Dichlorovinyl cysteine (DCVC) in the mouse kidney: tissue-binding and toxicity after glutathione depletion and probenecid treatment

The kidney binding of dichloro[¹⁴C]vinyl cysteine (¹⁴C-DCVC, 8 mg/kg body wt) and the kidney histopathology of DCVC (5 mg/kg body wt) were examined and compared in female C57BL mice subjected to various treatments. To evaluate the roles of organic anion transport and glutathione (GSH) status, mice were pretreated with probenecid (inhibitor of organic anion transport), l-buthionine-S,R-sulfoximine (BSO; inhibitor of GSH synthesis) or with diethyl maleate (DEM; GSH-depleting agent). In addition, the sites of ¹⁴C-DCVC binding in BSO-treated and control mice were monitored by microautoradiography. Probenecid was found to inhibit both kidney binding and toxicity of DCVC. In BSO-treated mice, DCVC binding remained roughly unchanged, whereas nephrotoxicity was severely increased and topographically extended to the subcapsular region. Microautoradiography showed that the site of DCVC binding in the straight portion of the proximal tubule was not changed by BSO. In DEM-treated mice, a clearly decreased DCVC binding was observed, while the effect on nephrotoxicity was minute. The effects of probenecid on DCVC binding and toxicity support a role for carriermediated transport of DCVC equivalents into the target cells. The BSO result suggests a protective function of GSH towards the nephrotoxicity of DCVC. Moreover, they support our previous contention that a primary lesion occurs at the site of DCVC binding, followed by a secondary, dose-dependent lesion localized outside the DCVC-binding region. In the case of DEM it is proposed that a DEM-GSH conjugate might compete for the uptake and/or activation of DCVC in the target cells.Part of this study was presented at the 10th European Drug Metabolism Workshop, Guildford, England, 6–11 July, 1986     

Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine.

The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.     

Pharmacokinetic procedures for the estimation of organ clearances for the formation of short-lived metabolites. Acetaminophen-induced glutathione depletion in hamster liver

Several approaches have been developed to estimate in vivo the intrinsic clearances of enzymes that catalyze the formation of chemically reactive metabolites that do not escape the organs in which they are formed. Two basic models are considered. Model 1 is a general model in which the chemically reactive metabolite is inactivated by a combination of a pseudo-first order reaction, such as a reaction with large pools of protein, and a second order reaction with a depletable endogenous substance, such as glutathione or an enzyme. Model 2 is a special case, in which at low doses of the parent compound the reactive metabolite preferentially reacts with a depletable endogenous substance, such as glutathione. In developing both models we have assumed that the rate of formation of reactive metabolite follows first order kinetics and that the concentration of reactive metabolite in liver reaches a steady state almost instantaneously. In developing Model 2 we also have assumed that the depletion of hepatic reduced glutathione is due solely to the formation of glutathione conjugates. The uses of the approaches based on Model 2 were illustrated by studying the effects of a marginally toxic dose of acetaminophen on the depletion and subsequent repletion of hepatic glutathione in hamsters. From the calculated rate of synthesis of glutathione in liver, the fraction of the dose of acetaminophen converted to the glutathione conjugate in liver, and the clearance for the formation of glutathione conjugate in vivo was estimated and was found to be similar to that obtained with hepatic 9,000 g supernatant preparations. Other uses of the models are described.     

Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion

We reported previously that acetaminophen overdose interrupts the signaling pathway of Fas receptor-mediated apoptosis. The aim of our study was to investigate the mechanism of this effect. Male C3Heb/FeJ mice received a single dose of acetaminophen (300 mg/kg ip) and/or anti-Fas antibody Jo-2 (0.6 mg/kg iv). Some animals were treated with allopurinol (100 mg/kg po) 18 and 1 h before acetaminophen injection. After 90 min of Jo treatment, there was processing of procaspase-3 and a significant increase in liver caspase-3 activity, which is consistent with apoptotic cell death. Treatment with acetaminophen 2.5 h before Jo inhibited the increase in hepatic caspase-3 activity by preventing the processing of the proenzyme. When administered alone, acetaminophen did not induce caspase-3 activation but caused significant liver injury. Acetaminophen treatment alone caused mitochondrial cytochrome c release, depletion of the hepatic ATP content by 55%, and a 10-fold increase in mitochondrial glutathione disulfide levels. Pretreatment with allopurinol prevented the mitochondrial oxidant stress and liver injury due to acetaminophen toxicity but had no effect on Jo-mediated apoptosis. Allopurinol did not affect the initial glutathione depletion after acetaminophen. However, allopurinol restored the sensitivity of hepatocytes to Fas receptor signaling in acetaminophen-treated animals. Histochemical evaluation of DNA fragmentation with the TUNEL assay showed that acetaminophen eliminated Fas receptor-mediated apoptosis in all hepatocytes not just in the damaged cells of the centrilobular area. Our data suggest that acetaminophen-induced mitochondrial dysfunction and not the initial glutathione depletion is responsible for the interruption of Fas receptor-mediated apoptotic signaling in hepatocytes.     

Acetaminophen-induced hepatic glycogen depletion and hyperglycemia in mice

Two hours following administration of a hepatotoxic dose of acetaminophen (500 mg/kg, i.p.) to mice, liver sections stained with periodic acid Schiff reagent showed centrilobular hepatic glycogen depletion. A chemical assay revealed that following acetaminophen administration (500 mg/kg) hepatic glycogen was depleted by 65% at 1 hr and 80% at 2 hr, whereas glutathione was depleted by 65% at 0.5 hr and 80% at 1.5 hr. Maximal glycogen depletion (85% at 2.5 hr correlated with maximal hyperglycemia (267 mg/100 ml at 2.5hr). At 4.0 hr following acetaminophen administration, blood glucose levels were not significantly different from saline-treated animals; however, glycogen levels were still maximally depleted. A comparison of the dose-response curves for hepatic glycogen depletion and glutathione depletion showed that acetaminophen (50–500 mg/kg at 2.5 hr) depleted both glycogen and glutathione by similar percentages at each dose. Since acetaminophen (100 mg/kg at 2.5 hr) depleted glutathione and glycogen by approximately 30%, evidence for hepatotoxicity was examined at this dose to determine the potential importance of hepatic necrosis in glycogen depletion. Twenty-four hours following administration of acetaminophen (100 mg/kg) to mice, histological evidence of hepatic necrosis was not detected and serum glutamate pyruvate transaminase (SGPT) levels were not significantly different from saline-treated mice. The potential role of glycogen depletion in altering the acetaminophen-induced hepatotoxicity was examined subsequently. When mice were fasted overnight, hepatic glutathione and glycogen were decreased by 40 and 75%, respectively, and fasted animals showed a dramatic increase in susceptibility to acetaminophen-induced hepatotoxicity as measured by increased SGPT levels. Availability of glucose in the drinking water (5%) overnight resulted in glycogen levels similar to those in fed animals, whereas hepatic glutathione levels were not significantly different from those of fasted animals. Fasted animals and animals given glucose water overnight were equally susceptible to acetaminophen-induced hepatotoxicity, as quantitated by increases in SGPT levels 24 hr after drug administration. The potential role of a reactive metabolite in glycogen depletion was investigated by treating mice with N-acetylcysteine to increase detoxification of the reactive metabolite. N-Acetylcysteine treatment of mice prevented acetaminophen-induced glycogen depletion.     

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: effects of Ca(2+)-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death.

Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca2+ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a "cause-and-effect" manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22-26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca(2+)-endonuclease inhibitor, and EGTA, a chelator of Ca2+ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca2+ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.     

Glutathione depletion and recovery after acute ethanol administration in the aging mouse

Glutathione (GSH) plays an important role in the detoxification of ethanol (EtOH) and acute EtOH administration leads to GSH depletion in the liver and other tissues. Aging is also associated with a progressive decline in GSH levels and impairment in GSH biosynthesis in many tissues. Thus, the present study was designed to examine the effects of aging on EtOH-induced depletion and recovery of GSH in different tissues of the C57Bl/6NNIA mouse. EtOH (2-5 g/kg) or saline was administered i.p. to mice of ages 6 months (young), 12 months (mature), and 24 months (old); and GSH and cyst(e)ine concentrations were measured 0-24h thereafter. EtOH administration (5 g/kg) depleted hepatic GSH levels >50% by 6h in all animals. By 24h, levels remained low in both young and old mice, but recovered to baseline levels in mature mice. At 6h, the decrease in hepatic GSH was dose-dependent up to 3g/kg EtOH, but not at higher doses. The extent of depletion at the 3g/kg dose was dependent upon age, with old mice demonstrating significantly lower GSH levels than mature mice (P<0.001). Altogether these results indicate that aging was associated with a greater degree of EtOH and fasting-induced GSH depletion and subsequent impaired recovery in liver. An impaired ability to recover was also observed in young animals. Further studies are required to determine if an inability to recover from GSH depletion by EtOH is associated with enhanced toxicity.     

Acute kidney injury model established by systemic glutathione depletion in mice

Glutathione (GSH) is one of the most extensively studied tripeptides. The roles for GSH in redox signaling, detoxification of xenobiotics and antioxidant defense have been investigated. A drug‐induced rhabdomyolysis mouse model was recently established in L‐buthionine‐(S,R)‐sulfoximine (BSO; a GSH synthesis inhibitor)‐treated normal mice by co‐administration of antibacterial drug and statin. In these models, mild kidney injury was observed in the BSO only‐treated mice. Therefore, in this study, we studied kidney injury in the GSH‐depleted mouse. BSO was intraperitoneally administered twice a day for 7 days to normal mice. The maximum level of plasma creatine phosphokinase (351 487 ± 53 815 U/L) was shown on day 8, and that of aspartate aminotransferase was shown on day 6. Increased levels of blood urea nitrogen, plasma creatinine, urinary kidney injury molecule‐1 and urinary creatinine were observed. An increase of mRNA expression level of renal lipocalin 2/neutrophil gelatinase‐associated lipocalin was observed. Degeneration and necrosis in the skeletal muscle and high concentrations of myoglobin (Mb) in blood (347‐203 925 ng/mL) and urine (2.5‐68 583 ng/mL) with large interindividual variability were shown from day 5 of BSO administration. Mb‐stained regions in the renal tubule and renal cast were histologically observed. In this study, the GSH‐depletion treatment established an acute kidney injury mouse model due to Mb release from the damaged skeletal muscle. This mouse model would be useful for predicting potential acute kidney injury risks in non‐clinical drug development.     

Effect of ribose cysteine pretreatment on hepatic and renal acetaminophen metabolite formation and glutathione depletion

Ribose cysteine (2(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid) protects against acetaminophen-induced hepatic and renal toxicity. The mechanism for this protection is not known, but may involve inactivation of the toxic electrophile via enhancement of glutathione (GSH) biosynthesis. Therefore, the goal of this study was to determine if GSH biosynthesis was required for the ribose cysteine protection. Male CD-1 mice were injected with either acetaminophen or acetaminophen and ribose cysteine. The ribose cysteine cotreatment antagonized the acetaminophen-induced depletion of non-protein sulfhydryls in liver as well as GSH in kidney. Moreover, ribose cysteine cotreatment significantly increased the concentration of acetaminophen-cysteine, hepatic acetaminophen-mercapturate in liver and renal acetaminophen-GSH metabolites in kidney 4 hr after acetaminophen. To determine whether protection against acetaminophen-induced liver and kidney damage involved ribose cysteine dependent GSH biosynthesis, buthionine sulfoximine was used to selectively block gamma-glutamylcysteine synthetase (gamma-GCS). Plasma sorbitol dehydrogenase (SDH) activity and blood urea nitrogen from mice pretreated with buthionine sulfoximine and challenged with acetaminophen indicated that both liver and kidney injury had occurred. While co-treatment with ribose cysteine had previously protected against acetaminophen-induced liver and kidney injury, it did not diminish the acetaminophen-induced damage to either organ in the buthionine sulfoximine-treated mice. In conclusion, ribose cysteine serves as a cysteine prodrug that facilitates GSH biosynthesis and protects against acetaminophen-induced target organ toxicity.     

Potentiation of cisplatin-induced lipid peroxidation in kidney cortical slices by glutathione depletion

Effects of cis-diamminedichloroplatinum II (cisplatin), an antitumor agent with a dose-limiting effect of nephrotoxicity, on lipid peroxides and glutathione (GSH) were examined in rat kidney cortical slices treated with or without diethylmaleate (DEM), a GSH depletor, in vitro. DEM (3 mM) decreased the GSH level to about 16% of the control with a concomitant increase in lipid peroxides after 90 min of incubation. The same effects were obtained with 1 mM cisplatin 90 min later. Cisplatin (1 mM) with DEM (2 mM) stimulated both the decrease in GSH and the increase in lipid peroxides 90 min after incubation. However, cisplatin with DEM markedly stimulated lipid peroxidation with a small effect on the GSH decrease by cisplatin alone 30 min after incubation, while each drug by itself did not affect lipid peroxidation. The antioxidants N,N'-diphenyl-p-phenylene-diamine (DPPD), promethazine, and ascorbic acid abolished cisplatin-induced lipid peroxidation in the presence of DEM. DPPD had no effect on the depletion of GSH caused by cisplatin and DEM. Ascorbic acid and promethazine caused only a slight return towards the control level. The results suggested that cisplatin-induced lipid peroxidation is due to another mechanism in addition to the GSH depletion caused by the antitumor drug.     

Effects of D-limonene on hepatic microsomal monooxygenase activity and paracetamol-induced glutathione depletion in mouse

1. d-Limonene, a monoterpenoid constituent of citrus fruit oil, blocks tumour induction by chemical carcinogens in laboratory animals, apparently by preventing bioactivation of procarcinogens and by enhancing conjugation of proximal carcinogenic metabolites.

2. Inhibitory effects of d-limonene were measured in vitro using cytochrome P450 isoform-specific substrates. d-Limonene inhibited p-nitrophenol hydroxylase (pNP) activity in vitro in liver microsomes from acetone-, phenobarbital (PB)- and β-naphthoflavone (BNF)-treated mouse, and 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes from PB- and BNF-treated mouse. p-Nitrophenol and ethoxyresorufin are substrates for cytochromes P2E1 and P1A1, respectively. No inhibition of benzphetamine (BNZP) or aminopyrine (AP) demethylases by d-limonene was observed.

3. EROD, BNZP and AP activities in liver microsomes were increased 18 h after i.p. administration of d-limonene to acetone-induced mouse, while pNP activity was unchanged. The immunodetectable protein level of cytochrome P2B1 in non-acetone treated mouse was increased 18h after d-limonene, with no differences in P2E1 or P1A1.

4. Acute d-limonene did not protect against paracetamol (acetaminophen)-induced depletion of liver reduced glutathione (GSH). A prolonged paracetamol challenge (0–6% diet for 10 days) elevated liver cytosolic GSH-S-transferase activity (GST) two-fold and decreased liver GSH to 46% of control values. Dietary d-limonene (1–0% diet for 10 days) maintained liver GSH concentrations at 92% of control values in the paracetamol-challenged mouse without altering GST activity. d-Limonene also increased liver GSH concentration (23%) in mouse fed 1–0% d-limonene alone.     

Possible role of hepatic glutathione in transport of methylmercury into mouse kidney

The mechanism of the renal uptake of methylmercury was studied in mice. Preadministration of 1,2-dichloro-4-nitrobenzene (DCNB), which is a reagent that depletes hepatic glutathione (GSH) without affecting the renal GSH level, 30 min before injection of methylmercury significantly decreased the renal accumulation of mercury. The renal accumulation of mercury in mice receiving methylmercury-GSH intravenously was significantly higher than that in mice receiving methylmercuric chloride. These results suggest the possibility that hepatic GSH, as a source of extracellular GSH, plays an important role in the renal accumulation of methylmercury. No significant difference in renal mercury accumulation between bile duct-cannulated mice and normal mice was observed, indicating that the enterohepatic circulation of methylmercury is not an important factor in the renal accumulation of methylmercury in mice. Pretreatment of mice with acivicin, a potent inhibitor of gamma-glutamyl transpeptidase (gamma-GTP), significantly depressed the renal uptake of methylmercury and increased the urinary excretion of GSH and methylmercury. In in vitro reactions, methylmercury-GSH was degraded into methylmercury-cysteinylglycine by gamma-GTP, and this product was then converted to methylmercury-cysteine by dipeptidase. These results suggest that methylmercury is transported into the kidney as a complex with GSH, and then incorporated into the renal cells after degradation of the GSH moiety by gamma-GTP and dipeptidase, although the methylmercury bound to extracellular GSH can be reversibly transferred to plasma proteins in the bloodstream.     

Endrin-induced depletion of glutathione and inhibition of glutathione peroxidase activity in rats

1. Recent studies have shown that endrin induces lipid peroxidation and may produce toxicity through an oxidative stress. We have therefore examined the effect of endrin administration to rats on glutathione content and the activities of glutathione metabolizing enzymes. 2. The oral administration of endrin resulted in dose- and time-dependent decreases in hepatic and renal glutathione content with maximum depletion (90%) occurring in liver at approximately 24 hr post-treatment. 3. Decreases in glutathione content were also observed in lung, brain, spleen and heart. 4. Endrin (4 mg/kg) decreased selenium dependent glutathione peroxidase activity in liver and kidney by 64 and 50%, respectively, while small increases were observed in the activities of glutathione reductase and glutathione S-transferase. 5. The toxicity of endrin may be at least in part related to oxidative tissue damage associated with depletion of glutathione and inhibition of glutathione peroxidase activity.     

Porcine kidney microsomal cysteine S-conjugate N-acetyltransferase-catalyzed N-acetylation of haloalkene-derived cysteine S-conjugates.

N-Acetylation of xenobiotic-derived cysteine S-conjugates is a key step in the mercapturic acid pathway. The aim of this study was to investigate the N-acetylation of haloalkene-derived S-haloalkyl and S-haloalkenyl cysteine S-conjugates by porcine kidney cysteine S-conjugate N-acetyltransferase (NAcT). A radioactive assay for the quantification of NAcT activity was developed as a new method for partial purification of the enzyme, which was necessitated by the substantial loss of activity during the immunoaffinity chromatography method. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate, rather than N,N-bis[3-gluconamidopropyl]deoxycholamide, was used to solubilize the NAcT from porcine kidney microsomes in the revised procedure. The partially purified NAcT was free of detectable aminoacylase activity. Although low acetyl-coenzyme A hydrolase activity was observed, its effect on the assay was minimized by addition of excess acetyl-coenzyme A in the NAcT assay mixture. Attempts to separate the residual hydrolase activity from NAcT by different chromatographic procedures were either unsuccessful or lead to inactivation of NAcT. Most of the cysteine S-conjugates studied were N-acetylated by NAcT. Although the apparent K(m) values for the cysteine S-conjugates studied differed by a factor of approximately 2.5 (124-302 microM), a greater than 15-fold difference in the apparent V(max) (0.75-15.6 nmol/h) and V(max)/K(m) (0.008-0.126 x 10(-3) l h(-1)) values was observed. These data show that a range of haloalkene-derived cysteine S-conjugates serve as substrates for pig kidney NAcT. The significant differences in cytotoxicity of these conjugates may be a result of more variable deacetylation rates of the corresponding mercapturates.     

Pharmacokinetics and pharmacodynamics of mesna-mediated plasma cysteine depletion

Cellular glutathione (GSH) levels are related to the resistance of tumor cells to platinum and alkylating agents, and depletion of GSH may enhance the activity of these drugs. The pharmacodynamic effects of mesna on depleting plasma cysteine, a GSH precursor, were evaluated in 22 patients as part of a Phase I study. Escalating doses of ifosfamide and mesna were administered; carboplatin was administered to achieve an AUC of 4 mg x min/mL. Plasma samples were collected and assayed by reverse-phase high-performance liquid chromatography (HPLC) for total mesna and total cysteine concentrations at 0, 1, 3, 6, 24, 25, 28, and 48 hours. A one-compartment pharmacokinetic model was fit to the mesna plasma concentrations, using M.A.P. Bayesian estimation (ADAPT II). Pharmacodynamics were evaluated by fitting an inhibitory Emax model to the cysteine concentration data. Both the pharmacokinetic (median R2 = 0.95; range = 0.85-0.98) and pharmacodynamic (median R2 = 0.96; range = 0.74-1.0) models fit the data well. Mean (coefficient of variation [CV%]) mesna pharmacokinetic parameter estimates were as follows: Vss of 15.3 (29) L/m2, CL of 4.6 (29) L/h/m2, and half-life of 2.2 (37) hours. Mean (CV%) pharmacodynamic parameter estimates were as follows: Emax of 31.7 (19) microg/mL and EC50 of 10.3 (52) microg/mL. Mesna produced a rapid, concentration-dependent reduction in plasma cysteine concentrations that could be adequately characterized by an inhibitory Emax pharmacodynamic model. The depletion of plasma cysteine was facilitated by ifosfamide, suggesting a pharmacodynamic interaction between these two agents. Further increases in mesna doses beyond those administered in this study would be unlikely to provide additional benefit.     

Transfection with gamma-glutamyl transpeptidase enhances recovery from glutathione depletion using extracellular glutathione.

Glutathione (L-gamma-glutamyl-L-cysteinylglycine) is an important constituent of the antioxidant and detoxifying mechanisms of cells. The plasma membrane bound enzyme, gamma-glutamyl transpeptidase (GGT), catalyzes the first step in the degradation of extracellular glutathione, the components of which are then used for de novo glutathione synthesis. We tested the hypothesis that an increase in GGT activity would enhance the utilization of extracellular glutathione by cells challenged with a glutathione-depleting agent. A eukaryotic system stably overexpressing GGT (nearly 200-fold) was developed by transfection of NIH-3T3 fibroblasts with a human placental GGT cDNA. These cells and controls were incubated for 30 min with 1 mM diethyl maleate, which caused approximately 80% intracellular glutathione depletion. Glutathione was added to the medium and cells were allowed to resynthesize intracellular glutathione. The transfected cells used extracellular glutathione much more efficiently than controls in terms of both the concentration dependence and the rate of glutathione resynthesis. Serine-borate, a competitive inhibitor of GGT, blocked the restoration of intracellular glutathione. The results support the hypothesis that the increase in GGT activity that occurs in some toxicologic or pathologic conditions could provide protection against glutathione depletion.     

Renal glutathione depletion and nephrotoxicity of cadmium-metallothionein in rats

Renal glutathione (GSH) concentrations were reduced approximately 80% at 4 hr after a single injection of buthionine sulfoxime (BSO) (4 mmol/kg body wt) and remained reduced for at least 16 hr in male rats. Following BSO injection, rats were injected with a nephrotoxic dose of cadmium-metallothionein (Cd-MT) (0.3 mg Cd as Cd-MT/kg body wt) and killed 1, 4, or 12 hr later. Damage to the kidney was assessed histologically and by measurement of p-aminohippuric acid (PAH) uptake into renal cortical slices. Although the renal accumulation of Cd following Cd-MT injection was significantly lower in BSO-pretreated rats as compared to nonpretreated rats, the damage to kidney was more severe. At 4 and 12 hr, both Cd-MT-induced inhibition of PAH uptake and morphological damage were significantly increased in BSO-pretreated rats. In certain experiments, the induction of renal intracellular MT synthesis by zinc pretreatment slightly decreased the renal toxicity of Cd-MT in the BSO-treated rats. The results demonstrate that although GSH depletion decreases the renal accumulation of Cd in rats injected with Cd-MT, the nephrotoxicity of Cd-MT is increased. Preinduction of MT in the kidney can only partially overcome this increase in toxicity. Therefore both GSH and intracellular MT levels can influence the renal toxicity of injected Cd-MT.     

Effect of glutathione depletion on tissue deposition of methylmercury in rats.

Diethylmaleate and other compounds capable of being metabolized to mercapturic acid derivatives were administered to rats in order to deplete tissue stores of glutathione (GSH). Deposition of mercury was significantly decreased in kidney and erythrocytes of depleted animals when [²⁰³Hg]methylmercuric chloride was administered during the period of maximal depletion of GSH. Increasing doses of diethylmaleate resulted in steadily decreasing GSH concentrations in brain, kidney, erythrocytes, and liver which were accompanied by decreases in mercury deposition in kidney and brain. Increasing the time interval between diethylmaleate and subsequent methylmercury administration resulted in correspondingly greater concentrations of mercury in kidney that were associated with recovery of renal GSH concentrations. Disodium maleate was also effective in lowering GSH values and reducing mercury deposition in kidney and erythrocytes. A second dose of disodium maleate, administered 24 hr after the first, was less effective than the first in depleting renal GSH and reducing mercury deposition. However, a second dose of diethylmaleate was equally as effective as the first. A high degree of correlation (r = 0.85) was found between renal mercury accumulation and renal GSH concentration when data from all depletion experiments were considered. The results suggest that renal GSH may be a determinant in the deposition of mercury in the kidney.     

Effect of glutathione depletion on tissue and plasma prostacyclin and thromboxane in rats.

Experiments were designed to determine the effects of glutathione (GSH) depletion with L-buthionine sulfoximine (BSO) or diethyl maleate (DEM) on tissue and plasma prostacyclin (6-keto-PGF1 alpha) and thromboxane (TxB2) levels in male Sprague-Dawley rats. Despite depleting hepatic GSH to as much as 34% of control, BSO at various levels (0.4, 0.8 and 1.2 g/kg body wt) had no effect on hepatic, renal, pulmonary or cardiac tissue levels of 6-keto-PGF1 alpha and TxB2 or circulating levels of 6-keto-PGF1 alpha in portal or arterial plasma. When rats were pretreated with 3-methylcholanthrene (3-MC) to induce cytochrome P450, BSO (0.8 g/kg body wt) also had no effect on tissue or plasma prostanoid levels with the exception of a slight, but significant, increase in hepatic 6-keto-PGF1 alpha in non-induced rats. In contrast, depletions of hepatic, renal and pulmonary tissue GSH by DEM (1 mL/kg body wt) to 12, 50 and 30% of control, respectively, were associated with elevations of 6-keto-PGF1 alpha in these tissues and in plasma obtained by right ventricular heart puncture. Pretreatment of rats with 3-MC had no significant effect on tissue GSH or prostanoid levels in controls or DEM-treated rats but plasma levels of 6-keto-PGF1 alpha were lower in comparison to non-induced rats. DEM with or without 3-MC pretreatment was associated with increased TxB2 in renal tissue, whereas DEM elevated TxB2 only in pulmonary tissue from non-induced rats. It appears that factors besides GSH depletion may be required to raise plasma and/or tissue 6-keto-PGF1 alpha levels in vivo.