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.     

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.     

Glutathione depletion and cytotoxicity by naphthalene 1,2-oxide in isolated hepatocytes

The ability of naphthalene 1,2-oxide to diffuse across intact cellular membranes, the subsequent biotransformation of this epoxide and its potential to produce losses in cellular viability have been examined in incubations of isolated hepatocytes. Addition of 1R,2S- or 1S,2R-naphthalene oxide enantiomers (15, 30 and 60 microM) to isolated hepatocytes resulted in a rapid depletion of intracellular glutathione. Depletion of glutathione was concentration dependent and maximal at 5-15 min. Addition of either of the enantiomeric oxides at 60 microM resulted in the loss of more than 20 nmol glutathione/10(6) cells (1 ml cells); thus more than a third of the added epoxide was available for conjugation with intracellular glutathione. The time course and concentration dependence of glutathione depletion corresponded to the rapid, concentration-dependent formation of naphthalene oxide glutathione conjugates. The levels of glutathione adduct were highest 1 min after addition of naphthalene oxide and declined to 25% of this level after 30 min. Loss of glutathione conjugates from incubations correlated with the formation of N-acetylcysteine adducts. In contrast, the levels of glutathione adducts added exogenously to hepatocytes were relatively stable over a 120-min incubation suggesting that although further metabolism of naphthalene oxide glutathione adducts formed intracellularly is possible, extracellular glutathione adducts cannot penetrate the hepatocellular membrane. Small amounts of radiolabel from [3H]naphthalene 1,2-oxide were bound covalently to macromolecules in hepatocytes; the rate of this binding slowed rapidly after the first minute of incubation. Severe blebbing of the surface of the hepatocytes was noted in cells incubated for 30 min with 480 microM naphthalene oxide. Many of the cells were vacuolated at 60 min and progressed to frank necrosis with pyknotic nuclei and inability to exclude trypan blue. Cells incubated with 1-naphthol responded in a qualitatively similar fashion to those cells incubated with epoxide; however, hepatocytes incubated with 1-naphthol progressed to frank cellular necrosis at a slower rate. In hepatocytes partially depleted of glutathione by pretreatment with buthionine sulfoximine, addition of 1S,2R-naphthalene oxide at a rate of 1 nmol/min/10(6) cells resulted in significant losses in cell viability. In contrast, no losses in cell viability were observed with the enantiomer, 1R,2S-naphthalene oxide. Both epoxides produced similar losses in cellular glutathione levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes

The metabolism and toxic effects of eugenol (4-allyl-2-methoxyphenol) were studies in isolated rat hepatocytes. Incubation of hepatocytes with eugenol resulted in the formation of conjugates with sulfate, glucuronic acid and glutathione. The major metabolite formed was the glucuronic acid conjugate. Covalent binding to cellular protein was observed using [3H]eugenol. Loss of intracellular glutathione and cell death were also observed in these incubations. Concentrations of 1 mM eugenol caused a loss of over 90% of intracellular glutathione and resulted in approximately 85% cell death over a 5-h incubation period. The loss of the majority of glutathione occurred prior to the onset of cell death (2 h). The effects of eugenol were concentration dependent. The addition of 1 mM N-acetylcysteine to incubations containing 1 mM eugenol was able to completely prevent glutathione loss and cell death as well as inhibit the covalent binding of eugenol metabolites to protein. Conversely, pretreatment of hepatocytes with diethylmaleate to deplete intracellular glutathione increased the cytotoxic effects of eugenol. These results demonstrate that eugenol is actively metabolized in hepatocytes and suggest that the cytotoxic effects of eugenol are due to the formation of a reactive intermediate, possibly a quinone methide.     

ATP-generating glycolytic substrates prevent N-nitrosofenfluramine-induced cytotoxicity in isolated rat hepatocytes

The relationship between cytotoxicity induced by N-nitrosofenfluramine and mitochondrial or glycolytic adenosine triphosphate (ATP) synthesis-dependent intracellular bioenergetics was studied in isolated rat hepatocytes. The supplementation of fructose, an ATP-generating glycolytic substrate, to hepatocyte suspensions prevented N-nitrosofenfluramine-induced cell injury accompanied by the formation of cell blebs, abrupt loss of intracellular ATP and reduced glutathione and mitochondrial membrane potential (DeltaPsi), and the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation, during a 2h incubation period. Fructose (1-20mM) resulted in concentration-dependent protection against the cytotoxicity of N-nitrosofenfluramine at a concentration of 0.6mM, a low toxic dose. Pretreatment with xylitol, another glycolytic substrate, at concentration of 15mM also prevented the cytotoxicity caused by the nitroso compound, but neither glucose nor sucrose exhibited protective effects. In addition, fructose inhibited N-nitrosofenfluramine (0.5 and 0.6mM)-induced DNA damage, as evaluated in the comet assay, indicating that nuclei as well as mitochondria are target sites of the compound. These results indicate that (a) the onset of N-nitrosofenfluramine-induced cytotoxicity in rat hepatocytes is linked to mitochondrial failure, and that (b) the insufficient supply of ATP in turn limits the activities of all energy-requiring reactions and consequently leads to acute cell death.     

Dimerumic acid protected oxidative stress-induced cytotoxicity in isolated rat hepatocytes

Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.     

Mechanisms of chemical mediated cytotoxicity and chemoprotection in isolated rat hepatocytes

Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) cause injury and malondialdehyde formation in rat hepatocytes, MNNG toxicity is much more sensitive to inhibition by antioxidants. In order to quantify the relationship between toxicity and antioxidation potential, we compared 14 antioxidants that protected against MNNG and MMS toxicity. Chemoprotection was quantified as the concentration that delayed by 1 h the decline in trypan blue exclusion to less than or equal to 50%. While chemoprotection against MNNG and antioxidant efficacy were directly related (R = 0.86), chemoprotection against MMS and antioxidant efficacy were unrelated (R = 0.37). Since we hypothesized that protection against MMS involved stabilization of membranes, the capacity of the 14 compounds to stabilize membranes in an unrelated system (i.e. prevention of erythrocyte osmotic rupture) was assayed. Chemoprotection against both MNNG and MMS correlated with reduced RBC fragility (R = 0.97 and 0.70, respectively). One of the better protecting compounds, 4b,5,9b,10-tetrahydroindeno[1,2-b]indole, was also protective against hepatocellular toxicity mediated by acetaminophen, carbon tetrachloride and tert-butyl hydroperoxide, suggesting a fundamental basis in the mechanism of chemoprotection. We propose that methylating agents and perhaps other chemical toxicants destabilize cellular membranes resulting in hepatocellular injury. For MNNG, radical mediated events may result in membrane destabilization; for MMS, membranes are destabilized without concurrent radical events. The current studies provide a basis for future work to determine structure-activity relationships of chemoprotective agents, examine protection mechanisms, and develop better protective compounds.     

Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism

Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.     

Allyl alcohol cytotoxicity and glutathione depletion in isolated periportal and perivenous rat hepatocytes

The mechanism of the periportal (p.p.) toxicity of allyl alcohol (AlOH) was investigated in p.p. and perivenous (p.v.) hepatocytes isolated by digitonin-collagenase perfusion. The distinct origin of the cell preparations was confirmed by the p.p./p.v. ratios of alanine aminotransferase (p.p./p.v. = 1.8), lactate dehydrogenase (1.3) and glutamine synthetase (0.10). The activity of alcohol dehydrogenase (ADH) was not markedly different in p.p. and p.v. cells. Both types of cells oxidized AlOH at a high but equal rate of about 3 mumol/(min.g cells). Concomitantly with rapid oxidation of 0.7 mM AlOH, glutathione (GSH) was depleted by about 95% and its secretion was completely inhibited in both cell types. Although the GSH content was partially restored during a subsequent 3-h incubation, cellular ATP and K+ content gradually decreased and the leakage of lactate dehydrogenase increased in both types of cells. However, the p.p. cells tended to resist AlOH in vitro better, probably due to their 26% higher GSH content after preincubation with L-methionine. Altering the partial pressure of oxygen in physiological range had no effect on the toxicity of AlOH. The results are contrary to the suggestions that the p.p. location of AlOH liver injury is caused by higher ADH activity or higher oxygen tension in the p.p. zone. Rather, the regiospecificity of the injury may be due to rapid uptake and oxidation of AlOH in the p.p. region.     

Modulation of mercury-induced mitochondria-dependent apoptosis by glycine in hepatocytes

Mercury (Hg) is one of the universal environmental pollutants and is responsible for various organ pathophysiology including oxidative stress-induced hepatic disorders. In the present study, we aimed to explore the protective role of glycine in Hg-induced cytotoxicity and cell death in murine hepatocytes. Exposure of mercury (20 μM), in the form HgCl₂ for 1 h, significantly enhanced the ALT and ALP leakage, increased reactive oxygen species production, reduced cell viability and distorted the antioxidant status of hepatocytes. Flow cytometric analyses shows that Hg-induced apoptotic death in hepatocytes. Mechanism of this pathophysiology involves reduced mitochondrial membrane potential, variations in Bcl-2/Bad proteins, activation of caspases and cleavage of PARP protein. In addition, Hg distinctly increased NF-κB phosphorylation in association with IKKα phosphorylation and IκBα degradation. Concurrent treatment with glycine (45 mM), however, reduced Hg-induced oxidative stress, attenuated the changes in NF-κB phosphorylation and protects hepatocytes from Hg-induced apoptotic death. Hg also distinctly increased the phosphorylation of p38, JNK and ERK mitogen-activated protein kinase (MAPKs). Glycine treatment suppressed these apoptotic events, signifying its protective role in Hg-induced hepatocyte apoptosis as referred by reduction of p38, JNK and ERK MAPK signaling pathways. Results suggest that glycine can modulate Hg-induced oxidative stress and apoptosis in hepatocytes probably because of its antioxidant activity and functioning via mitochondria-dependent pathways and could be a beneficial agent in oxidative stress-mediated liver diseases.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

Modulation of gluconeogenesis by epinephrine in hepatocytes isolated from genetically obese (fa/fa) Zucker rats

The obese (fa/fa) Zucker rat shows an impaired sympathetic tone which is accompanied by an altered thermogenesis and changes in both lipid and carbohydrate metabolism. In this work, we have investigated the regulatory effects of epinephrine on the rate of gluconeogenesis from a mixture of [(14)C]lactate/pyruvate, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine caused a dose-dependent stimulation of the rate of [(14)C]glucose formation in both obese and lean rat hepatocytes, the maximal rates being five- and twofold higher than the corresponding basal values (0.50 +/- 0.06 and 1.96 +/- 0.15 micromol of lactate converted to glucose/g of cell x 20 min, respectively). No significant differences were found between the calculated half-maximal effective concentrations (EC(50)) for epinephrine in obese and lean rat liver cells. The stimulation of gluconeogenesis by epinephrine was accompanied by a decrease in the cellular concentration of fructose 2,6-bisphosphate, and an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, to similar extents in both types of hepatocytes. Epinephrine also significantly raised the hepatocyte content of cyclic AMP, with about a twofold increase at a saturating concentration of the catecholamine (1 microM), in both lean and obese rat liver cells. However, at suboptimal concentrations of epinephrine, the rise in cyclic AMP levels was significantly less marked in obese than in lean rat hepatocytes. Nevertheless, no significant differences were found in either the affinity or the number of beta-adrenergic receptors, in radioligand binding studies carried out in liver plasma membranes obtained from obese and lean Zucker rats. In conclusion, compared to the corresponding basal values, the response of gluconeogenesis from lactate to the stimulatory effect of epinephrine is higher in obese (fa/fa) than in lean (Fa/-) Zucker rat hepatocytes, with no significant differences in the calculated EC(50) values for this hormone. This occurs in spite of an apparent decreased sensitivity of the adenylate cyclase system to the stimulatory effect of epinephrine in obese rat liver cells.     

Studies on the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine cytotoxicity in isolated hepatocytes

Oxidative stress and covalent binding have been proposed as possible mechanisms involved in the cytotoxic effects of the parkinsonism-causing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the toxicity induced by MPTP in isolated rat hepatocytes seems to be relatively independent of oxygen radical-induced oxidative stress. Here we demonstrate that MPTP cytotoxicity is not potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, nor prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the iron-chelating agent desferrioxamine. Moreover, preincubation of hepatocytes with diethylmaleate to lower the level of intracellular reduced glutathione (to 20% of the initial value) did not affect either the rate or extent of MPTP cytotoxicity. Thus, nucleophilic soluble thiols do not seem to play a protective role against MPTP-induced cell damage, in contrast to what one would have expected if covalent protein binding and oxidative stress were involved as toxic mechanisms. On the other hand, MPTP cytotoxicity was potentiated by pretreatment of hepatocytes with cytochrome P-450 inhibitors (e.g., SKF 525A and metyrapone) and a more rapid depletion of ATP was observed in these experimental conditions. We conclude that mitochondrial damage and subsequent ATP depletion are likely to play a critical role in the toxicity of MPTP to isolated hepatocytes and that the metabolism of MPTP via the cytochrome P-450 monooxygenase system can be considered to be a detoxifying pathway.     

Enzyme-induced aziridine formation by isolated hepatocytes

The metabolism of 2-bromoethylaminonaphthoquinone in hepatocytes isolated from rats was studied. This compound was chemically inert in the reaction system used. However, in buffer solution containing isolated hepatocytes, it was gradually converted into aziridinylnaphthoquinone. Under the same reaction conditions, 4-chlorobutylaminonaphthoquinone also gave the cyclization products, pyrrolidinylnaphthoquinone. Cellular GSH decreased in both reactions.     

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes

Exposure of rat hepatocytes to cadmium below 50 μM for a short period (10 min) resulted in cellular acidification. Conversely, exposure to Cd more than 50 μM for a long period (60 min) caused cellular alkalinization accompanied by membrane damage as reflected by decrease in cellular K content and loss of intracellular lactic dehydrogenase. In hepatocytes exposed to 5 μM Cd, a concentration sufficient to induce acidification without cytotoxicity, the metal was preferentially associated with the crude nuclei and cell debris fractions, suggesting an interaction between Cd and cell membranes to cause acidification. Omission of bicarbonate from the incubation medium induced cellular acidification. The presence of Cd in this medium did not potentiate the medium-induced acidification. Mg-ATP (25 μM) induced cellular acidification in relation to an increase in the concentration of cytosolic free Ca. The coexistence of Mg-ATP and Cd at the concentrations which had no effect on cellular pH in the presence of either agants induced cellular acidification. These observations suggest that Cd induced cellular acidification by modulating the process connected with the rise in cytosolic free Ca via interaction with plasma membranes. This acidification had no strong immediate cytotoxic actions but led to subsequent cellular alkalinization accompanied with severe cytotoxicity and membrane breakage.     

Glucagon binding by isolated chick hepatocytes

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.     

Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated rat hepatocytes

Alkyl esters of gallic acid inhibited the respiration rate of mouse sarcoma 786A and mouse mammary adenocarcinoma TA3 cell lines and its multiresistant variant TA3-MTX-R more effectively than gallic acid, both in the absence and in the presence of the uncoupler CCCP. The order of inhibition of the respiration rate by gallates in intact cells was n-octyl- approximately iso-amyl- approximately n-amyl- approximately iso-butyl->n-butyl->iso-propyl->n-propyl-gallate>gallic acid. Sarcoma 786A was significantly more susceptible to all seven esters than the TA3 cell line. Respiration rates of the TA3-MTX-R cell line showed almost the same sensitivity to these esters as the TA3 cell line. However, hepatocytes were significantly less sensitive than all tumor cells tested. These alkyl gallates blocked mitochondrial electron flow, mainly at the NADH-CoQ segment, preventing ATP synthesis, which would lead to cellular death. These esters also inhibited, in the same order of potencies as respiration, the growth of 786A, TA3 and TA3-MTX-R cells in culture. In mice carrying TA3 or TA3-MTX-R tumor cells, an important decrease of the tumor growth rate and an increase of survival were observed when mice were treated with iso-butyl gallate alone or in combination with doxorubicin. These results indicate that alkyl gallates are selectively cytotoxic to tumor cells, which may be due to the mitochondrial dysfunctions of these cells.     

Biochemical changes in isolated hepatocytes exposed to tert-butyl hydroperoxide. implications for its cytotoxicity.

When isolated hepatocytes were exposed to tert-butyl hydroperoxide (tBOOH) they lost their cellular membrane integrity. Decreased levels of GSH, increased phosphorylase a activity (an indirect index of the amount of free cytosolic Ca²⁺), and increase in the formation of malondialdehyde (MDA)-like products (an index of lipid peroxidation) preceeded the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH), indicating that this later process was the consequence of the former intracellular events. While ATP levels were not modified during the incubation of cells with increasing concentrations of tBOOH, protein synthesis was decreased in a concentration-dependent manner. The glycogen content decreased at the same time as the increase in LDH leakage. The addition of promethazine (PMZ) an antioxidant molecule, prevented the lipid peroxidation, but did not protect cells against the oxidative effects of tBOOH, including loss of membrane integrity. Nevertheless, the addition of GSH to cell suspensions incubated with tBOOH, decreased the formation of MDA-like products, restored the protein synthesis rate, prevented partially the activation of phosphorylase a and preserved cell viability. On the basis of these results, we postulate that both GSH depletion and modification in phosphorylase a activity (Ca²⁺ levels) were the most relevant intracellular events to explain the cytotoxicity of tBOOH.Abbreviations tBOOH tert-butyl hydroperoxides - GSH reduced glutathione - LDH lactate dehydrogenase - MDA malondialdehyde - TBA thiobarbituric acid - PMZ promethazin - BSA bovine serum albumin     

The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes

Chromium (VI) is an environmental and occupational carcinogen, and it is accepted that intracellular reduction is necessary for DNA damage and cytotoxicity. We have investigated the interaction of Cr(VI) with hepatocytes in vitro to determine the contribution of various hepatic enzymes to the reduction of Cr(VI). Cr(VI) caused a dose-dependent decrease in cell viability and intracellular reduced glutathione (GSH) levels between 100 and 500 microM within 3 h exposure of hepatocytes. Both DT-diaphorase and cytochrome P450 play only a minor role in detoxifying Cr(VI) and/or its metabolites. (GSH) appears to act as a non-enzymatic reductant, reducing Cr(VI) to a toxic form. The evidence for this is two-fold. Firstly, GSH was depleted during the metabolism of Cr(VI) and, secondly, pretreatment of the cells with diethylmaleate to deplete GSH levels, partially protected the cells from Cr(VI) toxicity. Glutathione reductase appears to play an important role in the enzymatic reduction of Cr(VI) as inhibition of this enzyme by carmustine (BCNU) markedly protected the cells from cytotoxicity.