Enantioselective Depletion of Mitochondrial Glutathione

(R,S)-3-Hydroxy-4-pentenoate rapidly and selectively depletes the mitochondrial glutathione pool in rat hepatocytes, but shows little cytotoxicity and...
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Chem. Res. Toxicol. 1996, 9, 361-364

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Articles Enantioselective Depletion of Mitochondrial Glutathione Concentrations by (S)- and (R)-3-Hydroxy-4-pentenoate Mazzaz Hashmi,†,‡ Silke Gra¨f,§ Manfred Braun,§ and M. W. Anders*,† Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, and Institut fu¨ r Organische Chemie und Makromolekulare Chemie, Universita¨ t Du¨ sseldorf, Universita¨ tsstrasse 1, 40225 Du¨ sseldorf, Federal Republic of Germany Received April 24, 1995X

(R,S)-3-Hydroxy-4-pentenoate rapidly and selectively depletes the mitochondrial glutathione pool in rat hepatocytes, but shows little cytotoxicity and does not induce mitochondrial dysfunction [Shan, X., et al. (1993) Chem. Res. Toxicol. 6, 75-81]. The objective of the present studies was to investigate the 3-hydroxybutanoate dehydrogenase-dependent oxidation of (R)and (S)-3-hydroxy-4-pentenoate and the enantioselectivity of 3-hydroxy-4-pentenoate-induced depletion of mitochondrial glutathione concentrations in isolated rat liver mitochondria and hepatocytes. (S)-3-Hydroxy-4-pentenoate, but not (R)-3-hydroxy-4-pentenoate, was a substrate for 3-hydroxybutanoate dehydrogenase. Incubation of rat liver mitochondria or hepatocytes with (S)-3-hydroxy-4-pentenoate resulted in a time- and concentration-dependent depletion of mitochondrial glutathione concentrations, whereas (R)-3-hydroxy-4-pentenoate produced little depletion. These results show that (S)-3-hydroxy-4-pentenoate is a substrate for 3-hydroxybutanoate dehydrogenase and is converted to the Michael acceptor 3-oxo-4-pentenoate, which reacts with glutathione and thereby depletes the mitochondrial glutathione pool. (S)-3-Hydroxy4-pentenoate may find use in the study of mitochondrial glutathione homeostasis and the role of mitochondrial glutathione in cellular protection.

Introduction Glutathione plays important roles in antioxidant defense and in the detoxification of xenobiotics. The hepatocellular glutathione content is largely distributed between cytosolic (∼85%) and mitochondrial pools (∼15%) (1-3), although a small (∼2%) nuclear pool has also been identified (4). Previous studies indicate that the mitochondrial glutathione pool plays a critical role in cytoprotection against xenobiotic-induced cell damage: the onset of cell injury induced by ethacrynic acid or 1,3bis(2-chloroethyl)-1-nitrosourea and adriamycin correlates with the depletion of the mitochondrial glutathione pool (5, 6). Although diethyl maleate depletes cytosolic glutathione concentrations (6) and ethacrynic acid depletes both cytosolic and mitochondrial glutathione concentrations (3, 6), (R,S)-3-hydroxy-4-pentenoate potently and selectively depletes the mitochondrial glutathione pool in rat hepatocytes (7). Moreover, (R,S)-3-hydroxy-4-pentenoate potentiates the cytotoxicity of tert-butyl hydroperoxide, ethacrynic acid, and menadione, but does not induce mitochondrial dysfunction or cytotoxicity. These properties of (R,S)-3-hydroxy-4-pentenoate make it a candidate for further development in exploring the role of mitochondrial glutathione in cellular homeostasis. Chirality plays an important role in the absorption, protein binding, metabolism, and excretion of xenobiotics †

University of Rochester. Present address: Department of Radiology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642. § Universita ¨ t Du¨sseldorf. X Abstract published in Advance ACS Abstracts, December 1, 1995. ‡

0893-228x/96/2709-0361$12.00/0

(8). The oxidation of (R,S)-3-hydroxy-4-pentenoate is catalyzed by 3-hydroxybutanoate dehydrogenase (7), which shows high selectivity for (R)-3-hydroxybutanoate as a substrate (9). The objective of the present work was to study the enantioselectivity of the oxidation of 3-hydroxy-4-pentenoate by 3-hydroxybutanoate dehydrogenase and to evaluate the enantiomers of (R,S)-3-hydroxy4-pentenoate as depletors of the mitochondrial glutathione pool. (S)-3-Hydroxy-4-pentenoate proved to be a selective depletor of the mitochondrial glutathione pool, whereas (R)-3-hydroxy-4-pentenoate was relatively inactive.

Experimental Procedures Materials. Male Fischer rats (150-200 g) were purchased from Charles River Laboratories (Wilmington, MA). (R,S)-3Hydroxy-4-pentenoic acid was obtained by synthesis (10). Glutathione, glutathione disulfide, digitonin, and 3-hydroxybutanoate dehydrogenase (from Pseudomonas lemoignei, type IV) were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase H was obtained from Boehringer Mannheim Corp. (Indianapolis, IN). All other chemicals were obtained from commercial sources in the highest purity available. Instrumental Analyses. 1H NMR spectra were recorded with a Bruker WP270 spectrometer operating at 270.13 MHz. Chemical shifts are expressed in ppm downfield from tetramethylsilane. HPLC analyses were performed with a Gilson 305/306 pump system and a Perkin-Elmer LC-235 diode array detector; the chromatographic conditions are described below. Preparation of (R)- and (S)-3-Hydroxy-4-pentenoic Acid. (R)-3-Hydroxy-4-pentenoic acid, [R]17D ) -26.0° (c ) 1.0 in 95% aqueous ethanol), was obtained by stereoselective aldol reaction of commercially available (R)-2-hydroxy-1,2,2-triphenylethyl

© 1996 American Chemical Society

362 Chem. Res. Toxicol., Vol. 9, No. 2, 1996 acetate (11) with acrolein (12). In an analogous way, (S)-3hydroxy-4-pentenoic acid ([R]17D ) +26.0°) was prepared from commercial (S)-2-hydroxy-1,2,2-triphenylethyl acetate. 3-Hydroxybutanoate Dehydrogenase Assay. The activities of (R,S)-, (S)-, and (R)-3-hydroxy-4-pentenoate as substrates for 3-hydroxybutanoate dehydrogenase were measured by monitoring NADH formation at 340 nm (13). Isolation of Liver Mitochondria and Incubation Conditions. Liver mitochondria were isolated as described previously (14) from male Fischer rats. Protein concentrations were measured by the method of Bradford (15) with bovine serum albumin as the standard. Liver mitochondria (5 mg of protein/mL) were incubated with 0.05 or 0.15 mM (R,S)-, (S)-, or (R)-3-hydroxy-4-pentenoate in the presence of 0.5 mM NAD+ and 50 mM Tris buffer (pH 7.4) in a total reaction volume of 1 mL; the reaction mixture was incubated for the indicated times at 37 °C. The reaction was stopped by the addition of 0.05 mL of 70% perchloric acid. Glutathione concentrations were quantified as described below. Isolation and Incubation of Hepatocytes. Hepatocytes were isolated from male Fischer rats by the collagenase perfusion method of Molde´us et al. (16). Approximately 85% of the hepatocytes excluded trypan blue. Cells (106 cells/mL) were incubated with 0.15 mM (R,S)-, (S)-, or (R)-3-hydroxy-4-pentenoate, Krebs-Henseleit buffer (118 mM NaCl, 24 mM NaHCO3, 4.8 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, and 2.6 mM CaCl2), and 25 mM Hepes buffer (pH 7.2) in a final volume of 1.0 mL at 37 °C under an atmosphere of air. Digitonin Fractionation of Hepatocytes. Mitochondrial and cytosolic glutathione pools were separated as described previously (17). Plastic 1.5-mL centrifuge tubes containing, from the bottom, 0.1 mL of 20% (v/v) perchloric acid, 0.5 mL of a 6:1 (v/v) mixture of silicone oil (d ) 1.050) and white, light, domestic paraffin oil (Saybolt viscosity 125/135), and a 0.1-mL top layer of 19.8 mM EDTA, 19.8 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and 250 mM mannitol in 19.8 mM 3-morpholinosulfonic acid buffer (pH 7.4) were prepared. A 0.05-mL sample of the incubation mixture was rapidly mixed with the top layer, and the tubes were centrifuged within 1 min at 13300g (Fisher microcentrifuge, Model 59A) at room temperature for 3 min. Cytosolic and mitochondrial fractions were separated by including 0.02 mg of digitonin in the 3-morpholinosulfonic acid buffer. A 0.05-mL sample was taken from the supernatant, and the silicone-paraffin oil mixture was removed by aspiration. Krebs-Henseleit buffer (0.8 mL) and 0.1 mL of 6% Triton X-100 were added to the bottom layer. Samples from the supernatant and the bottom layer were assayed for glutathione concentrations. Quantification of Glutathione Concentrations. Glutathione concentrations were measured by the HPLC method of Reed et al. (18). Samples (1 mL) were brought to pH 8.0 with 1 M K2CO3 (cresol red indicator, 5 mg/L) and derivatized by the addition of 0.08 mL of iodoacetic acid (20 mg/mL). After 60 min, 0.5 mL of 20% 2,4-dinitrofluorobenzene in ethanol was added to the samples, which were kept overnight at room temperature in the dark. Prior to HPLC analysis, the samples were centrifuged at 13300g (Fisher microcentrifuge, Model 59A) for 5 min at 5 °C to remove precipitated KClO4. Glutathione concentrations were determined by HPLC on a Lichrosorb-NH2 column (5 µm, 250 × 4 mm, E. Merck, Gibbstown, NJ) with a 80% methanol-sodium acetate buffer (pH 4.56) as the eluent. The composition of the gradient was adjusted to maintain a constant retention time for glutathione. Control experiments in which no iodoacetic acid was added showed that the glutathione adduct of 3-oxo-4-pentenoate [S-(3-oxo-4-carboxybutyl)glutathione] did not interfere with the analysis of glutathione. Statistical Analyses. Data in Figures 2 and 3 were analyzed by a two-way analysis of variance with PRISM v1.03 or INSTAT v2.05a (GraphPad Software, Inc., San Diego, CA). A level of p < 0.05 was chosen for acceptance or rejection of the null hypothesis.

Hashmi et al.

Figure 1. Oxidation of (R,S)-, (S)-, and (R)-3-hydroxy-4pentenoate by 3-hydroxybutanoate dehydrogenase. (R,S)-3hydroxy-4-pentenoate (0.05 mM, b; 0.15 mM, O), (S)-3-hydroxy4-pentenoate (0.05 mM, 2; 0.15 mM, 4), and (R)-3-hydroxy-4pentenoate (0.05 mM, [; 0.15 mM, ]) were incubated with 50 milliunits of 3-hydroxybutanoate dehydrogenase in the presence of 0.5 mM NAD+ at 37 °C for the indicated times, and NADH formation was determined as described in Experimental Procedures. Data are shown as mean ( SD (n ) 3).

Results 3-Hydroxybutanoate Dehydrogenase Assay. Incubation of 0.05 or 0.15 mM (S)-3-hydroxy-4-pentenoate with 3-hydroxybutanoate dehydrogenase resulted in a time-dependent increase in NADH formation (Figure 1). NADH formation was greatest with (S)-3-hydroxy-4pentenoate as the substrate. Little NADH formation was observed with (R)-3-hydroxy-4-pentenoate as the substrate, and an intermediate rate of NADH formation was seen with (R,S)-3-hydroxy-4-pentenoate as the substrate. Effect of (R,S)-, (S)-, or (R)-3-Hydroxy-4-pentenoate on Hepatic Mitochondrial Glutathione Concentrations. Incubation of isolated rat liver mitochondria with 0.05 and 0.15 mM (R,S)- or (S)-3-hydroxy-4pentenoate resulted in a time- and concentration-dependent reduction in mitochondrial glutathione concentrations (Figure 2A,B). Glutathione depletion with 0.05 and 0.15 mM (S)-3-hydroxy-4-pentenoate was greater than that observed with (R,S)-3-hydroxy-4-pentenoate. (R)-3-Hydroxy-4-pentenoate produced little depletion of mitochondrial glutathione concentrations (Figure 2C). Effect of (R,S)-, (S)-, or (R)-3-Hydroxy-4-pentenoate on Mitochondrial and Cytosolic Glutathione Concentrations in Rat Hepatocytes. Hepatocyte mitochondrial and cytosolic fractions were separated by treatment of the cell suspension with digitonin (18). Incubation of rat hepatocytes with 0.15 mM (R,S)- or (S)3-hydroxy-4-pentenoate resulted in a time-dependent depletion of mitochondrial glutathione concentrations (Figure 3A) that was significantly greater at all times than that observed in cytosolic fractions (Figure 3B), and greater depletion was observed with the (S)-enantiomer than with the racemic modification in both mitochondria and cytosol at all times, except at 15 min in cytosolic fractions. (R)-3-Hydroxy-4-pentenoate did not deplete mitochondrial or cytosolic glutathione concentrations (Figure 3A,B).

Discussion Previous studies showed that (R,S)-3-hydroxy-4-pentenoate (1) is a substrate for 3-hydroxybutanoate dehy-

Enantioselective Depletion of Mitochondrial GSH

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Figure 2. Time- and concentration-dependent depletion of rat hepatic mitochondrial glutathione concentrations by (R,S)-, (S)-, and (R)-3-hydroxy-4-pentenoate. Mitochondria were incubated with 0 (b), 0.05 (0), or 0.15 (2) mM (R,S)- (panel A), (S)- (panel B), or (R)-3-hydroxy-4-pentenoate (panel C) for 0, 5, 15, or 30 min at 37 °C. Mitochondrial glutathione concentrations were measured as described in the Experimental Procedures. Data are shown as mean ( SD (n ) 3). The depletion of mitochondrial glutathione concentrations by both 0.05 and 0.15 mM (S)-3-hydroxy-4-pentenoate was significantly greater (p < 0.05) than that produced by respective concentrations of (R,S)-3-hydroxy-4-pentenoate.

Figure 3. Time-dependent depletion of rat hepatocyte mitochondrial (panel A) or cytosolic (panel B) glutathione concentrations by (R,S)-, (S)-, or (R)-3-hydroxy-4-pentenoate. Hepatocytes were incubated with 0.15 mM (R,S)- (b), (S)- (2), or (R)- ([) 3-hydroxypentenoate for 0, 5, 15, or 30 min at 37 °C. Glutathione concentrations were measured as described in Experimental Procedures. Control mitochondrial and cytosolic glutathione concentrations at 0, 5, 15, and 30 min were 4.3 ( 0.5, 4.13 ( 0.3, 3.5 ( 0.3, and 3.8 ( 0.1 and 26.64 ( 3.2, 28.2 ( 2.6, 26.0 ( 2.9, and 26.5 ( 4.0 nmol of glutathione/106 cells, respectively. Data are shown as mean ( SD (n ) 3). The depletion of mitochondrial glutathione concentrations by (R,S)- or (S)-3hydroxy-4-pentenoate was significantly greater (p < 0.05) than the depletion observed in cytosol at all times, and the depletion of glutathione concentrations by (S)-3-hydroxy-4-pentenoate was greater than that by (R,S)-3-hydroxy-4-pentenoate, except at 15 min in cytosolic fractions.

Figure 4. Postulated mechanism for the 3-hydroxybutanoate dehydrogenase-dependent oxidation of (S)-3-hydroxy-4-pentenoate (1); [2, 3-oxo-4-pentenoate; 3, (S)-(3-oxo-4-carboxybutyl)glutathione; GSH, glutathione; 3-HBD, 3-hydroxybutanoate dehydrogenase]. The structure of (R)-3-hydroxybutanoate is also shown.

drogenase and is oxidized to 3-oxo-4-pentenoate (2), which reacts with glutathione to give the conjugate S-(3oxo-4-carboxybutyl)glutathione (3) (7) (Figure 4). The bioactivation of (R,S)-3-hydroxy-4-pentenoate and the selective depletion of mitochondrial glutathione concentrations require mitochondria and are inhibited by (R)3-hydroxybutanoate (7). In the present work, these studies were extended to investigate the stereoselective oxidation of 3-hydroxy-4-pentenoate enantiomers by 3-hydroxybutanoate dehydrogenase and the depletion of mitochondrial glutathione in the isolated liver mitochon-

dria and in rat hepatocytes by (S)- and (R)-3-hydroxy-4pentenoate. Time- and concentration-dependent increases in NADH formation were observed when (R,S)- or (S)-3-hydroxy4-pentenoate was incubated with 3-hydroxybutanoate dehydrogenase; greater NADH formation was observed with (S)-3-hydroxy-4-pentenoate than with (R,S)-3-hydroxy-4-pentenoate. (R)-3-Hydroxy-4-pentenoate was almost inactive at the concentrations studied (Figure 1). (R)-3-Hydroxybutanoate is the preferred substrate for 3-hydroxybutanoate dehydrogenase (9). In the present studies, (S)-3-hydroxy-4-pentenoate was the preferred substrate. Although (R)-3-hydroxybutanoate and (S)-3hydroxy-4-pentenoate have opposite absolute stereochemical configurations according to sequence rule, they have the same relative configuration (19). Incubation of (R,S)- or (S)-3-hydroxy-4-pentenoate with rat liver mitochondria resulted in the depletion of mitochondrial glutathione concentrations (Figure 2). The depletion of mitochondrial glutathione concentrations by (S)-3-hydroxy-4-pentenoate was greater than that produced by (R,S)-3-hydroxy-4-pentenoate. (R)-3-Hydroxy4-pentenoate did not deplete mitochondrial glutathione concentrations. A time-dependent decrease in mitochondrial glutathione concentrations was observed when isolated rat hepatocytes were incubated with (S)-3-hydroxy-4-pentenoate, indicating that this enantiomer selectively depletes the mitochondrial glutathione pool relative to the cytosolic glutathione pool (Figure 3). (R)-3-Hydroxy-4pentenoate did not deplete mitochondrial glutathione concentrations, and (R,S)-3-hydroxy-4-pentenoate produced an intermediate depletion, as reported previously (7). (S)-3-Hydroxy-4-pentenoate decreased cytosolic glutathione concentrations, but the decrease was significantly smaller than that seen in mitochondria. The mechanism of the depletion of cytosolic glutathione concentrations by (S)-3-hydroxy-4-pentenoate, which is bioactivated by the mitochondrial enzyme 3-hydroxybutanoate dehydrogenase (7), is not presently understood, but may be due to the leakage of 3-oxo-4-pentenoate from its site of formation in the mitochondria into the cytosol. Alternatively, the profound decrease in mitochondrial glutathione concentrations produced by (S)-3-hydroxy4-pentenoate may have created a sink that allowed cytosolic glutathione to flow into the mitochondria, thereby depleting the cytosolic glutathione pool. Glutathione is actively taken up by mitochondria (20-25),

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and these transport systems may contribute to the observed decrease in cytosolic glutathione concentrations produced by (S)-3-hydroxy-4-pentenoate. The results presented in this paper show that the mitochondrial glutathione depletion produced by (R,S)3-hydroxy-4-pentenoate is due to the enantioselective oxidation of (S)-3-hydroxy-4-pentenoate to 3-oxo-4-pentenoate by 3-hydroxybutanoate dehydrogenase. (S)-3Hydroxy-4-pentenoate may find use as a tool to manipulate mitochondrial glutathione concentrations and to explore the role of mitochondrial glutathione in cytoprotection.

Acknowledgment. This research was supported by National Institutes of Environmental Health Sciences Grant ES03127 (M.W.A.) and the Fonds der Chemischen Industrie (M.B.). The authors thank Ms. Sandra E. Morgan for her assistance in preparing the manuscript.

References (1) Jocelyn, P. C., and Kamminga, A. (1974) The non-protein thiol of rat liver mitochondria. Biochim. Biophys. Acta 343, 356-362. (2) Jocelyn, P. C. (1975) Some properties of mitochondrial glutathione. Biochim. Biophys. Acta 369, 427-436. (3) Wahlla¨nder, A., Soboll, S., and Sies, H. (1979) Hepatic mitochondrial and cytosolic glutathione content and the subcellular distribution of GSH-S-transferases. FEBS Lett. 97, 138-140. (4) Jevtovic-Todorovic, V., and Guenthner, T. M. (1992) Depletion of a discrete nuclear glutathione pool by oxidative stress, but not by buthionine sulfoximine. Correlation with enhanced alkylating agent cytotoxicity to human melanoma cells in vitro. Biochem. Pharmacol. 44, 1383-1393. (5) Meredith, M. J., and Reed, D. J. (1982) Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and 1,3-bis(2-chloroethyl)-1nitrosourea (BCNU). Biochem. Pharmacol. 32, 1383-1388. (6) Meredith, M. J., and Reed, D. J. (1982) Status of the mitochondrial pool of glutathione in the isolated hepatocyte. J. Biol. Chem. 257, 3747-3753. (7) Shan, X., Jones, D. P., Hashmi, M., and Anders, M. W. (1993) Selective depletion of mitochondrial glutathione concentrations by (R,S)-3-hydroxy-4-pentenoate potentiates oxidative cell death. Chem. Res. Toxicol. 6, 75-81. (8) Williams, K. M. (1991) Molecular asymmetry and its pharmacological consequences. Adv. Pharmacol. 22, 58-135.

Hashmi et al. (9) Lehninger, A. L., Sudduth, H. C., and Wise, J. B. (1960) D-βHydroxybutyric dehydrogenase of mitochondria. J. Biol. Chem. 235, 2450-2455. (10) Zibuck, R., and Streiber, J. M. (1989) A new preparation of ethyl 3-oxo-4-pentenoate: A useful annelating reagent. J. Org. Chem. 54, 4717-4719. (11) Braun, M., Gra¨f, S., and Herzog, S. (1993) (R)-(+)-2-Hydroxy-1,2,2triphenylethyl acetate. Org. Synth. 72, 32-37. (12) Gra¨f, S., and Braun, M. (1993) De-novo synthesis of enantiomerically pure deoxy- and aminodeoxyfuranosides. Liebigs Ann. Chem., 1091-1098. (13) Williamson, D. H., and Mellanby, J. (1974) D(-)-3-Hydroxybutyrate. In Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.) pp 1836-1839, Academic Press, New York. (14) Johnson, D., and Lardy, H. (1967) Isolation of liver and kidney mitochondria. Methods Enzymol. 10, 94-96. (15) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (16) Molde´us, P., Ho¨gberg, J., and Orrenius, S. (1978) Isolation and use of liver cells. Methods Enzymol. 52, 60-71. (17) Andersson, B. S., and Jones, D. P. (1985) Use of digitonin fractionation to determine mitochondrial transmembrane ion distribution in cells during anoxia. Anal. Biochem. 146, 164172. (18) 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. (19) Cahn, R. S. (1964) An introduction to the sequence rules. J. Chem. Educ. 41, 116-125. (20) Griffith, O. W., and Meister, A. (1985) Origin and turnover of mitochondrial glutathione. Proc. Natl. Acad. Sci. U.S.A. 82, 46684672. (21) Ma˚rtensson, J., Lai, J. C. K., and Meister, A. (1990) High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function. Proc. Natl. Acad. Sci. U.S.A. 87, 7185-7189. (22) Kurosawa, K., Hayashi, N., Sato, N., Kamada, T., and Tagawa, K. (1990) Transport of glutathione across the mitochondrial membranes. Biochem. Biophys. Res. Commun. 167, 367-372. (23) Schnellmann, R. G. (1991) Renal mitochondrial glutathione transport. Life Sci. 49, 393-398. (24) McKernan, T. B., Woods, E. B., and Lash, L. H. (1991) Arch. Biochem. Biophys. 288, 653-663. (25) Lash, L. H. (1995) Mitochondrial uptake of glutathione and nephrotoxicity. Toxicologist 15, 213.

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