Renal and hepatic glutathione pool modifications in response to depletion treatments

In this study we examined the response of the renal and hepatic glutathione (GSH) pool in rats to drastic GSH depletion treatments. For this purpose, we used a protein-free diet, starvation, and the injection of varying doses of diethyl maleate as depleting agents. We analysed GSH levels in both kidney and liver tissue homogenates after rats were fed a protein-free diet for 2 or 7 days or starved for 1, 2, or 3 days, as well as after diethyl maleate administration in a single maximal dose or in varying doses. The results indicated that the liver GSH pool was always more labile than the kidney GSH pool. Moreover, kidney GSH levels were almost unchanged after 7 days on a protein-free diet or after 2 days of starvation, while liver showed significant changes in GSH levels. When we analysed the repletion rate, kidney had higher kinetic parameters (k = 0.148 h-1) than liver (0.097 h-1). We conclude that efficient mechanisms of maintaining GSH levels exist in the kidney and these may serve to avoid GSH diminution and hence preserve renal function during states of GSH depletion.     

Erythrocyte and plasma glutathione levels in patients with chronic renal insufficiency

Reduced glutathione (GSH) levels were investigated in the erythrocytes and plasma of nondialyzed patients with varying degrees of renal insufficiency and also of patients on regular hemodialysis treatment. GSH levels were from 19 to 70% higher in the erythrocytes of examined patients as compared to their corresponding age-matched controls. A correlation was found between the degree of renal insufficiency and the erythrocyte GSH level. No variations in plasma GSH levels which could be related to the degree of renal deterioration were observed. A routine hemodialysis did not significantly affect erythrocyte and plasma GSH levels. No significant differences in GSH levels between anemic and nonanemic uremic patients were observed.     

Mechanism of renal peritubular extraction of plasma glutathione. The catalytic activity of contralumenal gamma-glutamyltransferase is prerequisite to the apparent peritubular extraction of plasma glutathione

To clarify the peritubular mechanism for renal handling of plasma glutathione (GSH), variation of GSH levels in plasma, urine, kidney and liver was examined after intravenous administration of GSH to three groups of animals; control, acivicin-treated and rats treated with buthionine sulfoximine (BSO). Treatment of animals with BSO, a potent inhibitor of de novo GSH synthesis, markedly reduced hepatorenal GSH levels. Acivicin did not affect these levels. Upon intravenous injection of GSH (0.1 mmol/kg), renal GSH levels did not appreciably change in any of three animal groups. The rate of GSH disappearance from the circulation was rapid in control and BSO-treated rats, while it was markedly retarded in animals whose renal gamma-glutamyltransferase was extensively inactivated by acivicin. At 30 min after administration a significant amount of injected GSH was localized extracellularly (urine and plasma) in acivicin-treated animals. By contrast, most of the GSH rapidly disappeared from the extracellular space in control and BSO-treated animals. Together with the immunocytochemical evidence for the peritubular gamma-glutamyltransferase [Spater, H.W., Poruchynsky, M.S., Quintana, N., Inoue, M. & Novikoff, A.B. (1982) Proc. Natl Acad. Sci. USA 79, 3547-3550] the present results are fully consistent with the contention that the catalytic function of this enzyme is principally responsible for the peritubular mechanism for the renal handling of plasma GSH.     

Elevation of renal glutathione enhances ischemic injury.

In a previous study, we tested the hypothesis that an elevated level of renal glutathione (GSH) would protect the kidney from ischemic injury. However, prior elevation of GSH with GSH monoethylester enhanced then injury induced by 35 min of ischemia and blood reflow [Scaduto RC Jr, Gattone VH, Grotyohann LW, et al; Effect of an altered glutathione content on renal ischemic injury. Am J Physiol 1988;255:F911-F921]. Additionally, GSH monoethylester produced morphologic alterations in the absence of ischemia. Thus the greater ischemic injury observed after GSH ester pretreatment could have been due to a synergistic effect between the events caused by ischemia and the pretreatment. The present study was conducted to evaluate the utility of elevating renal GSH levels by administration of GSH. Administration of GSH (1 mmol/kg body weight) caused a 3-fold elevation of renal GSH levels and a 6-fold elevation of renal cysteine levels after 60 min without causing changes in renal morphology or GFR. After 35 min of renal artery occlusion and 90 min of blood reflow, animals pretreated with GSH had a much greater decline in GFR than untreated control animals. This enhancement of renal ischemic injury in GSH-treated animals was similar to that observed following administration of GSH monoethylester. We conclude that administration of GSH is the method of choice for elevation of renal GSH and that elevation of renal GSH leads to an enhanced ischemia-induced injury which is independent of the method employed to elevate renal GSH.     

Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione

The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.     

Inhaled glutathione decreases PGE2 and increases lymphocytes in cystic fibrosis lungs

Reduced glutathione (GSH), a major antioxidant and modulator of cell proliferation, is decreased in the bronchoalveolar lavage fluid (BALF) of cystic fibrosis (CF) patients. We previously have shown that GSH inhalation in CF patients significantly increased GSH levels in BALF and improved lung function (M. Griese et al., 2004, Am. J. Respir. Crit. Care Med.169, 822-828). GSH depletion in vitro enhances susceptibility to oxidative stress, increases inflammatory cytokine release, and impairs T cell responses. We therefore hypothesized that an increase in GSH in BALF reduces oxidative stress, decreases inflammation, and modulates T cell responses in lungs of CF patients. BALF from 17 CF patients (median FEV1 67% (43-105%) of predicted) was assessed before and after GSH inhalation for total protein, markers of oxidative stress (8-isoprostane, myeloperoxidase, and ascorbic and uric acid), pattern of protein oxidation, prostaglandin E2 (PGE2), and proinflammatory cytokines. BALF cells were differentiated using cytospin slides, and lymphocytes were further analyzed by flow cytometry. Inhalation of GSH decreased BALF levels of PGE2 and increased CD4+ and CD8+ lymphocytes in BALF significantly but had no effect on markers of oxidative stress. BALF lymphocytes correlated positively with lung function, whereas levels of PGE2 showed an inverse correlation. The patients with the greatest improvement in lung function after GSH treatment also had the largest decline in PGE2 levels. We conclude that GSH inhalation in CF patients increases lymphocytes and suppresses PGE2 in the bronchoalveolar space. Thus, GSH primarily affected the pulmonary immune response rather than the oxidative status in CF patients. The effect of GSH inhalation on PGE2 levels and lymphocytes in CF warrants further investigation.     

Sulfur-containing amino acids that increase renal glutathione protect the kidney against papillary necrosis induced by 2-bromoethylamine

Papillary necrosis was observed in the kidneys of rats, 72 h after receiving a single injection of bromoethylamine (BEA). This effect was associated with renal glutathione (GSH) depletion 1 h after the administration of BEA. Stimulation of renal GSH synthesis by pretreatment of the animals either with glutamine + glycine + cystine or N-acetyl-L-cysteine was attempted. Low doses of these precursors administered previously to BEA, respectively, decreased or abolished the GSH depletion. Nevertheless, both pretreatments failed to modify the magnitude of renal papillary necrosis. High doses of these precursors did not modify the BEA-induced GSH depletion, but they significantly increased GSH levels 24 h after BEA administration. At this time, although a smaller intensity of renal papillary necrosis was observed with the amino acid mixture pretreatment, N-acetyl-L-cysteine pretreated rats showed no papillary necrosis. It is suggested that the observed protective effects against BEA-induced renal papillary injury may be ascribed in some measure, to a mechanism independent of GSH.     

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.     

Copper deficiency and tissue glutathione concentration in the rat

Copper deficiency in rats increased renal vein and arterial (heart) plasma GSH concentration by approximately 50%. There was no change in plasma GSSG concentration. Renal vein plasma GSSG/GSH ratio was decreased in copper deficiency, which is consistent with previous reports showing a copper-dependent thiol oxidase activity in the renal basement membrane. No change occurred in arterial plasma GSSG/GSH ratio. Hepatic GSH concentrations were also elevated by 50% in copper deficiency, GSSG concentrations were unaffected, but GSSG/GSH ratio was depressed. Renal and cardiac tissue GSH and GSSG were unaffected by copper deficiency. The decreased SOD activity and GSH-Px activity observed in copper deficiency may contribute to increased hepatic and plasma GSH concentrations.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Low content of hepatic reduced glutathione in patients with Wilson's disease

In five of six patients with symptomatic Wilson's disease (WD) with increased hepatic copper content, increased renal copper excretion, and decreased serum concentrations of ceruloplasmin, significantly low levels of hepatic reduced glutathione (GSH) were found. Three of these patients showed increased levels of oxidized glutathione which in part could account for the missing GSH. These changes may result from increased lipid peroxidation due to the rise of intracellular copper concentration. Furthermore, WD patients showed a 50% decrease in the activity of hepatic GSH S-transferases. From these results we conclude that the disturbance in the hepatic glutathione system of patients with symptomatic WD may contribute to the perpetuation of liver damage. These patients, additionally, may be predisposed to an increased sensitivity to drugs interacting with glutathione.     

Elevation of glutathione levels by ammonium ions in primary cultures of rat astrocytes

It is well established that ammonia is detoxified in the brain to form glutamine and that astrocytes play a major role in this process. The synthesis of glutamine requires glutamate and ATP. Since glutamate and ATP are also required for the synthesis of glutathione (GSH), we examined the effect of pathophysiological concentrations of ammonia on levels of GSH in primary cultures of astrocytes. GSH content in the medium increased in a dose- and time-dependent manner in the presence of ammonia. After an initial decrease, cellular GSH content increased in a similar manner. The levels of glutathione disulfide (GSSG) were also increased. A linear relationship was observed between ammonia concentration and the increase in GSH levels. An increase in the efflux of GSH from cells into medium was also observed under these conditions. Buthionine sulfoximine and acivicin, but not methionine sulfoximine, blocked the ammonia induced increase in GSH levels. No, or minor, changes in the activities of enzymes (gamma-glutamyl transpeptidase, GSH reductase and GSH-peroxidase) that might influence GSH levels were identified and thus could not account for the ammonia induced increase in GSH levels in astrocytes. These findings indicate that pathophysiological concentrations of ammonium ions result in increased astroglial levels of GSH which may affect the metabolism and function of astrocytes.     

Renal mitochondrial glutathione transport

Freshly isolated tightly coupled rabbit renal cortical mitochondria rapidly accumulated glutathione (GSH) against an electrical and concentration gradient, and in the presence and absence of pyruvate/malate, succinate, antimycin A, or FCCP. Mitochondrial GSH uptake was dependent on medium GSH concentration, was not saturable, and reached equilibrium within 1 min of addition. Mitochondrial GSH uptake was partially inhibited by glycine, ophthalmic acid, and serine but not glutamate, cysteine, gamma-glutamyl-glutamate, or proline. These results show that 1) mitochondrial GSH uptake is by both a carrier-mediated process and by diffusion, and 2) the GSH carrier system has structural specificity with the glycine residue being a recognition site.     

Time course effects of vanadium supplement on cytosolic reduced glutathione level and glutathione S-transferase activity

The influence of vanadium, an important dietary micronutrient, was evaluated on the cytosolic reduced glutathione (GSH) content and glutathione S-transferase (GST) activity in several rat target tissues. Supplementation of drinking water with vanadium at the level of 0.2 or 0.5 ppm for 4, 8, or 12 wk was found to increase the GSH level with a concomitant elevation in GST activity in the liver followed by small intestine mucosa, large intestine mucosa, and kidney. The results were almost dose-dependent and mostly pronounced with 0.5 ppm vanadium after 12 wk of its continuous supplementation. Neither the GSH level nor GST activity was significantly altered in forestomach and lung following vanadium supplementation throughout the study. The levels of vanadium that were found to increase the content of GSH and activity of GST in the liver, intestine, and kidney did not exert any toxic manifestation was evidenced from water and food consumption as well as the growth responses of the experimental animals. Moreover, these doses of vanadium did not impair either hepatic or renal functions as they did not alter the serum activities of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), sorbitol dehydrogenase (SDH), as well as serum urea and creatinine levels. All these results clearly indicate that vanadium under the doses employed in our study has a significant inducing role on GSH content with a concurrent elevation in GST activity in the liver and specific extrahepatic tissues without any apparent sign of cytotoxicity. This attribute of vanadium may have a greater importance in terms of biotransformation and detoxification of xenobiotics, including carcinogens. In addition, since the ability to afford an increment in the endogenous GSH-GST pool by anticarcinogenic natural substances has been found to correlate with their activity to inhibit neoplastic transformation, the trace element vanadium may be considered as a novel anticancer agent.     

A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung.     

Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs

Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.     

Lung glutathione adaptive responses to cigarette smoke exposure

Background

Smoking tobacco is a leading cause of chronic obstructive pulmonary disease (COPD), but although the majority of COPD cases can be directly related to smoking, only a quarter of smokers actually develop the disease. A potential reason for the disparity between smoking and COPD may involve an individual''s ability to mount a protective adaptive response to cigarette smoke (CS). Glutathione (GSH) is highly concentrated in the lung epithelial lining fluid (ELF) and protects against many inhaled oxidants. The changes in GSH that occur with CS are not well investigated; therefore the GSH adaptive response that occurs with a commonly utilized CS exposure was examined in mice.

Methods

Mice were exposed to CS for 5 h after which they were rested in filtered air for up to 16 h. GSH levels were measured in the ELF, bronchoalveolar lavage cells, plasma, and tissues. GSH synthesis was assessed by measuring γ-glutamylcysteine ligase (GCL) activity in lung and liver tissue.

Results

GSH levels in the ELF, plasma, and liver were decreased by as much as 50% during the 5 h CS exposure period whereas the lung GSH levels were unchanged. Next, the time course of rebound in GSH levels after the CS exposure was examined. CS exposure initially decreased ELF GSH levels by 50% but within 2 h GSH levels rebound to about 3 times basal levels and peaked at 16 h with a 6-fold increase and over repeat exposures were maintained at a 3-fold elevation for up to 2 months. Similar changes were observed in tissue GCL activity which is the rate limiting step in GSH synthesis. Furthermore, elevation in ELF GSH levels was not arbitrary since the CS induced GSH adaptive response after a 3d exposure period prevented GSH levels from dropping below basal levels.

Conclusions

CS exposures evoke a powerful GSH adaptive response in the lung and systemically. These data suggests there may be a sensor that sets the ELF GSH adaptive response to prevent GSH levels from dipping below basal levels. Factors that disrupt GSH adaptive responses may contribute to the pathophysiology of COPD.     

Uptake of glutathione by renal cortical mitochondria.

Transport of GSH into renal cortical mitochondria was studied. Mitochondria were highly enriched with little contamination from other subcellular organelles (as assessed by marker enzymes), they exhibited coupled respiration (respiratory control ratio greater than 3.0), and they had initial GSH concentrations of 5.71 +/- 0.65 nmol/mg protein (n = 47). Incubation of mitochondria with GSH in a triethanolamine, pH 7.4, buffer containing sucrose, potassium phosphate, MgCl2, and KCl, produced time- and concentration-dependent increases in intramitochondrial GSH content. Uptake was linear versus time for at least 2 min and exhibited kinetics consistent with one low-affinity, high-capacity process (Km = 1.3 mM, Vmax = 5.59 nmol/min per mg protein), although the results cannot exclude the presence of other, less quantitatively significant pathways. The initial rate of uptake of 5 mM GSH was not significantly altered by uncouplers (0.1 mM 2,4-dinitrophenol and 25 microM carbonyl cyanide m-chlorophenylhydrazone) or by 1 mM ADP. In contrast, incubation with 1 mM ATP, 1 mM KCN, 0.1 mM or 1 mM CaCl2 inhibited uptake by 41, 39, 43, or 55%, respectively. GSH uptake was markedly inhibited by gamma-glutamylglutamate and by a series of S-alkyl GSH derivatives. Strong interactions (i.e., both cis and trans effects) were observed with other dicarboxylates (i.e., succinate, malate, glutamate) but not with monocarboxylates (i.e., lactate, pyruvate). Preincubation of mitochondria with GSH protected against tert-butyl hydroperoxide- or methyl vinyl ketone-induced inhibition of state 3 respiration. These results demonstrate uptake of GSH into renal cortical mitochondria that appears to involve electroneutral countertransport (exchange) with other dicarboxylates. Functionally, GSH uptake into mitochondria can protect these organelles from various forms of injury, such as oxidative stress.     

Methods for depleting brain glutathione

To search for a technique to deplete reduced glutathione (GSH) in brain, the influence of various types of compounds on brain GSH levels was investigated in mice. Of the compounds tested, cyclohexene-1-one, cycloheptene-1-one and diethyl maleate were shown to be potent GSH depletors in brain as well as in liver. The depletion of cerebral GSH ranged about 40-60% of control levels at 1 and 3 hr after intraperitoneal injection. Cyclohexene, cycloheptene, phorone, acetaminophen, and benzyl chloride caused mild depletion of cerebral GSH, but buthionine sulfoximine did not alter cerebral GSH levels. Further, intracerebroventricular injection of cyclohexene-1-one and cycloheptene-1-one caused depletion of brain GSH to about 60-80% of control levels at 1 hr after injection, and the effects persisted for at least 6 hr. Under these conditions, hepatic GSH was not altered. These results demonstrated that cyclohexene-1-one and cycloheptene-1-one can cause not only a marked depletion of brain GSH by systemic administration, but also depletion of cerebral GSH by intracerebroventricular injection by virtue of being water-soluble compounds. Thus, methods for depleting brain GSH employing both compounds are available for exploring possible functions of cerebral GSH in in vivo systems.     

Relationships between alterations in glutathione metabolism and the disposition of inorganic mercury in rats: effects of biliary ligation and chemically induced modulation of glutathione status

Influences of biliary ligation and systemic depletion of glutathione (GSH) or modulation of GSH status on the disposition of a low, non-nephrotoxic i.v. dose of inorganic mercury were evaluated in rats in the present study. Renal and hepatic disposition, and the urinary and fecal excretion, of inorganic mercury were assessed 24 h after the injection of a 0.5-micromol/kg dose of mercuric chloride in control rats and rats pretreated with acivicin (two 10-mg/kg i.p. doses in 2 ml/kg normal saline, 90 min apart, 60 min before mercuric chloride), buthionine sulfoximine (BSO; 2 mmol/kg i.v. in 4 ml/kg normal saline, 2 h before mercuric chloride) or diethylmaleate (DEM; 3.37 mmol/kg i.p. in 2 ml/kg corn oil, 2 h before mercuric chloride) that either underwent or did not undergo acute biliary ligation prior to the injection of mercury. Among the groups that did not undergo biliary ligation, the pretreatments used to alter GSH status systemically had varying effects on the disposition of inorganic mercury in the kidneys, liver, and blood. Biliary ligation caused the net renal accumulation of mercury to decrease under all pretreatment conditions. By contrast, biliary ligation caused significant increases in the hepatic burden of mercury in all pretreatment groups except in theacivicin-pretreated group. Blood levels of mercury also increased as a result of biliary ligation, regardless of the type of pretreatment used. The present findings indicate that biliary ligation combined with methods used to modulate GSH status systemically have additive effects with respect to causing reductions in the net renal accumulation of mercury. Additionally, the findings indicate that at least some fraction of the renal accumulation of inorganic mercury is linked mechanistically to the hepato-biliary system.