Lipid peroxidation induced by N-acetylcysteine in isolated rat hepatocytes

In isolated rat hepatocytes N-acetylcysteine induces an increase of lipid peroxidation, as evaluated by the malondialdehyde production and diene conjugation. Lipid peroxidation did not result in increased cell mortality. Antioxidants and free radicals scavengers completely protect toward lipid peroxidation induced by N-acetylcysteine.     

NAD+ depletion and cytotoxicity in isolated hepatocytes

Activation of poly(ADP-ribose)polymerase by DNA damaging agents causes a depletion of intracellular NAD+ and subsequent lowering of ATP pools, which if extensive may lead to cell death. We have studied the cytotoxicity to isolated hepatocytes of dimethyl sulphate, a direct-acting carcinogen and mutagen, hydrogen peroxide, generated by glucose/glucose oxidase, and menadione (2-methyl-1,4-naphthoquinone) in relation to their effects on intracellular NAD+ and ATP levels. Both dimethyl sulphate and glucose/glucose oxidase caused a depletion of NAD+, which was apparently due to an activation of poly(ADP-ribose)polymerase as it was prevented by inhibitors of the polymerase, i.e. 3-aminobenzamide and nicotinamide. This protection of intracellular NAD+ was accompanied by a prevention of the cytotoxicity of both dimethyl sulphate and glucose/glucose oxidase, while it did not alter the decrease in intracellular ATP they induced. This apparent dissociation of effects on ATP from NAD+ does not support the suggestion that activation of poly(ADP-ribose)polymerase leads to a decrease in cellular ATP as a consequence of NAD+ depletion. Menadione also caused a depletion of NAD+ which preceded cytotoxicity, but in contrast to dimethyl sulphate and H2O2 this depletion did not involve poly(ADP-ribose)polymerase as it was not prevented by inhibitors of the enzyme. Our results also indicate that the cytotoxicity of menadione is not mediated by H2O2 alone. Marked depletion of intracellular NAD+ prior to toxicity and a protection against toxicity associated with maintenance of NAD+ suggest a possible role for the maintenance of intracellular NAD+ in cellular integrity.     

Lipid peroxidation in isolated rat hepatocytes measured by ethane and n-pentane formation

Isolated rat hepatocytes (1×10⁷ cells/ml) were aerobically incubated in Eagle's Minimum Essential Medium which contained 2.0% albumin. As potential parameters of lipid peroxidation ethane and n-pentane formed were measured in samples obtained from the gas phase above the incubation mixture. 15–30 nmol ethane or n-pentane were produced by 10⁷ hepatocytes within 90 min. Carbon tetrachloride (CCl₄) or ADP-complexed ferrous ions stimulated ethane and n-pentane formation considerably, depending on the concentrations of the compounds. With CCl₄ 10⁷ cells formed max 180 nmol ethane and 140 nmol n-pentane within 90 min incubation, whereas with Fe(II) max 130 nmol ethane and 220 nmol n-pentane could be detected.When n-pentane was added to the gas phase above the incubation mixture containing either medium or medium plus hepatocytes its amount decreased by 30% within the first 5 min of incubation. However, afterwards only minor amounts of n-pentane disappeared, even in the presence of hepatocytes. This indicates that n-pentane equilibrates with the cell suspension under the conditions used.Cell viability, as determined by the release of lactate dehydrogenase into the medium and by the uptake of trypan blue by the cells, and the recovery of the cells decreased only in presence of relatively high concentrations of CCl₄, or Fe(II) respectively. However, a maximal effect on ethane and n-pentane formation was reached already with lower concentration.The results have been presented in part during the 22nd Spring Meeting of the Deutsche Pharmakologische Gesellschaft [de Ruiter N, Ottenwälder H (1981) Naunyn-Schmiedebergs Arch Pharmacol (Suppl) 316: R 20]     

N-acetyl-p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes.

Incubation of isolated rat hepatocytes with N-acetyl-p-benzoquinone imine (NAPQI) or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) resulted in a concentration-dependent decrease in the protein thiol content of the mitochondrial, cytosolic and microsomal fractions. On a concentration basis, 3,5-Me2-NAPQI induced a more marked depletion of protein thiols than did NAPQI. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic separation of the proteins of each fraction showed that different proteins had different susceptibilities to modification of their cysteine residues by the quinone imines. A few protein bands showed a decreased protein thiol content following incubation with non-toxic concentrations of quinone imines, whereas other proteins were affected by higher concentrations. Concentrations of quinone imines that were highly cytotoxic induced a general loss of protein thiols. NAPQI-induced protein thiol depletion occurred within 5 min and remained essentially unchanged for at least 30 min. In contrast, protein thiol depletion induced by 3,5-Me2-NAPQI increased over the 30-min time course of the experiment. Toxic concentrations of 3,5-Me2-NAPQI caused the formation of high molecular mass aggregates in all three subcellular fractions after 30 min of incubation. The observed crosslinking was not due to protein disulfide formation. However, no aggregate formation was observed after exposure of hepatocytes to NAPQI. One of the major target proteins of quinone imine-induced protein thiol depletion was a 17 kDa microsomal protein that was identified as the microsomal glutathione S-transferase. Exposure of hepatocytes and isolated liver microsomes to the quinone imines resulted in an up to four-fold increase in the specific activity of the microsomal glutathione S-transferase. In conclusion, our results are consistent with the suggestion of a critical role of protein thiol depletion in quinone imine-induced cytotoxicity.     

Relative contributions of protein sulfhydryl loss and lipid peroxidation to allyl alcohol-induced cytotoxicity in isolated rat hepatocytes

The time course of allyl alcohol-induced toxicity was studied in freshly isolated rat hepatocytes. The sequence of events was as follows: an initial, rapid depletion of glutathione (GSH), a subsequent increase in malondialdehyde (MDA) and decrease in protein sulfhydryl groups (PSH), and the eventual loss of membrane integrity. The sulfhydryl compounds, N-acetylcysteine and dithiothreitol, markedly delayed the depletion of GSH, prevented significant loss of PSH, and protected the cells against viability loss. In contrast, the antioxidants, butylated hydroxytoluene and Trolox C, and the iron-chelating agent, deferoxamine, suppressed allyl alcohol-induced MDA production without affecting the depletion of cellular thiols or the loss of viability. The results suggest that the inactivation of protein thiol groups is critical for allyl alcohol toxicity whereas lipid peroxidation is not essential to the toxic process.     

Early effects of 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene on mitochondrial outer membrane enzyme activities of rat liver

During the early stages of the carcinogen 2-acetylaminofluorene (2-AAF) feeding, there were marked decreases of hepatic mitochondrial type B monoamine oxidase (MAO) and kynurenine 3-hydroxylase activities in male rats, but not in female rats. The administration of N-hydroxy-2-AAF (N-OH-2-AAF), a proximate carcinogenic metabolite of 2-AAF, to male rats also resulted in the decrease of both enzyme activities. The findings indicate that type B MAO and kynurenine 3-hydroxylase which reside in the outer mitochondrial membranes are susceptible to 2-AAF during the early stages of its hepatocarcinogenesis.     

Lipid peroxidation in isolated hepatocytes from rats ingesting ethanol chronically

Summary Isolated hepatocytes from rats consuming ethanol (8.5 g/kg) daily produce malondialdehyde in significantly higher amounts than liver cells from control animals. The release of LDH and the uptake of trypan blue in both types of hepatocytes do not differ during the incubation period of 2 h. GLDH, however, is only set free into the medium from liver cells of ethanol drinking rats, indicating that mitochondrial alterations are involved.Bomotrichloromethane (CBrCl₃) promotes lipid peroxidation in hepatocytes from ethanol drinking rats in a much higher degree than in cells from control rats. The cell damage induced by CBrCl₃ and indicated by a release of LDH and GLDH from the hepatocytes and their uptake of trypan blue is also much more pronounced in liver cells from ethanol drinking animals.The stronger action of CBrCl₃ cannot be explained by an enhanced microsomal metabolism, because no increase of drug metabolizing enzymes could be observed. The relatively low ethanol consumption did not influence body growth and liver weight and did not evoke any triglyceride accumulation.The normal balance between processes favouring lipid peroxidations and reactions protecting the liver cells seems to be shifted to a state during alcohol intake which promotes formation of radicals.     

Lipid peroxidation and cell damage in isolated hepatocytes due to bromotrichloromethane

Small amounts of malondialdehyde (MDA, about 0.5 nmol/10⁶ cells) were produced during 120-min incubation of isolated rat hepatocytes indicating lipid peroxidation in the cells. Trypan blue uptake and lactate dehydrogenase release followed MDA formation. MDA increased to about 3.5 nmol/10⁶ cells in the presence of 2 mm bromotrichloromethane (CBrCl₃). A parallelism of MDA production with trypan blue uptake and lactate dehydrogenase release was observed. The MDA production depended on the CBrCl₃ concentration in the incubation medium. A higher oxygen consumption of cells incubated in the presence of CBrCl₃ was detectable compared to controls. During incubation no increase in glutamate dehydrogenase release could be observed, even if 2 mm CBrCl₃ was present. This led us to conclude that lipid peroxidation induced by CBrCl₃ destroys the plasma membrane of the hepatocytes without seriously damaging mitochondria.     

Selective inhibition of glucuronidation by 2,2,2-triphenylethyl-UDP in isolated rat hepatocytes: conjugation of harmol, 3,3',5-triiodothyronine, and N-hydroxy-2-acetylaminofluorene

2,2,2-Triphenylethyl-UDP (TPEU) was synthesized as an analogue of the transition state of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase; it contains both a uridine and an acceptor substrate moiety. It inhibits rat liver microsomal UDP-glucuronosyltransferase [Eur. J. Biochem. 188:309-312 (1990)]. In the present work, TPEU was tested as an inhibitor of glucuronidation in intact rat hepatocytes. Two phenols (harmol and 3,3',5-triiodothyronine) and a hydroxamic acid (N-hydroxy-2-acetylaminofluorene) were used as substrates for glucuronidation. The glucuronidation of these substrates was strongly decreased by TPEU at 0.3-5 mM. Up to 5 mM TPEU did not kill the cells, as shown by unimpaired trypan blue exclusion at the end of the incubation. When glucuronidation was inhibited, the sulfation of harmol increased, as did the production of reactive species generated from N-hydroxy-2-acetylaminofluorene that bind to cellular macromolecules. This indicates that a decreased substrate consumption by loss of glucuronidation leads to increased conversion by competing pathways. The results show, therefore, that TPEU is an effective inhibitor of glucuronidation in this cellular system in vitro.     

Effect of 2-acetylaminofluorene on intracellular free Ca2+ in isolated rat hepatocytes.

The effect of 2-acetylaminofluorene (2-AAF) on the intracellular free Ca2+ ([Ca2+]i) and viability of isolated rat hepatocytes has been investigated using the fluorescent probes quin 2 and propidium iodide respectively. At the highest concentration tested (224 microM), 2-AAF produces an elevation of [Ca2+]i which shows a biphasic profile. A small initial increase is observed during the first 5 min; this is followed by a considerable rise which reaches up to 2.5 times the control value at 15 min. These changes in intracellular calcium are not accompanied by detectable alterations in cell viability. In order to determine the mechanisms by which this effect of 2-AAF takes place, three calcium antagonists, namely verapamil, TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) and ruthenium red (RuR), have been used. The results suggest that the first phase is dependent upon internal Ca2+ store mobilization, while the second phase seems to be related to Ca2+ entry from the extracellular space. The data obtained with RuR further indicate that mitochondria may be involved in the perturbation of calcium homeostasis caused by 2-AAF. In addition, in the experiments involving antagonists, no consistent pattern emerges that suggests a close relationship between intracellular Ca2+ levels and cell viability. The present study provides further information on the mechanisms by which these well-known hepatotoxin 2-AAF may interact with liver cells. It also shows that when these cells are exposed to a toxin, short-term changes in [Ca2+]i may not be accompanied by loss of cell viability, and conversely, that changes in cell viability may occur without alterations in [Ca2+]i.     

Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes

The toxicity of cadmium may be due to alteration in membrane structure which may be caused by peroxidation of the composite lipids. Isolated hepatocytes provide a suitable system to examine the role of lipid peroxidation in a toxic response at the cellular level. Therefore, isolated rat hepatocytes were incubated with varying cadmium concentrations (50–400 μm) for up to 75 min. An increase in lipid peroxidation due to cadmium was observed. The integrity of the cell membrane, as measured by loss of intracellular potassium ion and leakage of aspartate aminotransferase, was adversely affected in the presence of cadmium. The lactate to pyruvate ratio of hepatocyte suspensions was increased upon incubation with cadmium. Several chelating compounds were found to reduce intracellular accumulation of cadmium, cellular toxicity and lipid peroxidation. However, amelioration of toxicity was not consistently associated with inhibition of the lipid peroxidation response. The antioxidant compounds, sodium diethyldithiocarbamate and N,N′-diphenyl-p-phenylene-diamine were found to inhibit the lipid peroxidation attributable to cadmium, but did not have any consistent protective effect against loss of intracellular potassium ion. The results of this study show that the toxicity induced by cadmium in isolated rat hepatocytes can be dissociated from the concurrently observed lipid peroxidation, which indicates that the toxic response is not caused by the lipid peroxidation.     

Nitrofurantoin-mediated oxidative stress cytotoxicity in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism(s) of toxicity of the antimicrobial drug nitrofurantoin. This 5-nitrofuran derivative stimulated hepatocyte oxygen uptake in the presence of the mitochondrial respiration inhibitors KCN or antimycin A. This could indicate the formation of O2- and H2O2, following intracellular nitrofurantoin reduction. Addition of nitrofurantoin to suspensions of isolated rat hepatocytes produced a dose- and time-dependent decrease of cell viability. H2O2 probably plays a significant role in the cytotoxic effects of nitrofurantoin as the catalase inhibitors azide or aminotriazole markedly enhanced cytotoxicity. The loss of cell viability was preceded by glutathione (GSH) depletion and a concomitant and nearly stoichiometric formation of oxidised glutathione (GSSG) that did not occur in hepatocytes lacking glutathione peroxidase activity isolated from rats fed a low-selenium diet. This indicates that H2O2 and the seleno-enzyme glutathione peroxidase are responsible for GSH oxidation. Furthermore, addition of nitrofurantoin to isolated rat hepatocytes produced a reversible inactivation of hepatocyte glutathione reductase activity and explains the maintenance of high GSSG levels. The compromised hepatocytes were also highly susceptible to H2O2. The hepatocyte toxicity of nitrofurantoin may, therefore, be attributed to oxidative stress caused by redox-cycling mediated oxygen activation.     

Cytotoxic and cytoprotective activities of curcumin. Effects on paracetamol-induced cytotoxicity, lipid peroxidation and glutathione depletion in rat hepatocytes

The cytoprotective effect of curcumin, a natural constituent of Curcuma longa, on the cytotoxicity of paracetamol in rat hepatocytes was studied. Paracetamol was selected as a model-toxin, since it is known to be bioactivated by 3-methylcholanthrene inducible cytochromes P450 presumably to N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which upon overdosage causes protein- and non-protein thiol-depletion, lipid peroxidation and cytotoxicity measured as LDH-leakage. At low concentrations curcumin was found to protect significantly against paracetamol-induced lipid peroxidation, without protection against paracetamol-induced LDH-leakage and without protection against paracetamol-induced GSH-depletion. At a 100 times higher concentration of curcumin the observed protective effect on lipid peroxidation was accompanied with a tendency to increase cellular GSH-depletion and LDH-leakage. No time-dependency was found as to the curcumin-induced effects: treatment of the hepatocytes 1 hr before, concomitantly or 1 hr after the addition of paracetamol to the cells had similar effects. In contrast to what was expected on the basis of previous in vivo experiments, at higher concentrations curcumin itself was found to be slightly cytotoxic. Curcumin-induced LDH-leakage was accompanied by a significant depletion of GSH. It has been concluded that the observed cytoprotective and cytotoxic activities of curcumin may be explained by a strong anti-oxidant capacity of curcumin and the capability of curcumin to conjugate with GSH. Furthermore, it has been concluded that lipid peroxidation is not playing a causal role in cell-death induced by paracetamol or by curcumin.     

Lipid peroxidation, protein synthesis, and protection by calcium EDTA in paracetamol injury to isolated hepatocytes

Hepatocytes from rats treated with phenobarbitone were exposed to 10 mM paracetamol for 1 hr and then incubated in buffered Ringer solution. Enzyme leakage and trypan blue entry became severe in the paracetamol treated cells some 2 hr after the end of exposure. These signs of cell injury could be blocked by 4 mM CaEDTA added during or after paracetamol exposure. CaEDTA did not alter covalent binding of [14C]paracetamol. Ca2+ free media did not prevent paracetamol injury. Lipid peroxidation was observed in cells but could be blocked without protecting the cells. Protein synthesis was depressed early on in cells previously exposed to paracetamol, CaEDTA did not protect against this inhibition. These observations suggest that an early cytoplasmic lesion develops into a later lethal lesion at the cell surface.     

Lipid peroxidation and irreversible cell damage: synergism between carbon tetrachloride and 1,2-dibromoethane in isolated rat hepatocytes

The combination of 1,2-dibromoethane (DBE) with carbon tetrachloride (CCl4) in the isolated rat hepatocyte model produces a significant potentiation of both lipid peroxidation and plasma membrane damage induced by the latter compound. The increase in malondialdehyde production precedes the hepatocyte damage, evaluated in terms both of lactate dehydrogenase leakage and trypan blue exclusion. When hepatocytes are isolated from vitamin E pretreated rats, both the prooxidant and the cytotoxic effects of CCl4 are prevented. Also the synergism between CCl4 and DBE on lipid peroxidation disappears completely while that on cell damage is strongly reduced. The increased lipid peroxidation appears to be one of the mechanisms of the observed synergism between CCl4 and DBE on hepatocyte damage. Regarding the antioxidant status of the hepatocyte challenged with CCl4 and DBE, an early and significant consumption of vitamin E is observed only in the presence of the mixture of these xenobiotics. Total nonprotein thiol content is not significantly modified by CCl4 poisoning while DBE, alone and in association with CCl4, markedly decreases it. Vitamin E supplementation does not prevent but moderately delays total nonprotein thiol depletion due to DBE or to the mixture. Finally, glutathione transferase activity is significantly reduced by CCl4 treatment and not by DBE, and vitamin E supplementation totally prevents such inhibition. The increased prooxidant effect of CCl4 plus DBE compared to CCl4 alone seems related to the shift in DBE metabolism consequent to the CCl4-dependent inactivation of glutathione transferase.     

Molecular mechanisms for bromotrichloromethane cytotoxicity in isolated rat hepatocytes

1. Bromotrichloromethane added to isolated rat hepatocytes resulted in increased cell death as determined by trypan blue uptake. Toxicity increased in a concentration-dependent fashion between 2.0-5.0 M bromotrichloromethane. 2. Lipid peroxidation (malondialdehyde) increased in a time-dependent fashion but in contrast to toxicity reached a maximum level at 2.0 mM bromotrichloromethane. 3. Hypoxia increased the toxicity of bromotrichloromethane three-fold but only decreased the amount of lipid peroxidation to a small degree. 4. In spite of this poor correlation between toxicity and lipid peroxidation, the antioxidant butylated hydroxyanisole and the iron chelator desferal protected the cells from toxicity under both aerobic and hypoxic conditions and prevented lipid peroxidation. 5. During treatment with bromotrichloromethane, cellular glutathione levels slowly decreased and oxidized glutathione appeared in the media. The addition of cystine to the incubation media prevented the formation of extracellular oxidized glutathione, indicating that cellular glutathione had leaked from the cell during treatment and was oxidized in the incubation media. Although this suggested that glutathione does not play a protective role against bromotrichloromethane toxicity, diethyl maleate-pretreatment of the cells to decrease glutathione levels markedly increased bromotrichloromethane toxicity. 6. The addition of ascorbic acid to the incubation media increased bromotrichloromethane toxicity. This was attributed to the reductive activation of bromotrichloromethane in an iron and oxygen-dependent reaction. 7. It was concluded that peroxidation of essential phospholipids contributes to bromotrichloromethane-induced hepatocyte cytotoxicity.     

Protective effect of aloe extract against the cytotoxicity of 1,4-naphthoquinone in isolated rat hepatocytes involves modulations in cellular thiol levels

Aloe is a familiar ingredient in a wide range of health care and cosmetic products and has been reported to possess various physiological effects, antioxidative, anticarcinogenic, antiinflammatory and laxative. Aloe has also been reported to have an effect on liver function. The cytoprotective effect of aloe extract against 1,4-naphthoquinone-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. After exposure to 1,4-naphthoquinone (100 microM), a decrease in cell viability measured as >60% lactate dehydrogenase depletion was induced. Cellular glutathione (GSH) and protein-SH levels were also significantly decreased in a time-dependent manner. However addition of aloe extract resulted in a dose-dependent improvement of these effects. This cytoprotective effect of aloe could be attributed to its inhibition of GSH and protein-SH depletions. The effect of the aloe extracts were also dose-dependent. Addition of diethyl maleate (1 mM), a cellular glutathione-depleting agent, to hepatocytes treated with both 1,4-naphthoquinone and aloe extract, induced depletion of GSH, but did not affect protein-SH or lactate dehydrogenase. These results suggest that the 1,4-naphthoquinone-induced toxicity in rat hepatocytes was inhibited by aloe extract, and that this protective effect was due to the maintenance of cellular thiols, especially protein-SH.