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Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice
Mechanisms of Ageing and Development 120 (2000) 127 – 139 www.elsevier.com/locate/mechagedev
Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice Theresa S. Chen a,*, John P. Richie Jr. b, Herbert T. Nagasawa c, Calvin A. Lang d a
Department of Pharmacology and Toxicology, School of Medicine, Uni6ersity of Louis6ille, Louis6ille, KY 40292, USA b American Health Foundation, Valhalla, NY, USA c VA Medical Center and Department of Medicinal Chemistry, Uni6ersity of Minnesota, Minneapolis, MN, USA d Department of Biochemistry and Molecular Biology, Uni6ersity of Louis6ille, Louis6ille, KY 40292, USA Received 26 May 2139; received in revised form 11 September 2000; accepted 12 September 2000
Abstract Our previous results indicated that glutathione (GSH) and/or cysteine (Cys) deficiency occurs in many aging tissues and also after acetaminophen (APAP) administration. The aim of this study was to investigate whether GSH monoethyl ester (GSH-OEt) can correct these deficiencies. Mice of different ages (3 – 31 months) through the life span were sacrificed 2 h after i.p. injection of GSH-OEt (10 mmol/kg). In separate experiments, old mice (30 – 31 months) received the same dose of ester 30 min before the administration of APAP (375 mg/kg) or buthionine sulfoximine (BSO, 4 mmol/kg), an inhibitor of GSH biosynthesis. Liver and kidney samples were analyzed for GSH and Cys by HPLC. The hepatic GSH and renal cortical GSH and Cys concentrations were about 30% lower in old mice (30 – 31 months) compared to mature mice (12 months). GSH-OEt corrected these aging-related decreases. APAP decreased both hepatic and renal cortical GSH and Cys concentrations in old mice, but GSH-OEt prevented these decreases. GSH-OEt also prevented the BSO-induced decreases in hepatic and renal GSH concentrations. The results demonstrated that GSH-OEt
* Corresponding author. Tel.: +1-502-8527887; fax: +1-502-8527868. E-mail address: [email protected] (T.S. Chen). 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 4 - 1
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protected against GSH deficiency due to biological aging as well as APAP-induced decreases in old mice. © 2000 Published by Elsevier Science Ireland Ltd. Keywords: Glutathione; Acetaminophen; Glutathione monoethyl ester; Liver; Kidney; Aging
1. Introduction Glutathione (GSH), the major cellular antioxidant, has diverse biological functions including protection of cells from damage by substances such as reactive oxygen species, free radicals, and reactive metabolites of acetaminophen (APAP) as well as other drugs (Meister and Anderson, 1983). Our previous results indicated that GSH deficiency is found in many tissues in old animals, including liver, kidney, blood, and brain (Chen et al., 1989, 1990; Richie et al., 1992). Also, low levels of GSH were manifested in various chronic diseases (Harding et al., 1996). In view of the biological importance of GSH, there is considerable merit in therapeutic intervention strategies to correct the GSH deficiencies in such situations. Since intact GSH does not enter cells readily, administration of exogenous GSH is not a viable means of increasing tissue GSH levels (Puri and Meister, 1983). In contrast, precursors of GSH or cysteine (Cys), such as GSH esters or 2-substituted thiazolidine-4-carboxylic acids (Cys prodrugs) enter cells more readily (Goldman, 1976; Nagasawa et al., 1984; Anderson et al., 1985; Roberts et al., 1987), and thus may provide more effective means to increase tissue GSH levels. Earlier work (Richie et al., 1987; Richie and Lang, 1988) demonstrated that feeding adult mosquitoes with magnesium thiazolidine-4-carboxylate, a Cys precursor, increased both total body GSH content and longevity. Puri and Meister (1983) reported that administration of the monomethyl ester of GSH (GSH-OMe), a GSH precursor, to fasting adult mice led to substantial increases in liver and kidney GSH concentrations. Whether glutathione monoethyl ester (GSH-OEt) can also correct the GSH deficiencies in aging has not been investigated. Also, the effects of the ester on possible aging-related changes in glutathione status (i.e., the concentrations of GSH and the related compounds glutathione disulfide (GSSG), Cys, and cystine) in the liver and kidney have not been reported. Acetaminophen (APAP) is a widely used antipyretic and analgesic drug, which in overdose, can cause extensive hepatic and renal damage (Boyer and Rouff, 1971; Kleinman et al., 1980). APAP-induced tissue necrosis is believed to result form the covalent binding of reactive intermediates to cellular macromolecules. GSH plays an important role in the detoxification of these reactive intermediates in both the liver and the kidney (Mitchell et al., 1973; Mudge et al., 1978). Further, older animals are known to be more susceptible to the toxic effects of drugs such as APAP (Tarloff et al., 1996). We showed previously that the extent of APAP-induced GSH depletion and the recovery from depletion were age-dependent and that detoxification capacities in both liver and kidneys of old mice were compromised (Chen et al., 1990; Richie et al., 1992). GSH-OMe has been shown to enhance liver GSH levels and prevent APAP-induced hepatotoxicity in fasting adult mice (Puri and Meister, 1983). Whether GSH-OEt acts similarly in old animals has not been reported.
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The objectives of the present study were to determine (1) whether GSH-OEt can enhance tissue GSH and Cys levels at different stages in the life span of mice; (2) whether GSH-OEt can correct the GSH deficiencies due to old age and to APAP; and (3) whether the enhancement of GSH levels requires breakdown of GSH-OEt to constituent amino acids followed by de novo biosynthesis of GSH through studies with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis.
2. Materials and methods
2.1. Chemicals APAP, GSH, GSSG, Cys, cystine, BSO, monochloroacetic acid, and heptanesulfonic acid were obtained from Sigma Chemical (St. Louis, MO). Metaphosphoric acid was obtained from Aldrich Chemical (Milwaukee, WI). GSH-OEt (free base) was synthesized by the method of Campbell and Griffith (1989).
2.2. Animals Male C57BL/6 mice, 3 – 31-month-old, were obtained from the National Institute on Aging colony at Charles River Breeding Laboratories (Wilmington, MA). The ages studied represented the growth (3–6 months), mature (12–24 months), and aging or old (30 – 31 months) stages of the life span of this mouse strain (Chen et al., 1989, 1990). In this paper, mice in the 30–31-month-old age group are referred to as ‘old’ mice. All mice were acclimated for a week in our animal care facility in rooms on a 12 h light – dark cycle and received food and water ad libitum.
2.3. Treatments Mice of different ages (3 – 31 months) were sacrificed 2 h after i.p. injection of 10 mmol/kg of GSH-OEt in 0.9% NaCl (8 ml/kg). In separate experiments, old mice (30– 31 months) were sacrificed 4 h after i.p. injection of 375 mg/kg APAP in ethanol:propylene glycol (1:4). In experiments on the protective effects of GSHOEt, old mice received an i.p. dose of 10 mmol/kg of the ester 30 min before the administration of APAP (375 mg/kg). The GSH-OEt solution was freshly prepared and injected immediately after preparation. To determine the effects of GSH-OEt on de novo GSH biosynthesis, old mice received the same dose of GSH-OEt 30 min after i.p. injection of 4 mmol/kg BSO in 0.9% NaCl. Mice were sacrificed 2 h after BSO treatment. In control experiments, mice received comparable volumes of the vehicle alone.
2.4. Tissue preparation Mice were sacrificed by cervical dislocation and decapitation. Liver and kidneys were excised, rinsed in ice-cold 0.9% NaCl, trimmed of adherent tissues, and
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weighed. Samples were kept on ice and processed as quickly as possible to prevent oxidation and degradation of sulfhydryl compounds. A 10% tissue homogenate (w/v) of liver or kidney cortex was prepared in 5% MPA, using an all-glass Tenbroeck homogenizer, and kept on ice. After standing for 20–40 min, the homogenate was centrifuged for 1 min (10 000× g) and the acid-soluble fraction was collected for measurement of sulfhydryl and disulfide compounds.
2.5. Analysis of glutathione and cyst(e)ine GSH, GSSG, Cys, and cystine were quantified by HPLC with dual electrochemical detection (Richie and Lang, 1987). In brief, 40-ml samples were injected onto a 250 ×4.6 mm, 5 mm, C-18 column, eluted isocratically with a mobile phase consisting of 0.1 M monochloroacetic acid, 2 mM heptane sulfonic acid, and 4% methanol at pH 3.0, delivered at a flow rate of 1 ml/min. The compounds were detected in the eluant with a Bioanalytical Systems model LC4B dual electrochemical detector using two Au/Hg electrodes in series with potentials of − 1.00 and 0.15 V for the upstream and downstream electrodes, respectively. Current (nA) was measured at the downstream electrode. Analytes were quantified from peak area measurements using external standards.
2.6. Statistical analyses The data were analyzed using the SPSS for Windows computer program (SPSS, Chicago, IL). Differences between the means of two groups were evaluated by a two-tailed t-test for independent samples. Differences between the means of three or more groups were evaluated by an ANOVA. A value of PB 0.05 was considered statistically significant.
3. Results
3.1. Effects of aging and GSH-OEt on hepatic GSH status The hepatic GSH concentrations in 30–31-month-old mice were significantly lower than those in 3-, 12- and 24-month-old mice (Fig. 1, upper panel). The values were 6.3590.19 mmol/g in 12-month-old mice and 4.539 1.22 mmol/g in 30–31month-old mice. In contrast, there were no significant changes in hepatic Cys (Fig. 1, lower panel), or in cystine and GSSG concentrations throughout the life span (data not shown). The effects of GSH-OEt on hepatic GSH status at different ages are also shown in Fig. 1. GSH-OEt (10 mmol/kg) caused small but statistically insignificant increases in hepatic GSH concentration in mice aged 3, 12, and 24 months. It caused a 41% increase (P B0.05) in hepatic GSH concentration in old (30–31 months) mice. The ester caused approximately twofold increases in Cys concentrations in 3-, 12- and 24-month-old mice, but did not have a significant effect in
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Fig. 1. Effects of aging and GSH-OEt on hepatic GSH and Cys concentrations in mice. GSH and Cys concentrations were determined in samples from mice of different ages throughout the life span (3 – 31-month-old). GSH-OEt (10 mmol/kg) was administered i.p. and mice were sacrificed 2 h later. Data bars represent mean9 S.D. (n= 3 to 8). * Significantly different from controls of the respective age group. a Significantly different from 3-, 12-, and 24-month-old controls.
30– 31-month-old mice. Furthermore, there were no significant effects on hepatic cystine or GSSG concentrations in any of the age groups of mice (data not shown).
3.2. Effects of aging and GSH-OEt on renal cortical GSH status Renal cortical GSH and Cys concentrations ranged from 1.59 to 2.66, and 0.75 to 1.27 mmol/g, respectively, in all age groups (Fig. 2). The concentrations of GSH in 30 – 31-month-old mice were significantly lower than in 3-month-old mice (Fig. 2,
Fig. 2. Effects of aging and GSH-OEt on renal cortical GSH and Cys concentrations in mice. Data presentation as described in legend for Fig. 1. Data bars represent mean 9S.D. (n =4 to 7). (*) Significantly different from controls of the respective age group. a Significantly different from 3-monthold control; b significantly different from 3- and 24-month-old controls.
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Fig. 3. Hepatic effects of APAP and GSH-OEt in old (30 – 31 months) mice. APAP (375 mg/kg) was administered i.p. and mice were sacrificed 4 h later. APAP+ GSH-OEt denotes group receiving GSH-OEt (10 mmol/kg) 30 min before the administration of APAP. Data bars represent mean 9S.D. (n = 4 to 5). a Significantly different from the 30 – 31-month-old controls; b significantly different from APAP alone.
upper panel), and the concentrations of Cys in 30–31-month-old mice were significantly lower than that in both the 3- and 24-month-old groups (Fig. 2, lower panel). The renal cortical cystine and GSSG concentrations ranged from 0.07 to 0.10 mmol/g and 0.06 to 0.07 mmol/g, respectively, in all age groups of mice and neither of these disulfides changed with age. GSH-OEt (10 mmol/kg) significantly increased the renal cortical GSH and Cys concentrations in mice of different ages throughout their life span. In each age group, GSH concentrations were increased to 2–3-fold and Cys concentrations were increased to 3 – 4-fold over controls (Fig. 2). Interestingly, the ester also produced significant increases in renal cortical cystine (0.23–0.29 mmol/g) and GSSG concentrations (0.17 – 0.32 mmol/g) in each age group.
3.3. Hepatic effects of APAP and GSH-OEt in old (30 – 31 months) mice APAP at a dose of 375 mg/kg produced significant decreases in hepatic GSH and Cys concentrations 4 h after treatment; the concentrations of GSH and Cys were 30% and 15%, respectively, of control values in old mice (Fig. 3). APAP did not affect cystine or GSSG concentrations. GSH-OEt (10 mmol/kg) administered 30 min before APAP, prevented the APAP-induced decreases in hepatic GSH and Cys concentrations. Hepatic GSH and Cys concentrations in mice receiving the ester plus APAP were 117% and 126%, respectively, of the values in untreated
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controls. The hepatic concentration of GSSG was also significantly increased in animals receiving APAP plus GSH-OEt (0.899 0.32 mmol/g) as compared to untreated controls (0.16 90.11 mmol/g) and mice receiving APAP alone (0.06 9 0.04 mmol/g).
3.4. Renal effects of APAP and GSH-OEt in old (30 – 31 months) mice APAP caused significant decreases in renal cortical GSH and Cys concentrations at 4 h to 36% and 15%, respectively, of control values in old mice (Fig. 4). GSH and Cys concentrations were decreased to 0.5790.20 and 0.119 0.05 mmol/g, respectively, after APAP administration. GSH-OEt attenuated the APAP-induced decreases in renal GSH and Cys concentrations. Renal cortical GSH and Cys concentrations in mice receiving the ester plus APAP were 289% and 214%, respectively, of the values in untreated controls. Renal cortical GSSG concentration was also significantly higher in mice receiving GSH-OEt plus APAP (0.419 0.21 mmol/g) than in untreated controls (0.079 0.08 mmol/g) and mice receiving APAP alone (0.029 0.01 mmol/g).
3.5. Effects of GSH-OEt in BSO-treated old (30 – 31 months) mice The hepatic concentrations of GSH and Cys were decreased to 56% and 67%, respectively, of the control values 2 h after BSO (4 mmol/kg) administration. However, only the decrease in GSH concentration was statistically significant. GSH-OEt administered 30 min after BSO prevented the BSO-induced depletion of
Fig. 4. Renal effects of APAP and GSH-OEt in old (30 – 31 months) mice. Data presentation as described in the legend for Fig. 3. a Significantly different from the 30 – 31-month-old controls; b significantly different from APAP alone.
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Fig. 5. Hepatic effects of GSH-OEt in BSO-treated old (30 – 31 months) mice. BSO (4 mmol/kg), dissolved in 0.9% NaCl, was administered i.p.; controls received an equivalent volume of 0.9% NaCl. BSO+ GSH-OEt denotes group receiving BSO 30 min before the administration of GSH-OEt (10 mmol/kg). Data bars represent mean 9S.D. (n =4 to 5). a Significantly different from the 30 – 31-monthold controls; b significantly different from BSO alone.
sulfhydryl compounds (Fig. 5); hepatic GSH was maintained at the control level and Cys was 59% higher than the control value. BSO also caused significant decreases in the renal cortical concentrations of GSH and Cys at 4 h to 17% and 28%, respectively, of the control values (Fig. 6). GSH-OEt prevented the BSO-induced decreases; renal cortical GSH and Cys concentrations in mice receiving BSO plus GSH-OEt were 210% and 255%, respectively, of values in untreated controls.
Fig. 6. Renal effects of GSH-OEt in BSO-treated old (30 – 31 months) mice. Data presentation as described in the legend for Fig. 5. a Significantly different from the 30 – 31-month-old controls; b significantly different from BSO alone.
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4. Discussion
4.1. GSH-OEt corrects aging-related deficiency of GSH Our earlier studies showed that GSH deficiencies occur in many aging tissues (Hazelton and Lang, 1980; Richie and Lang, 1985; Chen et al., 1989, 1990). Similar deficiency of hepatic GSH and renal GSH and Cys in old (30–31 months) mice were observed in the present study, but there were no differences in cystine and GSSG concentrations. Others have also shown that hepatic GSH and Cys decreased significantly in 24-month-old mice compared to young mice without affecting cystine or GSSG contents of the liver (Nakata et al., 1996). In contrast, some investigators reported no change or a slight increase in GSH levels with age (Beierschmitt and Weiner, 1986; Rikans and Moore, 1988). The present study demonstrated for the first time that GSH-OEt, a GSH prodrug, is an effective enhancer of tissue sulfhydryl levels in old (30–31 months) mice. Thus, the administration of this ester corrected the GSH deficiency in liver and kidney of the old mouse and the Cys levels in the kidney and liver increased by 308% and 43%, respectively, over controls. Others have shown that GSH-OMe increased hepatic and renal GSH levels in fasting adult mice (Puri and Meister, 1983). GSH-OEt also increased GSH levels in human lymphoid cells and human skin fibroblasts in vitro (Wellner et al., 1984). In adult mosquitoes fed magnesium thiazolidine-4-carboxylic acid, a Cys precursor, both GSH content and longevity of the mosquitoes were greatly increased (Richie et al., 1987; Richie and Lang, 1988). In the present study, only the acute effects of GSH-OEt were determined. It is not known whether chronic administration of the ester would produce a sustained effect on GSH levels in old animals or increase longevity.
4.2. GSH-OEt pre6ents APAP-induced GSH and Cys depletion in old (30 – 31 months) mice Enhanced susceptibility to toxic challenge has been demonstrated in old animals for a variety of pharmacological agents including APAP. Earlier results demonstrated that the toxicity of APAP in the mosquito increased 7-fold, as assessed by LD50 values in senescence, and was highly correlated with a lower GSH content (Richie and Lang, 1985). This was further confirmed in aging (31-month-old) C57BL/6 mice where loss of APAP detoxification capacity in the liver and kidneys was correlated with the GSH deficiency of aging (Chen et al., 1990; Richie et al., 1992). It is known that older (18-month-old) rats are more susceptible to APAP-induced hepatic and renal damage than are younger (3-month-old) rats (Tarloff et al., 1996). Similarly, clinical studies suggest an increased risk of APAP toxicity in the elderly (Miners et al., 1988). The present study demonstrated that APAP depleted not only GSH but also Cys in the liver and kidneys of 31-month-old mice. The proportionately greater depletion of Cys (85%) than GSH (64–70%) observed in 30–31-month-old mice receiving APAP, suggest possible utilization of Cys for GSH biosynthesis following GSH
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depletion. However, it cannot be ruled out that the depletion of Cys may be due to direct Cys conjugation with the activated metabolite of APAP. The toxicity of APAP involves its metabolic conversion to a chemically reactive intermediate, N-acetyl-p-benzoquinone imine (Dahlin et al., 1984) that interacts rapidly with GSH and vital macromolecules (Mitchell et al., 1973). It is well established that APAP-induced hepatotoxicity is associated with depletion of hepatic GSH levels to about 30% or less of normal adult levels and can be attenuated by compounds that replenish tissue GSH levels (Mitchell et al., 1973). L-2-oxothiazolidine-4-carboxylate, a Cys prodrug, as well as other 2-substituted thiazolidine-4-carboxylic acids, are known to promote GSH biosynthesis and to protect against APAP toxicity by decreasing mortality in mice receiving LD90 doses of APAP (Williamson et al., 1982; Nagasawa et al., 1984; Roberts et al., 1987). The present study is the first report of means to increase tissue sulfhydryl levels in old (30 – 31 months) mice. The importance of this finding is that GSH-OEt may also be effective in preventing APAP-induced hepatotoxicity in old animals if administered acutely after APAP. Further, it is conceivable that long term correction of hepatic GSH deficiency (if possible) could reverse the increased susceptibility of old animals to APAP. In addition, tissue differences in the effects of GSH-OEt were observed. Renal GSH levels were increased to 2 –3-fold above control values at all ages studied, while hepatic GSH was increased only modestly and only in old mice. In studies with fasting adult mice, Anderson et al. (1985) also found that renal GSH levels were increased to a much greater extent than those of liver after administration of GSH-OEt. The observed tissue differences in the effects of GSH-OEt could be due to differences in the uptake of the ester. In fact, Anderson et al. (1985) demonstrated that the 35S incorporated into kidney GSH was about 4-fold that in liver after i.p. injection of 35S-labeled GSH monoethyl ester to mice.
4.3. Effects of GSH-OEt in BSO-treated old (30 – 31 months) mice BSO produced quantitatively different effects on the sulfhydryl levels in the liver and kidney of old mice. The extent of GSH and Cys depletion was greater in the kidneys than in the liver (Figs. 5 and 6). These data are in agreement with findings of others in adult mice (Griffith and Meister, 1979) and in the rat (Standeven and Wetterhahn, 1991). The differential effects of BSO can be attributed to a greater turnover of GSH in the kidneys due to high g-glutamyl transpeptidase activity in that organ (Griffith and Meister, 1979; Potter and Tran, 1993). BSO decreased not only tissue GSH but also Cys concentrations in old mice (Figs. 5 and 6). This effect of BSO on the Cys levels may be secondary to GSH depletion, or possibly a direct effect of BSO on the uptake of L-Cys, the rate-limiting substrate for GSH biosynthesis. Indeed, Griffith et al. (1979) showed that BSO inhibited the uptake of g-glutamyl amino acids in mouse kidney. Also, BSO is known to inhibit the uptake of L-Cys in cultured rat lenses (Murray and Rathbun, 1990).
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4.4. Possible mechanism of GSH-OEt-induced increases in GSH le6els in old (30 – 31 months) mice The mechanism by which GSH-OEt enhances tissue GSH levels is not clear. Two of the possible mechanisms include (1) cellular uptake of the ester followed by its hydrolysis to form GSH and (2) extracellular hydrolysis of the ester followed by g-glutamyl transpeptidase-catalyzed breakdown of the derived GSH to GSH precursors that are taken up by cells and utilized for GSH biosynthesis. Some evidence consistent with each of these two mechanisms is available from reported studies and the present results. Evidence supporting the first mechanism includes demonstrations of (1) rapid hydrolysis of GSH-OMe by mouse liver and kidney homogenates (Puri and Meister, 1983; Anderson et al., 1985); (2) greater effectiveness of GSH-OMe over GSH in replenishing APAP-induced depleted hepatic GSH stores in adult mice (Puri and Meister, 1983); and (3) greater permeability of rat liver lysosomal membranes to esters than to the corresponding free amino acids and dipeptides (Goldman and Kaplan, 1973; Goldman and Naider, 1974). Also, we found that the GSH-OEt was effective in increasing GSH levels in mice in which GSH biosynthesis was inhibited by BSO. On the other hand, evidence supporting the second mechanism includes the finding of Grattagliano et al. (1995) that GSH-OEt was not readily taken up by rat liver but was hydrolyzed by plasma esterases thereby slowly releasing GSH into the extracellular space. Our finding that the GSH-OEt also increased hepatic and renal Cys concentrations in old mice treated with APAP or BSO raises the possibility that some fraction of the ester may undergo hydrolysis to GSH followed by further breakdown to form Cys, as would be expected in the second mechanism.
Acknowledgements The authors thank Dr Walter M. Williams for helpful discussions and Marcia C. Liu for technical assistance. This work was supported in part by the National Institute on Aging.
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Richie, J.P., Jr., Lang, C.A., 1988. A decrease in cysteine levels causes the glutathione deficiency of aging in the mosquito. Proc. Soc. Exp. Biol. Med. 187, 235 – 240. Richie, J.P., Jr., Lang, C.A., Chen, T.S., 1992. Acetaminophen-induced depletion of glutathione and cysteine in the aging mouse kidney. Biochem. Pharmacol. 44, 129 – 135. Rikans, L.E., Moore, D.R., 1988. Acetaminophen hepatotoxicity in aging rats. Drug Chem. Toxicol. 11, 237–247. Roberts, J.C., Nagasawa, H.T., Zera, R.T., Fricke, R.F., Goon, D.J.W., 1987. Prodrugs of L-cysteine as protective agents against acetaminophen-induced hepatotoxicity. 2-(Polyhydroxyalkyl)- and 2-(polyacetoxyalkyl) thiazolidine-4(R)-carboxylic acids. J. Med. Chem. 30, 1891 – 1896. Standeven, A.M., Wetterhahn, K.E., 1991. Tissue-specific changes in glutathione and cysteine after buthionine sulfoximine treatment of rats and the potential for artifacts in thiol levels resulting from tissue preparation. Toxicol. Appl. Pharmacol. 107, 269 – 284. Tarloff, J.B., Khairallah, E.A., Cohen, S.D., Goldstein, R.S., 1996. Sex- and age-dependent acetaminophen hepato-and nephrotoxicity in Sprague-Dawley rats: role of tissue accumulation, nonprotein sulfhydryl depletion, and covalent binding. Fundam. Appl. Toxicol. 30, 13 – 22. Wellner, V.P., Anderson, M.E., Puri, R.N., Jensen, G.L., Meister, A., 1984. Radioprotection by glutathione ester: transport of glutathione ester into human lymphoid cells and fibroblasts. Proc. Natl. Acad. Sci. USA 81, 4732–4735. Williamson, J.M., Boettcher, B., Meister, A., 1982. Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis. Proc. Natl. Acad. Sci. USA 79, 6246 – 6249.
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Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice Theresa S. Chen a,*, John P. Richie Jr. b, Herbert T. Nagasawa c, Calvin A. Lang d a
Department of Pharmacology and Toxicology, School of Medicine, Uni6ersity of Louis6ille, Louis6ille, KY 40292, USA b American Health Foundation, Valhalla, NY, USA c VA Medical Center and Department of Medicinal Chemistry, Uni6ersity of Minnesota, Minneapolis, MN, USA d Department of Biochemistry and Molecular Biology, Uni6ersity of Louis6ille, Louis6ille, KY 40292, USA Received 26 May 2139; received in revised form 11 September 2000; accepted 12 September 2000
Abstract Our previous results indicated that glutathione (GSH) and/or cysteine (Cys) deficiency occurs in many aging tissues and also after acetaminophen (APAP) administration. The aim of this study was to investigate whether GSH monoethyl ester (GSH-OEt) can correct these deficiencies. Mice of different ages (3 – 31 months) through the life span were sacrificed 2 h after i.p. injection of GSH-OEt (10 mmol/kg). In separate experiments, old mice (30 – 31 months) received the same dose of ester 30 min before the administration of APAP (375 mg/kg) or buthionine sulfoximine (BSO, 4 mmol/kg), an inhibitor of GSH biosynthesis. Liver and kidney samples were analyzed for GSH and Cys by HPLC. The hepatic GSH and renal cortical GSH and Cys concentrations were about 30% lower in old mice (30 – 31 months) compared to mature mice (12 months). GSH-OEt corrected these aging-related decreases. APAP decreased both hepatic and renal cortical GSH and Cys concentrations in old mice, but GSH-OEt prevented these decreases. GSH-OEt also prevented the BSO-induced decreases in hepatic and renal GSH concentrations. The results demonstrated that GSH-OEt
* Corresponding author. Tel.: +1-502-8527887; fax: +1-502-8527868. E-mail address: [email protected] (T.S. Chen). 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 4 - 1
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protected against GSH deficiency due to biological aging as well as APAP-induced decreases in old mice. © 2000 Published by Elsevier Science Ireland Ltd. Keywords: Glutathione; Acetaminophen; Glutathione monoethyl ester; Liver; Kidney; Aging
1. Introduction Glutathione (GSH), the major cellular antioxidant, has diverse biological functions including protection of cells from damage by substances such as reactive oxygen species, free radicals, and reactive metabolites of acetaminophen (APAP) as well as other drugs (Meister and Anderson, 1983). Our previous results indicated that GSH deficiency is found in many tissues in old animals, including liver, kidney, blood, and brain (Chen et al., 1989, 1990; Richie et al., 1992). Also, low levels of GSH were manifested in various chronic diseases (Harding et al., 1996). In view of the biological importance of GSH, there is considerable merit in therapeutic intervention strategies to correct the GSH deficiencies in such situations. Since intact GSH does not enter cells readily, administration of exogenous GSH is not a viable means of increasing tissue GSH levels (Puri and Meister, 1983). In contrast, precursors of GSH or cysteine (Cys), such as GSH esters or 2-substituted thiazolidine-4-carboxylic acids (Cys prodrugs) enter cells more readily (Goldman, 1976; Nagasawa et al., 1984; Anderson et al., 1985; Roberts et al., 1987), and thus may provide more effective means to increase tissue GSH levels. Earlier work (Richie et al., 1987; Richie and Lang, 1988) demonstrated that feeding adult mosquitoes with magnesium thiazolidine-4-carboxylate, a Cys precursor, increased both total body GSH content and longevity. Puri and Meister (1983) reported that administration of the monomethyl ester of GSH (GSH-OMe), a GSH precursor, to fasting adult mice led to substantial increases in liver and kidney GSH concentrations. Whether glutathione monoethyl ester (GSH-OEt) can also correct the GSH deficiencies in aging has not been investigated. Also, the effects of the ester on possible aging-related changes in glutathione status (i.e., the concentrations of GSH and the related compounds glutathione disulfide (GSSG), Cys, and cystine) in the liver and kidney have not been reported. Acetaminophen (APAP) is a widely used antipyretic and analgesic drug, which in overdose, can cause extensive hepatic and renal damage (Boyer and Rouff, 1971; Kleinman et al., 1980). APAP-induced tissue necrosis is believed to result form the covalent binding of reactive intermediates to cellular macromolecules. GSH plays an important role in the detoxification of these reactive intermediates in both the liver and the kidney (Mitchell et al., 1973; Mudge et al., 1978). Further, older animals are known to be more susceptible to the toxic effects of drugs such as APAP (Tarloff et al., 1996). We showed previously that the extent of APAP-induced GSH depletion and the recovery from depletion were age-dependent and that detoxification capacities in both liver and kidneys of old mice were compromised (Chen et al., 1990; Richie et al., 1992). GSH-OMe has been shown to enhance liver GSH levels and prevent APAP-induced hepatotoxicity in fasting adult mice (Puri and Meister, 1983). Whether GSH-OEt acts similarly in old animals has not been reported.
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The objectives of the present study were to determine (1) whether GSH-OEt can enhance tissue GSH and Cys levels at different stages in the life span of mice; (2) whether GSH-OEt can correct the GSH deficiencies due to old age and to APAP; and (3) whether the enhancement of GSH levels requires breakdown of GSH-OEt to constituent amino acids followed by de novo biosynthesis of GSH through studies with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis.
2. Materials and methods
2.1. Chemicals APAP, GSH, GSSG, Cys, cystine, BSO, monochloroacetic acid, and heptanesulfonic acid were obtained from Sigma Chemical (St. Louis, MO). Metaphosphoric acid was obtained from Aldrich Chemical (Milwaukee, WI). GSH-OEt (free base) was synthesized by the method of Campbell and Griffith (1989).
2.2. Animals Male C57BL/6 mice, 3 – 31-month-old, were obtained from the National Institute on Aging colony at Charles River Breeding Laboratories (Wilmington, MA). The ages studied represented the growth (3–6 months), mature (12–24 months), and aging or old (30 – 31 months) stages of the life span of this mouse strain (Chen et al., 1989, 1990). In this paper, mice in the 30–31-month-old age group are referred to as ‘old’ mice. All mice were acclimated for a week in our animal care facility in rooms on a 12 h light – dark cycle and received food and water ad libitum.
2.3. Treatments Mice of different ages (3 – 31 months) were sacrificed 2 h after i.p. injection of 10 mmol/kg of GSH-OEt in 0.9% NaCl (8 ml/kg). In separate experiments, old mice (30– 31 months) were sacrificed 4 h after i.p. injection of 375 mg/kg APAP in ethanol:propylene glycol (1:4). In experiments on the protective effects of GSHOEt, old mice received an i.p. dose of 10 mmol/kg of the ester 30 min before the administration of APAP (375 mg/kg). The GSH-OEt solution was freshly prepared and injected immediately after preparation. To determine the effects of GSH-OEt on de novo GSH biosynthesis, old mice received the same dose of GSH-OEt 30 min after i.p. injection of 4 mmol/kg BSO in 0.9% NaCl. Mice were sacrificed 2 h after BSO treatment. In control experiments, mice received comparable volumes of the vehicle alone.
2.4. Tissue preparation Mice were sacrificed by cervical dislocation and decapitation. Liver and kidneys were excised, rinsed in ice-cold 0.9% NaCl, trimmed of adherent tissues, and
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weighed. Samples were kept on ice and processed as quickly as possible to prevent oxidation and degradation of sulfhydryl compounds. A 10% tissue homogenate (w/v) of liver or kidney cortex was prepared in 5% MPA, using an all-glass Tenbroeck homogenizer, and kept on ice. After standing for 20–40 min, the homogenate was centrifuged for 1 min (10 000× g) and the acid-soluble fraction was collected for measurement of sulfhydryl and disulfide compounds.
2.5. Analysis of glutathione and cyst(e)ine GSH, GSSG, Cys, and cystine were quantified by HPLC with dual electrochemical detection (Richie and Lang, 1987). In brief, 40-ml samples were injected onto a 250 ×4.6 mm, 5 mm, C-18 column, eluted isocratically with a mobile phase consisting of 0.1 M monochloroacetic acid, 2 mM heptane sulfonic acid, and 4% methanol at pH 3.0, delivered at a flow rate of 1 ml/min. The compounds were detected in the eluant with a Bioanalytical Systems model LC4B dual electrochemical detector using two Au/Hg electrodes in series with potentials of − 1.00 and 0.15 V for the upstream and downstream electrodes, respectively. Current (nA) was measured at the downstream electrode. Analytes were quantified from peak area measurements using external standards.
2.6. Statistical analyses The data were analyzed using the SPSS for Windows computer program (SPSS, Chicago, IL). Differences between the means of two groups were evaluated by a two-tailed t-test for independent samples. Differences between the means of three or more groups were evaluated by an ANOVA. A value of PB 0.05 was considered statistically significant.
3. Results
3.1. Effects of aging and GSH-OEt on hepatic GSH status The hepatic GSH concentrations in 30–31-month-old mice were significantly lower than those in 3-, 12- and 24-month-old mice (Fig. 1, upper panel). The values were 6.3590.19 mmol/g in 12-month-old mice and 4.539 1.22 mmol/g in 30–31month-old mice. In contrast, there were no significant changes in hepatic Cys (Fig. 1, lower panel), or in cystine and GSSG concentrations throughout the life span (data not shown). The effects of GSH-OEt on hepatic GSH status at different ages are also shown in Fig. 1. GSH-OEt (10 mmol/kg) caused small but statistically insignificant increases in hepatic GSH concentration in mice aged 3, 12, and 24 months. It caused a 41% increase (P B0.05) in hepatic GSH concentration in old (30–31 months) mice. The ester caused approximately twofold increases in Cys concentrations in 3-, 12- and 24-month-old mice, but did not have a significant effect in
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Fig. 1. Effects of aging and GSH-OEt on hepatic GSH and Cys concentrations in mice. GSH and Cys concentrations were determined in samples from mice of different ages throughout the life span (3 – 31-month-old). GSH-OEt (10 mmol/kg) was administered i.p. and mice were sacrificed 2 h later. Data bars represent mean9 S.D. (n= 3 to 8). * Significantly different from controls of the respective age group. a Significantly different from 3-, 12-, and 24-month-old controls.
30– 31-month-old mice. Furthermore, there were no significant effects on hepatic cystine or GSSG concentrations in any of the age groups of mice (data not shown).
3.2. Effects of aging and GSH-OEt on renal cortical GSH status Renal cortical GSH and Cys concentrations ranged from 1.59 to 2.66, and 0.75 to 1.27 mmol/g, respectively, in all age groups (Fig. 2). The concentrations of GSH in 30 – 31-month-old mice were significantly lower than in 3-month-old mice (Fig. 2,
Fig. 2. Effects of aging and GSH-OEt on renal cortical GSH and Cys concentrations in mice. Data presentation as described in legend for Fig. 1. Data bars represent mean 9S.D. (n =4 to 7). (*) Significantly different from controls of the respective age group. a Significantly different from 3-monthold control; b significantly different from 3- and 24-month-old controls.
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Fig. 3. Hepatic effects of APAP and GSH-OEt in old (30 – 31 months) mice. APAP (375 mg/kg) was administered i.p. and mice were sacrificed 4 h later. APAP+ GSH-OEt denotes group receiving GSH-OEt (10 mmol/kg) 30 min before the administration of APAP. Data bars represent mean 9S.D. (n = 4 to 5). a Significantly different from the 30 – 31-month-old controls; b significantly different from APAP alone.
upper panel), and the concentrations of Cys in 30–31-month-old mice were significantly lower than that in both the 3- and 24-month-old groups (Fig. 2, lower panel). The renal cortical cystine and GSSG concentrations ranged from 0.07 to 0.10 mmol/g and 0.06 to 0.07 mmol/g, respectively, in all age groups of mice and neither of these disulfides changed with age. GSH-OEt (10 mmol/kg) significantly increased the renal cortical GSH and Cys concentrations in mice of different ages throughout their life span. In each age group, GSH concentrations were increased to 2–3-fold and Cys concentrations were increased to 3 – 4-fold over controls (Fig. 2). Interestingly, the ester also produced significant increases in renal cortical cystine (0.23–0.29 mmol/g) and GSSG concentrations (0.17 – 0.32 mmol/g) in each age group.
3.3. Hepatic effects of APAP and GSH-OEt in old (30 – 31 months) mice APAP at a dose of 375 mg/kg produced significant decreases in hepatic GSH and Cys concentrations 4 h after treatment; the concentrations of GSH and Cys were 30% and 15%, respectively, of control values in old mice (Fig. 3). APAP did not affect cystine or GSSG concentrations. GSH-OEt (10 mmol/kg) administered 30 min before APAP, prevented the APAP-induced decreases in hepatic GSH and Cys concentrations. Hepatic GSH and Cys concentrations in mice receiving the ester plus APAP were 117% and 126%, respectively, of the values in untreated
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controls. The hepatic concentration of GSSG was also significantly increased in animals receiving APAP plus GSH-OEt (0.899 0.32 mmol/g) as compared to untreated controls (0.16 90.11 mmol/g) and mice receiving APAP alone (0.06 9 0.04 mmol/g).
3.4. Renal effects of APAP and GSH-OEt in old (30 – 31 months) mice APAP caused significant decreases in renal cortical GSH and Cys concentrations at 4 h to 36% and 15%, respectively, of control values in old mice (Fig. 4). GSH and Cys concentrations were decreased to 0.5790.20 and 0.119 0.05 mmol/g, respectively, after APAP administration. GSH-OEt attenuated the APAP-induced decreases in renal GSH and Cys concentrations. Renal cortical GSH and Cys concentrations in mice receiving the ester plus APAP were 289% and 214%, respectively, of the values in untreated controls. Renal cortical GSSG concentration was also significantly higher in mice receiving GSH-OEt plus APAP (0.419 0.21 mmol/g) than in untreated controls (0.079 0.08 mmol/g) and mice receiving APAP alone (0.029 0.01 mmol/g).
3.5. Effects of GSH-OEt in BSO-treated old (30 – 31 months) mice The hepatic concentrations of GSH and Cys were decreased to 56% and 67%, respectively, of the control values 2 h after BSO (4 mmol/kg) administration. However, only the decrease in GSH concentration was statistically significant. GSH-OEt administered 30 min after BSO prevented the BSO-induced depletion of
Fig. 4. Renal effects of APAP and GSH-OEt in old (30 – 31 months) mice. Data presentation as described in the legend for Fig. 3. a Significantly different from the 30 – 31-month-old controls; b significantly different from APAP alone.
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Fig. 5. Hepatic effects of GSH-OEt in BSO-treated old (30 – 31 months) mice. BSO (4 mmol/kg), dissolved in 0.9% NaCl, was administered i.p.; controls received an equivalent volume of 0.9% NaCl. BSO+ GSH-OEt denotes group receiving BSO 30 min before the administration of GSH-OEt (10 mmol/kg). Data bars represent mean 9S.D. (n =4 to 5). a Significantly different from the 30 – 31-monthold controls; b significantly different from BSO alone.
sulfhydryl compounds (Fig. 5); hepatic GSH was maintained at the control level and Cys was 59% higher than the control value. BSO also caused significant decreases in the renal cortical concentrations of GSH and Cys at 4 h to 17% and 28%, respectively, of the control values (Fig. 6). GSH-OEt prevented the BSO-induced decreases; renal cortical GSH and Cys concentrations in mice receiving BSO plus GSH-OEt were 210% and 255%, respectively, of values in untreated controls.
Fig. 6. Renal effects of GSH-OEt in BSO-treated old (30 – 31 months) mice. Data presentation as described in the legend for Fig. 5. a Significantly different from the 30 – 31-month-old controls; b significantly different from BSO alone.
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4. Discussion
4.1. GSH-OEt corrects aging-related deficiency of GSH Our earlier studies showed that GSH deficiencies occur in many aging tissues (Hazelton and Lang, 1980; Richie and Lang, 1985; Chen et al., 1989, 1990). Similar deficiency of hepatic GSH and renal GSH and Cys in old (30–31 months) mice were observed in the present study, but there were no differences in cystine and GSSG concentrations. Others have also shown that hepatic GSH and Cys decreased significantly in 24-month-old mice compared to young mice without affecting cystine or GSSG contents of the liver (Nakata et al., 1996). In contrast, some investigators reported no change or a slight increase in GSH levels with age (Beierschmitt and Weiner, 1986; Rikans and Moore, 1988). The present study demonstrated for the first time that GSH-OEt, a GSH prodrug, is an effective enhancer of tissue sulfhydryl levels in old (30–31 months) mice. Thus, the administration of this ester corrected the GSH deficiency in liver and kidney of the old mouse and the Cys levels in the kidney and liver increased by 308% and 43%, respectively, over controls. Others have shown that GSH-OMe increased hepatic and renal GSH levels in fasting adult mice (Puri and Meister, 1983). GSH-OEt also increased GSH levels in human lymphoid cells and human skin fibroblasts in vitro (Wellner et al., 1984). In adult mosquitoes fed magnesium thiazolidine-4-carboxylic acid, a Cys precursor, both GSH content and longevity of the mosquitoes were greatly increased (Richie et al., 1987; Richie and Lang, 1988). In the present study, only the acute effects of GSH-OEt were determined. It is not known whether chronic administration of the ester would produce a sustained effect on GSH levels in old animals or increase longevity.
4.2. GSH-OEt pre6ents APAP-induced GSH and Cys depletion in old (30 – 31 months) mice Enhanced susceptibility to toxic challenge has been demonstrated in old animals for a variety of pharmacological agents including APAP. Earlier results demonstrated that the toxicity of APAP in the mosquito increased 7-fold, as assessed by LD50 values in senescence, and was highly correlated with a lower GSH content (Richie and Lang, 1985). This was further confirmed in aging (31-month-old) C57BL/6 mice where loss of APAP detoxification capacity in the liver and kidneys was correlated with the GSH deficiency of aging (Chen et al., 1990; Richie et al., 1992). It is known that older (18-month-old) rats are more susceptible to APAP-induced hepatic and renal damage than are younger (3-month-old) rats (Tarloff et al., 1996). Similarly, clinical studies suggest an increased risk of APAP toxicity in the elderly (Miners et al., 1988). The present study demonstrated that APAP depleted not only GSH but also Cys in the liver and kidneys of 31-month-old mice. The proportionately greater depletion of Cys (85%) than GSH (64–70%) observed in 30–31-month-old mice receiving APAP, suggest possible utilization of Cys for GSH biosynthesis following GSH
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depletion. However, it cannot be ruled out that the depletion of Cys may be due to direct Cys conjugation with the activated metabolite of APAP. The toxicity of APAP involves its metabolic conversion to a chemically reactive intermediate, N-acetyl-p-benzoquinone imine (Dahlin et al., 1984) that interacts rapidly with GSH and vital macromolecules (Mitchell et al., 1973). It is well established that APAP-induced hepatotoxicity is associated with depletion of hepatic GSH levels to about 30% or less of normal adult levels and can be attenuated by compounds that replenish tissue GSH levels (Mitchell et al., 1973). L-2-oxothiazolidine-4-carboxylate, a Cys prodrug, as well as other 2-substituted thiazolidine-4-carboxylic acids, are known to promote GSH biosynthesis and to protect against APAP toxicity by decreasing mortality in mice receiving LD90 doses of APAP (Williamson et al., 1982; Nagasawa et al., 1984; Roberts et al., 1987). The present study is the first report of means to increase tissue sulfhydryl levels in old (30 – 31 months) mice. The importance of this finding is that GSH-OEt may also be effective in preventing APAP-induced hepatotoxicity in old animals if administered acutely after APAP. Further, it is conceivable that long term correction of hepatic GSH deficiency (if possible) could reverse the increased susceptibility of old animals to APAP. In addition, tissue differences in the effects of GSH-OEt were observed. Renal GSH levels were increased to 2 –3-fold above control values at all ages studied, while hepatic GSH was increased only modestly and only in old mice. In studies with fasting adult mice, Anderson et al. (1985) also found that renal GSH levels were increased to a much greater extent than those of liver after administration of GSH-OEt. The observed tissue differences in the effects of GSH-OEt could be due to differences in the uptake of the ester. In fact, Anderson et al. (1985) demonstrated that the 35S incorporated into kidney GSH was about 4-fold that in liver after i.p. injection of 35S-labeled GSH monoethyl ester to mice.
4.3. Effects of GSH-OEt in BSO-treated old (30 – 31 months) mice BSO produced quantitatively different effects on the sulfhydryl levels in the liver and kidney of old mice. The extent of GSH and Cys depletion was greater in the kidneys than in the liver (Figs. 5 and 6). These data are in agreement with findings of others in adult mice (Griffith and Meister, 1979) and in the rat (Standeven and Wetterhahn, 1991). The differential effects of BSO can be attributed to a greater turnover of GSH in the kidneys due to high g-glutamyl transpeptidase activity in that organ (Griffith and Meister, 1979; Potter and Tran, 1993). BSO decreased not only tissue GSH but also Cys concentrations in old mice (Figs. 5 and 6). This effect of BSO on the Cys levels may be secondary to GSH depletion, or possibly a direct effect of BSO on the uptake of L-Cys, the rate-limiting substrate for GSH biosynthesis. Indeed, Griffith et al. (1979) showed that BSO inhibited the uptake of g-glutamyl amino acids in mouse kidney. Also, BSO is known to inhibit the uptake of L-Cys in cultured rat lenses (Murray and Rathbun, 1990).
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4.4. Possible mechanism of GSH-OEt-induced increases in GSH le6els in old (30 – 31 months) mice The mechanism by which GSH-OEt enhances tissue GSH levels is not clear. Two of the possible mechanisms include (1) cellular uptake of the ester followed by its hydrolysis to form GSH and (2) extracellular hydrolysis of the ester followed by g-glutamyl transpeptidase-catalyzed breakdown of the derived GSH to GSH precursors that are taken up by cells and utilized for GSH biosynthesis. Some evidence consistent with each of these two mechanisms is available from reported studies and the present results. Evidence supporting the first mechanism includes demonstrations of (1) rapid hydrolysis of GSH-OMe by mouse liver and kidney homogenates (Puri and Meister, 1983; Anderson et al., 1985); (2) greater effectiveness of GSH-OMe over GSH in replenishing APAP-induced depleted hepatic GSH stores in adult mice (Puri and Meister, 1983); and (3) greater permeability of rat liver lysosomal membranes to esters than to the corresponding free amino acids and dipeptides (Goldman and Kaplan, 1973; Goldman and Naider, 1974). Also, we found that the GSH-OEt was effective in increasing GSH levels in mice in which GSH biosynthesis was inhibited by BSO. On the other hand, evidence supporting the second mechanism includes the finding of Grattagliano et al. (1995) that GSH-OEt was not readily taken up by rat liver but was hydrolyzed by plasma esterases thereby slowly releasing GSH into the extracellular space. Our finding that the GSH-OEt also increased hepatic and renal Cys concentrations in old mice treated with APAP or BSO raises the possibility that some fraction of the ester may undergo hydrolysis to GSH followed by further breakdown to form Cys, as would be expected in the second mechanism.
Acknowledgements The authors thank Dr Walter M. Williams for helpful discussions and Marcia C. Liu for technical assistance. This work was supported in part by the National Institute on Aging.
References Anderson, M.E., Powrie, F., Puri, R.N., Meister, A., 1985. Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione. Arch. Biochem. Biophys. 239, 538 – 548. Beierschmitt, W.P., Weiner, M., 1986. Age-related changes in renal metabolism of acetaminophen in male Fischer 344 rats. Age 9, 7–13. Boyer, T.D., Rouff, S.L., 1971. Acetaminophen-induced hepatic necrosis and renal failure. J. Am. Med. Assoc. 218, 440–441. Campbell, E.B., Griffith, O.W., 1989. Glutathione monoethyl ester: high performance liquid chromatographic analysis and direct preparation of the free base form. Anal. Biochem. 183, 21 – 25.
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