Fate and Toxicity of 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A)-derived Mercapturates in Male, Fischer 344 Rats 

2-Fluoromethoxy-1,1,3,3,3-pentafluoro-1-propene (Figure 1, compound 1) was provided by Abbott Laboratories (Abbott Park, IL). S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine were synthesized as described previously. [21]S-(1,2-Dichlorovinyl)-N-acetyl-L-cysteine and S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine were obtained by synthesis. [17,22]Porcine kidney acylase I (grade III), L-methionine, and fluorescamine were purchased from Sigma Chemical Company (St. Louis, MO). An acylase was purified from rat kidney cytosol. [20]Acetic anhydride-d⁶was purchased from Cambridge Isotope Laboratories (Andover, MA). S-Benzyl-N-acetyl-L-cysteine and silica gel (Merck, West Point, PA; grade 60, 240-400 mesh, 60 Angstrom) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Thin-layer chromatography (TLC) plates (silica gel, 250 [micro sign]m, AL SIL G/UV; Whatman, Hillsboro, OR) were purchased from VWR Scientific (Rochester, NY). Tetrahydrofuran was dried over sodium metal and freshly distilled before use. All other reagents were obtained from commercial suppliers and were used without further purification, except as noted.

Male Fischer 344 rat kidney cytosol and human kidney cytosol were prepared by homogenization of the kidneys in 50 mM potassium phosphate buffer (pH 7.4) at 4 [degree sign]C. The homogenates were centrifuged at 9,000g, and the supernatants were centrifuged at 100,000g to obtain the cytosolic fractions. Protein concentrations were determined by the method of Bradford with bovine serum albumin used as the standard. [23] 

Melting points were determined with a Mel-Temp melting point apparatus and are uncorrected.¹H nuclear magnetic resonance (NMR) and (¹⁹) F NMR spectra were recorded with a Bruker 270n MHz spectrometer operating at 270 MHz for¹H and 254 MHz for¹⁹F. Chemical shifts, [small delta, Greek], are reported in parts per million (ppm). The internal standard ([small delta, Greek]= 0.0 ppm) for¹H NMR with CDCl³as the solvent was tetramethylsilane. The solvent resonance peak at 2.1 ppm was used as the internal standard for¹H NMR spectra when acetone-4⁶was the solvent. Trifluoroacetamide ([small delta, Greek]= 0.0 ppm) was used as the external standard for¹⁹F NMR spectra.

Mass spectra were recorded with a Hewlett-Packard 5890 series II gas chromatograph (25 m x 0.2 mm, 0.5-[micro sign]m film thickness, HP-1 crosslinked methyl siloxane column; Hewlett-Packard, Wilmington, DE) coupled to a Hewlett-Packard 5972 series II mass selective detector; the injector and transfer-line temperatures were 200 [degree sign]C and 240 [degree sign]C, respectively. The methyl esters of compounds 3, 5, 3-d³, 5-d³, or [acetyl-²H³]S-benzyl-N-acetyl-L-cysteine were analyzed with a temperature program of 50 [degree sign]C for 1 min followed by a linear gradient of 10 [degree sign]C/min to 240 [degree sign]C. Methyl esters of compounds 3 and 5 and [acetyl-²H³]S-benzyl-N-acetyl-L-cysteine were prepared for analysis by gas chromatography-mass spectrometry (GC-MS) by reaction with an ethereal solution of the acids with diazomethane. (Caution: Diazomethane is toxic and mutagenic and should be used with care in an efficient fume hood.)

Silica gel was used for column chromatography, and the columns were eluted by gravity flow. Cysteine S-conjugates on TLC plates were detected with a spray reagent of 0.3% ninhydrin in n-butanol/acetic acid (97:3).

[acetyl-²H³] S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d³). A solution of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine 220 mg, 0.73 mM) in 2 N sodium hydroxide (5 ml) was cooled to 0 [degree sign]C in an ice bath. Acetic anhydride-d⁶(340 mg, 3.1 mM) was added in 50-[micro sign]l portions to the reaction mixture to give a pH of 6. The ice bath was removed, and the reaction mixture was stirred at room temperature for 10 min. The solution was brought to pH 2 by addition of concentrated HCl and extracted with ethyl acetate (3 x 30 ml). The organic layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield a pale yellow liquid. The product was loaded onto a silica gel column, which was eluted with dichloromethane/ethyl acetate (4:6) to give [acetyl-²H³]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine as a pale yellow solid (150 mg, 59%): TLC, R^f= 0.2 (methanol/ethyl acetate/acetic acid, 1:9:0.01); H NMR (CDCL³)[small delta, Greek] 8.5 (bs, 1H, COOH), 6.90 (d, 1H, NH), 5.5 (d of d, 2H, OCH²F), 4.90-5.10 (m, 1H, CF²CH(CF³)OCH²F), 4.42-4.57 (m, 1H, CH2 (²) CH (NH²) COOH), 3.37-3.50 and 3.52-3.72 (d of m, 2H, SCH², CH(NH²)COOH);¹⁹F NMR (CDCl³)[small delta, Greek] 3.77-3.89 (m, 3F, CF²CH(CF²CH(CF³), -5.90 to -0.90 (m, 2F, CF²CH(CF³)OCH²F), -77.20 (t, 1F, J = 54 Hz, OCH²F); GC-MS (methyl ester) t^R17.4 min, m/z (%) 360 (M⁺, 0.3), 301 (M⁺-COOCH³, 6.5%), 179 (C⁶H⁷D³NO³S⁺, 38), 147 (C⁵H³D³NO²S⁺, 25), 89 C³H⁵DNO (²)⁺, 100).

[acetyl-²H³]S-Benzyl-N-acetyl-L-cysteine. A solution of S-benzyl-L-cysteine (2.11 g, 10 mM) in water (20 ml) containing sodium hydroxide (2.2 g, 55 mM) was cooled to 0 [degree sign]C in an ice bath. Acetic anhydride-d⁶(2.3 g, 21 mM) was added in 100-[micro sign]l portions to he reaction mixture to give a pH of 6. The ice bath was removed, and the reaction mixture was stirred at room temperature for 10 min. The solution was brought to pH 2 by the addition of concentrated HCl and extracted with ethyl acetate (3 x 75 ml). The organic layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield [acetyl-²H³]S-benzyl-N-acetyl-L-cysteine as a white solid (2.4 g, 94%): mp 142-146 [degree sign]C; TLC, R^f= 0.67 (ethyl acetate/methanol/acetic acid, 8:2:0.01);¹H NMR (acetone-d⁶)[small delta, Greek] 7.65 (d, 1H, NH), 7.36-7.60 (m, 5H, C⁶H⁵), 4.82-4.94 (m, 1H, [small alpha, Greek]-CH), 3.95 (s, 2H, C⁶H⁵CH²), 2.90-3.15 (m, 2H, [small beta, Greek]-CH²); GC-MS (methyl ester) t^R21.2 min, m/z (%) 270 (M⁺, 1.8), 208 (M^+-NHCOCD3, 12), 179 (M^+-C7H⁷, 13), 147 (M^+-SC7H⁷, 7), 91 (C⁷H⁷⁺, 100).

[acetyl-²H³]N-Acetyl-L-cysteine. [acetyl-²H³]S-Benzyl-N-acetyl-L-cysteine (2.3 g, 8.9 mM), which was used from the previous step without further purification, was dissolved in liquid ammonia (50 ml), and sodium metal (1.5 g, 62 mM) was added to give disodium [acetyl-(²) H³]N-acetyl-L-cysteine. The liquid ammonia was allowed to evaporate, and the resulting solid was dissolved in 20 ml water and brought to pH 2 by the addition of concentrated HCI. The aqueous solution was extracted with ethyl acetate (3 x 50 ml). The organic layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield a white solid. The crude product was recystallized from ethyl acetate/petroleum ether to give [acetyl-²H³]N-acetyl-L-cysteine as a white crystalline solid (1.1 g, 74%): mp 108-109 [degree sign]C; TLC, R^f= 0.3 (ethyl acetate/methanol, 8:2);¹H NMR (acetone-d⁶)[small delta, Greek] 7.65 (d, 1H, NH), 4.82-4.90 (m, 1H, [small alpha, Greek]-CH), 3.10-3.22 (m, 2H, [small beta, Greek]-CH²).

[acetyl-²H³]S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5-d³). Triethylamine (1.51 g, 15 mM) was added to a stirred solution of [acetyl-²H³]N-acetyl-L-cysteine (830 mg, 5 mM) in THF (25 ml) in a flask that was purged with nitrogen, and the solution was cooled to 0 [degree sign]C. 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound 1, 1.08 g, 6 mM) in 5 ml THF was added by drops to the reaction mixture over 10 min, and the reaction was stirred for 1.5 h at 0 [degree sign]C. After addition of 30 ml water, the reaction was brought to pH 2 with 5N HCI, and the mixture was extracted with ethyl acetate (3 x 50 ml). The organic layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield a yellow viscous oil. The product was loaded onto a silica gel column, which was eluted with dichloromethane/ethyl acetate (6:4) to give [acetyl-²H³]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine as a yellow solid (389 mg, 25%): TLC, R^f= 0.21 (methanol/ethyl acetate/acetic acid, 1:9:0.01);¹H NMR (CDCl³)[small delta, Greek] 6.77 (d, 1H, CHNHCOCH³), 5.30-5.50 (d of d, 2H, J = 54 Hz, OCH (²) F), 4.65-4.80 (m, 1H, CH²CH(NHCOCH³)COOH), 3.25-3.70 (m, 2H, SCH²CH(NHCOCH³)COOH);¹⁹F NMR (CDCl³)[small delta, Greek] 10.90- 11.20 (m, 3F, CF = C(CF³)OCH²F), -32.20 to -32.0 (m, 1F, CF = C(CF³)OCH²F), -74.90 (t, 1F, J = 54 Hz, OCH²F); GC-MS (methyl ester) m/z (%) 340 (M⁺, 0.3), 281 (M⁺-COOCH (³), 7.7), 147 (C⁵H³D³NO²S⁺, 88), 89 (C³H⁵DNO²⁺, 100).

In Vivo Toxicity Studies. Male Fischer 344 rats (weigh, 180-200 g; Charles River Laboratories, Wilmington, MA) in groups of three were given 125, 250, or 500 [micro sign]mol/kg compound 3; 62.5, 125, or 250 [micro sign]mol/kg compound 5[double vertical bar]; 125 [micro sign]mol/kg compound 3-d³; or 125 [micro sign]mol/kg compound 5-d³intraperitoneally. The mercapturic acids were dissolved in 0.9% saline, which was given in a volume of 6.6 ml/kg for compounds 3 and 3-d³and 8 ml/kg for compounds 5 or 5-d³. Control animals were given saline. Animals were placed in individual metabolism cages and housed with a 12-h light-dark cycle and provided with food and water ad libitum immediately after compound administration. The urine was collected from 0-24 h in the presence of sodium azide (10 mg).

After 24 h, the rats were anestherized with ether and killed by cardiac exsanguination. The blood was collected and centrifuged to obtain serum. The left kidney was removed, trimmed of fat, and the capsule was removed. Liver and kidney tissues were fixed in 0.2-cm transverse blocks and embedded in paraffin; kidney and liver tissue were sectioned at 3 and 5 [micro sign]m, respectively. The sections were stained with hematoxylin and eosin. The entire nephron was examined microscopically with specific severity scoring of the proximal convoluted tubules, differentiated by location into juxtamedullary, paracortical, or cortical regions. Lesions from each region were given scores of 0 (no significant lesions), 0.5, 1.0, or 1.5 to 4+(maximal severity). The amount of proteinaceous material in the collecting ducts was also scored from 0 (no protein casts) to 4+(abundant protein casts). The results are reported as the sum of the individual scores for each region, including protein casts; thus scores can range from 0 to 16. In the liver, the extent of inflammation of the portal triads, together with the severity of necrosis, was evaluated. All slides were read by a pathologist (R.B.B.) and were coded as to the experimental treatment.

Urine and serum glucose concentrations, serum glutamate-pyruvate transaminase activities, and blood urea nitrogen concentrations were measured with Sigma Kits 115, 505, and 535, respectively (Sigma Chemical Co.). Urine protein concentrations were measured by the method of Bradford (Bio-Rad Protein Assay Dye Reagent Concentrate; Bio-Rad, Richmond, CA) with bovine serum albumin as the standard. [23] 

Results were evaluated statistically by analysis of variance with Dunnett's multiple comparison test (InStat; GraphPad Software, San Diego, CA). A level of P<.05 was chosen for acceptance or rejection of the null hypothesis.

Analysis of Rat Urine by¹⁹F NMR Spectroscopy and Gas Chromatography-Mass Spectrometry. For¹⁹F NMR spectroscopic analysis, rat urine (0.6 ml) was centrifuged to remove insoluble material, and the supernatant was mixed with 100 [micro sign]l deuterium oxide and placed in a 5-mm NMR tube. The urine was then analyzed by¹⁹F NMR spectroscopy.

For GC-MS analysis, S-(2,2-dibromo-1,1-difluoroethyl)-N-acetyl-L-cysteine (0.1 mg) was added to each urine sample (2 ml) as an internal standard, and the samples were brought to pH 1.4 with 5 N hydrochloric acid. The samples were extracted with ethyl acetate (3 x 2 ml); the organic layers were separated and combined, dried over anhydrous sodium sulfate, and evaporated to dryness under a stream of nitrogen. The dried samples were treated with excess diazomethane in ether, evaporated to dryness, and dissolved in 200 [micro sign]l dichloromethane. To prepare the standard curves, a mixture of synthetically prepared compounds 3 and 3-d³or 5 and 5-d³were added to control urine samples and subjected to the extraction procedure described above. Samples (1 [micro sign]l) were analyzed by GC-MS, as described in Materials and Methods. The mercapturic acids in urine were analyzed by selective ion monitoring of ions characteristic for unlabeled mercapturic acids (m/z 144, C (⁵) H⁶NO²S⁺, or 176, C⁶H¹⁰NO³S⁺) and deuterium labeled (m/z 147, C⁵H³²H³NO²S⁺, or 179, C⁶H⁷²H³NO³S⁺). [24] 

Acylase Assays. Acylase activity with compound 3 as the substrate was determined by measuring the amount of the deacetylated product, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (Figure 1, compound 2) formed. Compound 2 concentrations were determined by reaction with fluorescamine. [25]The limit of detection of deacetylated mercapturates was 1 pM. The reaction mixtures (1 ml) contained human or rat kidney cytosol (2 or 3 mg protein), acylase I or purified rat kidney acylase (2 [micro sign]g protein), and 4 [micro sign]mol substrate in 50 mM potassium phosphate buffer (pH 7.4). The reaction mixtures were incubated at 37 [degree sign]C for 30 min, and the reaction was stopped by the addition of 0.2 ml 20% trichloroacetic acid. The mixtures were allowed to stand for 10 min in an ice bath and were then centrifuged (at 500g for 10 min). A sample (40 [micro sign]l) of the supernatant was added to 3.6 ml of 50 mM potassium phosphate buffer (pH 7.4), and the volume was brought to 4 ml by the addition of water. Fluorescamine (300 [micro sign]l of a solution containing 10 mg fluorescamine dissolved in 33 ml acetone) was added to the sample, and the fluorescence intensity (390 nm excitation, 475 emission) was measured with a Perkin-Elmer LS-5 Fluorescence Spectrophotometer (Norwalk, CT). Acylase activity with S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine and S-(1,2-dichlorovinyl)N-acetyl-L-cysteine, which are known acylase substrates, [16,26]was determined similarly.

Previous studies show that compound 4 undergoes rapid cyclization with the stoichiometric release of fluoride at pH 7.4. [21]Thus the deacetylation of compound 5 was determined by quantifying the fluoride ion release. The reaction mixtures were prepared as described before for compound 3. Fluoride ion concentrations were measured with a fluoride-specific electrode (ATI Orion, Boston, MA) and a Corning pH 3meter (Corning Glass Works, Medfield, MA). A standard curve was prepared with sodium fluoride.

Compound 3 given intraperitoneally to male Fischer 344 rats at doses of 125, 250, and 500 [micro sign]mol/kg did not cause significant changes in serum glucose concentrations, blood urea nitrogen concentrations, serum glutamate-pyruvate transaminase activities, in urine volumes, or in urine glucose and protein concentrations compared with control animals (data not shown).

Intraperitoneal administration of compound 5 to male Fischer 344 rats at 62.5 or 125 [micro sign]mol/kg did not produce significant changes in blood or urine clinical chemical parameters compared with control animals (data not shown). Two of the three rats given 250 [micro sign]mol/kg compound 5 showed increases in urine glucose concentrations of 114 and 76 mg at 0-24 h (control urine glucose, 6.54 +/− 1.38; mean +/− SD) but showed no significant increases in urine volumes and protein concentrations. In addition, no significant increases in blood urea nitrogen concentrations and serum glutamate-pyruvate transaminase activities were observed in rats given any dose of compound 5 compared with controls (data not shown).

Histopathologic studies of kidneys from rats given compound 3 showed no significant increase in necrosis at the corticomedullary junction compared with control animals (Figure 2): the histopathologic scores for rats given 250 and 500 [micro sign]mol/kg compound 3 were 0.66 +/− 0.57 and 1.33 +/− 1.44, which were not significantly different from those of control animals (control = 0;Table 1). [3]Histopathologic studies of the kidneys from two of three rats given 250 [micro sign]mol/kg compound 5 showed necrosis at the corticomedullary junction (scores = 4.0 and 4.5). With the exception of injury attributed to an apparent intrahepatic injection of 250 [micro sign]mol/kg compound 5, which produced hepatic necrosis that was accompanied by maximal renal juxtamedullary necrosis, no morphologic evidence of hepatic injury was observed in rats given compounds 3 or 5.

The¹⁹F NMR spectroscopic analysis of urine from rats given 250 [micro sign]mol/kg compound 3 showed resonances assigned only to unchanged compound 3 (Figure 3). Although¹⁹F NMR spectroscopic analysis of urine from rats given 250 [micro sign]mol/kg compound 5 showed resonances assigned mainly to unchanged compound 5, resonances assigned to 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (Figure 1, compound 6), 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid (the cyclization product of compound 4;Figure 1, compound 7), and inorganic fluoride were also observed (Figure 4).

To study the in vivo hydrolysis and reacetylation of compound A-derived mercapturates, rats were given 125 [micro sign]mol/kg compounds 3-d (³) or 5-d³, and the amounts of compounds 3 and 3-d³or 5 and 5-d (³), respectively, in urine were quantified by GC-MS analysis. Unchanged compounds 3-d³and 5-d³accounted for about 60% of the administered dose, whereas compounds 3 and 5 accounted for about 14% and 5%, respectively, of the administered dose (Figure 5).

No hydrolysis of compound 3 was detected with human or rat kidney cytosol or with acylase I and purified rat kidney acylase (Table 2). Hydrolysis of compound 5 was observed with human or rat kidney cytosol or with acylase I and purified rat kidney acylase, but the rates were much lower than with S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine or S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine, which are known acylase substrates. [16,26] 

The objective of this work was to study the fate and nephrotoxicity of compound A-derived mercapturates. Many nephrotoxic haloalkenes are metabolized to mercapturates (S-substituted N-acetyl-L-cysteines), which cannot undergo cysteine conjugate [small beta, Greek]-lyase-catalyzed bioactivation because substrates for the pyridoxal phosphate-dependent [small beta, Greek]-lyase must possess a primary amino group. [27]Mercapturates may, however, undergo acylase-catalyzed deacetylation to regenerate the corresponding cysteine S-conjugates (e.g., compound 3 [arrow right] 2 and 5 [arrow right] 4), [19]which may undergo [small beta, Greek]-lyase-dependent bioactivation. Thus acetylation of cysteine S-conjugates and deacetylation of mercapturates may constitute detoxication or bioactivation reactions for nephrotoxic haloalkenes, depending on the relative rates of acetylation and deacetylation. Many haloalkene-derived mercapturates are nephrotoxic or cytotoxic in kidney cells, indicating that deacetylation reactions do serve as bioactivation reactions. [13-18,28,29] 

Metabolism of compound A by the mercapturic acid pathway, which includes glutathione transferase-catalyzed glutathione S-conjugate formation, hydrolysis of the glutathione S-conjugates to the corresponding cysteine S-conjugates, and N-acetyltransferase-catalyzed N-acetylation of the cysteine S-conjugates, has been demonstrated. [6]The fate and nephrotoxicity of compound A-derived mercapturates have not, however, been investigated, and it is not known whether mercapturate formation constitutes a detoxication pathway or whether the mercapturates may undergo hydrolysis to the corresponding cysteine S-conjugates and bioactivation by the [small beta, Greek]-lyase pathway.

Compound A is metabolized to compounds 3 and 5 (Figure 1). [6]In the present studies, little metabolism of compound 3 was detected:[19]F NMR spectra of the urine of rats given compound 3 showed excretion of only unchanged mercapture (Figure 3). Compound 3-d³was, however, deacetylated and reacetylated in vivo as shown by the presence of compound 3 in the urine of rats given compound 3-d³(Figure 5), indicating that compound 3-d³was hydrolyzed to compound 2, which was acetylated to give compound 3 (Figure 1, compound 3-d³[arrow right] compound 2 [arrow right] compound 3). No acylase-catalyzed hydrolysis of compound 3 was observed (Table 1). Previous studies also show deacetylation and reacetylation of mercapturates: About 60% of the administered dose of [acetyl-(²) H³]S-(2,2-dichloro-1,1-difluoroethyl)-N-acetyl-L-cysteine and [acetyl-(²) H³]S-(2,2-dibromo-1, 1-difluoro-ethyl)-N-acetyl-L-cysteine is excreted in the urine, and 17% and 31% is excreted as the unlabeled mercapturate. [28]The observation of deacetylation of compound 3 in vivo, but not in vitro, may indicate that its hydrolysis is catalyzed by hydrolases in intestinal bacteria. [30]Finally, compound 3 was not nephrotoxic or hepatotoxic, as demonstrated by clinical chemical studies and by morphologic examination of the kidneys and livers of rats given compound 3 (Figure 2). These data show that formation of compound 3 constitutes a detoxication pathway for compound A.

Compound 5 was also deacetylated and reacetylated in vivo, as shown by the presence of compound 5 in the urine of rats given compound 5-d (³)(Figure 5)(Figure 1, compound 5-d³[arrow right] compound 4 [arrow right] compound 5). In addition, the acylase-catalyzed hydrolysis of compound 5 was detected (Table 1), but at low rates compared with S-(1,2-dichlorovinyl)-L-cysteine and S-(2-chloro-1, 1,2-trifluoroethyl)-L-cysteine, which are known acylase substrates. [16,26] 

(¹⁹) F NMR spectroscopic analysis of the urine of animals given compound 5 showed mainly resonances assigned to the intact mercapturate along with minor resonances assigned to inorganic fluoride and compounds 6 and 7 (Figure 4). These data show that compound 5 is hydrolyzed to give compound 4. Compound 4 may undergo cyclization to give the nontoxic compound 7. [21,31]Alternatively, compound 4 may undergo [small beta, Greek]-lyase-catalyzed metabolism to compound 6. [11,21]Fluoride may be formed by the cyclization of compound 4 to compound 7 or by its [small beta, Greek]-lyase-catalyzed metabolism. Compound 5 was nephrotoxic, as shown by increases in urine glucose concentrations and by morphologic examinations (Figure 2). Morphologic changes were, however, seen in only two of three animals studied, indicating that little compound 4 was available for [small beta, Greek]-lyase-catalyzed bioactivation. Compound 5 was not hepatotoxic.

Results from the present study show that mercapturate formation contributes more to the detoxication of compound A than to its bioactivation. The compound A - derived mercapturate compound 3 was not nephrotoxic and underwent little metabolism in vivo or in vitro. Compound 5 was mildly nephrotoxic and was deacetylated in vivo and in vitro. These findings are significant because compounds 3 and 5 are metabolites of compound A in rats and in humans exposed to sevoflurane and thereby exposed to compound A. [6,12]Thus the formation of mercapturates of compound A would reduce its bioactivation through the [small beta, Greek]-lyase pathway. Compound A is nephrotoxic in rats, [4-9,32,33]and evidence supporting a role for the [small beta, Greek]-lyase-dependent bioactivation of compound A has been presented. [6,9-11,21,31,34]Although considerable evidence implicates the [small beta, Greek]-lyase pathway in the nephrotoxicity of compound A, reports that support to show that the [small beta, Greek]-lyase pathway is not involved have been presented. [33,35] 

The authors thank Sandra E. Morgan for her assistance in preparing the manuscript.

[double vertical bar] The solubility of compound 5 prevented administration of doses > 250 [micro sign]mol/kg.