Mechanisms of Phenytoin‐Induced Toxicity in Freshly Isolated Rat Hepatocytes and the Protective Effects of Taurine and/or Melatonin

Phenytoin is a widely used antiepileptic drug. However, hepatotoxicity is one of its adverse effects reported in some patients. The mechanism(s) by which phenytoin causes hepatotoxicity is not clear yet. This study was designed to evaluate the cytotoxic mechanism(s) of phenytoin toward rat hepatocytes (whose cytochrome P450 enzymes had been induced by Phenobarbital). Furthermore, the effect of taurine and/or melatonin on this toxicity was investigated. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and mitochondrial depolarization were monitored as toxicity markers. Results showed that phenytoin caused an elevation in ROS formation, depletion of intracellular reduced glutathione, increase in cellular oxidized glutathione, enhancement of LPO, and mitochondrial damage. Taurine (1 mM) and/or melatonin (1 mM) administration decreased the intensity of cellular injury caused by phenytoin. This study suggests the protective role of taurine and/or melatonin against phenytoin‐induced cellular damage probably through their reactive radical scavenging properties and their effects on mitochondria.     

Dexamethasone protects cultured rat hepatocytes against cadmium toxicity: involvement of cellular thiols

In the present study, the effects of dexamethasone on cadmium-induced toxicity were evaluated in isolated rat hepatocytes. Hepatocytes were cultured for 24 h in William’s E medium containing fetal calf serum (10%), insulin (0.1 IU/ml), and glucagon (0.01 μM) in the absence or presence of 0.1 μM dexamethasone. Cadmium chloride, 5 or 10 μM, was added to the medium and the toxicity was evaluated for up to 48 h after treatment. Lactate dehydrogenase (LDH) release, the reduced and oxidized glutathione ratio (GSH/GSSG), protein-SH groups, and lipid peroxidation levels were evaluated. Cadmium induced a dose- and time-dependent LDH release in control hepatocytes at 24 h (Cd 10 μM 42%) while hepatocytes pretreated with dexamethasone showed lower necrosis (Cd 10 μM 12% at 24 h). GSH/GSSH ratio and protein-SH groups were higher while lipid peroxidation was lower in dexamethasone-treated hepatocytes as compared with untreated cells. In conclusion, cadmium toxicity was associated with an increase in intracellular oxidative stress responsible for accelerated cell death. The use of dexamethasone prevented cadmium damage, suggesting that the cytoprotective action of this hormone is related to its effect in preventing changes in thiols such as glutathione and protein-SH groups.     

Mechanism of sulfite cytotoxicity in isolated rat hepatocytes

Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity.     

Mechanisms of the Statins Cytotoxicity in Freshly Isolated Rat Hepatocytes

Statins are potent drugs, used as lipid‐lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin‐treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin‐induced toxicity. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:287‐294, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21485     

Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism

Vitamin E protection against chemical-induced toxicity to isolated hepatocytes was examined during an imbalance in the thiol redox system. Intracellular reduced glutathione (GSH) was depleted by two chemicals of distinct mechanisms of action: adriamycin, a cancer chemotherapeutic agent that undergoes redox cycling, producing reactive oxygen species that consume GSH, and ethacrynic acid, a direct depleter of GSH. The experimental system used both nonstressed vitamin E-adequate isolated rat hepatocytes and compromised hepatocytes subjected to physiologically induced stress, generated by incubation in calcium-free medium. At doses whereby intracellular GSH was near total depletion, cell injury induced by either chemical was found to follow the depletion of cellular alpha-tocopherol, regardless of the status of the GSH redox system. Changes in protein thiol contents of the cells closely paralleled the changes in alpha-tocopherol contents throughout the incubation period. Supplementation of the calcium-depleted hepatocytes with alpha-tocopheryl succinate (25 microM) markedly elevated their alpha-tocopherol content and prevented the toxicities of both drugs. The prevention of cell injury and the elevation in alpha-tocopherol contents were both associated with a prevention of the loss in cellular protein thiols in the near total absence of intracellular GSH. The mechanism of protection by vitamin E against chemical-induced toxicity to hepatocytes may therefore be an alpha-tocopherol-dependent maintenance of cellular protein thiols.     

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.     

Protective effects of intracellular reactive oxygen species generated by 6-formylpterin on tumor necrosis factor-alpha-induced apoptotic cell injury in cultured rat hepatocytes

The effects of 6-formylpterin on tumor necrosis factor (TNF)-alpha-induced apoptotic cell injury were studied in cultured rat hepatocytes. The incubation of the hepatocytes with TNF-alpha and actinomycin D (ActD) induced the apoptotic cell injury. The level of aspartate transaminase (AST) in the culture supernatant increased, and the cell viability, estimated by mitochondrial respiration (MTT assay), decreased. The DNA fragmentation and the caspase 3-like activity, which are characterized to apoptosis, increased. When the hepatocytes were incubated with 100-500 microM 6-formylpterin, the intracellular formation of reactive oxygen species (ROS) was observed, and the ratio of reduced and oxidized glutathione (GSH/GSSG) of whole cell lysate decreased. The co-incubation of the TNF-alpha/ActD-treated hepatocytes with 100-500 microM 6-formylpterin attenuated the TNF-alpha/ActD-induced apoptotic cell injury. The level of AST decreased and the cell viability increased. Both the DNA fragmentation and the caspase 3-like activity decreased. The caspases, executors of apoptosis, are known to require a reduced cystein in their active site to function, and the intact intracellular GSH/GSSG is essential for the caspase activation. Therefore, our findings suggest that intracellular ROS generated by 6-formylpterin decline the intracellular redox state to an oxidant state, which suppresses the caspase activity and prevents the apoptotic cell injury of hepatocytes.     

Thiols and polyamines in the cytoprotective effect of taurine on carbon tetrachloride‐induced hepatotoxicity

The mechanism by which taurine (2‐aminoethanesulfonic acid) protects hepatocytes injury induced by carbon tetrachloride (CCl₄) is not fully understood. In a previous study, we reported that cellular polyamines play an important role in this mechanism. The relationship between cellular glutathione (GSH), protein‐SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamine on the cytoprotective ability of taurine in CCl₄‐induced toxicity in isolated rat hepatocytes, was examined. CCl₄ induced a LDH release and decreased cellular thiols and polyamine levels. Treating with taurine reversed these depletions. The effect of CCl₄ was also reversed by the addition of exogenous polyamines. Pretreating with α‐difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, which is a key enzyme in polyamine biosynthesis and therefore used to deplete cellular polyamine, prevented the protective effect of taurine. Adding diethyl maleate, a cellular glutathione‐depleting agent, reduced the effect of exogenous polyamines. The role of polyamine in the cytoprotective effect of taurine in CCl₄‐induced toxicity may therefore be by preventing, among others, GSH and protein‐SH depletions. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 71–76, 1999     

Cocaine-induced biochemical changes and cytotoxicity in hepatocytes isolated from both mice and rats

The mechanism of cocaine-induced cytotoxicity was investigated in hepatocytes isolated from both male C3H mice and male Sprague-Dawley rats. Cocaine was more cytotoxic to mouse hepatocytes than rat and induced reduced glutathione (GSH) depletion prior to marked increases in cytotoxicity in both systems. In both mouse and rat cells, GSH depletion was accompanied by GSSG production, but in rat cells, quantitative measures suggested that other mechanisms contributed to GSH depletion. No cocaine-induced depletion of protein-thiol groups or generation of protein-glutathione mixed disulfides could be detected in rat cells. Cocaine induced lipid peroxidation, using malondialdehyde (MDA) production as an index of the peroxidation process, in both mouse and rat hepatocytes. Inhibition of MDA production to below control levels using the antioxidant N,N'-diphenyl-phenylene diamine (DPPD) however, had no inhibitory effect on cocaine-induced cytotoxicity in either mouse or rat cells. These data suggest that neither generalized protein thiol depletion nor lipid peroxidation are critical determinants of cocaine-induced cytotoxicity in cellular systems.     

Measurement of reduced and oxidized glutathione in cultures of adult rat hepatocytes

A reversed-phase ion-exchange high-performance liquid chroamtographic technique, suitable for the separate measurement of reduced (GSH) and oxidized (GSSG) glutathione in cultures of adult rat hepatocytes, is described. A commercially available Nucleosil 120-7NH₂ column was used. A complete run took ca. 22 min. The retention times for GSH and GSSG were 10.6 and 12.7 min, respectively, providing a resolution coefficient of 1.4. The coefficients of variation for GSH and GSSG were ca. 5 and 25%, respectively, for freshly isolated hepatocytes, and 16 and 15%, respectively, for 24-h cultured hepatocytes. The detector response was linear as a function of GSH and GSSG concentration and the hepatocytes concentration studied. Addition of up to 1.5 mg/ml bovine serum albumin to the culture medium had no effect on the linearity. The recovery for standards, ranging from 0 to 150 nmol of GSH or GSSG per millilitre in the presence of hepatocytes, was 98% for GSH and 80% for GSSG. The detection limit of the method was between 0.5 and 1.0 nmol of GSH and GSSG per millilitre. In cultured rat hepatocytes, the GSH content increased during the first 24 h of culture, followed by a slow decrease. After six days of culture, the GSH content was less than 50% of the value found for freshly isolated hepatocytes. GSSG was present in cultured rat hepatocytes in only small amounts and becomes unmeasurable after four days of culture.     

tert-Butylhydroperoxide-induced toxicity in isolated hepatocytes: contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide-induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Dithiothreitol (DTT) rescues mitochondria from nitrofurantoin‐induced mitotoxicity in rat

Nitrofurantoin (N‐(5‐nitro‐2‐furfurylidine) 1‐amino‐hydantoine; NIT) is mainly used for the treatment of acute urinary tract infections. However, its administration can be associated with liver failure or cirrhosis. The aim of this study was to determine whether NIT is a mitochondrial toxicant, if so, what mechanism(s) is involved. The rat liver mitochondria were isolated and treated with different doses of NIT alone or in combination with a reagent of choice for protecting thiol groups, dithiothreitol (DTT). Several mitochondrial parameters, including succinate dehydrogenase activity (also called 3‐(4,5‐dimethylthiazol‐2‐yl) 2,5‐diphenyl tetrazolium bromide assay), lipid peroxidation, superoxide dismutase activity, Reduced glutathione (GSH), and oxidized glutathione (GSSG), and GSSG (oxidized glutathione) levels were determined. The results from this study showed that simultaneous treatment of mitochondria with NIT and DTT significantly reduces the toxicity. Here, we provide evidence that mitochondrial dysfunction followed by depletion of reduced glutathione can be reversed by DTT administration.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.     

Mitochondrial poisons cause depletion of reduced glutathione in isolated hepatocytes.

Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 mM cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 mM fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.     

Prevention of nitrofurantoin-induced cytotoxicity in isolated hepatocytes by fructose

Nitrofurantoin is a widely utilized urinary antimicrobial drug which has been associated with pulmonary fibrosis, neuropathy, and hepatitis as well as hemolytic anemia in glucose-6-phosphate dehydrogenase-deficient individuals. Incubation of freshly isolated rat hepatocytes with nitrofurantoin caused oxygen activation as a result of futile redox cycling. Glutathione disulfide (GSSG) was formed and rapidly exported from the cell resulting in complete glutathione (GSH) depletion followed by cell death. However, fructose prevented the export of GSSG from the cell and GSH levels recovered rapidly without cytotoxicity occurring. Fructose did not affect nitrofurantoin metabolism but rapidly depleted cellular ATP levels by approximately 80% which remained depressed during the incubation period. Fructose, however, did not protect hepatocytes from nitrofurantoin-induced cytotoxicity if GSH was depleted beforehand. Protection by fructose only occurred at concentrations which caused ATP depletion. These results suggest that fructose prevents nitrofurantoin-induced toxicity by depleting ATP and thereby preventing the ATP-dependent GSSG efflux. GSSG is retained enabling NADPH and glutathione-reductase to reduce the GSSG back to GSH, thereby protecting the cell from nitrofurantoin-induced oxidative stress.     

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.     

Protective mechanisms of N‐acetyl‐cysteine against pyrrolizidine alkaloid clivorine‐induced hepatotoxicity

Pyrrolizidine alkaloid (PA) clivorine, isolated from traditional Chinese medicinal plant Ligularia hodgsonii Hook, has been shown to induce apoptosis in hepatocytes via mitochondrial‐mediated apoptotic pathway in our previous research. The present study was designed to observe the protection of N‐acetyl‐cysteine (NAC) on clivorine‐induced hepatocytes apoptosis. Our results showed that 5 mM NAC significantly reversed clivorine‐induced cytotoxicity via MTT and Trypan Blue staining assay. DNA apoptotic fragmentation analysis and Western‐blot results showed that NAC decreased clivorine‐induced apoptotic DNA ladder and caspase‐3 activation. Further results showed that NAC inhibited clivorine‐induced Bcl‐xL decrease, mitochondrial cytochrome c release and caspase‐9 activation. Intracellular glutathione (GSH) is an important ubiquitous redox‐active reducing sulfhydryl (? SH) tripeptide, and our results showed that clivorine (50 µM) decreased cellular GSH amounts and the ratio of GSH/GSSG in the time‐dependent manner, while 5 mM NAC obviously reversed this depletion. Further results showed that GSH synthesis inhibitor BSO augmented clivorine‐induced cytotoxicity, while exogenous GSH reversed its cytotoxicity on hepatocytes. Clivorine (50 µM) significantly induced cellular reactive oxygen species (ROS) generation. Further results showed that 50 µM Clivorine decreased glutathione peroxidase (GPx) activity and increased glutathione S transferase (GST) activity, which are both GSH‐related antioxidant enzymes. Thioredoxin‐1 (Trx) is also a ubiquitous redox‐active reducing (? SH) protein, and clivorine (50 µM) decreased cellular expression of Trx in a time‐dependent manner, while 5 mM NAC reversed this decrease. Taken together, our results demonstrate that the protection of NAC is major via maintaining cellular reduced environment and thus prevents clivorine‐induced mitochondrial‐mediated hepatocytes apoptosis. J. Cell. Biochem. 108: 424–432, 2009. © 2009 Wiley‐Liss, Inc.     

Investigation of rifampicin-induced hepatotoxicity in rat hepatocytes maintained in gel entrapment culture

Rifampicin-induced hepatotoxicity has been well recognized in animals and patients. However, it is undetectable in cultured hepatocyte monolayers in vitro at the equivalent toxic concentration in vivo. This study investigated the rifampicin-induced toxicity on rat hepatocytes in gel entrapment vs. in monolayer culture. Thiazolyl tetrazolium reduction and albumin secretion were routinely detected to identify the toxic responses of rat hepatocytes to rifampicin, while reactive oxygen species (ROS) accumulation and intracellular glutathione (GSH) content were assayed as biomarkers of oxidative stress. In addition, Nile red staining and malondialdehyde (MDA) generation were, respectively, used as endpoints for lipid accumulation and peroxidation. After treatment of hepatocytes for 96 h at a serum rifampicin concentration (12 μM), gel-entrapped rat hepatocytes showed significant cellular damage indicated by alternations of all parameters indicated above, while hepatocyte monolayers did not show severe responses. In contrast to a lack of protections by cytochrome P 450 inhibitors, the ROS scavenger (glycyrrhizic acid) and thiol compounds (N-acetylcysteine and GSH) significantly reduced rifampicin toxicity in gel-entrapped hepatocytes. It appears that gel-entrapped rat hepatocytes reflected significant hepatotoxicity of rifampicin in vivo, and this toxicity was most possibly associated with oxidative stress and lipid accumulation.     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.