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Meeting Abstracts  |   November 1998
Fate and Toxicity of 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A)-derived Mercapturates in Male, Fischer 344 Rats 
Author Notes
  • (Uttamsingh) Predoctoral Trainee, Department of Pharmacology and Physiology.
  • (Iyer) Postdoctoral Research Fellow, Department of Pharmacology and Physiology. Current address: Bristol-Meyers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08453.
  • (Baggs) Associate of Laboratory Animal Medicine and of Environmental Medicine.
  • (Anders) Lewis Pratt Ross Professor and Chair, Department of Pharmacology and Physiology, Professor of Environmental Medicine and of Anesthesiology.
Article Information
Meeting Abstracts   |   November 1998
Fate and Toxicity of 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A)-derived Mercapturates in Male, Fischer 344 Rats 
Anesthesiology 11 1998, Vol.89, 1174-1183. doi:
Anesthesiology 11 1998, Vol.89, 1174-1183. doi:
2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A;Figure 1, compound 1) is the major degradation products of sevoflurane formed in anesthesia circuits in the presence of soda lime or Baralyme (Chemetum, St. Louis, MO). [1-3] Compound A is nephrotoxic when given by inhalation or intraperitoneally to rats. [4-9] It undergoes glutathione-dependent metabolism to yield glutathione S-conjugates, which are hydrolyzed by [small gamma, Greek]-glutamyltransferase and dipeptidases to give the cysteine S-conjugates S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (Figure 1, compound 2) and S-[2-fluoromethoxy-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (Figure 1, compound 4). [10] Compounds 2 and 4 undergo [small beta, Greek]-lyase-dependent biotransformation to nephrotoxic metabolites. [9-12] The compound A-derived mercaptures S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (Figure 1, compound 3) and S-[2-fluoromethoxy-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-cysteine (Figure 1, compound 5) are excreted in the urine of rats given compound A and in urine of humans anesthetized with sevoflurane. [10,12] 
Figure 1. The proposed pathway for biosynthesis and hydrolysis of compound A-derived mercapturates. Compound 1, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A); compound 2, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine; compound 3, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine; compound 3-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]N-acetyl-L-cysteine; compound 4, S-[2-(fluoromethoxy)-tetrafluoro-1-propenyl]-L-cysteine; compound 5, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 5-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 6, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; compound 7, 2[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid; GST, glutathione S-transferase; GSH, glutathione; GGT, [small gamma, Greek]-glutamyltransferase; DP, dipeptidase, [small beta, Greek]-lyase.
Figure 1. The proposed pathway for biosynthesis and hydrolysis of compound A-derived mercapturates. Compound 1, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A); compound 2, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine; compound 3, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine; compound 3-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]N-acetyl-L-cysteine; compound 4, S-[2-(fluoromethoxy)-tetrafluoro-1-propenyl]-L-cysteine; compound 5, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 5-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 6, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; compound 7, 2[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid; GST, glutathione S-transferase; GSH, glutathione; GGT, [small gamma, Greek]-glutamyltransferase; DP, dipeptidase, [small beta, Greek]-lyase.
Figure 1. The proposed pathway for biosynthesis and hydrolysis of compound A-derived mercapturates. Compound 1, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A); compound 2, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine; compound 3, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine; compound 3-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]N-acetyl-L-cysteine; compound 4, S-[2-(fluoromethoxy)-tetrafluoro-1-propenyl]-L-cysteine; compound 5, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 5-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 6, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; compound 7, 2[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid; GST, glutathione S-transferase; GSH, glutathione; GGT, [small gamma, Greek]-glutamyltransferase; DP, dipeptidase, [small beta, Greek]-lyase.
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Mercapturates of several haloalkenes that are structurally similar to compound A are nephrotoxic in vivo and cytotoxic in kidney cells. [13-18] The [small beta, Greek]-lyase inhibitor (aminooxy)acetic acid reduces the nephrotoxicity of these haloalkene-derived mercapturates, indicating hydrolysis of the mercapturates and [small beta, Greek]-lyase-dependent bioactivation of the released cysteine S-conjugates.
The deacylation of N-acyl-L-amino acids is catalyzed by acylases. [19] Acylases I and III catalyze the deacetylation of several haloalkene-derived mercapturates that are nephrotoxic, cytotoxic, and mutagenic cysteine S-conjugates. [20] Thus acylases play important roles in the [small beta, Greek]-dependent bioactivation of haloalkenes and, therefore, contribute to the conjugation-dependent toxicity of haloalkenes.
The current experiments were designed to study the role of acylases in the [small beta, Greek]-lyase-dependent metabolism and the nephrotoxicity of compound A - derived mercapturates. The nephrotoxicity of compound A - derived mercapturates 3 and 5 was studied in male Fischer 344 rats. In addition, the acylase-catalyzed deacetylation of mercapturates 3 and 5 was studied in rat and human kidney cytosol and with acylase I and purified rat kidney acylase III.
Materials and Methods
Materials
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] 
Methods
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).
Synthesis
[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, Rf= 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) tR17.4 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, Rf= 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) tR21.2 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, Rf= 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, Rf= 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.
Results
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.
Figure 2. Kidneys from rats given (A) saline;(B) 500 [micro sign]mol/kg compound 3; or (C) 250 [micro sign]mol/kg compound 5. (A) Control: juxtamedullary region: note the proximal convoluted tubules (P) with abundant brush border. Bar = 50 [micro sign]m. (B) Juxtamedullary region with proximal convoluted tubular epithelium (P) demonstrating focal loss of brush border with excessive intracellular lipid accumulation and occasional pyknotic nucleus (arrow head). Toxicity score of 0.5, PCT. Bar = 50 [micro sign]m. (C) Proximal convoluted tubules (P) in the juxtamedullary region with frank necrosis of the tubular epithelial cell, as indicated by loss of cellular brush border, cytoplasmic basophilia, and loss of nuclei. The necrotic cells have separated from each other and are lifted off the underlying basement membrane. Although the lesion is severe in the affected tubules, some adjacent tubules in the region are less damaged (L), thus a PCT score of 2. Bar = 50 [micro sign]m.
Figure 2. Kidneys from rats given (A) saline;(B) 500 [micro sign]mol/kg compound 3; or (C) 250 [micro sign]mol/kg compound 5. (A) Control: juxtamedullary region: note the proximal convoluted tubules (P) with abundant brush border. Bar = 50 [micro sign]m. (B) Juxtamedullary region with proximal convoluted tubular epithelium (P) demonstrating focal loss of brush border with excessive intracellular lipid accumulation and occasional pyknotic nucleus (arrow head). Toxicity score of 0.5, PCT. Bar = 50 [micro sign]m. (C) Proximal convoluted tubules (P) in the juxtamedullary region with frank necrosis of the tubular epithelial cell, as indicated by loss of cellular brush border, cytoplasmic basophilia, and loss of nuclei. The necrotic cells have separated from each other and are lifted off the underlying basement membrane. Although the lesion is severe in the affected tubules, some adjacent tubules in the region are less damaged (L), thus a PCT score of 2. Bar = 50 [micro sign]m.
Figure 2. Kidneys from rats given (A) saline;(B) 500 [micro sign]mol/kg compound 3; or (C) 250 [micro sign]mol/kg compound 5. (A) Control: juxtamedullary region: note the proximal convoluted tubules (P) with abundant brush border. Bar = 50 [micro sign]m. (B) Juxtamedullary region with proximal convoluted tubular epithelium (P) demonstrating focal loss of brush border with excessive intracellular lipid accumulation and occasional pyknotic nucleus (arrow head). Toxicity score of 0.5, PCT. Bar = 50 [micro sign]m. (C) Proximal convoluted tubules (P) in the juxtamedullary region with frank necrosis of the tubular epithelial cell, as indicated by loss of cellular brush border, cytoplasmic basophilia, and loss of nuclei. The necrotic cells have separated from each other and are lifted off the underlying basement membrane. Although the lesion is severe in the affected tubules, some adjacent tubules in the region are less damaged (L), thus a PCT score of 2. Bar = 50 [micro sign]m.
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Table 1. Morphological Assessment of Kidney and Liver Damage in Rats Given S-[2-(Fluoromethoxy)]1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3) and S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5)
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Table 1. Morphological Assessment of Kidney and Liver Damage in Rats Given S-[2-(Fluoromethoxy)]1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3) and S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5)
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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).
Figure 3.19F NMR of the urine of a rat given 500 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 3.19F NMR of the urine of a rat given 500 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 3.19F NMR of the urine of a rat given 500 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
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Figure 4.19F NMR of the urine of a rat given 250 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1 -propenyl]N-acetyl-L-cysteine (compound 5) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 4.19F NMR of the urine of a rat given 250 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1 -propenyl]N-acetyl-L-cysteine (compound 5) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 4.19F NMR of the urine of a rat given 250 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1 -propenyl]N-acetyl-L-cysteine (compound 5) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
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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).
Figure 5. Excretion of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) and [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine and of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L -cysteine (compound 5) and [acetyl-(2) H3]S-[2(fluoromethoxy)-1,3,3,3 -tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (compound 5-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Rats were given 125 [micro sign]mol/kg compound 3-d3or 5-d3intraperitoneally, and the fraction of the administered dose excreted from 0-24 h as compounds 3 and 3-d3or compounds 5 and 5-d3, respectively, was quantified by gas chromatography--mass spectrometry, as described in the Materials and Methods section. Data are shown as mean +/− SEM (n = 3).
Figure 5. Excretion of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) and [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine and of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L -cysteine (compound 5) and [acetyl-(2) H3]S-[2(fluoromethoxy)-1,3,3,3 -tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (compound 5-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Rats were given 125 [micro sign]mol/kg compound 3-d3or 5-d3intraperitoneally, and the fraction of the administered dose excreted from 0-24 h as compounds 3 and 3-d3or compounds 5 and 5-d3, respectively, was quantified by gas chromatography--mass spectrometry, as described in the Materials and Methods section. Data are shown as mean +/− SEM (n = 3).
Figure 5. Excretion of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) and [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine and of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L -cysteine (compound 5) and [acetyl-(2) H3]S-[2(fluoromethoxy)-1,3,3,3 -tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (compound 5-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Rats were given 125 [micro sign]mol/kg compound 3-d3or 5-d3intraperitoneally, and the fraction of the administered dose excreted from 0-24 h as compounds 3 and 3-d3or compounds 5 and 5-d3, respectively, was quantified by gas chromatography--mass spectrometry, as described in the Materials and Methods section. Data are shown as mean +/− SEM (n = 3).
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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] 
Table 2. Biotransformation of S-[2-(Fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3), S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5), S-(2-Chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine by Rat and Human Kidney Cytosol, Acylase I, and Acylase
Image not available
Table 2. Biotransformation of S-[2-(Fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3), S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5), S-(2-Chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine by Rat and Human Kidney Cytosol, Acylase I, and Acylase
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Discussion
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.
REFERENCES
Frink EJ Jr, Malan TP, Morgan SE, Brown EA, Malcomson M, Brown BR Jr: Quantification of the degradation products of sevoflurane in two CO2absorbants during low-flow anesthesia in surgical patients. Anesthesiology 1992; 77:1064-9
Fang ZX, Eger EI II: Factors affecting the concentration of compound A resulting from the degradation of sevoflurane by soda lime and Baralyme[registered sign] in a standard anesthetic circuit. Anesth Analg 1995; 81:564-8
Bito H, Ikeda K: Effect of total flow rate on the concentration of degradation products generated by the reaction between sevoflurane and soda lime. Br J Anaesth 1995; 74:667-9
Gonsowski CT, Laster MJ, Eger II EI, Ferrell LD, Kerschmann RL: Toxicity of compound A in rats. Effect of a 3-hour administration. Anesthesiology 1994; 80:556-65
Gonsowski CT, Laster MJ, Eger II EI, Ferrell LD, Kerschmann RL: Toxicity of compound A in rats. Effect of increasing duration of administration. Anesthesiology 1994; 80:566-73
Jin L, Baillie TA, Davis MR, Kharasch ED: Nephrotoxicity of sevoflurane compound A [fluoromethyl-2,2-difluoro-1-(trifluoromethyl-)vinyl ether] in rats: Evidence for glutathione and cysteine conjugate formation and the role of renal cysteine conjugate [small beta, Greek]-lyase. Biochem Biophys Res Commun 1995; 210:498-506
Kandel L, Laster MJ, Eger EI II, Kerschmann RL, Martin J: Nephrotoxicity in rats undergoing a one-hour exposure to compound A. Anesth Analg 1995; 81:559-63
Keller KA, Callan C, Prokocimer P, Delgado-Herrera L, Friedman MB, Hoffman GM, Wooding WL, Cusick PK, Krasula RW: Inhalation toxicity study of a haloalkene degradant of sevoflurane, compound A (PIFE), in Sprague-Dawley rats. Anesthesiology 1995; 83:1220-32
Kharasch ED, Thorning D, Garton K, Hankins DC Kilty GC: Role of renal cysteine conjugate [small beta, Greek]-lyase in the mechanism of compound A nephrotoxicity in rats. Anesthesiology 1997; 86:160-71
Jin L, Davis MR, Kharasch ED, Doss GA, Baillie TA: 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 1996; 9:555-61
Spracklin DK, Kharasch ED: Evidence for metabolism of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A), a sevoflurane degradation product, by cysteine conjugate [small beta, Greek]-lysate. Chem Res Toxicol 1996; 9:696-702
Iyer RA, Frink EJ Jr, Ebert TJ, Anders MW: Cysteine conjugate [small beta, Greek]-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 1998; 88:611-8
Pratt IS, Lock EA: Deacetylation and further metabolism of the mercapturic acid of hexachloro-1,3-butadiene by rat kidney cytosol in vitro. Arch Toxicol 1988; 62:341-5
Vamvakas S, Dekant W, Berthold K, Schmidt S, Wild D, Henschler D: Enzymatic transformation of mercapturic acids derived from halogenated alkenes to reactive and mutagenic intermediates. Biochem Pharmacol 1987; 36:2741-8
Ishmael J, Lock EA: Nephrotoxicity of hexachlorobutadiene and its glutathione-derived conjugates. Toxicol Pathol 1986; 14:258-62
Boogaard PJ, Commandeur JNM, Mulder GJ, Vermeulen NPE, Nagelkerke JF: Toxicity of the cysteine-S-conjugates 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 1989; 38:3731-41
Commandeur JNM, Brakenhoff JPG, De Kanter FJJ, Vermeulen NPE: Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Biochem Pharmacol 1988; 37:4495-504
Wolfgang GHI, Gandolfi AJ, Stevens JL, Brendel K: N-Acetyl S-(1,2-dichlorovinyl)-L-cysteine produces a similar toxicity to S-(1,2-dichlorovinyl)-L-cysteine in rabbit renal slices: Differential transport and metabolism. Toxicol Appl Pharmacol 1989; 101:205-19
Anders MW, Dekant W: Aminoacylases. Adv Pharmacol 1994; 27:433-50
Uttamsingh V, Anders MW: Purification and characterization of an aminoacylase catalyzing the hydrolysis of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine. Fundam Appl Toxicol 1996; 30(Suppl 1, Part 2):314
Iyer RA, Anders MW: Cysteine conjugate [small beta, Greek]-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A). Chem Res Toxicol 1997; 10:811-9
Dekant W, Metzler M, Henschler D: Identification of S-1,2-dichlorovinyl-N-acetyl-cysteine as a urinary metabolite of trichloroethylene: A possible explanation for its nephrocarcinogenicity in male rats. Biochem Pharmacol 1986; 35:2455-8
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-54
Onkenhout W, Vermeulen NPE, Luijten WCMM, de Jong HJ: Electron impact mass spectrometric analysis of mercapturic acid methyl esters. Biomed Mass Spectrom 1983; 10:614-9
Udenfriend S, Stein S, Bohlen P, Dairman W, Leimgruber W, Weigele M: Fluoroscamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 1972; 178:871-2
Commandeur JNM, Boogaard PJ, Mulder GJ, Vermeulen NPE: Mutagenicity and cytotoxicity of two regioisomeric mercapturic acids and cysteine S-conjugates of trichloroethylene. Arch Toxicol 1991; 65:373-80
Cooper AJL: Enzymology of cysteine S-conjugate [small beta, Greek]-lyases. Adv Pharmacol 1994; 27:73-115
Commandeur JNM, Stijntjes GJ, Wijngaard J, Vermeulen NPE: Metabolism of L-cysteine S-conjugates and N-(trideuteroacetyl)-L-cysteine S-conjugates of four fluoroethylenes in the rat. Role of balance of deacetylation and acetylation in relation to the nephrotoxicity of mercapturic acids. Biochem Pharmacol 1991; 42:31-8
Stijntjes GJ, Commandeur JNM, te Koppele JM, McGuinness S, Gandolfi AJ, Vermeulen NPE: Examination of structure-toxicity relationships of L-cysteine-S-conjugates of halogenated alkenes and their corresponding mercapturic acids in rat renal tissue slices. Toxicology 1993; 79:67-79
Larsen GL, Bakke JE: Metabolism of mercapturic acid-pathway metabolites of 2-chloro-N-isopropylacetanilide (propachlor) by gastrointestinal bacteria. Xenobiotica 1983; 13:115-26
Iyer RA, Baggs RB, Anders MW: Nephrotoxicity of the glutathione and cysteine conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) in male, Fischer 344 rats. J Pharmacol Exp Ther 1997; 283:1544-51
Morio M, Fujii K, Satoh N, Imai M, Kawakami T, Negishi A, Kumagai Y, Kawai T: Reaction of sevoflurane and its degradation products with soda lime. Toxicity of the byproducts. Anesthesiology 1992; 77:1155-64
Martin JL, Kandel L, Laster MJ, Kerschmann RL, Eger EI II: Studies on the mechanism of nephrotoxicity of compound A in rats. J Anesthesiol 1997; 11:32-7
Iyer RA, Anders MW: Cysteine conjugate [small beta, Greek]-lyase-dependent bio-transformation of the cysteine S-conjugates of the sevoflurane degradation product compound A in human, nonhuman primate, and rat renal cytosol and mitochondria. Anesthesiology 1996; 85:1454-61
Martin JL, Laster MJ, Kandel L, Kerschmann RL, Reed GF, Eger II EI: Metabolism of compound A by renal cysteine-S-conjugate [small beta, Greek]-lyase is not the mechanism of compound A-induced renal injury in the rat. Anesth Analg 1996; 82:770-4
Figure 1. The proposed pathway for biosynthesis and hydrolysis of compound A-derived mercapturates. Compound 1, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A); compound 2, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine; compound 3, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine; compound 3-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]N-acetyl-L-cysteine; compound 4, S-[2-(fluoromethoxy)-tetrafluoro-1-propenyl]-L-cysteine; compound 5, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 5-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 6, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; compound 7, 2[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid; GST, glutathione S-transferase; GSH, glutathione; GGT, [small gamma, Greek]-glutamyltransferase; DP, dipeptidase, [small beta, Greek]-lyase.
Figure 1. The proposed pathway for biosynthesis and hydrolysis of compound A-derived mercapturates. Compound 1, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A); compound 2, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine; compound 3, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine; compound 3-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]N-acetyl-L-cysteine; compound 4, S-[2-(fluoromethoxy)-tetrafluoro-1-propenyl]-L-cysteine; compound 5, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 5-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 6, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; compound 7, 2[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid; GST, glutathione S-transferase; GSH, glutathione; GGT, [small gamma, Greek]-glutamyltransferase; DP, dipeptidase, [small beta, Greek]-lyase.
Figure 1. The proposed pathway for biosynthesis and hydrolysis of compound A-derived mercapturates. Compound 1, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A); compound 2, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine; compound 3, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine; compound 3-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]N-acetyl-L-cysteine; compound 4, S-[2-(fluoromethoxy)-tetrafluoro-1-propenyl]-L-cysteine; compound 5, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 5-d3, [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; compound 6, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; compound 7, 2[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3 -thiazole-4-carboxylic acid; GST, glutathione S-transferase; GSH, glutathione; GGT, [small gamma, Greek]-glutamyltransferase; DP, dipeptidase, [small beta, Greek]-lyase.
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Figure 2. Kidneys from rats given (A) saline;(B) 500 [micro sign]mol/kg compound 3; or (C) 250 [micro sign]mol/kg compound 5. (A) Control: juxtamedullary region: note the proximal convoluted tubules (P) with abundant brush border. Bar = 50 [micro sign]m. (B) Juxtamedullary region with proximal convoluted tubular epithelium (P) demonstrating focal loss of brush border with excessive intracellular lipid accumulation and occasional pyknotic nucleus (arrow head). Toxicity score of 0.5, PCT. Bar = 50 [micro sign]m. (C) Proximal convoluted tubules (P) in the juxtamedullary region with frank necrosis of the tubular epithelial cell, as indicated by loss of cellular brush border, cytoplasmic basophilia, and loss of nuclei. The necrotic cells have separated from each other and are lifted off the underlying basement membrane. Although the lesion is severe in the affected tubules, some adjacent tubules in the region are less damaged (L), thus a PCT score of 2. Bar = 50 [micro sign]m.
Figure 2. Kidneys from rats given (A) saline;(B) 500 [micro sign]mol/kg compound 3; or (C) 250 [micro sign]mol/kg compound 5. (A) Control: juxtamedullary region: note the proximal convoluted tubules (P) with abundant brush border. Bar = 50 [micro sign]m. (B) Juxtamedullary region with proximal convoluted tubular epithelium (P) demonstrating focal loss of brush border with excessive intracellular lipid accumulation and occasional pyknotic nucleus (arrow head). Toxicity score of 0.5, PCT. Bar = 50 [micro sign]m. (C) Proximal convoluted tubules (P) in the juxtamedullary region with frank necrosis of the tubular epithelial cell, as indicated by loss of cellular brush border, cytoplasmic basophilia, and loss of nuclei. The necrotic cells have separated from each other and are lifted off the underlying basement membrane. Although the lesion is severe in the affected tubules, some adjacent tubules in the region are less damaged (L), thus a PCT score of 2. Bar = 50 [micro sign]m.
Figure 2. Kidneys from rats given (A) saline;(B) 500 [micro sign]mol/kg compound 3; or (C) 250 [micro sign]mol/kg compound 5. (A) Control: juxtamedullary region: note the proximal convoluted tubules (P) with abundant brush border. Bar = 50 [micro sign]m. (B) Juxtamedullary region with proximal convoluted tubular epithelium (P) demonstrating focal loss of brush border with excessive intracellular lipid accumulation and occasional pyknotic nucleus (arrow head). Toxicity score of 0.5, PCT. Bar = 50 [micro sign]m. (C) Proximal convoluted tubules (P) in the juxtamedullary region with frank necrosis of the tubular epithelial cell, as indicated by loss of cellular brush border, cytoplasmic basophilia, and loss of nuclei. The necrotic cells have separated from each other and are lifted off the underlying basement membrane. Although the lesion is severe in the affected tubules, some adjacent tubules in the region are less damaged (L), thus a PCT score of 2. Bar = 50 [micro sign]m.
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Figure 3.19F NMR of the urine of a rat given 500 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 3.19F NMR of the urine of a rat given 500 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 3.19F NMR of the urine of a rat given 500 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
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Figure 4.19F NMR of the urine of a rat given 250 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1 -propenyl]N-acetyl-L-cysteine (compound 5) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 4.19F NMR of the urine of a rat given 250 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1 -propenyl]N-acetyl-L-cysteine (compound 5) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
Figure 4.19F NMR of the urine of a rat given 250 [micro sign]mol/kg S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1 -propenyl]N-acetyl-L-cysteine (compound 5) intraperitoneally. The urine samples were analyzed as described in the Materials and Methods section. The spectrum shown is typical of the spectra recorded in three rats.
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Figure 5. Excretion of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) and [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine and of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L -cysteine (compound 5) and [acetyl-(2) H3]S-[2(fluoromethoxy)-1,3,3,3 -tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (compound 5-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Rats were given 125 [micro sign]mol/kg compound 3-d3or 5-d3intraperitoneally, and the fraction of the administered dose excreted from 0-24 h as compounds 3 and 3-d3or compounds 5 and 5-d3, respectively, was quantified by gas chromatography--mass spectrometry, as described in the Materials and Methods section. Data are shown as mean +/− SEM (n = 3).
Figure 5. Excretion of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) and [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine and of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L -cysteine (compound 5) and [acetyl-(2) H3]S-[2(fluoromethoxy)-1,3,3,3 -tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (compound 5-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Rats were given 125 [micro sign]mol/kg compound 3-d3or 5-d3intraperitoneally, and the fraction of the administered dose excreted from 0-24 h as compounds 3 and 3-d3or compounds 5 and 5-d3, respectively, was quantified by gas chromatography--mass spectrometry, as described in the Materials and Methods section. Data are shown as mean +/− SEM (n = 3).
Figure 5. Excretion of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (compound 3) and [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (compound 3-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine and of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L -cysteine (compound 5) and [acetyl-(2) H3]S-[2(fluoromethoxy)-1,3,3,3 -tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (compound 5-d3) in rats given [acetyl-(2) H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Rats were given 125 [micro sign]mol/kg compound 3-d3or 5-d3intraperitoneally, and the fraction of the administered dose excreted from 0-24 h as compounds 3 and 3-d3or compounds 5 and 5-d3, respectively, was quantified by gas chromatography--mass spectrometry, as described in the Materials and Methods section. Data are shown as mean +/− SEM (n = 3).
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Table 1. Morphological Assessment of Kidney and Liver Damage in Rats Given S-[2-(Fluoromethoxy)]1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3) and S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5)
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Table 1. Morphological Assessment of Kidney and Liver Damage in Rats Given S-[2-(Fluoromethoxy)]1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3) and S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5)
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Table 2. Biotransformation of S-[2-(Fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3), S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5), S-(2-Chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine by Rat and Human Kidney Cytosol, Acylase I, and Acylase
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Table 2. Biotransformation of S-[2-(Fluoromethoxy)-1,1,3,3,3 -pentafluoropropyl]-N-acetyl-L-cysteine (Compound 3), S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine (Compound 5), S-(2-Chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine by Rat and Human Kidney Cytosol, Acylase I, and Acylase
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