min, and then increased to 35% acetonitrile over 0.5 min and held for 2.5 min. The column was briefly washed with 90% acetonitrile and reequilibrated back to 10% acetonitrile. Using the above conditions, the retention time of (Z)-N-AcFFVC-SO was 7.5 min, the two diastereomeric peaks for N-AcDFEC-SO eluted at 8.2 and 8.5 min, and DFEC-SO eluted as three peaks at 5.3, 5.5, and 5.9 min, which were presumed to represent diastereomers and quantified together. (Z)-N-AcFFVC-SO, N-Ac-DFEC-SO, and DFEC-SO were quantified by selected ion monitoring ([M + H]+ m/z 340, 360, and 318, respectively) using standard curves (r2 > 0.99) of peak area vs concentration generated using synthetic sulfoxide standards. Limits of quantification were 20, 20, and 8 ng/mL for the (Z)N-Ac-FFVC-SO, N-Ac-DFEC-SO, and DFEC-SO, respectively. Unless otherwise indicated, both N-Ac-DFEC-SO diastereomers were quantified together and formation rates were reported as the sum. 2. LC-MS/MS. Accurate mass verification of the synthetic standards and metabolically generated (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO was done using a quadrupole time-of-flight

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Figure 2. Electrospray LC-MS chromatograms obtained by selected ion monitoring. (A) Synthetic standard of (Z)-N-AcFFVC-SO, monitored at m/z 340. (B) Synthetic standard of N-AcDFEC-SO, monitored at m/z 360. Peaks 1 and 2 eluted at 8.2 and 8.5 min, respectively. The area ratio of peak 1 to peak 2 in the synthetic standard was 0.3, and it was 1.4-2.3 in enzymatically generated samples. tandem hybrid mass spectrometer (QTOF, Waters-Micromass, Manchester, U.K.) equipped with the CapLC system (Waters, Milford, MA). The stream select module was configured with an OPTI-PAK Symmetry 300 C18 trap column (Waters) connected in series with a nanoscale analytical column (75 µm i.d. × 15 cm, packed with 5 µm Jupiter C18 particles (Phenomenex, Torrance, CA). The samples (5 µL) were injected onto the trap column at 10 µL/min, desalted, and back-flushed to the analytical column at 0.5 µL/min using gradient elution. The gradient started at 5% B for 5 min and then went from 5 to 90% B in 5 min, followed by 90% B for 35 min (A ) 5% acetonitrile and 0.1% formic acid in water; B ) 95% acetonitrile with 0.1% formic acid in water). The (Z)-N-Ac-FFVC-SO eluted at 17.2 min, and the N-Ac-DFEC-SO eluted in two peaks at 13.4 and 15.8 min. The QTOF parameters were set as follows: the electrospray potential was set to 3.5 kV, the cone voltage was set to 40 V, the extraction cone was set to 2 V, and the source temperature was set to 80 °C. The instrument was operated in the MS/MS mode with the quadrupole isolation width set to include only the monoisotopic peak of each compound (the low mass resolution and high mass resolution parameters were set to 15 resulting in a mass window of 1 m/z centered about the [M + H]+ ion). The TOF scan range was from m/z 10-500, and the collision energy was set to 5 eV. The instrument was tuned to obtain a resolving power of 6000 for the corresponding mass range.

Results Formation of Sulfoxide Metabolites in Vitro. When the mercapturates (Z)-N-Ac-FFVC and N-AcDFEC were incubated with human liver and kidney microsomes and NADPH, the corresponding sulfoxides, (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO, were detected (Figure 2). The identity of the sulfoxides resulting from metabolism of (Z)-N-Ac-FFVC and N-Ac-DFEC was verified by consistent retention times, mass spectral characteristics, and the mass accuracy of the protonated monoisotopic molecular ion when compared to those of the synthetic compounds using electrospray MS/MS (Figures 3 and 4, respectively). N-Ac-DFEC-SO eluted as two peaks (Figure 2), whose mass spectra were identical (Figure 4), presumed to represent two diastereomers. Further structural identification of the diastereomers was not pursued.

Altuntas et al.

Incubation of DFEC with liver microsomes formed DFEC-SO. The identity of this metabolite was confirmed using electrospray LC-MS by comparison with the retention time and spectrum of the synthetic compound (Figure 5). Reaction components required for sulfoxidation of (Z)N-Ac-FFVC, N-Ac-DFEC, and DFEC were evaluated using human liver microsomes (Table 1). Sulfoxidation of both (Z)-N-Ac-FFVC and N-Ac-DFEC was negligible in the absence of NADPH or microsomal protein. In contrast, sulfoxidation of the DFEC S-conjugate at 37 °C proceeded equally well in the absence or presence of microsomal protein, and substantial amounts of DFECSO were formed without NADPH. Minimal DFEC sulfoxidation was observed at room temperature. These results suggested that (Z)-N-Ac-FFVC-SO and N-AcDFEC-SO formation was enzymatic, while DFEC sulfoxidation occurred nonenzymatically. Species and Tissue Differences in the Formation of (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO. Formation of (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO was compared in human and rat liver and kidney microsomes (Figure 6). Both (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO were formed by human liver microsomes, and considerable variability was observed between the three livers. Both (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO were also formed by rat liver microsomes, although the rates were substantially greater (2-30-fold) than with human liver microsomes (Figure 6A). Phenobarbital induction approximately doubled rat liver microsomal formation of both (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO (Table 1). Human kidney microsomes catalyzed the formation of (Z)-N-Ac-FFVC-SO. Rat kidney microsomes also catalyzed the sulfoxidation of (Z)-N-Ac-FFVC, at rates comparable to human liver microsomes. In contrast to human and rat livers, there was no difference in human and rat kidney (Z)-N-Ac-FFVC-SO formation. Phenobarbital induction had little effect on rat kidney microsomal (Z)-NAc-FFVC sulfoxidation. Any formation of N-Ac-DFECSO by either human or rat kidney microsomes, if at all present, was below the limit of quantification. Overall, FDVE mercapturate sulfoxidation was substantially greater in hepatic as compared with renal microsomes. FDVE mercapturate sulfoxidation was greater in rat than human livers. Enzymes Catalyzing (Z)-N-Ac-FFVC, N-Ac-DFEC, and DFEC Sulfoxidation. Thermal inactivation of heat labile FMO while leaving P450 activities unaffected, accomplished by incubating microsomes for 5 min at 45 °C in the absence of the NADPH, is an excellent method to discriminate between the participation of FMO and P450 enzymes in microsomal reactions (34). Heat inactivation of human liver microsomes resulted in no reduction in sulfoxide formation from (Z)-N-Ac-FFVC and only a 30% reduction in sulfoxide formation from N-Ac-DFEC (Table 2). The nonselective P450 inhibitor 1-aminobenzotriazole essentially prevented (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO formation. The FMO alternate substrate inhibitor methimazole decreased (Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation 60-80%. In contrast to (Z)-NAc-FFVC and N-Ac-DFEC sulfoxidation, 1-aminobenzotriazole and methimazole decreased the formation of DFEC-SO by only 25 and 15%, respectively (Table 2). Together, these results suggested a predominant role for P450 enzymes in (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-

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Figure 3. Electrospray LC-MS/MS spectra of (Z)-N-Ac-FFVC-SO obtained with a QTOF mass spectrometer. (A) Synthetic standard of (Z)-N-Ac-FFVC-SO (mass accuracy for the measured protonated monoisotopic molecular ion, m/z 340, was 21 ppm). (B) Extracted incubation of phenobarbital-induced rat liver microsomes with (Z)-N-Ac-FFVC as substrate (mass accuracy for the measured protonated monoisotopic molecular ion was 9 ppm).

Figure 4. Electrospray LC-MS/MS spectra of N-Ac-DFEC-SO obtained with a QTOF mass spectrometer. (A) Synthetic standard of N-Ac-DFEC-SO (mass accuracy for the measured protonated monoisotopic molecular ion, m/z 360, was 17 ppm). (B) Spectrum of the first chromatographic peak of N-Ac-DFEC-SO formed by incubation of phenobarbital-induced rat liver microsomes with N-Ac-DFEC as substrate (mass accuracy for the measured protonated monoisotopic molecular ion was 33 ppm). (C) Spectrum obtained from the second chromatographic peak of N-Ac-DFEC-SO formed by incubation of phenobarbital-induced rat liver microsomes with N-AcDFEC as substrate (mass accuracy for the measured protonated monoisotopic molecular ion was 31 ppm). Additional background ions are visible due to the low peak intensity.

SO formation and confirmed the nonenymatic nature of DFEC sulfoxidation. (Z)-N-Ac-FFVC and N-Ac-DFEC Sulfoxidation Catalyzed by Human and Rat Expressed 450s and FMOs. Because the use of P450 and FMO inhibitors suggested that P450 enzymes mediate the majority of (Z)N-Ac-FFVC and N-Ac-DFEC sulfoxidation, additional

experiments were directed toward identifying the P450 enzyme(s) involved. Therefore, 10 different expressed human P450 isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5) and three human FMO isoforms (FMO1, FMO3, and FMO5) were evaluated (Figure 7). P450 3A4 catalyzed the greatest (Z)-N-Ac-FFVC sulfoxidation, which was markedly enhanced by coexpressed

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Figure 5. Electrospray LC-MS analysis of DFEC-SO. (A) Chromatogram obtained by selected ion monitoring of m/z 318 and (B) spectrum from a synthetic standard of DFEC-SO. Table 1. Effect of Phenobarbital Induction on FDVE Mercapturatic Acid Sulfoxidation by Rat Microsomesa sulfoxide formation (pmol/mg/min) liver (control). liver (phenobarbital pretreated) kidney (control) kidney (phenobarbital pretreated)

(Z)-N-Ac-FFVC-SO

N-Ac-DFEC-SO

32.6 ( 0.7 122.4 ( 4.5

56.6 ( 6.8 256.7 ( 8.0

0.66 ( 0.15 1.20 ( 0.44

ND ND

a Incubations contained 2 mM substrate, 2 mM NADPH, and microsomes (4 mg). Results are the mean ( SD (n ) 3). ND, not detectable or below the limit of quantification.

Figure 6. Rates of sulfoxide formation from (Z)-N-Ac-FFVC and N-Ac-DFEC by (A) human (HLM) and rat (RLM) liver microsomes and (B) human (HKM) and rat (RKM) kidney microsomes. Rat tissues were from phenobarbital-induced (PB) and control (C) animals. Incubations contained 2 mM substrate, 2 mM NADPH, and microsomes (4 mg). Results are the mean ( SD (n ) 3). No sulfoxide formation from N-Ac-DFEC was observed with kidney microsomes from either humans or rats.

cytochrome b5. P450 3A5 also catalyzed (Z)-N-Ac-FFVC sulfoxidation, at a rate approximately half that of P450 3A4. P450 3A5 with coexpressed b5 was not available for direct comparison to P450 3A4 with coexpressed b5. P450 3A enzymes (without coexpressed b5) obtained from

a second source (PanVera) confirmed that P450s 3A5, and more so 3A4, had significant (Z)-N-Ac-FFVC sulfoxidation activity. The other P450 isoforms catalyzed comparatively minimal (Z)-N-Ac-FFVC sulfoxidation, and FMOs were relatively inactive. N-Ac-DFEC sulfoxidation by expressed enzymes was substantially less than that of (Z)-N-Ac-FFVC. Essentially, only human P450s 3A4 and 3A5 catalyzed the sulfoxidation of N-Ac-DFEC. No or negligible (Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation was detected in incubations without NADPH. Comparison of the P450 3A-catalyzed sulfoxidation of (Z)-N-Ac-FFVC and N-Ac-DFEC with rat and human enzymes is shown in Figure 8. With coexpressed b5, P450 3A4-catalyzed (Z)-N-Ac-FFVC sulfoxidation was 6-fold greater than that of N-Ac-DFEC. Without b5, N-AcDFEC sulfoxidation by P450s 3A4 and 3A5 was substantially less than that of (Z)-N-Ac-FFVC (using enzyme from PanVera) or undetectable (using enzyme from BD Gentest). Overall rates of sulfoxidation were greater with PanVera as compared with BD Gentest enzyme. Rates of (Z)-N-Ac-FFVC sulfoxidation by human P450 3A4 were comparable to those by rat P450s 3A1 and 3A2. In contrast, whereas human P450s 3A4 and 3A5 catalyzed negligible N-Ac-DFEC sulfoxidation, rat P450s 3A1 and 3A2 formed N-Ac-DFEC-SO at rates equal to (Z)-N-AcFFVC. Thus, there are apparent species differences in P450 3A-catalyzed sulfoxidation of N-Ac-DFEC but not (Z)-N-Ac-FFVC. Stereochemical aspects of N-Ac-DFEC sulfoxidation were evaluated using liver microsomes and expressed P450s. N-Ac-DFEC-SO eluted as two diastereomeric peaks with identical mass spectra (Figure 2). The peak 1:peak 2 area ratio was 0.3 in synthetic standards and >1 in enzymatically generated samples (containing sufficient amounts for quantification) (Table 3). The peak 1:2 area ratio was 2.0-2.5 in human liver microsomes and expressed P450 3A4 and 1.3-1.7 in rat liver microsomes and expressed P450 3A. Thus, there is an additional species difference in FDVE conjugates sulfoxidation. Inhibition of FDVE Mercapturates Sulfoxidation by Troleandomycin and Ketoconazole. (Z)-N-AcFFVC sulfoxide formation by expressed P450s 3A4 and 3A5 was completely inhibited by the mechanism-based P450 3A inhibitor troleandomycin (Figure 9). The competitive inhibitor ketoconazole reduced P450 3A4- and P450 3A5-catalyzed (Z)-N-Ac-FFVC-SO formation 7095% and 35-95%, respectively. Human liver microsomal (Z)-N-Ac-FFVC sulfoxidation was inhibited 85-100% by troleandomycin and 80-90% by ketoconazole at the highest inhibitor concentrations (Figure 10A). Microsomal N-Ac-DFEC sulfoxidation was generally inhibited 70-80% by troleandomycin and ketoconazole (Figure 10B), although the liver with the lowest uninhibited rate (human liver no. 140) showed little inhibition by troleandomycin.

Discussion The first objective of this investigation was to test the hypothesis that FDVE-cysteine and -mercapturic acid S-conjugates undergo metabolism in human and rat liver and kidney microsomes to sulfoxide metabolites. The results show that liver and kidney microsomes from rats and humans are capable of oxidizing the mercapturic acid conjugates (Z)-N-Ac-FFVC and N-Ac-DFEC to their cor-

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Table 2. Effect of Various FMO and P450 Inhibitors on the Sulfoxidation of FDVE Cysteine and Mercapturic Acid Conjugates by Human Liver Microsomesa sulfoxide formation (pmol/mg/min) incubation conditions

(Z)-N-Ac-FFVC-SO

N-Ac-DFEC-SO

DFEC-SO

complete system NADPH protein heat inactivation + methimazole (0.1 mM) + 1-aminobenzotriazole (0.5 mM)

31.8 ( 0.01 ND ND 30.8 ( 0.3 12.9 ( 0.2 ND

31.0 ( 0.9 ND 0.39 ( 0.48 22.0 ( 1.6 5.8 ( 0.3 0.49 ( 0.13

20.5 ( 0.1 11.8 ( 1.4 20.1 ( 0.6 15.6 ( 1.1 17.4 ( 0.1 15.0 ( 0.02

a Incubations were carried out with substrate (2 mM), microsomes from human liver no. 158 (1 mg of protein), inhibitors, and NADPH (2 mM). Results are the mean ( SD of two determinations. ND, not detectable.

Table 3. Stereoselectivity in N-Ac-DFEC Sulfoxides Formation by Microsomes and Expressed P450 3Aa species

enzyme preparation

peak 1/peak 2a

human

liver microsomes (no. 140) liver microsomes (no. 158) liver microsomes (no. 167) P4503A4+b5b P4503A4c liver microsomes (control) liver microsomes (phenobarbital pretreated) P4503A1b P4503A2b

2.0 ( 0.1 2.0 ( 0.03 2.3 ( 0.2 2.3 ( 0.05 2.5 ( 0.2 1.6 ( 0.01 1.7 ( 0.02

rat

1.4 ( 0.09 1.3 ( 0.02

Figure 7. Rates of sulfoxide formation from (Z)-N-Ac-FFVC and N-Ac-DFEC by expressed P450 (CYP) and FMO isoforms. Incubations were carried out with substrate (2 mM), NADPH (2 mM), P450 (10 pmol/mL), or FMO supersomes (200 µg protein/mL) for 30 min at 37 °C. Results are the mean ( SD (n ) 3). Results for FMO supersomes are pmol/mg/min. Absolute formation rates (pmol/min) with FMOs were comparable to or less than those with non-P4503A isoforms. Asterisks denote enzymes from PanVera Co; all others were from BD Gentest.

a Incubations were carried out with N-Ac-DFEC-SO (2 mM), microsomes (4 mg/mL protein) or expressed P450 3A (10 pmol/ mL), and NADPH (2 mM). Results are the mean ( SD of three determinations. a Results are the ratio of integrated areas for the diastereomers identified as peaks 1 and 2, shown in Figure 2. b Human and rat expressed P450 3A was obtained from BD Gentest. c Human expressed P450 3A was obtained from PanVera.

Figure 8. Sulfoxidation of (Z)-N-Ac-FFVC and N-Ac-DFEC by expressed human P450s 3A4 and 3A5 and rat P450s 3A1 and 3A2. Incubations contained substrate (2 mM), NADPH (2 mM), and P450 (10 pmol/mL) (30 min, 37 °C). Results are the mean ( SD (n ) 3). Asterisks denote enzymes from PanVera Co; all others were from BD Gentest.

Figure 9. Effect of P450 3A inhibitors on (Z)-N-Ac-FFVC sulfoxidation by expressed P450s 3A4 and 3A5. Results are expressed as activity remaining relative to controls (without inhibitor). Incubations contained substrate (2 mM), P450 (10 pmol/mL), NADPH (2 mM), and inhibitors [30 and 100 µM troleandomycin (TAO); 1 and 5 µM ketoconazole]. Results are the mean ( SD (n ) 3).

responding sulfoxides. In addition, the cysteine conjugate DFEC underwent facile nonenzymatic autoxidation to the respective sulfoxide. Enzymatic and nonenzymatic sulfoxidation of FDVE S-conjugates represent a novel biotransformation pathway of FDVE, which has not previously been described. This is in addition to FDVEcysteine conjugates metabolism by renal β-lyase and N-acetylation and cleavage of FDVE mercapturates by acylases (12, 14, 17, 18, 21, 22, 35). Accordingly, the metabolic scheme for FDVE can be revised to incorporate these new routes of biotransformation (Figure 11).

(Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation was predominantly hepatic. (Z)-N-Ac-FFVC-SO formation was approximately 10-fold greater (2-17 vs 0.2-1.8 pmol/min/mg, respectively) in human liver as compared with human kidney microsomes. In uninduced and phenobarbital-induced rats, (Z)-N-Ac-FFVC-SO formation was approximately 50- and 100-fold greater, respectively, in liver as compared with kidney microsomes. N-AcDFEC-SO formation was detected only in human and rat liver but not renal microsomes. Similar organ differ-

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Figure 10. Effect of P4503A inhibitors on human liver microsomal sulfoxidation of (A) (Z)-N-Ac-FFVC and (B) N-AcDFEC. Results are expressed as activity remaining relative to controls (without inhibitor). Incubations contained substrate (2 mM), microsomes (4 mg), NADPH (2 mM), and inhibitors. Results are the mean ( SD (n ) 3).

ences in N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation were also observed, whereby sulfoxidation by human kidney microsomes was not detected (30). Sulfoxidation of cysteine and mercapturic acid conjugates of cis- and trans-1,3-dichloropropene was not detected in rat kidney microsomes, whereas pig liver was active in the sulfoxidation of both cysteine S-conjugates and Nacetyl cysteine S-conjugates (23). Thus, FDVE mercapturates, like others, undergo sulfoxidation primarily in liver. The second objective of this investigation was to identify the enzyme(s) involved in FDVE S-conjugate sulfoxidation. DFEC sulfoxidation was deemed predominantly nonenzymatic, since it was not dependent on microsomal protein or NADPH and minimally affected by P450 and FMO inhibitors. Human liver microsomal sulfoxidation of (Z)-N-Ac-FFVC and N-Ac-DFEC was minimally affected by heat inactivation of FMO, and expressed human FMOs 1, 3, and 5 formed negligible amounts of (Z)-NAc-FFVC-SO and N-Ac-DFEC-SO. These results suggest minimal involvement of FMO in FDVE mercapturates sulfoxidation. At variance with this conclusion, however, was the inhibitory effect of methimazole on FDVE mercapturates sulfoxidation, although methimazole can affect P450 activity (36). Human liver microsomal sulfoxidation of (Z)-N-Ac-FFVC and N-Ac-DFEC was essentially prevented by the nonselective P450 inhibitor 1-aminobenzotriazole and substantially decreased by the P450 3A inhibitors troleandomycin and ketoconazole, and expressed P450 3A4 (and to a lesser extent P450 3A5) formed the greatest amounts of (Z)-N-Ac-FFVC-SO among the various P450 isoforms examined. These results suggest that P450 3A isoforms are the predominant catalysts

Altuntas et al.

of human liver microsomal FDVE mercapturates sulfoxidation. Rat liver microsomal (Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation was significantly enhanced by phenobarbital induction, and expressed rat P450s 3A1 and 3A2 catalyzed substantial amounts of (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO formation. Phenobarbital induces rat P450s 2A, 2B, and 2C, in addition to P450s 3A1 and 3A2 (37). However, the more selective P4503A1/2 inducer dexamethasone also increased FDVE mercapturates sulfoxidation (results not shown). These results suggest that P450 3A isoforms are also the predominant catalysts of rat liver microsomal FDVE mercapturates sulfoxidation. Two aspects of expressed P450 3A-catalyzed FDVE mercapturates sulfoxidation are notable. First, (Z)-N-AcFFVC-SO was formed by P450 3A5, although at rates approximately half that of P450 3A4. This is similar to previously reported differences in the metabolic capacities of P450s 3A4 and 3A5 (38). Because P450 3A5 is polymorphically expressed (39), there may be pharmacogenetic differences in FDVE mercapturates sulfoxidation. The existence of these differences, and any pharmacokinetic or toxicologic significance, remain unknown. In contrast, P450 3A5 formed negligible amounts of N-AcDFEC-SO. Second, P450 3A-catalyzed sulfoxidation differed markedly, depending on the enzyme source, with 5-10-fold greater sulfoxide formation with P450s 3A4 and 3A5 obtained from PanVera as compared with BD Gentest. Greater activity with PanVera enzymes resulted in detection of N-Ac-DFEC-SO, which was not observed at meaningful rates with P450s from BD Gentest. Differences in turnover may be due to coexpression of rabbit rather than human P450 reductase in PanVera as compared with BD Gentest P4503As. The role of P4503A1/2 and P4503A4/5 in FDVE mercapturates sulfoxidation was consistent with previous observations with other haloalkyl mercapturates. NAcetyl-S-(pentachlorobutadienyl)-L-cysteine, N-acetyl-S(1,2,2-trichlorovinyl)-L-cysteine, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, and N-acetyl-S-(2,2-dichlorovinyl)-Lcysteine sulfoxidation were greater in liver microsomes from phenobarbital- and dexamethasone-induced rats (26, 27). N-Acetyl-S-(pentachlorobutadienyl)-L-cysteine sulfoxidation was catalyzed predominantly by human liver microsomal and expressed P450s 3A4 and 3A5 (25). Sulfoxides formation from N-acetyl-S-(pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in rat liver microsomes was catalyzed mainly by P4503A1/2 (26, 27). Thus, P4503A isoforms are, in general, the major enzymes responsible for haloalkyl mercapturates sulfoxidation. The relative contribution of FMOs toward microsomal cysteine S-conjugate S-oxidation clearly depends on the conjugate structure. Generally, nucleophilic sulfur atoms are oxidized preferentially by FMO, whereas nonnucleophilic sulfur atoms are preferentially oxidized by P450 (29, 40). Cysteine conjugates with more nucleophilic sulfur atoms, S-allyl-L-cysteine and S-benzyl-L-cysteine, were much better human kidney and liver and rabbit liver microsomal FMO substrates than those with less nucleophilic sulfur atoms [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2,2-trichlorovinyl)-L-cysteine] (29, 30). This is likely attributed to the sulfur of allyl and benzyl compounds being more nucleophilic than that of vinyl compounds and the tendency for FMOs to oxidize strong

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Figure 11. Revised pathway of FDVE metabolism in rats and humans. Nomenclature is the same as in Figure 1. Additional compounds are (10) DFEC-SO; (11) (E) and (Z)-FFVC-SO; (12) N-Ac-DFEC-SO; and (13) (E)- and (Z)-N-Ac-FFVC-SO).

nucleophiles (40). Lipophilicity may also affect haloalkene S-conjugates sulfoxidation by FMO. S-Benzyl-L-cysteine is relatively lipophilic, with a nuclephilic sulfur atom, and has been shown to be a selective substrate for FMO (41). (Z)-N-Ac-FFVC-SO has a vinylic sulfur atom as well as strong electron-withdrawing fluorine atoms, which make the sulfur atom much less nucleophilic than that of S-allyl-L-cysteine, S-benzyl-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(1,2,2-trichlorovinyl)-L-cysteine. (Z)-N-Ac-FFVC and N-Ac-DFEC are less lipophilic then S-benzyl-L-cysteine, rendering them theoretically less susceptible to FMO sulfoxidation. This may partly explain the lack of FMO activity toward (Z)-N-Ac-FFVCSO and N-Ac-DFEC-SO formation. The third objective of this investigation was to determine whether species differences exist in FDVE S-conjugate sulfoxidation. Although FDVE is nephrotoxic in rats (3-8), sevoflurane (the parent drug), under conditions in which patients are exposed to FDVE, has been used extensively in patients without evidence of nephrotoxicity (42-46), although nephrotoxicity in healthy volunteers has been reported (47, 48) but not substantiated (49, 50). The mechanism(s) for this species difference in FDVE nephrotoxicity remains incompletely elucidated. The present results show that overall, FDVE mercapturates sulfoxidation was greater in rat than human tissues. Specifically, formation of both (Z)-N-Ac-FFVCSO and N-Ac-DFEC-SO was substantially (2-30-fold) greater in rat than human liver microsomes. In addition, N-Ac-DFEC-SO formation by rat P450s 3A1 and 3A2 was substantially greater than by human P450 3A4 and 3A5. Last, although the absolute configurations of the two

N-Ac-DFEC-SO diastereomers are unknown, there was a species difference in their relative formation by both liver microsomes and expressed P4503As. Thus, species differences in FDVE S-conjugates sulfoxidation might explain, in part, apparent differences in susceptibility to FDVE nephrotoxicity. There are other known interspecies differences in FDVE metabolism and/or toxification in rats vs humans, including (i) greater rates of FDVE-GSH conjugate formation in hepatic microsomes and cytosol (20), (ii) greater β-lyase-catalyzed metabolism of FDVE-cysteine conjugates in vitro in rat kidneys (17), (iii) greater excretion of N-Ac-DFEC relative to (E,Z)-N-Ac-FFVC in rats (18, 22), (iv) greater excretion of 3,3,3-trifluoro-2-fluoromethoxypropanoic acid (reflecting β-lyase-catalyzed FDVE cysteine conjugates metabolism) in vivo in rats (18, 22), (v) greater ratio of 3,3,3-trifluoro-2-fluoromethoxypropanoic acid (toxification) to mercapturates (detoxication) in urine in rats (18, 22), and (vi) relative resistance of human proximal tubular cells to the cytotoxic effects of FDVE-cysteine S-conjugates (51). The toxicologic significance of FDVE S-conjugates sulfoxidation remains unknown. Sulfoxidation represents an alternative route of metabolism for DFEC (alternative to β-lyase-catalyzed toxification) and an alternative to deacetylation back to potentially toxic cysteine conjugates for the nontoxic mercapturates N-Ac-DFEC and (E,Z)N-Ac-FFVC. Recent experiments showed that DFEC-SO and (Z)-N-Ac-FFVC-SO were more toxic in a human proximal tubular cell line in culture than the corresponding parent cysteine and mercapturic acid FDVE conjugates, and DFEC-SO was more toxic than (Z)-N-Ac-

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FFVC-SO (51). Nevertheless, it is not known whether FDVE conjugates sulfoxidation occurs in vivo, either in rats or in humans, or whether FDVE conjugates sulfoxides are nephrotoxic in vivo. The significance of the newly identified sulfoxidation pathway will depend on the relative toxicities of the various alternative routes of metabolism and merits further investigation. In summary, sulfoxidation of the mercapturates (Z)N-Ac-FFVC and N-Ac-DFEC is a newly revealed biotransformation pathway in the GSH-dependent metabolism of FDVE. DFEC sulfoxidation also occurs but via autoxidation. P4503A4/5 and P4503A1/2 are the major enzymes responsible for (Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation in human and rat liver microsomes. FDVE mercapturates sulfoxidation was greater in rat as compared with human liver microsomes. This could contribute to species differences in FDVE nephrotoxicity.

Acknowledgment. This investigation was supported by NIH Grants R01DK53765 and P30ES07033. We thank Pam Sheffels for her outstanding experimental contributions and Catalin Doneanu from the University of Washington Department of Medicinal Chemistry Mass Spectrometry Center for QTOF analysis.

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