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.     

Stimulatory effect of calcitonin on calcium uptake and glucose production in isolated rat hepatocytes

The biological effect of calcitonin (CT) was investigated in isolated rat hepatocytes. Addition of synthetic [Asu1,7] eel CT (20 and 40 ng/ml of medium with 2.5 mg dry weight of cells) into the medium containing calcium ion produced a marked elevation of calcium uptake in the hepatocytes, when calcium concentrations in the medium were monitored spectrophotometrically using arsenazo III. Intracellular calcium contents in the mitochondria and microsomes were significantly increased by CT addition. Meanwhile, glycogenolysis and gluconeogenesis in the hepatocytes were significantly stimulated by addition of CT (10, 10(2), and 10(3) ng/ml of medium with 5 mg dry weight of cells) into the medium containing calcium, although CT effects were less than those effects of 10(-7) M glucagon and 10(-5) M phenylephrine. These data clearly indicate that CT stimulates calcium uptake and glucose production in the hepatocytes, suggesting that CT has an effect in the metabolic function of rat liver cells.     

Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.     

Oxidative stress studied in intact mammalian cells

Exposure of isolated rat hepatocytes to toxic doses of menadione (2-methyl-1,4-naphthoquinone) results in enhanced formation of active oxygen species, depletion of cellular glutathione and protein thiols, and perturbation of intracellular calcium ion homeostasis. An increase in cytosolic Ca2+ concentration, resulting from inhibition of the plasma membrane Ca2+ translocase by menadione metabolism, appears to be critically involved in the development of cytotoxicity.     

Evaluation of calcium channel blockers as potential hepatoprotective agents in oxidative stress injury of perfused hepatocytes

The aim of this study was to investigate the effects of calcium channel blockers on tertbutyl hydroperoxide (TBH) induced liver injury using isolated perfused rat hepatocytes. Rat hepatocytes were immobilized in agarose threads and perfused with Williams E medium. Hepatocyte injury was induced by the addition of tertbutyl hydroperoxide (1 mM) to the perfusion medium 30 min after the addition of either verapamil or diltiazim. Hepatocyte injury was observed by monitoring the functional and metabolic competence of hepatocytes or by ultrastructural morphological examination of hepatocytes. Verapamil (0.5 mM) reduced lactate dehydrogenase leakage in TBH-injured hepatocytes as compared to the controls (154+/-11% vs. 247+/-30%). Lipid peroxides production was reduced after verapamil pretreatment as compared to the controls and oxygen consumption was increased by pretreatment of hepatocytes with verapamil. Verapamil pretreatment increased the protein synthesis activity at both levels of granular endoplasmic reticulum and free polysomes in cytoplasm and decreased ATPase activity. Diltiazem was qualitatively effective as verapamil. It is concluded that in hepatocyte oxidative injury, calcium channel blockers exhibited hepatoprotective properties. The hepatoprotective effect of calcium channel blockers was accompanied by a decrease in ATPase activity, which may implicate a normalization of Ca2+i after TBH intoxication.     

Role of cellular calcium homeostasis in toxic liver injury induced by the pyrrolizidine alkaloid senecionine and the alkenal trans-4-OH-2-hexenal

The pyrrolizidine alkaloid senecionine has been shown to be hepatotoxic, genotoxic, and cytotoxic. However, the biochemical mechanism by which senecionine produces hepatocellular toxicity remains to be elucidated. The role of calcium homeostasis in toxic liver injury was examined in isolated rat hepatocytes treated with senecionine and trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and appropriate cofactors. Hepatocytes treated with senecionine and t-4HH demonstrated greater cytotoxicity (leakage of lactate dehydrogenase) when incubated in the absence of extracellular Ca²⁺ than in its presence. Both compounds elicited an increase in cytosolic Ca²⁺ levels of isolated hepatocytes in the presence of extracellular Ca²⁺ In the following study, senecionine and t-4HH depleted intracellular glutathione levels and induced lipid peroxidation and cytotoxicity in isolated hepatocytes. Pretreatment with the thiolgroup reducing agent dithiothreitol prevented depletion of intracellular glutathione and protected hepatocytes against senecionine and t-4HH-induced lipid peroxidation and cytotoxicity. Both compounds also depleted intracellular ATP and NADPH levels. These results suggest that hepatotoxocity induced by senecionine and t-4HH is not dependent on the influx of extracellular Ca²⁺; however, alterations in intracellular Ca²⁺, possibly associated with depletion of intracellular glutathione, NADPH, and ATP, may play a critical role.     

Production of intracellular 35S-glutathione by rat and human hepatocytes for the quantification of xenobiotic reactive intermediates

The quantification and identification of xenobiotic reactive intermediates is difficult in the absence of highly radiolabeled drug. We have developed a method for identifying these intermediates by measuring the formation of adducts to intracellularly generated radiolabeled glutathione (GSH). Freshly isolated adherent rat and human hepatocytes were incubated overnight in methionine and cystine-free ('thio-free') medium. They were then exposed to 100 microM methionine and 10 microCi 35S-labeled methionine in otherwise thio-free medium to replete cellular GSH pools with intracellularly generated 35S-labeled GSH. After 3 h, acetaminophen was added as a test compound and the cells were incubated for an additional 24 h. Intracellular GSH and its specific activity were quantified after reaction with monobromobimane followed by HPLC analysis with fluorescence and radiochemical detection. Radiolabeled GSH was detectable at 3 h and maintained high specific activity and physiological concentrations for up to 24 h. Incubation medium from acetaminophen treated and nontreated hepatocytes were analyzed for radiolabeled peaks by HPLC using radiochemical detection. Radiolabeled peaks not present in nontreated hepatocytes were identified as acetaminophen GSH adducts by LC-MS. Formation of acetaminophen 35S-GSH adducts by rat hepatocytes containing endogenously synthesized 35S-GSH was increased with acetaminophen concentrations ranging from 500 to 2 mM.     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

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.     

Glutathione conjugation of synthetic steroids in isolated rat hepatocytes

When designing steroid drugs with multiple double bonds, the influence of glutathione conjugation on the pharmacodynamics of drug action should be considered. We have examined the effect of canrenone, a mineralocorticoid receptor antagonist, on isolated rat hepatocytes and found that 1 mM canrenone injured the hepatocytes during shortterm incubation at 37 C, while an analogue of canrenone which bears 4 double bonds (delta 1,11-CAN) did not manifest such toxicity. To further pursue this, we prepared testosterone analogues comprising multiple double bonds as model compounds, and incubated them with freshly isolated rat hepatocytes. The viability of the hepatocytes was not influenced by any of the steroids, but some of them having a double bond at the C6-C7 position reduced the cellular glutathione levels. This was found to be due to conjugation of glutathione to the C7 position of the steroid molecule, and the rate of conjugation was accelerated when an additional double bond was introduced at C1-C2 or C11-C12 positions. The finding is interesting as glucuronidation or sulfation are common as conjugation processes of steroids.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Metabolism of hydralazine by rat hepatocytes

The metabolism of hydralazine and its pyruvic acid hydrazone was studied in a medium containing isolated adult rat hepatocytes. Hydralazine undergoes conversion to 3-methyltriazolophthalazine, while hydralazine pyruvic acid hydrazone is not metabolized in rat hepatocytes.     

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.     

Metabolic activation and hepatotoxicity. Metabolism of bromobenzene in isolated hypatocytes.

Rat liver microsomes and isolated rat hepatocytes metabolized bromobenzene to watersoluble and protein-bound metabolites. The latter fraction—which normally accounted for 2–5% of the total products—was slightly increased when 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase, was added to the microsomal incubate. The presence of reduced glutathione (GSH), on the other hand, caused an almost complete inhibition of the formation of protein-bound metabolites from bromobenzene in microsomes. The rates of bromobenzene metabolism were similar in liver microsomes and hepatocytes, and increased severalfold after phenobarbital pretreatment of the rats. Metyrapone and SKF 525-A were inhibitory in both systems. Bromobenzene metabolism in hepatocytes isolated from phenobarbital-treated rats was associated with a rapid and marked decrease in the level of intracellular GSH. When the cells were incubated in a complete medium, however, the decrease in GSH leveled off at about 40% of the original concentration and there was no evidence of any accelerated rate of cell death even when the incubation with bromobenzene was prolonged to 10 h. This was most probably due to resynthesis of GSH by the hepatocytes, which partly compensated for the loss of this thiol associated with bromobenzene metabolism. Accordingly, in a deficient medium (lacking amino acids), the cytotoxic effect of bromobenzene metabolism was pronounced—less than 5% of the zerotime level of GSH and only 25% cell viability remaining after 5 h of incubation. It is concluded that the intracellular level of GSH is of major importance in regard to the cytotoxic effect of bromobenzene metabolism and that hepatocytes incubated in a complete medium are protected against toxicity by their ability to resynthesize this thiol.     

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes

mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.     

Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.     

Relationship between cellular calcium and vitamin E metabolism during protection against cell injury

The extent of chemically induced injury to isolated hepatocytes has been previously shown to depend on the content of alpha-tocopherol in the cells, the levels of which are influenced by the concentration of extracellular calcium. Investigations into the effect of calcium on the alpha-tocopherol content of nonchemically exposed cells demonstrated that incubation of isolated hepatocytes in a calcium-deficient medium decreased cell calcium content to 10% of initial levels, and resulted in the depletion of endogenous alpha-tocopherol. This loss in alpha-tocopherol was not accounted for by alpha-tocopherylquinone formation. After supplementation of the cell incubation medium with alpha-tocopheryl succinate, the decreased cell calcium content was associated with higher levels of cellular alpha-tocopherol than in calcium-adequate cells. This was the result of greater intracellular hydrolysis of the tocopheryl ester in the calcium-depleted cells, and not an effect of extracellular calcium concentration on the uptake of alpha-tocopheryl succinate into the cells or on the extracellular hydrolysis of the ester. Uptake studies indicated a much greater achievable level of alpha-tocopherol in hepatocytes after incubation with alpha-tocopherol than with the alpha-tocopheryl ester. These data provide substantial support for the hypotheses that the content of extracellular calcium per se is not the determinant in toxic injury to hepatocytes, but that cell calcium content affects the intracellular metabolism of alpha-tocopherol and its esters, which may subsequently govern the outcome of a toxic challenge.     

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