Cysteine Conjugate β-Lyase-dependent Biotransformation of the Cysteine S-Conjugates of the Sevoflurane Degradation Product Compound A in Human, Nonhuman Primate, and Rat Kidney Cytosol and Mitochondria

Compound A was provided by Abbott Laboratories, Abbott Park, Illinois. (Aminooxy)acetic acid and 2-keto-4-methylthiolbutyric acid were obtained from Sigma Chemical Company, St. Louis, Missouri. Cysteine conjugates 4, 5, and 11 were synthesized by published procedures, [19]and the identity and purity of the compounds were established by¹Hydrogen and¹⁹Fluorine nuclear magnetic resonance spectroscopy. The diastereomeric configuration of synthetic conjugate 4 and the geometric configuration of synthetic conjugate 5 have not been established. (Detailed descriptions of the syntheses and characterization will be reported in another publication.)

Male Fischer 344 rats (weighing 200-220 g; Charles River Laboratories, Wilmington, MA) were used. The protocol for animal use was reviewed and approved by the University Committee on Animal Resources. The rats were anesthetized with ether and killed by cardiac puncture. The kidneys were removed and homogenized in 0.1 M phosphate buffer (pH 7.4) and fractionated according to published procedures. [16]The cytosolic and mitochondrial fractions were dialyzed in Spectrapor dialysis membranes (molecular weight cutoff of MW 3000; Spectrum Medical Laboratories, Houston, TX) for 36 h at 4 degrees Celsius in 0.1 M phosphate buffer (pH 7.4). Protein concentrations were determined by the Bradford assay with bovine serum albumin as the standard. [20]Frozen (-80 degrees Celsius) human kidney tissues (provided by Dr. Evan Kharasch, University of Washington, Seattle, WA) were thawed before homogenization; the fractionation and dialysis were similar to the procedures used for the rat kidney fractions. The monkey kidney tissues were obtained from Analytical Biological Services (Wilmington, DE) and from BioWittaker (Walkersville, MD). The kidneys were transported on dry ice and stored at -80 degrees Celsius; the fractionation and dialysis procedures were the same as just described.

Reaction mixtures contained cytosol or mitochondria (2 mg protein/ml), cysteine S-conjugates (the concentrations are shown in the tables), and 0.1 M phosphate buffer (pH 7.4) in a total volume of 0.5 ml (rat tissue) or 0.25 ml (human and nonhuman primate tissues). The reaction mixtures were incubated for 30 to 45 min, as indicated in the tables, at 37 degrees Celsius with constant shaking. The reaction was terminated by adding a 30% (vol/vol) solution of trichloroacetic acid in water to give a pH 2.0. [12]The precipitated proteins were removed by centrifugation. The pyruvate concentrations in the supernatant were quantified enzymatically or chromatographically. In the enzymatic method, the pyruvate released was converted to alanine by alanine dehydrogenase in the presence of reduced nicotinamide adenine dinucleotide. [21]The progress of the reaction was monitored by measuring the decrease in absorbance at 340 nm in a Beckman DU-64 spectrophotometer (Beckman Instruments, Fullerton, CA). In the high-pressure liquid chromatography (HPLC) method, the pyruvate formed was derivatized by reaction with 0.1 M o-phenylenediamine in water and analyzed by high-pressure liquid chromatography. [22]Samples were analyzed on a Hewlett-Packard (Wilmington, DE) 1090 liquid chromatograph fitted with a Radial-Pak cartridge packed with Resolve C-18 RCM column (8 mm inner diameter x 100 mm; Millipore Corp., Milford, MA). The eluent was methanol:water:acetic acid (45:54:1) at a flow rate of 1 ml/min, and the fluorescence intensity of the eluate was measured with a Gilson model 121 fluorometer (Gilson International, Madison, WI). Heat-inactivated cytosol or mitochondria was prepared by heating the cytosolic or mitochondrial fractions in a boiling water bath for 3 or 4 min. Heat-inactivated proteins were used as a negative control. (Aminooxy)acetic acid (1 to 40 micro Meter), a beta-lyase inhibitor, [23]was used to determine the kinetics of the inhibition of beta-lyase activity.

Kinetic constants were calculated with the E-Z FIT enzyme kinetic model fitting program (version 2, Perrella Scientific, Springfield, PA). The data in Table 1and Table 2were analyzed by analysis of variance. For each fraction, a two-factor (species and conjugate) analysis of variance, which provides tests for each of the two factors separately as well as a test for interaction, was used. In addition, three pairwise comparisons (t tests), which compared the three species separately for each of the three conjugates, were performed.

Cysteine conjugates 4, 5, and 11 were biotransformed to pyruvate by rat, human, and cynomolgus-monkey kidney cytosol and mitochondria (Table 1). No activity was observed with heat-inactivated cytosol or mitochondria. The observation that the cysteine S-conjugates were converted to pyruvate, which is the product of a beta-lyase-catalyzed beta-elimination reaction of cysteine S-conjugates, [24]indicates a role for beta-lyase. Furthermore, (aminooxy)acetic acid, a known inhibitor of beta-lyase, [23]inhibited the formation of pyruvate. The inhibition constant (Kⁱ) for the inhibition by (aminooxy)acetic acid of the biotransformation of conjugates 4, 5, and 11 in rat kidney cytosol were 10.3 +/- 0.88, 6.75, and 7.00 +/- 0.58 micro Meter, respectively (mean +/- SE; n = 3 for conjugates 4 and 11; average, n = 2 for conjugate 5). The biotransformation of conjugates 4, 5, and 11 was increased 1.4 to 2 times in human kidney cytosol in the presence of 5 mM 2-keto-4-methylthiolbutyric acid, which had little effect on biotransformation in rat kidney cytosol (data not shown). Cysteine conjugate 11 is a known substrate for beta-lyase and was used as a positive control [12]; the specific activity of beta-lyase with conjugate 11 as the substrate in rat kidney cytosol was similar to that reported in the literature. [25] 

Beta-Lyase activity with conjugates 4, 5, and 11 as substrates was quantified in rat, human, and cynomolgus-monkey kidney cytosol (Table 1). Analysis of the data in Table 1by analysis of variance showed that the rate of biotransformation of conjugates 4, 5, and 11 was lower in human kidney cytosol than in rat kidney cytosol (P < 0.0001) and that the rate of biotransformation of conjugates 4 and 11 in cytosol from cynomolgus monkey was higher than in human cytosol (P < 0.0001). The rate of biotransformation of conjugate 5 in human kidney cytosol was not statistically different (P < 0.055) from the rate in monkey kidney cytosol.

Beta-Lyase activity with conjugates 4, 5, and 11 as substrates was measured in rat, human, and nonhuman-primate mitochondria (Table 1). Analysis of variance showed that the rates of biotransformation of conjugates 4, 5, and 11 were significantly lower in human kidney mitochondria than in rat kidney mitochondria (P < 0.0001) and that rates in cynomolgus-monkey kidney mitochondria were significantly higher than in human kidney mitochondria (P < 0.0001).

The biotransformation of conjugates 4, 5, and 11 was also studied in rhesus monkey (Macaca mulatta) and marmoset (Callithrix jacchus) cytosol and mitochondria. In rhesus monkey kidney cytosol, the rates of biotransformation of conjugates 4, 5, and 11 were 0.12 (0.12, 0.12), 0.11 (0.11, 0.12) and 0.64 (0.77, 0.51) nmol pyruvate/mg protein/min, respectively (n = 2, individual values are shown in parentheses; see Table 1for details of the experiments). The rates for rhesus monkey kidney mitochondria were 0.043 (0.015, 0.072), 0.07 (0.048, 0.098), and 0.25 (0.23, 0.28) nmol pyruvate/mg protein/min, respectively (n = 2). In marmoset kidney cytosol, the rates of biotransformation of conjugates 4, 5, and 11 were 0.063, 0.097, and 0.26 nmol pyruvate/mg protein/min, respectively (n = 1; see Table 1for details of the experiments). The rates for marmoset kidney mitochondria were 0 015, 0.089, and 0.18, respectively (n = 1). Although the small sample size precludes statistical analysis, the data for the rhesus monkeys and for the marmoset were similar to those observed in the cynomolgus monkey.

The rate of biotransformation of conjugates 4 and 5 was low compared with conjugate 11 in all species and in both mitochondria and cytosol. In addition, the specific activity in mitochondria was as much as five times lower than that seen in the cytosol with all substrates and in all species tested.

The kinetics (K^m, V^max, and V^max/K^m) of the biotransformation of conjugates 4, 5, and 11 in rat and human kidney cytosol was also studied (Table 2). The V^maxvalues obtained with rat kidney beta-lyase were higher for conjugates 4, 5, and 11 compared with human tissue, whereas the K^mvalues were similar in rat and human kidney cytosol for all three conjugates. The V^max/K^maxratios for the cysteine conjugates 4, 5, and 11 in human kidney cytosol were 2 to 25 times lower than in rat kidney cytosol (analysis of variance; P < 0001, P = 0.0009, P < 0.0001, respectively).

The present investigations were designed to test the hypothesis that the compound A-derived cysteine conjugates 4 and 5 are substrates for kidney cysteine conjugate beta-lyase and to compare the rates of biotransformation of the three cysteine S-conjugates in human, nonhuman primate, and rat kidney tissue. The biotransformation of cysteine conjugate 11, which is a known substrate for beta-lyase, [12]was used as a positive control.

The beta-lyase pathway was established as an important bioactivation mechanism for a range of nephrotoxic halogenated alkenes. [9-11]This pathway includes hepatic glutathione transferase-catalyzed glutathione S-conjugate formation and renal beta-lyase-dependent bioactivation of halogenated alkene-derived cysteine conjugates. Tetrafluoroethylene, chlorotrifluoroethylene, hexafluoropropene, and 2-bromo-2-chloro-1,1-difluoroethylene, which is a degradation product of halothane, [3]are toxic to the kidneys, [26,27]and all undergo bioactivation by the beta-lyase pathway. [12,28-30]Compound A is an analog of several nephrotoxic fluorinated alkenes that undergo glutathione transferase-catalyzed glutathione S-conjugate formation and beta-lyase-dependent bioactivation of the corresponding cysteine S-conjugates. Evidence that compound A is a substrate for hepatic glutathione transferases was recently reported: Compound A-derived glutathione conjugates are present in the bile of rats given compound A, and the corresponding mercapturic acids are excreted in urine. [8,14]Furthermore, the nephrotoxicity of compound A was inhibited by the beta-lyase inhibitor (aminooxy)acetic acid. Recent studies also show that cysteine S-conjugates 4 and 5 are substrates for rat kidney beta-lyase. [15] 

The results presented here show that the compound A-derived cysteine S-conjugates 4 and 5 are substrates for human, nonhuman primate, and rat kidney beta-lyase. The cysteine S-conjugates were biotransformed to pyruvate, which is a known product of a beta-lyase-catalyzed beta-elimination reaction of cysteine S-conjugates. [24,31]The failure to observe pyruvate formation when heat-inactivated tissue fractions were used indicates that pyruvate formation was enzyme catalyzed. In addition, the biotransformation of conjugates 4, 5, and 11 was inhibited by the beta-lyase inhibitor (aminooxy)acetic acid. [23]Finally, the biotransformation of conjugates 4, 5, and 11 by human kidney cytosol was stimulated by 2-keto-4-methylthiobutyric acid. Beta-Lyase catalyzes both transamination and beta-elimination reactions of cysteine S-conjugates, and beta-lyase-catalyzed transamination reactions convert enzyme-bound pyridoxal phosphate to pyridoxamine phosphate. [11,32]The stimulation of beta-lyase activity by 2-keto-4-methylthiobutyric acid can be attributed to the conversion of the pyridoxamine form of the beta-lyase to the pyridoxal phosphate form of the beta-lyase, which is competent to catalyze beta-elimination reactions. 2-Keto-4-methylthiobutyric acid stimulated cysteine S-conjugates biotransformation in human, but not in rat, kidney cytosol. The reason for this difference is not known but may be due to different rates of transamination reactions between the two species or to the contribution of L-amino acid oxidase activity, which converts cysteine S-conjugates to alpha-keto acids. [33] 

The demonstration of a role for beta-lyase in the biotransformation of compound A-derived cysteine conjugates allows the proposal of a scheme for the metabolism of compound A. Compound A may undergo glutathione transferase-catalyzed glutathione-conjugate formation to yield S-[2(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]glutathione 2 and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]glutathione 3. [8,14]gamma Glutamyltransferase- and dipeptidase-catalyzed hydrolysis of the glutathione conjugates would produce cysteine S-conjugates 4 and 5. Acetylation of conjugates 4 and 5 would give the corresponding mercapturic acids, which have been detected in the urine of rats given compound A. [8,14]The beta-lyase-catalyzed transformation of conjugate 4 would be expected to afford 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropanethiol 6, which may lose fluoride to yield 2-(fluoromethoxy)-3,3,3-trifluorothiopropanoyl fluoride 8 (Figure 1). The expected product obtained from the beta-lyase-catalyzed biotransformation of conjugate 5 is 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenethiol 7, which may tautomerize to give the same thioacylating agent 8 as that obtained from conjugate 4. Thioacylating agent 8 may react with tissue nucleophiles or may undergo hydrolysis to give 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid 9. Examination of reaction mixtures containing conjugates 4 and 5 and rat kidney tissue by¹⁹F nuclear magnetic resonance spectroscopy showed the formation of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid 9 as a stable, terminal product (R. Iyer and M. W. Anders, unpublished observations); acid 9 has been identified by gas chromatography/mass spectrometry as a product of the metabolism of conjugates 4 and 5. [15]Beta-Lyase activity with cysteine conjugates 4, 5, and 11 was detected in human, nonhuman primate, and rat kidney tissue. With all substrates and in all species, kidney beta-lyase activity was greater in cytosol than in mitochondria, which corresponds to previous observations. [16] 

With all substrates studied, activities were highest in rat kidney cytosol and mitochondria (Table 1). With conjugate 4 as the substrate, the rank order of activities in kidney cytosol was rat > cynomolgus > human; in kidney mitochondria, the rank order of activities was rat > cynomolgus > human. Similar results were obtained with conjugate 5 as the substrate: the rank order of activities in kidney cytosol was rat > cynomolgus > human, and the rank order in kidney mitochondria was rat > cynomolgus > human. These results confirm previous observations that show that human kidney beta-lyase activity is much lower than that present in rat kidney. [16-18] 

Kinetic analysis confirms the observation that kidney cytosolic beta-lyase activity is lower in humans than in rats. The K^mvalues for conjugates 4, 5, and 11 were similar in rat and human kidney cytosol. The V^maxfor conjugate 4 biotransformation in rat kidney cytosol was about seven times higher than in human kidney cytosol, whereas the V^maxfor conjugate 5 biotransformation in rat kidney cytosol was about four times higher than in human kidney cytosol. The V^maxfor conjugate 11 was 10 to 15 times higher than that of conjugates 4 and 5. Thus the kinetic data and specific activity measurements both show that beta-lyase activity is lower in human kidney tissue compared with rat kidney tissue. The ratio V^max/K^mapproximates the first-order rate constant for an enzyme-catalyzed reaction and provides an estimate of rates of biotransformation at low substrate concentrations. [34]The V^max/K sub m ratios for conjugates 4 and 5 were significantly lower in human kidney cytosol than in rat kidney cytosol (Table 2). The V^max/K^mratio for conjugate 11 in rat kidney cytosol is similar to that reported previously. [35] 

These data are important for understanding the potential nephrotoxic effects of compound A. The beta-lyase-catalyzed biotransformation of cysteine S-conjugates is a key step in the bioactivation of halogenated alkenes. [9-11]Although compound A is nephrotoxic in rats, [4-8]compound A-related nephrotoxicity has not been observed in humans given sevoflurane anesthesia. [2,36-40]Similarly, nephrotoxic effects were not observed in cynomolgus monkeys exposed to sevoflurane for 3 h per day, 3 days per week for 8 weeks, but compound A concentrations were not quantified. [41]Although the present results and work from other laboratories [8,14,15]indicate involvement of the beta-lyase pathway in the bioactivation of compound A, the role of the beta-lyase in compound A-induced nephrotoxicity in rats has been challenged, [42]and more studies are needed to elaborate the mechanism of compound A-induced nephrotoxicity.

Halothane undergoes base-catalyzed degradation to afford 2-bromo-2-chloro-1,1-difluoroethylene, [3]which is a potent nephrotoxin in rats. [26]S-(2-Bromo-2-chloro-1, 1-difluoroethyl)-N-acetyl-L-cysteine, the mercapturic acid of 2-bromo-2-chloro-1,1-difluoroethylene, is excreted in the urine of humans anesthetized with halothane, [43,44]indicating that 2-bromo-2-chloro-1,1-difluoroethylene undergoes glutathione-dependent metabolism. Furthermore, S-(2-bromo-2-chloro-1,1-difluoroethyl)glutathione and S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, the glutathione and cysteine conjugates of 2-bromo-2-chloro-1,1-difluoroethylene, are nephrotoxic in rats and cytotoxic in kidney-derived, cultured LLC-PK1 cells. [30,45]S-(2-Bromo-2-chloro-1,1-difluoroethyl)-L-cysteine is a substrate for beta-lyase. [46]Although 2-bromo-2-chloro-1,1 difluoroethylene is nephrotoxic and undergoes glutathione- and beta-lyase-dependent bioactivation, nephrotoxicity attributable to 2-bromo-2-chloro-1,1-difluoroethylene formation and bioactivation has not been observed after decades of widespread use of halothane as an anesthetic in humans. The concentration of 2-bromo-2-chloro-1,1-difluoroethylene in the expired gases of patients anesthetized with halothane amounts to 4 to 5 ppm, [3]and its median lethal concentration in mice is 250 ppm. [26]The low concentrations of 2-bromo-2-chloro-1,1-difluoroethylene in the anesthetic circuit, along with low human kidney beta-lyase activities, may explain the lack of 2-bromo-2-chloro-1,1-difluoroethylene-induced nephrotoxic effects in humans anesthetized with halothane. Thus the formation of fluorinated alkenes as degradation products of halogenated anesthetics does not necessarily lead to human toxicity. Thorough clinical evaluation of humans anesthetized with sevoflurane is needed to establish its safety.

The authors thank Evan D. Kharasch, MD, PhD, Departments of Anesthesiology and Medicinal Chemistry, University of Washington, Seattle, Washington, for providing samples of human kidney tissue; Christopher Cox, PhD, Department of Biostatistics and Environmental Health Sciences Center, University of Rochester, for statistical analyses; and Ramez Awwad for technical assistance.