1332
Chem. Res. Toxicol. 1998, 11, 1332-1338
Glutathione Transferase Zeta-Catalyzed Biotransformation of Dichloroacetic Acid and Other r-Haloacids Zeen Tong,†,‡ Philip G. Board,§ and M. W. Anders*,† Department of Pharmacology & Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, and Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, GPO Box 34, Canberra ACT 2601, Australia Received June 18, 1998
Dichloroacetic acid (DCA) is a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clinically in the management of lactic acidosis. Recent studies show that glutathione transferase Zeta (GSTZ) catalyzes the oxygenation of DCA to glyoxylic acid [Tong et al. (1998) Biochem. J. 331, 371-374]. In the present studies, the substrate selectivity of GSTZ, the kinetics of DCA metabolism, and the fate of DCA and glutathione were investigated. The results showed that GSTZ catalyzed the oxygenation of bromochloro-, bromofluoro-, chlorofluoro-, dibromo-, and dichloroacetic acid, but not difluoroacetic acid, to glyoxylic acid. GSTZ also catalyzed the biotransformation of fluoroacetic acid to S-(carboxymethyl)glutathione, and of (R,S)-2-bromopropionic acid, (R)-, (S)-, and (R,S)-2chloropropionic acid, and (R,S)-2-iodopropionic acid, but not (R,S)-2-fluoropropionic acid, to S-(R-methylcarboxymethyl)glutathione; and of 2,2-dichloropropionic acid to pyruvate. No biotransformation of 3,3-dichloropropionic acid was detected, and no GSTZ-catalyzed fluoride release from ethyl fluoroacetate and fluoroacetamide was observed. The relative rates of DCA biotransformation by hepatic cytosol were mouse > rat > human. Immunoblotting showed the presence of GSTZ in mouse, rat, and human liver cytosol. 13C NMR spectroscopic studies showed that [2-13C]glyoxylic acid was the only observable, stable metabolite of [2-13C]DCA. Also, glutathione was required, but was neither consumed nor oxidized to glutathione disulfide, during the oxygenation of DCA to glyoxylic acid. These results are consistent with a reaction mechanism that involves displacement of chloride from DCA by glutathione to afford S-(Rchlorocarboxymethyl)glutathione, which may undergo hydrolysis to give the hemithioacetal S-(R-hydroxycarboxymethyl)glutathione. Elimination of glutathione from the hemithioacetal would give glyoxylic acid.
Introduction Dichloroacetic acid (DCA)1 is a contaminant in chlorinated drinking water consumed by an estimated 170 million individuals in the USA (1, 2). DCA is hepatocarcinogenic in male B6C3F1 mice and Fischer 344 rats (3-6) and is a metabolite of tetrachloroethene, trichloroethene, trichloroacetic acid, and chloral hydrate (79). DCA, which activates pyruvate dehydrogenase secondary to its inhibition of pyruvate dehydrogenase kinase, is used clinically in the management of lactic acidosis and has been proposed for use as a neuroprotective agent (10, 11). [14C]DCA is metabolized to chloroacetic acid, glyoxylic acid, glycolic acid, oxalic acid, glycine, hippuric acid, and carbon dioxide (12-14). Previous studies showed that the oxygenation of DCA to glyoxylic acid is catalyzed by * Address correspondence to this author at the Department of Pharmacology & Physiology, University of Rochester Medical Center, 601 Elmwood Ave., Box 711, Rochester, NY 14642. Voice: 716-2751681. Fax: 716-244-9283. E-mail:
[email protected]. † University of Rochester Medical Center. ‡ Present address: Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543-8000. § Australian National University. 1 Abbreviations: DCA, dichloroacetic acid; GST, glutathione transferase; GSTZ, rat liver glutathione transferase Zeta.
cytosolic enzymes that require glutathione for activity (15, 16). Recent studies from this laboratory show that the glutathione-dependent oxygenation of DCA to glyoxylic acid is catalyzed by the newly discovered glutathione transferase Zeta (17, 18). Previous studies showed that R-haloacids in addition to DCA undergo glutathione-dependent biotransformation: the defluorination of fluoroacetic acid in rodents is catalyzed by glutathione-dependent cytosolic enzymes (19-22). In addition, the neurotoxic R-haloacid 2-chloropropionic acid undergoes glutathione-dependent biotransformation to S-(R-methylcarboxymethyl)glutathione (23). The present experiments were designed to study the GSTZ-catalyzed biotransformation of DCA and other R-haloacids. The substrate selectivity of the enzyme, the kinetics of the reaction in human, rat, and mouse liver tissue, and the fate of [2-13C]DCA and glutathione were investigated.
Materials and Methods Materials. Dichloroacetic acid, ethyl bromofluoroacetate, (R,S)-2-bromopropionic acid, (S)-2-bromopropionic acid, (R)-, (S)-, and (R,S)-2-chloropropionic acid, sodium 2,2-dichloropropionate, 30% pyridine-70% hydrogen fluoride, (R,S)-alanine,
10.1021/tx980144f CCC: $15.00 © 1998 American Chemical Society Published on Web 10/08/1998
Biotransformation of Haloacids by GST Zeta cis-3-chloroacrylic acid, and 2,4-dinitrofluorobenzene were obtained from Aldrich Chemical Co. (Milwaukee, WI). Dibromoacetic acid was purchased from Spectrum Chemical (New Brunswick, NJ). Bromochloroacetic acid was obtained from Supelco (Bellefonte, PA). Ethyl chlorofluoroacetate was obtained from Lancaster (Windham, NH). [2-13C]Dichloroacetic acid was obtained from Isotec Inc. (Miamisburg, OH). Fluoroacetic acid sodium salt, sodium pyruvate, and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit anti-human GSTZ1-1 antibodies were prepared as described by Board et al. (17). Western blot kits were purchased from BioRad (Hercules, CA). GSTZ was purified as previously reported (18); 10% glycerol was present in the buffers used for enzyme purification. Cytosolic fractions were prepared and dialyzed as previously described (18) from male, Fischer 344 rats (200-225 g, Charles River Laboratories, Wilmington, MA), male, B6C3F1 mice (4 weeks old, Charles River Laboratories) and male and female human liver (International Institute for the Advancement of Medicine, Scranton, PA). Chlorofluoroacetic acid and bromofluoroacetic acid were obtained by hydrolysis of their ethyl esters. The ethyl ester (1 g) was stirred with 20 mL of 1 N HCl for about 3 h at 40 °C. 19F NMR spectroscopy was used to monitor the course of the reaction. After the hydrolysis was complete, the reaction mixture was concentrated under vacuum. Distillation of the crude product gave chlorofluoroacetic acid and bromofluoroacetic acid that were 99% and 97% pure, respectively, by 19F and 1H NMR spectroscopy. (R,S)-2-Fluoropropionic acid was synthesized as described by Olah et al. (24). (R,S)-Alanine (1.34 g, 15 mmol) was dissolved in 40 mL of 30% pyridine-70% hydrogen fluoride. The mixture was purged with dry nitrogen and cooled on ice. Sodium nitrite (2.1 g, 30 mmol) was added slowly to the mixture with stirring over 20 min, the ice bath was removed, and the mixture was stirred at room temperature for another 6 h. The reaction mixture was poured into 80 mL of ice-water and extracted 3 times with ether. The ether layers were pooled, extracted with 10% hydrogen chloride, and dried with anhydrous sodium sulfate. The ether was evaporated in vacuo, and vacuum distillation of the residue gave a slightly yellow liquid (0.6 g, 43%) that was 92% pure by 1H and 19F NMR spectroscopy. 1H NMR (DMSO): δ 5.18 (d of q, 1H, J ) 50 Hz, CH3CHFCOOH), 1.56 (d of d, 3H, J ) 24 Hz, CH3CHFCOOH). 19F NMR (DMSO): δ -106.8 to -107.4 (m). (R,S)-2-Iodopropionic acid was synthesized as described by Belletire and Fry (25). (R,S)-2-Bromopropionic acid (1.53 g, 10 mmol) was refluxed with potassium iodide (2.4 g, 15 mmol) in 10 mL of acetone for 22 h. The acetone was evaporated in vacuo, and the residue was dissolved in water and extracted 3 times with chloroform. The organic layers were pooled, extracted with sodium bisulfite solution and then water, and dried with anhydrous sodium sulfate. After removal of the solvent under vacuum, Kugelrohr distillation of the residue gave a crude product. Recrystallization from petroleum ether gave a white solid (1.6 g, 80%); mp 42-44 °C. 1H NMR spectroscopy showed the product was pure. 1H NMR (CDCl3): δ 4.52 (q, 1H, CH3CHICOOH), 1.98 (d, 3H, CH3CHICOOH). The method described by Eliel and Traxler for the synthesis of 3-chloroisovaleric acid was used to synthesize 3,3-dichloropropionic acid (26). cis-3-Chloroacrylic acid (1.07 g, 10 mmol) was dissolved in 50 mL of absolute ether and cooled on ice. The solution was saturated with dry hydrogen chloride gas at 0 °C and then allowed to come to room temperature in a sealed flask. After standing for 10 days at room temperature, the ether was evaporated in vacuo. Kugelrohr distillation gave a colorless liquid (1.38 g, 96.5%). 1H NMR showed the product was pure. 1H NMR (CDCl ): δ 6.07 (t, 1H, CHCl CH COOH), 3.33 (d, 2H, 3 2 2 CHCl2CH2COOH). S-Carboxymethylglutathione was synthesized as previously reported by Reed et al. (27). Glutathione (3.07 g, 10 mmol) was dissolved in 20 mL of water in a flask purged with nitrogen. Potassium carbonate (2.7 g, 20 mmol) was added, and, after the
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1333 potassium carbonate had dissolved, iodoacetic acid (1.97 g, 10.5 mmol) was slowly added to the stirred mixture, which was kept under reduced light. The reaction mixture was stirred under reduced light for 1 h. Silica gel TLC was used to monitor the course of the reaction. When the reaction was complete, the reaction mixture was brought to about pH 2 with concentrated HCl and concentrated under vacuum. The crude product was dissolved in 0.1% acetic acid and loaded onto a C18 column, which was eluted with acetonitrile/water/acetic acid (5:94:1). Fractions were collected and analyzed by silica gel TLC (1butanol/water/acetic acid, 5:3:2) with ninhydrin as the visualizing agent. The fractions containing product were pooled, and removal of the solvents under vacuum gave a white solid (1.4 g, 26.9%) that was >95% pure by 1H NMR spectroscopy: mp 77-79 °C; TLC Rf ) 0.5 (1-butanol/water/acetic acid, 5:3:2); 1H NMR (D2O) δ 4.53-4.61 (m, 1H, R-CH of Cys), 3.96 (s, 2H, R-CH2 of Gly), 3.89 (t, 1H, R-CH of Glu), 3.38 (s, 2H, S-CH2), 2.893.14 (m, 2H, β-CH2 of Cys), 2.47-2.57 (m, 2H, γ-CH2 of Glu), 2.10-2.22 (m, 2H, β-CH2 of Glu). S-(R-Methylcarboxymethyl)glutathione was synthesized by reaction of glutathione and 2-bromopropionic acid. Glutathione (1.54 g, 5 mmol) was dissolved in 20 mL of water in a flask purged with nitrogen. The solution was brought to about pH 9.5 with 5 N NaOH. Methanol (15 mL) was added followed by addition of (S)-2-bromopropionic acid (0.84 g, 5.5 mmol) in 10 mL of methanol over 15 min to the stirred reaction mixture kept under nitrogen at room temperature. The reaction was continued at room temperature for another 3 h, and the course of the reaction was monitored with TLC. The reaction mixture was concentrated under vacuum, and the residue was dissolved in 0.1% acetic acid and applied to a C18 column, which was eluted with acetonitrile/water/acetic acid (5:94:1). Fractions were collected and analyzed by silica gel TLC [1-butanol/water/acetic acid (5:3:2)] with ninhydrin as the visualizing agent. Evaporation of the solvent gave a white solid (1.16 g, 61.2%) that was about 99% pure by 1H NMR spectroscopy; mp 75-77 °C; TLC Rf ) 0.55 (1-butanol/water/acetic acid, 5:3:2); 1H NMR (D2O) δ 4.51-4.60 (m, 1H, R-CH of Cys), 3.94 (s, 2H, R-CH2 of Gly), 3.83 (t, 1H, R-CH of Glu), 3.5-3.6 (m, 1H, S-CH), 2.91-3.15 (m, 2H, β-CH2 of Cys), 2.44-2.55 (m, 2H, γ-CH2 of Glu), 2.08-2.19 (m, 2H, β-CH2 of Glu), 1.37 (d, 3H, R-CH3). Instrumental Analyses. 1H and 19F NMR spectra were acquired with a Bruker 270 MHz spectrometer operating at 270 MHz for 1H and 254 MHz for 19F. Chemical shifts (δ) are referenced to tetramethylsilane (δ ) 0.0 ppm) for 1H NMR (D2O, CDCl3, or DMSO) and to trifluoroacetamide (δ ) 0.0 ppm) for 19F NMR (D O). Proton-decoupled 13C NMR spectra were 2 obtained with a General Electric Omega-400 spectrometer equipped with a multinuclear probe and operating at 100.7 MHz for 13C. The spectral width was 25 000 Hz. Chemical shifts (δ) are referenced to tetramethylsilane (δ ) 0.0 ppm) for 13C NMR spectra (D2O). Biotransformation Studies. (A) Substrate Selectivity Studies. Enzyme activity with bromochloro-, bromofluoro-, chlorofluoro- dibromo-, dichloro-, and difluoroacetic acid as substrates was measured by quantifying glyoxylic acid formation, as previously described (18). Incubation mixtures contained substrate (0.5 mM), glutathione (1 mM), and purified GSTZ (0.6 µg for bromofluoroacetic and chlorofluoroacetic acids, 1.3 µg for DCA and bromochloroacetic acid, and 2.7 µg for dibromoacetic and difluoroacetic acids) in 0.1 M phosphate buffer (pH 7.4) and were incubated at 37 °C for 20 min. Control incubation mixtures lacked enzyme. The defluorination of fluoroacetic acid, ethyl fluoroacetate, and fluoroacetamide was studied by 19F NMR spectroscopy. Reaction mixtures were prepared in 5-mm NMR tubes and contained substrate (2 mM), glutathione (4 mM), and 10 µg of purified GSTZ in 0.5 mL of 0.1 M phosphate buffer (pH 7.4) prepared with 10% D2O. The reaction mixtures were incubated at 37 °C, and the 19F NMR spectrum was recorded after incubation for 2 h with fluoroacetic acid and fluoroaceta-
1334 Chem. Res. Toxicol., Vol. 11, No. 11, 1998 mide as the substrates and for 30 min with ethyl fluoroacetate as the substrate. Controls lacked glutathione or enzyme. Glutathione conjugate formation was used to measure the activity of purified GSTZ with fluoroacetic acid and 2-halopropionic acids as substrates. Reaction mixtures contained fluoroacetic acid (0.5 mM) or 2-halopropionic acid (0.5 mM), glutathione (1 mM), and 5 µg (with fluoroacetic acid as substrate) or 1.5 µg (with 2-halopropionic acids as substrates) of purified GSTZ in 0.5 mL of 0.1 M phosphate buffer (pH 7.4) and were incubated at 37 °C for 20 min. With fluoroacetic acid as the substrate, a sample (200 µL) of the reaction mixture was collected at the end of the incubation time for quantification of fluoride formation, as described previously (28). To the remaining 300 µL of reaction mixture were added 20 µL of potassium carbonate solution (6.2 g in 7.5 mL of water) and 300 µL of 2,4dinitrofluorobenzene solution (0.1 mL in 5 mL of ethanol). The mixture was mixed and kept at room temperature overnight. With 2-halopropionic acids as substrates, 20 µL of potassium carbonate solution and 300 µL of 2,4-dinitrofluorobenzene solution were added to the reaction mixture, which was kept overnight at room temperature. The samples were analyzed with the HPLC system described below for the quantification of glutathione and glutathione disulfide concentrations. Product formation from fluoroacetic acid and 2-halopropionic acids was determined from standard curves prepared with synthetic S-carboxymethylglutathione and S-(R-methylcarboxymethyl)glutathione, respectively. Enzyme activity with 2,2-dichloropropionic acid as the substrate was determined by quantifying pyruvate formation, as described by Stijntjes et al. (29). 2,2-Dichloropropionic acid (0.5 mM) was incubated with glutathione (1 mM) and 4.5 µg of purified GSTZ in 0.5 mL of 0.1 M phosphate buffer (pH 7.4) at 37 °C for 20 min. At the end of the incubation time, 200 µL of 1 M o-phenylenediamine solution was added to the reaction mixture, which was heated at 80 °C for 30 min. Samples were analyzed on a Hewlett-Packard 1090 liquid chromatograph equipped with a Radial-Pak cartridge packed with a Resolve C-18 RCM column (8 mm × 100 mm, Millipore Corp., Milford, MA). The column was eluted with methanol/water/acetic acid (45:54:1) at a flow rate of 1 mL/min. The fluorescence intensity of the eluate was monitored with a Gilson Model 121 fluorometer. Pyruvate formation was quantified with a standard curve prepared under the same conditions. Enzyme activity with 3,3-dichloropropionic acid as the substrate was measured by the disappearance of substrate by GC/ MS. 3,3-Dichloropropionic acid (0.2 mM) was incubated with glutathione (1 mM) and purified GSTZ (6 µg) in 0.5 mL of 0.1 M phosphate buffer (pH 7.4) for 20 min. The reaction was stopped by heat-inactivation (boiling water bath for 3 min) of the enzyme. A sample (450 µL) of the reaction mixture was transferred to a vial containing 200 µL of phosphate buffer (pH 9.0), 200 µL of 0.5 M tetrabutylammonium hydrogen sulfate, and 10 µL of 5 mM DCA (added as an internal standard). Methylene chloride (1 mL) and benzyl bromide (20 µL) were added to the vial, which was shaken for 1.5 h. The organic layer was transferred to a clean vial, extracted with water, and dried with anhydrous magnesium sulfate. The samples were analyzed on a Hewlett-Packard 5890 gas chromatograph (25 m × 0.2 mm, 0.5 µm film thickness, HP-1 cross-linked methyl silicon column, splitless injection) coupled to a Hewlett-Packard 5970B mass selective detector. The injector and transfer-line temperatures were 240 and 285 °C, respectively. The samples were analyzed with a temperature program of 70 °C for 1 min followed by a linear gradient of 10 °C/min to 220 °C, which was maintained for 3 min. The benzyl esters of DCA and 3,3dichloropropionic acid were quantified by monitoring the sum of ions from m/z 218-222 for DCA and from m/z 232-236 for 3,3-dichloropropionic acid. Ratios of the area of benzyl 3,3dichloropropionate to the area of benzyl dichloroacetate were used to quantify 3,3-dichloropropionic acid concentrations against a standard curve prepared under the same conditions.
Tong et al. (B) Fate of [2-13C]DCA and of Glutathione in the Oxygenation of DCA to Glyoxylic Acid. The fate of [2-13C]DCA was studied by 13C NMR spectroscopy. Reaction mixtures were prepared in 5-mm NMR tubes and contained [2-13C]DCA (10 mM), glutathione (10 mM), and purified GSTZ (20 µg) in 0.5 mL of 0.1 M phosphate buffer (pH 7.4) prepared with 10% D2O and were incubated at 37 °C. 13C NMR spectra were recorded after 30, 60, and 120 min of incubation. In other experiments, [2-13C]DCA (10 mM), glutathione (10 mM), and rat hepatic cytosolic protein (5.5 mg) in 1 mL of 0.1 M phosphate buffer (pH 7.4) prepared with 10% D2O were incubated for 2.5 h. The cytosolic proteins were removed by ultrafiltration in Centricon-10 concentrators (Amicon, Beverly, MA), the filtrate was transferred to a 5-mm NMR tube, and the 13C NMR spectrum was recorded. Controls lacked glutathione or enzyme. The fate of glutathione was studied after incubation of glutathione with DCA in the presence of purified GSTZ or rat liver cytosol. Glutathione (0.5 mM), DCA (0.5 mM), and 3 µg of purified GSTZ or 1.4 mg of cytosolic protein were incubated in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1 mL at 37 °C for 50 min. The reaction was stopped by addition of 50 µL of trifluoroacetic acid. The acidified reaction mixtures were placed on ice for 10 min and then centrifuged to remove precipitated proteins. The concentrations of glyoxylic acid and of glutathione and glutathione disulfide were measured as described previously (18, 27). A sample (100 µL) of the supernatant was mixed with 20 µL of potassium carbonate (6.2 g/7.5 mL) and 80 µL of iodoacetic acid (100 mg/5 mL) solutions, and the samples were kept in the dark at room temperature for 1 h. 2,4-Dinitrofluorobenzene (300 µL of a solution of 0.1 mL of fluorodinitrobenzene in 5 mL of ethanol) was added to the reaction mixture, which was kept overnight in the dark at room temperature. The reaction mixture was centrifuged, and a sample of the supernatant (50 µL) was analyzed on a HewlettPackard 1090 liquid chromatography equipped with a µBondapak NH2 column (Waters, Milford, MA). The absorbance of the eluate was measured at 352 nm with a Hewlett-Packard diodearray detector. Data were acquired and analyzed with a HP ChemStation for liquid chromatography. (C) Kinetic Studies. To investigate species differences in DCA metabolism, Km and Vmax values for DCA (0.05-1.0 mM) were determined at a fixed glutathione concentration of 1.0 mM with human, rat, and mouse liver cytosolic proteins (0.1 mg for mice, 0.2 mg for rats, and 0.8 mg for human) as previously described (18). The Km and Vmax were calculated with the EZFIT program (version 2.0; Perrella Scientific, Inc., Springfield, PA). The quality of the human liver tissues was assessed by measuring cytosolic glutathione transferase activity with cumene hydroperoxide as the substrate, as previously described (30). (D) Immunoblot Analysis. Human, rat, and mouse liver cytosolic fractions were also analyzed by SDS/PAGE and blotted with rabbit anti-human GSTZ1-1 antibodies, as described previously (18).
Results The substrate selectivity of the purified GSTZ was studied by measuring glyoxylic acid formation with DCA and other dihaloacetic acids as substrates. GSTZ catalyzed the formation of glyoxylic acid from bromochloro-, bromofluoro-, chlorofluoro-, dibromo-, and dichloroacetic acid, but no glyoxylic acid formation was detected with difluoroacetic acid as the substrate (Table 1). The relative rates of glyoxylic acid formation were bromofluoroacetic acid > chlorofluoroacetic acid > bromochloroacetic acid > dichloroacetic acid > dibromoacetic acid. The studies on the substrate selectivity of GSTZ were extended to other R-haloacids. 19F NMR spectroscopic studies showed that GSTZ catalyzed the glutathionedependent release of fluoride from fluoroacetic acid (Figure 1), but not from ethyl fluoroacetate or fluoroac-
Biotransformation of Haloacids by GST Zeta
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1335
Table 1. Substrate Selectivity of Purified Rat Liver GST Zetaa substrate
activity [nmol min-1 (mg of purified protein)-1]
bromochloroacetic acid bromofluoroacetic acid chlorofluoroacetic acid dibromoacetic acid dichloroacetic acid difluoroacetic acid fluoroacetic acid (R,S)-2-bromopropionic acid (R,S)-2-chloropropionic acid (R)-2-chloropropionic acid (S)-2-chloropropionic acid (R,S)-2-fluoropropionic acid (R,S)-2-iodopropionic acid 2,2-dichloropropionic acid 3,3-dichloropropionic acid
1411 ( 65 5028 ( 150 3883 ( 43 155 ( 4 1038 ( 20 ND 172 ( 16 2142 ( 108 1655 ( 66 318 ( 14 1809 ( 71 ND 2532 ( 43 244 ( 6 ND
a Substrates were incubated with purified GSTZ, and product formation was measured as described under Materials and Methods. Data are presented as means ( SD (n ) 3). ND, not detectable.
Figure 1. GST Zeta-catalyzed defluorination of fluoroacetic acid. Fluoroacetic acid was incubated with purified GST Zeta (A), with glutathione (B), or with GST Zeta and glutathione (C), and the incubation mixtures were examined by 19F NMR spectroscopy, as described under Materials and Methods.
Figure 2. GST Zeta-catalyzed biotransformation of fluoroacetic acid to S-(carboxymethyl)glutathione. Fluoroacetic acid was incubated with glutathione and purified GST Zeta (A) or with glutathione and heat-inactivated GST Zeta (B), and the formation of S-(carboxymethyl)glutathione was determined by HPLC, as described under Materials and Methods.
etamide (data not shown). The carbon-containing metabolite derived from fluoroacetic acid was identified as S-carboxymethylglutathione by HPLC (Figure 2). The activity of GSTZ toward fluoroacetic acid was lower than with DCA (Table 1). The ratio of fluoride:S-carboxy-
Figure 3. GST Zeta-catalyzed biotransformation of (S)-2chloropropionic acid to S-(R-methylcarboxymethyl)glutathione. (S)-2-Chloropropionic acid was incubated with glutathione and purified GST Zeta (A) or with glutathione and heat-inactivated GST Zeta (B), and the formation of S-(R-methylcarboxymethyl)glutathione was determined by HPLC, as described under Materials and Methods.
methylglutathione formation was 0.97 ( 0.04 (n ) 3) with fluoroacetic acid as the substrate. GSTZ also catalyzed the formation of S-(R-methylcarboxymethyl)glutathione from 2-halopropionates (Figure 3, Table 1); the relative rates of conjugate formation were (R,S)-2-iodopropionic acid > (R,S)-2-bromopropionic acid > (R,S)-2-chloropropionic acid. No product formation was detected with (R,S)-2-fluoropropionic acid as the substrate. The rate of conjugate formation with (S)-2-chloropropionic acid as the substrate was greater than with (R)-2-chloropropionic acid as the substrate (Table 1). GSTZ also catalyzed the biotransformation of 2,2-dichloropropionic acid to pyruvic acid, but the activity was much lower than with DCA as the substrate (Table 1). To study whether GSTZ is selective for R-haloacids, the biotransformation of the β-haloacid 3,3-dichloropropionic acid was also investigated, but no enzyme activity was detected by measuring substrate disappearance (Table 1). In addition, the R-substituted acetic acids (methylthio)acetic acid, thiodiglycolic acid, and methoxyacetic acid were studied as substrates; although the formation of S-(carboxymethyl)glutathione was detected with all three compounds with rat liver cytosol as the catalyst, the rates of biotransformation were very low [ chlorofluoroacetic acid > bromochloroacetic acid > DCA > dibromoacetic acid; no activity was detected with difluoroacetic acid as the substrate (Table 1). These results
Tong et al.
indicate that activity is affected by both steric and inductive effects, as has been observed in the displacement of halide from dihaloacetate esters by iodide (32). GSTZ also catalyzed the glutathione-dependent defluorination of fluoroacetic acid and the concomitant formation of S-(carboxymethyl)glutathione (Figures 1 and 2). Moreover, the GSTZ-catalyzed defluorination of fluoroacetic acid was accompanied by the stoichiometric formation of S-(carboxymethyl)glutathione and fluoride, indicating that the glutathione conjugate was the sole carboncontaining metabolite of fluoroacetic acid. Previous studies showed that the defluorination of fluoroacetic acid was catalyzed by glutathione-dependent rat and mouse liver cytosolic enzymes (19-22), but the identities of the enzymes and the carbon-containing metabolites were not established. No fluoride release was observed by 19F NMR spectroscopy with ethyl fluoroacetate or fluoroacetamide as substrates, indicating that the carboxyl group is required for enzyme activity. 2-Chloropropionic acid is biotransformed to S-(R-methylcarboxymethyl)glutathione by rat liver enzymes that do not adhere to a S-hexylglutathione affinity column, and it was proposed that the biotransformation of 2-chloropropionic acid is catalyzed by glutathione S-transferase theta (23). The present studies showed, however, that GSTZ catalyzed the biotransformation of 2-halopropionic acids to the glutathione conjugate S-(R-methylcarboxymethyl)glutathione. The relative rates of conjugate formation were (R,S)-2-iodopropionic acid > (R,S)-2bromopropionic acid > (R,S)-2-chloropropionic acid; no activity was detected with (R,S)-2-fluoropropionic acid as the substrate. GSTZ also catalyzed the enantioselective conjugation of 2-chloropropionates with glutathione: activity was greater with (S)-2-chloropropionic acid as the substrate than with (R)-2-chloropropionic acid as the substrate. The finding that GSTZ catalyzed the formation of a stable glutathione conjugate, i.e., S-(R-methylcarboxymethyl)glutathione, of 2-chloropropionic acid also provides a possible explanation for the observed depletion of hepatic glutathione concentrations by 2-chloropropionic acid (23). In addition, GSTZ catalyzed the biotransformation of 2,2-dichloropropionic acid to pyruvic acid, albeit at a low rate. In contrast, no biotransformation of 3,3dichloropropionic acid, as measured by loss of substrate, was observed, indicating that β-haloacids are not substrates for GSTZ. The substrate selectivity studies showed that GSTZ catalyzed the biotransformation of R-haloacids (fluoroacetic acid, dihaloacetic acids, 2-halopropionic acids, and 2,2-dichloropropionic acid), but not the biotransformation of a β-haloacid (3,3-dichloropropionic acid), an R-haloacid ester (ethyl fluoroacetate), or an R-haloacetamide (fluoroacetamide). The observed substrate selectivity indicates that GSTZ preferentially catalyzes the attack of glutathione at positions R to the carboxyl group. Recent studies show that GSTZ1-1 is identical with maleylacetoacetate isomerase (33), which catalyzes the attack of glutathione on the R-carbon of maleylacetoacetate. Hence, the enzyme may require the presence of a carboxyl group, which poises the substrate for R attack. Studies were conducted to provide information about the mechanism of the GSTZ-catalyzed oxygenation of DCA. Studies with [2-13C]DCA showed that [2-13C]glyoxylic acid was the only observable, stable metabolite formed and that the oxygenation of DCA to glyoxylic acid required active enzyme and was glutathione-dependent
Biotransformation of Haloacids by GST Zeta
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1337
Table 2. Fate of Glutathione after Incubation with DCA and Purified Rat Liver GST Zeta or Rat Liver Cytosola glyoxylic acid (nmol)
GSH (nmol)
GSSG (nmol)
enzyme
control
DCA
control
DCA
control
DCA
Cytosol GST Zeta
0(0 0(0
124 ( 1.2 160 ( 3.8
486 ( 19.7 454 ( 10.8
480 ( 13.1 454 ( 11.5
39.3 ( 0.6 16.9 ( 0.0
39.1 ( 0.3 16.9 ( 0.0
a DCA was incubated with glutathione and purified GST Zeta or rat liver cytosol, and the concentrations of glyoxylic acid, glutathione (GSH), and glutathione disulfide (GSSG) in the incubation mixtures were determined, as described under Materials and Methods. Data are presented as means ( SD (n ) 3).
Scheme 1
Table 3. Kinetics of the Biotransformation of DCA to Glyoxylic Acid by Human, Rat, and Mouse Liver Cytosola species
Km (µM)
Vmax -1 [nmol min-1 (mg of protein)-1]
Vmax/Km (×10-3)
human rat mouse
47.3 ( 6.7 70.1 ( 5.3* 81.9 ( 9.56*
0.39 ( 0.11 2.04 ( 0.27* 4.31 ( 0.42*†
8.25 ( 1.37 32.4 ( 4.87* 52.9 ( 2.46*†
a Human, rat, or mouse liver cytosol was incubated with DCA, and the formation of glyoxylic acid was quantified, as described under Materials and Methods. Individual kinetic constants for human subjects: Km, 2 males (40.1, 53.3), 1 female (48.7); Vmax, 2 males (0.31, 0.52), 1 female (0.35). Data are shown as means ( SD (n ) 3-4): (*) significantly different (p < 0.05) from kinetic constants for human cytosol; (†) significantly different (p < 0.05) from kinetic constants for rat cytosol. Data were analyzed by oneway ANOVA with Bonferroni post test (GraphPad Prism, version 2.01; GraphPad Software, San Diego, CA).
Figure 5. Immunoblot analysis of human, rat, and mouse liver cytosol. Samples (75 µg of cytosolic protein was loaded in each lane) were analyzed by SDS/PAGE and blotted with rabbit antihuman GSTZ1-1 antibodies, as described under Materials and Methods. Lanes 1-3, human liver cytosol; lanes 4-6, mouse liver cytosol; lanes 7-9, rat liver cytosol.
(Figure 4). Although S-(R-hydroxycarboxymethyl)glutathione, the hemithioacetal of glutathione and glyoxylic acid, was observed by 13C NMR spectroscopy, it may be formed as an intermediate in the reaction or by reaction of enzymatically formed glyoxylic acid and glutathione. Indeed, 13C NMR analysis of a mixture of glutathione and glyoxylic acids showed the same resonances (4a and 4b in Figure 4) that were observed in incubation mixtures (data not shown). Previous studies showed that [1,2-13C]DCA is biotransformed to [1,2-13C]glyoxylic acid by rat and human liver cytosol (16). The present studies showed that glutathione is required for the GSTZ-catalyzed oxygenation of DCA metabolism; moreover, glutathione cannot be replaced with L-methionine, N-acetyl-L-cysteine, or DTT (data not shown). To provide information about the mechanism of the GSTZ-catalyzed oxygenation of DCA to glyoxylic acid, the fate of glutathione in the oxygenation of DCA was studied (Table 2). Although 20-30% DCA was converted to glyoxylic acid, glutathione was completely recovered from the incubation mixtures as compared with controls (Table 2). Moreover, no glutathione disulfide formation was associated with DCA oxygenation (Table 2). The results demonstrated that glutathione was required, but was neither consumed nor oxidized, for the oxygenation of DCA to glyoxylic acid. These results are consistent with a reaction mechanism for the oxygenation of DCA that involves displacement of chloride by glutathione to afford S-(R-chlorocarboxymethyl)glutathione as an intermediate; hydrolysis of the intermediate R-chlorosulfide would give the hemithioacetal S-(R-hydroxycarboxymethyl)glutathione, which may
Scheme 2
Scheme 3
eliminate glutathione to give glyoxylic acid (Scheme 1). R-Chlorosulfides undergo rapid hydrolysis (34). Similarly, glutathione is required for the biotransformation of dibromomethane to formaldehyde, but is not consumed during the reaction (35). Also, S-(chloromethyl)glutathione is an intermediate in the glutathione transferase theta-catalyzed oxygenation of dichloromethane (36). With fluoroacetic acid or 2-halopropionic acids as substrates, stable glutathione conjugates were formed (Scheme 2). The biotransformation of 2,2-dichloropropionic acid may involve the intermediate formation of S-(Rchloro-R-methylcarboxymethyl)glutathione; hydrolysis of the R-chlorosulfide intermediate would give S-(R-hydroxyR-methylcarboxymethyl)glutathione, which is the hemithioacetal of pyruvic acid and glutathione. Elimination of glutathione from S-(R-hydroxy-R-methylcarboxymethyl)glutathione would give the observed product pyruvic acid (Scheme 3). DCA is hepatocarcinogenic in both male B6C3F1 mice and F344 rats (3-6). Estimation of the mean daily dose at which 50% of the animals exhibited liver neoplasia indicates that male, Fischer (F344) rats (mean daily dose is approximately 10 mg-1 kg-1 day-1) are about 10 times more sensitive than the male, B6C3F1 mice (mean daily dose is approximately 100 mg-1 kg-1 day-1) (6). The relationship of the GSTZ-dependent metabolism of DCA to its observed carcinogenicity has not been established. The present data showed, however, that rates of DCA biotransformation were much greater in B6C3F1 mice than in Fischer rats. Hence, the carcinogenicity of DCA does not appear to be directly related to its GSTZdependent biotransformation. Preliminary studies indicate that purified GSTZ was rapidly inactivated by incubation with 50 µM DCA, but not with glyoxylic acid, in the presence of glutathione; also, incubation of rat liver cytosol with bromochloroacetic acid, DCA, or dibromoacetic acid decreased the biotransformation of DCA to glyoxylate.2 In addition, the biotransformation of DCA to glyoxylic acid is decreased in rats given 0.3 mmol of DCA/kg.3 These observations 2 3
Z. Tong, B. Gargano, and M. W. Anders, unpublished observations. W. B. Anderson and M. W. Anders, unpublished observations.
1338 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
may offer an explanation for the finding that the halflife of DCA in humans is prolonged after repeated dosing with DCA and the reduced biotransformation of DCA in hepatic cytosol from DCA-treated rats (16, 37, 38). Further studies on the mechanism of the GSTZ-catalyzed biotransformation of R-haloacids are warranted as are studies on the relationship of the GSTZ-catalyzed biotransformation of DCA to its observed carcinogenicity and to the inactivation of GSTZ.
Acknowledgment. This research was supported by National Institute of Environmental Health Sciences grant ES03127 to M.W.A. We thank Dr. Stephen Curry, Astra Arcus USA, Rochester, NY, for the gift of [2-13C]DCA, Ms. Daria Krenitsky for technical assistance, Mr. Scott Kennedy for assistance with acquiring the 13C NMR spectra, and Ms. Sandra E. Morgan for her assistance in preparing the manuscript.
References (1) Uden, P. C., and Miller, J. W. (1983) Chlorinated acids and chloral in drinking water. J. Am. Water Works Assoc. 75, 524-527. (2) Craun, G. F. (1985) Epidemiological consideration for associations between the disinfection of water and cancer in humans. In Water Chlorination: Chemistry, Environmental Impact, and Health Effects (Jolley, R. L., Bull, R. J., Davis, W. P., Katz, S., Roberts, M. H., Jr., and Jacobs, V. A., Eds.) pp 131-141, Lewis Publishers, Chelsea, MI. (3) Herren-Freund, S. L., Pereira, M. A., Khoury, M. D., and Olson, G. (1987) The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver. Toxicol. Appl. Pharmacol. 90, 183-189. (4) Bull, R. J., Sanchez, I. M., Nelson, M. A., Larson, J. L., and Lansing, A. J. (1990) Liver tumor induction in B6C3F1 mice by dichloroacetate and trichloroacetate. Toxicology 63, 341-359. (5) DeAngelo, A. B., Daniel, F. B., Stober, J. A., and Olson, G. R. (1991) The carcinogenicity of dichloroacetic acid in the male B6C3F1 mouse. Fundam. Appl. Toxicol. 16, 337-347. (6) DeAngelo, A. B., Daniel, F. B., Most, B. M., and Olson, G. R. (1996) The carcinogenicity of dichloroacetic acid in the male Fischer 344 rat. Toxicology 114, 207-221. (7) Dekant, W., Metzler, M., and Henschler, D. (1984) Novel metabolites of trichloroethylene through dechlorination reactions in rats, mice and humans. Biochem. Pharmacol. 33, 2021-2027. (8) Larson, J. L., and Bull, R. J. (1992) Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate. Toxicol. Appl. Pharmacol. 115, 278-285. (9) Henderson, G. N., Yan, Z., James, M. O., Davydova, N., and Stacpoole, P. W. (1997) Kinetics and metabolism of chloral hydrate in children: Identification of dichloroacetate as a metabolite. Biochem. Biophys. Res. Commun. 235, 695-698. (10) Stacpoole, P. W. (1992) The pharmacology of dichloroacetate. Metabolism 38, 1124-1144. (11) Peeling, J., Sutherland, G., Brown, R. A., and Curry, S. (1996) Protective effect of dichloroacetate in a rat model of forebrain ischemia. Neurosci. Lett. 208, 21-24. (12) Larson, J. L., and Bull, R. J. (1992) Metabolism and lipoperoxidative activity of trichloroacetate and dichloroacetate in rats and mice. Toxicol. Appl. Pharmacol. 115, 268-277. (13) Lin, E. L. C., Mattox, J. K., and Daniel, F. B. (1993) Tissue distribution, excretion, and urinary metabolites of dichloroacetic acid in the male Fischer 344 rat. J. Toxicol. Environ. Health 38, 19-32. (14) Cornett, R., James, M. O., Rocca, J. R., Yan, Z., Henderson, G. N., and Stacpoole, P. W. (1997) Glycine conjugation in the biotransformation of dichloroacetate. ISSX Proc. 12, 142. (15) Lipscomb, J. C., Mahle, D. A., Brashear, W. T., and Barton, H. A. (1995) Dichloroacetic acid: Metabolism in cytosol. Drug Metab. Dispos. 23, 1202-1205. (16) James, M. O., Cornett, R., Yan, Z., Henderson, G. N., and Stacpoole, P. W. (1997) Glutathione-dependent conversion of glyoxylate, a major metabolite of dichloroacetate biotransformation in hepatic cytosol from humans and rats, is reduced in dichloroacetate-treated rats. Drug Metab. Dispos. 25, 1223-1227. (17) Board, P. G., Baker, R. T., Chelvanayagam, G., and Jermiin, L. S. (1997) Zeta, a novel class of glutathione transferases in a range of species from plants to humans. Biochem. J. 328, 929-935.
Tong et al. (18) Tong, Z., Board, P. G., and Anders, M. W. (1998) Glutathione transferase Zeta catalyzes the oxygenation of the carcinogen dichloroacetic acid to glyoxylic acid. Biochem. J. 331, 371-374. (19) Kostyniak, P. J., Bosmann, H. B., and Smith, F. A. (1978) Defluorination of fluoroacetate in vitro by rat liver subcellular fractions. Toxicol. Appl. Pharmacol. 44, 89-97. (20) Soiefer, A. I., and Kostyniak, P. J. (1983) The enzymatic defluorination of fluoroacetate in mouse liver cytosol: The separation of defluorination activity from several glutathione S-transferases of mouse liver. Arch. Biochem. Biophys. 225, 928-935. (21) Soiefer, A. I., and Kostyniak, P. J. (1984) Purification of a fluoroacetate-specific defluorinase from mouse liver cytosol. J. Biol. Chem. 259, 10787-10792. (22) Wang, S.-H., Rice, S. A., Serra, M. T., and Gross, B. (1986) Purification and identification of rat hepatic cytosolic enzymes responsible for defluorination of methoxyflurane and fluoroacetate. Drug Metab. Dispos. 14, 392-398. (23) Wyatt, I., Gyte, A., Mainwaring, G., Widdowson, P. S., and Lock, E. A. (1996) Glutathione depletion in the liver and brain produced by 2-chloropropionic acid: Relevance to cerebellar granule cell necrosis. Arch. Toxicol. 70, 380-389. (24) Olah, G. A., Welch, J. T., Vankar, Y. D., Nojima, M., Kerekes, I., and Olah, J. A. (1979) Synthetic Methods. 63. Pyridinium poly(hydrogen fluoride) (30% pyridine-70% hydrogen fluoride): A convenient reagent for organic fluorination reactions. J. Org. Chem. 44, 3872-3881. (25) Belletire, J. L., and Fry, D. F. (1987) Oxidative coupling of carboxylic dianions: The total synthesis of (()-hinokinin and (()fomentaric acid. J. Org. Chem. 52, 2549-2555. (26) Eliel, E. L., and Traxler, J. T. (1956) The mechanism of halide reductions with lithium aluminum hydride. V. Reduction of β-, γ-, δ- and -chloroacids and the corresponding chlorohydrins. J. Am. Chem. Soc. 78, 4049-4053. (27) Reed, D. J., Babson, J. R., Beatty, P. W., Brodie, A. E., Ellis, W. W., and Potter, D. W. (1980) High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106, 55-62. (28) Iyer, R. A., and Anders, M. W. (1997) Cysteine conjugate β-lyasedependent 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. 10, 811819. (29) Stijntjes, G. J., te Koppele, J. M., and Vermeulen, N. P. E. (1992) High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate β-lyase activity: Application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine. Anal. Biochem. 206, 334-343. (30) Igarashi, T., Satoh, T., Ueno, K., and Kitagawa, H. (1983) Species difference in glutathione level and glutathione related enzyme activities in rats, mice, guinea pigs and hamsters. J. Pharm. Dyn. 6, 941-949. (31) Slone, D. H., Gallagher, E. P., Ramsdell, H. S., Rettie, A. E., Stapleton, P. L., Berlad, L. G., and Eaton, D. L. (1995) Human variability in hepatic glutathione S-transferase-mediated conjugation of aflatoxin B1-epoxide and other substrates. Pharmacogenetics 5, 224-233. (32) McBee, E. T., Christman, D. L., Johnson, R. W., Jr., and Roberts, C. W. (1956) Rates of reaction of some halogen-containing esters with potassium iodide in dry acetone. J. Am. Chem. Soc. 78, 4595-4596. (33) Ferna´ndez-Can˜o´n, J. M., and Pen˜alva, M. A. (1998) Characterization of a fungal maleylacetoacetate isomerase gene and identification of its human homologue. J. Biol. Chem. 273, 329-337. (34) Bo¨hme, H., Fischer, H., and Frank, R. (1949) Darstellung und Eigenschaften der R-halogenierten Thioa¨ther. Justus Liebig’s Ann. Chem. 563, 54-72. (35) Ahmed, A. E., and Anders, M. W. (1978) Metabolism of dihalomethanes to formaldehyde and inorganic halide. II. Studies on the mechanism of the reaction. Biochem. Pharmacol. 27, 2021-2025. (36) Hashmi, M., Dechert, S., Dekant, W., and Anders, M. W. (1994) Bioactivation of [13C]dichloromethane in mouse, rat, and human liver cytosol: 13C Nuclear magnetic resonance spectroscopic studies. Chem. Res. Toxicol. 7, 291-296. (37) Curry, S. H., Chu, P.-I., Baumgartner, T. G., and Stackpoole, P. W. (1985) Plasma concentrations and metabolic effects of intravenous sodium dichloroacetate. Clin. Pharmacol. Ther. 37, 8993. (38) Curry, S. H., Lorenz, A., Chu, P.-I., Limacher, M., and Stacpoole, P. W. (1991) Disposition and pharmacodynamics of dichloroacetate (DCA) and oxalate following oral DCA doses. Biopharm. Drug Dispos. 12, 375-390.
TX980144F
