S-nitrosyl glutathione-mediated hepatocyte cytotoxicity

1. The addition of n-butyl nitrite (BN) to isolated rat hepatocytes caused rapid S-nitrosyl glutathione (GSNO) formation, then a concomitant decrease in protein thiols, followed by a marked ATP depletion. Cytotoxic concentrations of BN also caused lipid peroxidation after a long lag period but before cytotoxicity ensued.

2. Prior glutathione (GSH) depletion protected hepatocytes against the BN-induced decrease in protein thiols, ATP depletion, lipid peroxidation and cytotoxicity. Thus cytotoxic effects were thought to be mediated via GSNO formed by reaction of BN with GSH, a reaction catalysed by the cytosolic fraction.

3. Cytotoxicity and lipid peroxidation, but not depletion of GSH, protein thiols or ATP, could be averted by the subsequent addition of antioxidants or the iron chelator, desferoxamine.

4. Addition of the thiol reductant, dithiothreitol to BN-treated hepatocytes restored GSH and protein thiols and also prevented cytotoxicity.     

A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+

The molecular cytotoxic mechanisms of hepatocyte cell death induced by CuCl2, an essential redox transition metal has been compared with CdCl2, an environmental toxin. The ED50 concentrations found for Cu2+ and Cd2+ (i.e. 50% membrane lysis in 2 h) were 50 and 20 microM respectively. However reactive oxygen species ('ROS') formation, GSH oxidation and lipid peroxidation were induced by Cu2+ at these concentrations much more rapidly than by Cd2+. The decline of mitochondrial membrane potential though occurred at the same time and to the same extent for both metals. Furthermore the cytotoxicity and decline of mitochondrial membrane potential induced by these metals was prevented by the 'ROS' scavengers dimethyl sulfoxide, mannitol, catalase or SOD, as well as by desferoxamine, N,N diphenylphenylenediamine or alpha-tocopherol succinate. Hepatocyte GSH was protective as GSH depleted hepatocytes were much more susceptible to Cu2+ and Cd2+ than normal hepatocytes. It is concluded that Cu2+-induced cytotoxicity occurs as a result of a mitochondrial 'ROS' formation independently of cytosolic 'ROS' formation due to redox cycling.     

Reduction of vesicant toxicity by butylated hydroxyanisole in A-431 skin cells

Mechlorethamine (HN2) and 2-chloroethylethyl sulfide (CEES) are mustard vesicants. Butylated hydroxyanisole (BHA) is a phenolic antioxidant. In the present work, the ability of BHA to reduce the toxicity of HN2 and CEES was investigated using A-431 skin cells. HN2 toxicity was found to be dependent, at least in part, on the cellular glutathione (GSH) status. BHA also decreased HN2-induced DNA damage and lipid peroxidation. The cytoprotective activity of BHA was also compared with that of the structurally unrelated antioxidant ebselen. Whereas ebselen (30 microM) protected skin cells from the toxicity of both HN2 (10-80 microM) and CEES (10-40 microM), BHA (100 microM) reduced the toxicity of HN2 only. Taken together, these data suggest that antioxidants such as BHA or ebselen may serve as useful treatments for injury caused by blistering agents.     

The Involvement of Cytochrome P4502E1 in 2-Bromoethanol-Induced Hepatocyte Cytotoxicity

Abstract: The cytotoxicity of 2-bromoethanol towards hepatocytes isolated from rats was concentration-dependent (EC₅₀-100 μM, 2 hr). Bromoacetaldehyde was more toxic (EC₅₀-60 μM, 2 hr) and bromoacetic acid was less toxic (EC₅₀-150 μM, 2 hr). Glutathione (GSH) depletion occurred before cytotoxicity ensued and GSH depleted hepatocytes were more susceptible to 2-bromoethanol. Lipid peroxidation increased steadily 1 hr after 2-bromoethanol addition and antioxidants, iron chelators or hypoxia prevented 2-bromoethanol induced lipid peroxidation and cell lysis. Alcohol de-hydrogenase inhibitors, methyl pyrazole or dimethyl sulfoxide only partly prevented 2-bromoethanol induced GSH depletion, lipid peroxidation and cytotoxicity. However, cytochrome P4502E1 (CYP2EI) inhibitors/substrates were more effective at preventing 2-bromoethanol-induced GSH depletion, lipid peroxidation and cytotoxicity suggesting that 2-bromoethanol is mostly metabolically activated by CYP2E1. Also, hepatocytes isolated from CYP2E1 induced rats were more susceptible to 2-bromoethanol and hepatocytes isolated from rats pretreated with carbon disulfide to inactivate CYP2E1 were more resistant to 2-bromoethanol treatment. Formation of S-(formylmethyl)glutathione during 2-bromoethanol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by cytochrome P4502E1 inhibitors/substrates or by Anti-Rat CYP2E1. Furthermore, aldehyde dehydrogenase inhibitors-cyanamide or chloral hydrate increased 2-bromoethanol dependent hepatocyte susceptibility. This suggests that 2-bromoethanol is preferably metabolised by CYP2E1 dependent monoxygenase to form 2-bromoacetaldehyde which causes cell lysis as a result of GSH depletion and lipid peroxidation.     

Glyoxal markedly compromises hepatocyte resistance to hydrogen peroxide

Glyoxal is an interesting endogenous alpha-oxoaldehyde as it originates from pathways that have been linked to various pathologies, including lipid peroxidation, DNA oxidation and glucose autoxidation. In our previous study we showed that the LD(50) of glyoxal towards isolated rat hepatocytes was 5mM. However, 10microM glyoxal was sufficient to overcome hepatocyte resistance to H(2)O(2)-mediated cytotoxicity. Hepatocyte GSH oxidation, NADPH oxidation, reactive oxygen species formation, DNA oxidation, protein carbonylation and loss of mitochondrial potential were also markedly increased before cytotoxicity ensued. Cytotoxicity was prevented by glyoxal traps, the ferric chelator, desferoxamine, and antioxidants such as quercetin and propyl gallate. These results suggest there is a powerful relationship between H(2)O(2)-induced oxidative stress and glyoxal which involves an inhibition of the NADPH supply by glyoxal resulting in cytotoxicity caused by H(2)O(2)-induced mitochondrial oxidative stress.     

A comparison of hepatocyte cytotoxic mechanisms for chromate and arsenite

In the following, we have compared the cytotoxic mechanisms of the chromate CrO(4)(2-) and arsenite AsO(2)(-). Chromate (Cr (VI)) cytotoxicity was associated with reactive oxygen species (ROS) formation, lipid peroxidation and loss of mitochondrial membrane potential, which were prevented by catalase, antioxidants and ROS scavengers. Hepatocyte glutathione was also rapidly oxidized. Chromate reduction was inhibited in glutathione depleted hepatocytes, and glutathione depleted hepatocytes were also much more resistant to chromate induced cytotoxicity, ROS formation and lipid peroxidation. This suggests that chromate is reductively activated by glutathione. Chromate cytotoxicity also involved lysosomal injury and protease activation, which were prevented by lysosomotropic agents, endocytosis inhibitors, protease inhibitors and ROS scavengers. On the other hand, arsenite cytotoxicity was associated with much less oxidative stress, and lysosomal damage did not occur. However, arsenite cytotoxicity was also associated with loss of mitochondrial membrane potential, which in contrast to chromate cytotoxicity was inhibited by the ATP generators fructose, xylitol and glutamine. Arsenite induced cytotoxicity, mitochondrial membrane potential decline and also ROS formation were significantly increased by inactivating hepatocyte methionine synthase or hepatocyte methyl transferase. However, methyl donors such as betaine, methionine or folic acid prevented arsenite but not chromate cytotoxicity, and this suggests that arsenite is detoxified by reductive methylation. In conclusion, chromate induced cytotoxicity could be attributed to oxidative stress and lysosomal damage, whereas arsenite induced cytotoxicity could be attributed to mitochondrial toxicity and ATP depletion.     

Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro.

The present study was undertaken to find potent molecules against the toxicity of nitrogen mustard mechlorethamine (HN2) on respiratory epithelial cells, using a human bronchial epithelial cell line (16HBE14o-) as an in vitro model. The compounds examined included inhibitors of poly(ADP-ribose) polymerase (PARP), sulfhydryl-group donors as nucleophiles, and iron chelators and inhibitors of lipid peroxidation as antioxidants. Their effectiveness was determined upon observance of metabolic dysfunction induced by HN2 following a 4-h exposure, using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and ATP-level assays as indicators. Moreover, the fluorescent probe, monobromobimane (mBBr), and 2',7'-dichlorofluorescin-diacetate (H2DCF-DA) were used to assess intracellular sulfhydryl and peroxide level modifications by flow cytometry, respectively, following a 3-h exposure. At last, cell death was assessed by flow cytometry using the propidium iodide (PI)-dye-exclusion assay following 24-h exposure. PARP inhibitors (niacinamide, 3-aminobenzamide, 6(5H)-phenanthridinone), and two sulfhydryl-group donors (N-acetylcysteine, WR-1065) were found to be effective in preventing HN2-induced metabolic dysfunction when added in immediate or delayed treatment with HN2. Only N-acetylcysteine, however, was found to prevent cell death induced by HN2, though it must be present at the time of the HN2 challenge. Flow cytometric measurements of intracellular sulfhydryl levels strongly suggested that N-acetylcysteine and WR-1065 are preventive in alkylation of cellular compounds, mainly by direct extracellular interaction with HN2. PARP inhibitors prevent secondary deleterious effects induced by HN2, considering metabolism dysfunction as the endpoint. Elsewhere, the oxidative stress appears to be a side effect in HN2 toxicity only upon considering the inefficiency of several antioxidants.     

Accelerated cytotoxic mechanism screening of hydralazine using an in vitro hepatocyte inflammatory cell peroxidase model

Long-term treatment of hypertensive disorders with hydralazine has resulted in some patients developing hepatitis and lupus erythematosus, an autoimmune syndrome. The concentration of hydralazine required to cause 50% cytotoxicity in 2 h (LC(50)) toward isolated rat hepatocytes was found to be 8 mM. Cytotoxicity was delayed by the P450 inhibitor, 1-aminobenzotriazole, suggesting that P450 catalyzed the formation of toxic metabolites from hydralazine. No hydralazine-induced oxidative stress was apparent as there was little effect on hepatocyte lipid peroxidation, protein carbonyl formation, intracellular H(2)O(2), or hepatocyte GSH levels and no effect of butylated hydroxyanisole (BHA) on cytotoxicity. Drug-induced hepatotoxicity in vivo has often been attributed to infiltrating inflammatory cells, for example, neutrophils or resident Kupffer cells whose NADPH oxidase generates H(2)O(2), when activated. The effect of a nontoxic continuous infusion of H(2)O(2) on hydralazine cytotoxicity was investigated. It was found that H(2)O(2) increased hepatocyte susceptibility to hydralazine 4-fold (LC(50), 2 mM). Cytotoxicity was still prevented by the P450 inhibitor but now involved some oxidative stress as shown by increased protein carbonyls, endogenous H(2)O(2), and GSH oxidation. Lipid peroxidation was not increased, and cytotoxicity was not inhibited by BHA. Cytotoxicity, however, was inhibited by 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), a ROS scavenger. Because neutrophils or Kupffer cells release myeloperoxidase on activation, the effect of adding peroxidase to the hepatocytes exposed to H(2)O(2) on hydralazine cytotoxicity was investigated. It was found that peroxidase/H(2)O(2) increased hepatocyte susceptibility to hydralazine 80-fold (LC 50, 0.1 mM). Furthermore, cytotoxicity occurred following extensive oxidative stress that included lipid peroxidation, and cytotoxicity that was now prevented by the antioxidant BHA. These results indicate that three cytotoxic pathways exist for hydralazine: a P450-catalyzed pathway not involving oxidative stress, a P450/H(2)O(2)-catalyzed oxidative stress-mediated cytotoxic pathway not involving lipid peroxidation, and a peroxidase/H(2)O(2)-catalyzed lipid peroxidation-mediated cytotoxic pathway.     

过氧化氢对原代培养大鼠肝细胞的毒性作用及其机理

本文报道了过氧化氢诱发原代培养大鼠肝细胞毒性作用的可能机理．过氧化氢（０．２～１．０ｍｍｏｌ·Ｌ－１）温育６ｈ可以引起大鼠肝细胞坏死性损伤，导致谷丙转氨酶释放增加及细胞存活率下降，加入过氧化氢酶（２５０～１５００Ｕ·ｍＬ－１）及抗氧化剂五味子乙素（１０～１００μｍｏｌ·Ｌ－１）均可降低过氧化氢的毒性作用．加入过氧化氢（０．６和１．０ｍｍｏｌ·Ｌ－１）可在６ｍｉｎ内使大鼠肝细胞内钙从１８０ｎｍｏｌ·Ｌ－１明显持续升高至７００ｎｍｏｌ·Ｌ－１以上（约３．５倍）．过氧化氢与肝细胞作用３０ｍｉｎ至１ｈ既可导致细胞膜脂质过氧化，表现为丙二醛蓄积及膜流动性下降，明显早于肝细胞发生坏死性损伤的时间．肝细胞胞浆中还原型谷胱甘肽（ＧＳＨ）含量在加入过氧化氢３０ｍｉｎ后明显降低，可推测肝细胞在后面的温育中对过氧化氢毒性的敏感性增加．以上结果证实过氧化氢诱发的原代培养大鼠肝细胞致死性损伤可能与细胞内钙迅速持续增高，细胞膜脂质过氧化及ＧＳＨ含量下降有关．     

A 43 kDa protein from the herb Cajanus indicus L. protects thioacetamide induced cytotoxicity in hepatocytes.

The aim of this study was to investigate the role of a hepatoprotective protein isolated from the herb Cajanus indicus L. on thioacetamide (TAA) induced toxicity in isolated mouse hepatocytes. In vitro cell viability, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and total protein leakage were measured as the indicator of cell damage. The amount of glutathione (GSH) and lipid peroxidation were also measured to determine the oxidative status of the cells. The reduced cell viability in TAA treated hepatocytes was almost completely recovered upon protein treatment. LDH, ALT and total protein secretion outside the cells after TAA treatment confirmed the cell membrane damage. Incubation of hepatocytes with the protein prior to TAA administration significantly prevented the cell membrane damage as revealed from less LDH, ALT and total protein leakage. TAA depleted endogenous antioxidant GSH and increased membrane lipid peroxidation in hepatocytes. The protein had very prominent effect in altering the GSH level and lipid peroxidation. The protein exhibited all these cytoprotective effects in a dose-dependent manner. Besides, measurement of DPPH radical scavenging activity showed that the protein could scavenge free radicals. In addition, the protein resisted TAA induced alterations of various effects when applied in combination with TAA. The cytoprotective activity of the protein was found to be comparable with alpha-tocopherol, a well-known antioxidant. Results suggest that the protein from C. indicus can act as a hepatoprotector and primary antioxidant against TAA-induced cytotoxicity in mouse hepatocytes.     

Phenthoate induced-oxidative stress in fresh isolated mice hepatocytes: Alleviation by ascorbic acid

This study was conducted to determine the protective role of ascorbic acid (AA) against cytotoxicity of the phenthoate (PTA) on hepatocytes. Lipid peroxidation (LPO), nitric oxide (NO), antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, gamma glutamyl transferase and glutathione-S-transferase activities were measured. Non-enzymes antioxidant levels of reduced glutathione (GSH), ferric reducing antioxidant power (FRAP), AA and total proteins (TP) were determined. The results were demonstrated that the 2 hr-LC₅₀ obtained for PTA value was 79.94 mM. LPO and NO were increased in hepatocytes-treated with PTA. PTA-induced oxidative stress in the isolated hepatocytes by decreasing the levels of some antioxidant enzymes. GSH levels were also diminished in PTA treated-hepatocytes as compared to DMSO hepatocytes. FRAP, AA and total protein were also decreased following treatment with PTA. These findings suggest that cytotoxicity of PTA is mediated by increasing free radical formation and decreasing the antioxidants. PTA hepatotoxicity increased in a time- and concentration-dependent manner. The AA can be very effective in perhaps reducing the extent of injury and in overcoming oxidant damage caused by PTA.     

Molecular mechanisms of hydrogen sulfide toxicity

RATIONALE: The toxicity of H2S has been attributed to its ability to inhibit cytochrome c oxidase in a similar manner to HCN. However, the successful use of methemoglobin for the treatment of HCN poisoning was not successful for H2S poisonings even though the ferric heme group of methemoglobin scavenges H2S. Thus, we speculated that other mechanisms contribute to H2S induced cytotoxicity. Experimental procedure. Hepatocyte isolation and viability and enzyme activities were measured as described by Moldeus et al. (1978), and Steen et al. (2001). RESULTS: Incubation of isolated hepatocytes with NaHS solutions (a H2S source) resulted in glutathione (GSH) depletion. Moreover, GSH depletion was also observed in TRIS-HCl buffer (pH 6.0) treated with NaHS. Several ferric chelators (desferoxamime and DETAPAC) and antioxidant enzymes (superoxide dismutase [SOD] and catalase) prevented cell-free and hepatocyte GSH depletion. GSH-depleted hepatocytes were very susceptible to NaHS cytotoxicity, indicating that GSH detoxified NaHS or H2S in cells. Cytotoxicity was also partly prevented by desferoxamine and DETAPC, but it was increased by ferric EDTA or EDTA. Cell-free oxygen consumption experiments in TRIS-HCl buffer showed that NaHS autoxidation formed hydrogen peroxide and was prevented by DETAPC but increased by EDTA. We hypothesize that H2S can reduce intracellular bound ferric iron to form unbound ferrous iron, which activates iron. Additionally, H2S can increase the hepatocyte formation of reactive oxygen species (ROS) (known to occur with electron transport chain). H2S cytotoxicity therefore also involves a reactive sulfur species, which depletes GSH and activates oxygen to form ROS.     

Taurine, a conditionally essential amino acid, ameliorates arsenic-induced cytotoxicity in murine hepatocytes

Arsenic is a potent environmental toxin. Present study has been designed to evaluate the protective role of taurine (2-aminoethanesulfonic acid) against arsenic induced cytotoxicity in murine hepatocytes. Sodium arsenite (NaAsO₂) was chosen as the source of arsenic. Incubation of hepatocytes with the toxin (1 mM) for 2 h reduced the cell viability as well as intra-cellular antioxidant power. Increased activities of alanine transaminase (ALT) and alkaline phosphatase (ALP) due to toxin exposure confirmed membrane damage. Toxin treatment caused reduction in the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). In addition, the same treatment reduced the level of glutathione (GSH), elevated the level of oxidized glutathione (GSSG) and increased the extent of lipid peroxidation. Incubation of hepatocytes with taurine, both prior to and in combination with NaAsO₂, attenuated the extent of lipid peroxidation and enhanced the activities of enzymatic as well as non enzymatic antioxidants. Besides, taurine administration normalized the arsenic-induced enhanced levels of the marker enzymes ALT and ALP in hepatocytes. The cytoprotective activity of taurine against arsenic poisoning was found to be comparable to that of a known antioxidant, vitamin C. Combining all, the results suggest that taurine protects mouse hepatocytes against arsenic induced cytotoxicity.     

Molecular mechanism for prevention of N-acetyl-p-benzoquinoneimine cytotoxicity by the permeable thiol drugs diethyldithiocarbamate and dithiothreitol.

The present study was carried out to elucidate the mechanism by which the permeable thiol drug diethyldithiocarbamate (DEDC) exhibited an antidotal effect against acetaminophen-induced hepatotoxicity in vivo. DEDC was found to act as an antidote against acetaminophen-induced cytotoxicity in hepatocytes isolated from a pyrazole-pretreated rat without affecting cytochrome P-450 levels. The mechanism of protection exhibited against reactive intermediate N-acetyl-p-benzoquinoneimine (NAPQI)-induced cytotoxicity by DEDC was then investigated and compared with that exhibited by the permeable thiol-reductant dithiothreitol (DTT). Cytotoxicity induced by the dimethylated analogue 2,6-dimethyl-N-acetyl-p-benzoquinoneimine (2,6-diMeNAPQI) was prevented if the hepatocytes were preincubated with DEDC for 5 min and removed before addition of 2,6-diMeNAPQI. Both DEDC and DTT were also found to act as antidotes against NAPQI- and 2,6-diMeNAPQI-induced cytotoxicity in isolated rat hepatocytes if added within 2 min of the addition of the quinoneimines. However, the addition of DEDC or DTT 10 min after either quinoneimine did not prevent subsequent cytotoxicity or restore GSH levels, indicating that the alkylation of GSH and of protein thiols was irreversible at that time. Fast atom bombardment mass spectrometry was used to show that DEDC formed conjugates with both NAPQI and 2,6-diMeNAPQI. Furthermore, these conjugates were found to be nontoxic. This suggests that DEDC acts as a trap for the toxic quinoneimines, thus preventing alkylation of essential macromolecules. In contrast, DTT reduced the quinoneimines to their respective nontoxic parent compounds and presumably also reduced mixed-protein disulfides and GSSG, thereby regenerating protein thiols and GSH. Therefore, this study suggests that DEDC and DTT act as antidotes by two different mechanisms.     

tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals

tert-Butyl hydroperoxide (TBHP) mobilizes arachidonic acid (AA) from membrane phospholipids in rat hepatocytes under cytotoxic conditions, thus leading to an increase in intracellular AA, which precedes cell death. In the present work, the involvement of lipid peroxidation, thiol status, and reactive oxygen species (ROS) in the intracellular AA accumulation induced by 0.5 mM TBHP was studied in rat hepatocytes. Cells treated with TBHP maintained viability and energy status at 10 min. However, TBHP depleted GSH, as well as inducing lipid peroxidation and ROS formation, detected by dichlorofluorescein (DCF) fluorescence. TBHP also significantly increased (32.5%) the intracellular [14C]-AA from [14C]-AA-labelled hepatocytes. The phospholipase A(2) (PLA(2)) inhibitor, mepacrine, completely inhibited the [14C]-AA response. The addition of antioxidants to the cell suspensions affected the TBHP-induced lipid response differently. The [14C]-AA accumulation correlated directly with ROS and negatively with endogenous GSH. No correlation between [14C]-AA and lipid peroxidation was found. Promethazine prevented lipid peroxidation and did not affect the [14C]-AA increase. We conclude that TBHP stimulates the release of [14C]-AA from membrane phospholipids through a PLA(2)-mediated mechanism. Endogenous GSH and ROS play a major role in this effect, while lipid peroxidation-related events are unlikely to be involved. Results suggest that specific ROS generated in iron-dependent reactions, different from lipid peroxyl radicals, are involved in PLA(2) activation, this process being important in TBHP-induced hepatocyte injury.     

A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity

Addition of U(VI) (uranyl acetate) to isolated rat hepatocytes results in rapid glutathione oxidation, reactive oxygen species (ROS) formation, lipid peroxidation, decreased mitochondrial membrane potential, and lysosomal membrane rupture before hepatocyte lysis occurred. Cytotoxicity was prevented by ROS scavengers, antioxidants, and glutamine (ATP generator). Hepatocyte dichlorofluorescein oxidation was inhibited by mannitol (a hydroxyl radical scavenger) or butylated hydroxyanisole and butylated hydroxytoluene (antioxidants). Glutathione depleted hepatocytes were resistant to U(VI) toxicity and much less dichlorofluorescein oxidation occurred. Reduction of U(VI) by glutathione or cysteine in vitro was also accompanied by oxygen uptake and was inhibited by Ca(II) (a U(IV) or U(VI) reduction inhibitor). U(VI)-induced cytotoxicity and ROS formation was also inhibited by Ca(II), which suggests that U(IV) and U(IV) GSH mediate ROS formation in isolated hepatocytes. The U(VI) reductive mechanism required for toxicity has not been investigated. Cytotoxicity was also prevented by cytochrome P450 inhibitors, particularly CYP 2E1 inhibitors, but not inhibitors of DT diaphorase or glutathione reductase. This suggests that P450 reductase and reduced cytochrome P450 contributes to U(VI) reduction to U(IV). In conclusion, U(VI) cytotoxicity is associated with mitochondrial/lysosomal toxicity by the reduced biological metabolites and ROS.     

Role of glutathione depletion in the cytotoxicity of acetaminophen in a primary culture system of rat hepatocytes

A primary culture system of postnatal rat hepatocytes was utilized to study the cytotoxicity of acetaminophen and the toxicological significance of glutathione (GSH) depletion. The relative time of onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to gain insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time- and dose-dependent depletion of cellular GSH. The acetaminophen-induced GSH depletion and ensuing lactate dehydrogenase (LDH) leakage were quite modest and delayed in onset, in contrast to that caused by iodoacetamide (IAA) and by diethylmaleate (DEM), 2 well-known depletors of GSH. There was comparable LDH leakage, irrespective of drug treatment, when GSH levels decreased to about 20% of normal. Reduction of GSH levels below the 20% threshold by IAA treatment resulted in marked LDH leakage and loss of viability. Maximal LDH leakage in response to IAA and acetaminophen preceded maximal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence of cell damage as well as GSH depletion. IAA and DEM produced a comparable, modest accumulation of MDA, yet IAA was much more cytotoxic. These findings indicate that lipid peroxidation does not play a central role in hepatocellular injury by compounds which deplete GSH, although it may contribute to degeneration of the cell. As events in the cultured postnatal hepatocytes paralleled those reported in vivo, the system can be a useful and valid model with which to study mechanisms of chemical toxicity.     

Involvement of oxidative stress and mitochondrial/lysosomal cross-talk in olanzapine cytotoxicity in freshly isolated rat hepatocytes

1.?Olanzapine (OLZ) is a widely used atypical antipsychotic agent for the treatment of schizophrenia and other disorders. Serious hepatotoxicity and elevated liver enzymes have been reported in patients receiving OLZ. However, the cellular and molecular mechanisms of the OLZ hepatotoxicity are unknown.

2.?In this study, the cytotoxic effect of OLZ on freshly isolated rat hepatocytes was assessed. Our results showed that the cytotoxicity of OLZ in hepatocytes is mediated by overproduction of reactive oxygen species (ROS), mitochondrial potential collapse, lysosomal membrane leakiness, GSH depletion and lipid peroxidation preceding cell lysis. All the aforementioned OLZ-induced cellular events were significantly (p?<?0.05) prevented by ROS scavengers, antioxidants, endocytosis inhibitors and adenosine triphosphate generators. Also, the present results demonstrated that CYP450 is involved in OLZ-induced oxidative stress and cytotoxicity mechanism.

3.?It is concluded that OLZ hepatotoxicity is associated with both mitochondrial/lysosomal involvement following the initiation of oxidative stress in hepatocytes.     

Cytotoxicity of butylated hydroxyanisole and butylated hydroxytoluene in isolated rat hepatocytes

The effects of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on isolated rat hepatocytes were investigated. Both antioxidants were observed to be cytotoxic in a concentration-dependent manner at concentrations ranging from 100 to 750 microM. At equimolar concentrations BHT was more cytotoxic than BHA. Their toxicity appeared to be independent of their metabolism to reactive intermediates since inhibitors of cytochrome P-450 (metyrapone, SKF 525-A and piperonyl butoxide) had no effect on the cytotoxicity and N-acetylcysteine was also without protective effect. In addition, deuterated BHT was equitoxic with BHT. Only low temperature incubation (4 degrees), which has previously been shown to inhibit the insertion of these compounds into biomembranes, was effective in inhibiting the cytotoxic effects. Using isolated rat liver mitochondria we observed that both BHA and BHT inhibited respiratory control primarily by stimulating state 4 respiration and thus acting as membrane uncouplers. BHA and BHT also effectively dissipated membrane potential across the mitochondrial membrane and caused the release of calcium and mitochondrial swelling. These mitochondrial effects were reflected by a rapid decrease in ATP levels in intact hepatocytes which preceded cell death. These results suggest that the observed cytotoxicity of BHA and BHT to hepatocytes is related to their effects on biomembranes and mitochondrial bioenergetics.     

Molecular Mechanisms of Hydrogen Sulfide Toxicity

Rationale. The toxicity of H2S has been attributed to its ability to inhibit cytochrome c oxidase in a similar manner to HCN. However, the successful use of methemoglobin for the treatment of HCN poisoning was not successful for H2S poisonings even though the ferric heme group of methemoglobin scavenges H2S. Thus, we speculated that other mechanisms contribute to H2S induced cytotoxicity. Experimental procedure. Hepatocyte isolation and viability and enzyme activities were measured as described by , and . Results. Incubation of isolated hepatocytes with NaHS solutions (a H₂S source) resulted in glutathione (GSH) depletion. Moreover, GSH depletion was also observed in TRIS-HCl buffer (pH 6.0) treated with NaHS. Several ferric chelators (desferoxamime and DETAPAC) and antioxidant enzymes (superoxide dismutase [SOD] and catalase) prevented cell-free and hepatocyte GSH depletion. GSH-depleted hepatocytes were very susceptible to NaHS cytotoxicity, indicating that GSH detoxified NaHS or H₂S in cells. Cytotoxicity was also partly prevented by desferoxamine and DETAPC, but it was increased by ferric EDTA or EDTA. Cell-free oxygen consumption experiments in TRIS-HCl buffer showed that NaHS autoxidation formed hydrogen peroxide and was prevented by DETAPC but increased by EDTA. We hypothesize that H₂S can reduce intracellular bound ferric iron to form unbound ferrous iron, which activates iron. Additionally, H₂S can increase the hepatocyte formation of reactive oxygen species (ROS) (known to occur with electron transport chain). H₂S cytotoxicity therefore also involves a reactive sulfur species, which depletes GSH and activates oxygen to form ROS.