Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.     

Oxidant-induced changes in the cellular energy homeostasis. A study with 3,5-dimethyl N-acetyl-p-benzoquinone imine and isolated hepatocytes

Exposure of isolated hepatocytes to 400 microM 3,5-dimethyl N-acetyl-p-benzoquinone imine (3,5-diMe NAPQI), rapidly induced the formation of plasma membrane blebs. More than 50% of the viable cells were affected after 1 min incubation with 3,5-diMe NAPQI. Rapid loss of mitochondrial ATP, and sequential increases in ADP and AMP accompanied hepatocyte blebbing. 3,5-diMe NAPQI also induced a pronounced elevation of mitochondrial NADP level, whereas the NAD concentration was unaffected. Similar alterations in the adenine and pyridine nucleotide pools were found to occur in the cytosol, although at slower rates. During the initial phase of ATP loss and NADP production, there was also a concomitant decrease in the oxygen uptake of the hepatocytes. The decreases in energy substrates occurred in parallel to an increased uptake of trypan blue into the cells. Treatment of the hepatocytes with dithiothreitol, following 4 min exposure of the cells to 3,5-diMe NAPQI, reversed the quinone imine-induced changes in nucleotide levels and reduced the cytotoxicity. It is concluded that alteration of mitochondrial function, which results in changes in the cellular energy homeostasis, is an important event in the development of cytotoxicity caused by 3,5-diMe NAPQI.     

Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.     

Toxicity of ethacrynic acid in isolated rat hepatocytes.

Ethacrynic acid, a loop diuretic drug, caused lipid peroxidation in isolated rat hepatocytes. The thiobarbituric acid reactive substances (TBARS) formation showed a good correlation with the leakage of glutamic-oxaloacetic acid transaminase (GOT) from the hepatocytes. The addition of antioxidants such as N, N'-diphenyl-p-phenylenediamine (DPPD) and promethazine to the isolated rat hepatocyte suspension containing ethacrynic acid prevented the lipid peroxidation and decreased the GOT leakage to some extent. SKF-525A inhibited the oxidative metabolism of ethacrynic acid and decreased the TBARS formation, suggesting that the lipid peroxidation was caused by the oxidative metabolism. The intracellular reduced glutathione markedly decreased in the hepatocyte suspension containing ethacrynic acid and the hepatocellular protein sulfhydryls were decreased, which was negatively correlated with the GOT leakage. Thus the ethacrynic acid-induced hepatotoxicity was found to be related to the lipid peroxidation and the decrease of cellular protein sulfhydryls.     

Steroid metabolism in isolated rat hepatocytes

In vivo predictability of results obtained from studies of drug metabolism using isolated rat hepatocytes is questionable, mainly because of modeling difficulties due to the simultaneously occurring substrate transferring processes. In the present study, an attempt was made at simplifying the models used to describe the kinetics of biotransformation by enzymes enclosed in a cellular environment. Viability assessment of the cell preparation indicated that the cell membrane was intact and functional. Six corticosteroids were used in these studies. Simplifying assumptions concerning uptake and protein binding were confirmed by running independent experiments. Progress curves of unchanged steroid disappearance from the cell suspending medium at different initial concentrations were used to either confirm applicability or detect deviations from simple Micha?lis-Menten behavior and were fitted to the appropriate kinetic models by means of nonlinear least-squares regression analysis. As an example, corticosterone extraction ratio obtained in this study compared well with literature values from intact rats. A linear correlation was found between the logarithm of the apparent first order rate constant (Vm/Km) obtained at low substrate concentrations and the logarithm of the oil/water partition coefficients of 17 alpha-hydroxyprogesterone, corticosterone and hydrocortisone.     

Codeine-mediated hepatotoxicity in isolated rat hepatocytes

Administration of codeine to freshly isolated rat hepatocytes resulted in cytotoxicity characterized by a dose- and time-dependent leakage of lactate dehydrogenase (LDH) out of the cells. Codeine also caused a decrease in hepatic reduced sulfhydryl content. Cytochrome P-450 content and NADPH levels were not changed. Induction and inhibition studies of several potential pathways of codeine biotransformation were carried out in order to determine if codeine must be metabolized to a reactive intermediate to elicit these hepatotoxic effects. Codeine hepatotoxicity as measured by LDH release was not changed after induction of cytochrome P-450 by phenobarbital and was decreased after cytochrome P-448 induction by beta-naphthoflavone. However, codeine hepatotoxicity was inhibited when an inhibitor of cytochrome P-450 metabolism, metyrapone, was added. Inhibition of the other major hepatic oxidative enzyme system, flavin adenine dinucleotide (FAD)-containing monooxygenase, increased the cytotoxicity of codeine. Inhibition of alcohol dehydrogenase had no effect on codeine hepatotoxicity. These results indicate that codeine hepatotoxicity is caused by a cytochrome P-450-generated intermediate of codeine, whereas FAD-containing monooxygenase may metabolize codeine to a nontoxic intermediate.     

Copper toxicity in isolated rat hepatocytes

In relative excess, copper is a cytotoxic metal. The injury may be related to the process of lipid peroxidation. Isolated hepatocytes provide a suitable system for an examination of this aspect of copper toxicity. Furthermore, interactions between copper and agents that protect against its toxic effects in vivo can be examined at a cellular level by use of isolated hepatocytes. Therefore, isolated rat hepatocytes were incubated with varying cupric chloride concentrations (5–200 μm) for up to 90 min. The copper caused a concentration and time-related decrease in cell viability as assessed by loss of intracellular potassium ion (K⁺) and aspartate aminotransferase (AST). An increase in lipid peroxidation and a decrease in reduced glutathione were also observed in response to copper. Of several potentially interactant compounds tested, only chromic chloride, diethyldithiocarbamate, and penicillamine were found to reduce the loss of K⁺. Ammonium molybdate alone and in combination with sodium sulfate were able to markedly decrease the release of AST from the hepatocytes. While the antioxidants, butylated hydroxyanisole and N,N′-diphenyl-p-phenylenediamine decreased the lipid peroxidation attributable to copper, they had no protective effects against loss of cell viability. This suggests that lipid peroxidation is not the cause of the injurious effects of this metal in isolated rat hepatocytes.     

Acrylamide toxicity in isolated rat hepatocytes

Acrylamide (ACR) is an important industrial chemical used primarily in the production of polymers and co-polymers. Acrylamide is mainly neurotoxic to experimental animals as well as humans and has also been shown to be mutagenic and carcinogenic. The present study was designed to investigate the toxicity of ACR on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and were incubated with different concentrations of ACR (0.1, 1, 10 m ) for 2 hours. Cell viability by trypan blue exclusion and leakage of the enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Reduced glutathione (GSH), glutathione S-transferase (GST) activity were also measured. A significant decrease in the cell viability was observed after exposure to 10 m ACR for 30 min, while 1 m ACR caused a significant decrease in the viability after 60 min. ALT leakage was parallel to the cell viability. AST leakage was significantly increased at 30 min of incubation with 10 m ACR, whereas 2 hours of incubation was required for the leakage of AST from rats hepatocytes with 1 m ACR. 10 m ACR decreased significantly GSH as early as 30 min, while GSH level was decreased at 60 min after exposure to 1 m ACR. Also, the GST activity increased with increasing the dose of ACR. Cytochrome P450 concentration was decreased after exposure to 10 m ACR. The effect of ACR on cell viability, ALT and AST leakage, GSH and GST activity was time and dose dependent.     

Metabolism of acetaminophen by cultured rat hepatocytes. Depletion of protein thiol groups without any loss of viability

Over the course of 4 hr, the metabolism of acetaminophen (APAP) by cultured rat hepatocytes resulted in a depletion of protein thiols and an accumulation of oxidized glutathione (GSSG) in the medium. With 20 mM APAP, arylation and the formation of glutathione mixed disulfides accounted for a loss of 22% of the total protein thiols in the absence of any loss of viability. With 20 mM APAP and an inhibition of glutathione reductase by 1.3-(2-chloroethyl)-1-nitrosourea (BCNU), protein thiols were depleted by 40% by arylation and the formation of glutathione mixed disulfides, again without a loss of viability. With 20 mM APAP and BCNU in the presence of 20 mM deferoxamine, there was still little or no cell killing after 8 hr despite a loss now of almost 60% of the total protein thiols. These data do not support the hypothesis that a depletion of protein thiols is related to the toxicity of APAP. One millimolar APAP and BCNU killed 60% of the hepatocytes within 4 hr. In this circumstance, the loss of protein thiols was not attributable to either arylation by APAP metabolites or the formation of glutathione mixed disulfides. The antioxidant N,N'-diphenyl-phenylenediamine prevented the cell killing and the loss of protein thiols, a result implicating a role for lipid peroxidation in the depletion of protein-bound thiols. However, protein thiol depletion under these circumstances is not necessarily related to the lethal cell injury and most likely represents an epiphenomenon of the peroxidation of cellular lipids.     

Hepatotoxicity of trichlorfon and dichlorvos in isolated rat hepatocytes.

Hepatotoxicity of organophosphorus insecticides, trichlorofon and dichlorvos, a dechlorinated form of the former, was examined in isolated hepatocytes from untreated control and phenobarbital-pretreated (80 mg/kg, i.p., for 3 days) rats. These compounds produced toxic effects on hepatocytes as evidenced by malondialdehyde production and lactate dehydrogenase leakage in a dose-dependent manner up to the concentration of 2 mM, dichlorvos being more toxic than trichlorfon. Hepatocytes from phenobarbital-pretreated rats were more sensitive to these organophosphates than those from control rats. Dichloroacetaldehyde and dichloroacetic acid, metabolites of dichlorvos, did not injure hepatocytes. The toxic effects of dichlorvos on hepatocytes were enhanced by increasing oxygen concentration during the incubation, or by addition of glycolytic substrates (pyruvate, lactate or fructose) to the incubation mixtures. On the other hand, addition of antioxidants (diethyldithiocarbamate or N,N'-diphenyl-p-phenylenediamine), or cytochrome P-450 inhibitors (SKF-525A or metyrapone) to the incubation mixtures attenuated malondialdehyde production caused by dichlorvos and protected cells from death. Addition of dichlorvos to the incubation mixtures of hepatic microsomes stimulated lipid peroxidation in the presence of NADPH, which was inhibited by further addition of superoxide dismutase but not catalase. These results suggest that hepatotoxicity of trichlorfon and dichlorvos are related to their peroxidative property in microsomes which is accelerated by oxygen.     

Metabolism of procyclidine in isolated rat hepatocytes

1. The biotransformation of procyclidine in isolated hepatocytes, prepared from untreated and from phenobarbital-pretreated rats, is described. 2. Major metabolic pathways are ketone formation on carbon-4 and monohydroxylation in cis-4, trans-4 and (1R*, 3R*, 7S* (or R*))-trans-3 positions of the cyclohexyl ring. 3. Minor pathways consist of monohydroxylation in (1R*, 3S*, 7R*)- and (1R*, 3S*, 7S*)-cis-3 and vicinal diol formation in (1R*, 3R*, 4S*, 7R* (or S*))-cis-3, cis-4 and (1R*, 3S*, 4R*, 7S* (or R*))-trans-3, trans-4 positions of the cyclohexyl part of the molecule. 4. After phenobarbital treatment monohydroxylation in cis-4, trans-4 and trans-3 and vicinal diol formation in trans-3, trans-4 positions are significantly increased and the cis-4 to trans-3 ratio is reversed. 5. The hypothesis is made that the monohydroxylations in cis-3 and trans-3 represent an intermediate step in the formation of the dihydroxycyclohexyl metabolites, since this pathway is not observed in vivo. The hypothesis is supported by incubation experiments of synthetic monohydroxycyclohexyl derivates of procyclidine with isolated rat hepatocytes.     

Metabolism of procyclidine in isolated rat hepatocytes

1. The biotransformation of procyclidine in isolated hepatocytes, prepared from untreated and from phenobarbital-pretreated rats, is described.

2. Major metabolic pathways are ketone formation on carbon-4 and monohydroxyl-ation in cis-4, trans-4 and (1R*, 3R*, 7S*(or R*))-trans-3 positions of the cyclohexyl ring.

3. Minor pathways consist of monohydroxylation in (1R*, 3R*, 7R*)- and (1R*, 3S*, 7S*)-cis-3 and vicinal diol formation in (1R*, 3R*, 4S*, 7R*(or S*))-cis-3, cis-4 and (1R*, 3S*, 4R*, 7S*(or R*))-trans-3, trans-4 positions of the cyclohexyl part of the molecule.

4. After phenobarbital treatment monohydroxylation in cis-4, trans-4 and trans-3 and vicinal diol formation in trans-3, trans-4 positions are significantly increased and the cis-4 to trans-3 ratio is reversed.

5. The hypothesis is made that the monohydroxylations in cis-3 and trans-3 represent an intermediate step in the formation of the dihydroxycyclohexyl metabolites, since this pathway is not observed in vivo. The hypothesis is supported by incubation experiments of synthetic monohydroxycyclohexyl derivates of procyclidine with isolated rat hepatocytes.     

Covalent binding of morphine to isolated rat hepatocytes.

Incubation of [3H]morphine with isolated hepatocytes caused covalent binding of [3H]-morphine to hepatocellular proteins. Sulfhydryl compounds protected against morphine-induced toxicity and decreased covalent binding. Analysis of covalently bound proteins in the cytosol by electrophoresis indicated that covalently bound radiolabel was associated with macromolecules greater than 25 kDa and increased throughout the incubation. In contrast, covalent binding to the particulate fraction was highly selectively associated with three protein bands of 50-53 and 33 kDa. Covalent binding of morphine to particulate fraction proteins was observed in hepatocytes which exhibited cellular damage. We conclude that the covalent binding of morphine to protein is associated with morphine-induced cytotoxicity.     

Metabolism of juvenile hormone with isolated rat hepatocytes.

The metabolism of juvenile hormone I has been examined in rat hepatocyte suspensions. The hormone penetrates rapidly into the cells where its concentration becomes higher than in the incubation medium. About 25 per cent of the hormone is metabolized during min 1 of incubation, while at 30 min of incubation, essentially all the hormone has been metabolized into several organic-soluble and water-soluble metabolites. Eventually the organic-soluble metabolites (including the diol ester, the diol acid and the acid) are recycled by re-entry and are metabolized further into water-soluble metabolites. The most important water-soluble metabolite is the mercapturic acid followed by the glucuronide. These results suggest that the low toxicity of juvenile hormone in mammals may be explained, at least in part, by metabolic detoxication.     

Cytotoxicity of chloroquine in isolated rat hepatocytes

Chloroquine is a synthetic quinoline being used as an antimalaria and antirheumatoid agent. Several cases of hepatotoxicity have been reported with the use of chloroquine. However, the mechanism(s) of its hepatotoxic effect is unknown. The purpose of this study was to investigate the cytotoxic mechanism of chloroquine. Cytotoxicity was studied using freshly isolated rat hepatocytes incubated in Krebs-Henseleit buffer under a flow of 95% O(2) and 5% CO(2). Chloroquine was toxic towards hepatocytes and caused cell death with an ED(50) of about 100 mm in 2 h. The events before cell death were rapid GSH depletion and lipid peroxidation. Cytochrome P-450 inhibitors, troleandromycine, cimetidine and quinidine increased the cytotoxicity of chloroquine. Antioxidants significantly prevented the cytotoxicity of chloroquine. Depleting the hepatocyte GSH beforehand increased the chloroquine cytotoxicity. Preventing chloroquine metabolism by specific P-450 inhibitors increased its toxicity, suggesting that a major part of its toxicity is mediated by chloroquine and not by its metabolites. A depletion of the antioxidant defense system is involved in the mechanism of cytotoxicity.     

Cytotoxicity of menadione and related quinones in freshly isolated rat hepatocytes: effects on thiol homeostasis and energy charge

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione DMNQ) and in their potential to induce redox cycling (NQ menadione DMNQ). The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 M) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability: exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage. From our results we conclude that the arylation capacity of a quinone is a determining factor in the cytotoxic potential of such compounds and that the decrease of protein thiols and of the energy charge are critical events preceding loss of cell viability.     

Toxicity of heparin in isolated rat hepatocytes

The effect of heparin on isolated rat hepatocytes in monolayer culture was assessed to investigate the observed increase in serum aminotransferase activity in patients treated with heparin for thromboembolic disorders. Cells were treated with porcine intestinal mucosal heparin or beef lung heparin in concentrations ranging from 0.01 to 100 units/ml. Toxicity was evaluated based on cell damage or death measured by LDH release into the culture media as a fraction of total system LDH (LDH index). Toxicity appeared at concentrations between 1 and 10 units/ml (P less than 0.05). The uptake and binding of heparin by the hepatocyte were evaluated by addition of tritium-labeled heparin to the cultures. Sucrose gradient centrifugation with isolation of the liver plasma membranes (LPM) showed little membrane binding of heparin. The majority of intracellular heparin was located in the cytosol fraction. Heparin gains access to hepatocytes and causes a dose-related toxic effect resulting in cell damage and death. This investigation indicates that the increased serum aminotransferase concentrations seen with heparin treatment may be due to a direct hepatotoxic effect of heparin.     

Benoxaprofen induced toxicity in isolated rat hepatocytes

The toxicity of benoxaprofen, a non-steroidal anti-inflammatory compound was investigated using rat hepatic microsomal and isolated hepatocyte suspensions. In microsomes, benoxaprofen produced a Type I binding spectra and competitively inhibited (k_i 380 μM) the oxidative metabolism of aminopyrine. Marked toxicity was observed following incubation of benoxaprofen with isolated hepatocytes from either untreated, phenobarbitone (PB) or 3-methylcholanthrene (3-MC) pretreated male rats. In untreated hepatocytes increases in the intracellular lactate/pyruvate (L/P) ratio and alanine aminotransferase (ALT) release were related to the benoxaprofen concentration and duration of incubation. Alterations in L/P ratio preceded the release of cytosolic ALT and at 4 h a well defined dose-response relationship existed between the benoxaprofen concentration and the observed increases in the L/P ratio and ALT release. Pretreatment of animals with either PB or 3-MC did not affect the temporal nature nor the magnitude of the hepatocyte response to benoxaprofen. In addition, inhibitors of cytochrome P-450 isozymes (SKF-525A, metyrapone and -napthoflavone) were ineffective with regard to modifying the observed toxicity. The results of this study suggest that hepatic cytochrome P-450 mediated metabolism may not be implicated in the toxicity of benoxaprofen in isolated hepatocytes. However, alterations in the cellular redox state and evidence of plasma membrane bleb formation suggest that benoxaprofen may uncouple oxidative phosphorylation and disturb intracellular calcium ion homeostasis.     

Effects of silver in isolated rat hepatocytes

Addition of silver nitrate or silver lactate to freshly isolated hepatocytes caused dose-dependent loss of cell viability, measured by trypan blue exclusion, at concentrations within 30-70 microM. Silver cytotoxicity was accompanied by a decrease in hepatic thiol concentration and an increase in lipid peroxidation. Treatment of hepatocytes with the reduced glutathione (GSH)-depleting agent diethylmaleate markedly increased their vulnerability to silver toxicity whereas protective effects were produced by the thiol-reducing agent, dithiothreitol. Both alpha-tocopherol, which protected from the onset of silver-associated lipid peroxidation, and the iron chelator agent, deferoxamine failed to prevent loss of cell viability. These data suggest that perturbation of intracellular thiol homeostasis may play a critical role in the mechanism underlying silver-induced lethal damage to isolated rat hepatocytes.