Enalapril cytotoxicity in primary cultures of rat hepatocytes. I. Effects of cytochrome P450 inducers and inhibitors.

Enalapril maleate (EN) incubated with primary cultures of rat hepatocytes was cytotoxic in concentrations of 0.5 mM or greater. Toxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium at 24 h. SKF525A, alpha-naphthoflavone (alpha NF) and metyrapone (MTP) reduced the toxicity of EN. In vivo pretreatment with phenobarbital (PB) and beta-naphthoflavone (beta NF) had minor protective effects on the responses of hepatocytes to EN exposure. However, in vivo pretreatment with pregnenolone-16 alpha-carbonitrile (PCN) substantially potentiated EN cytotoxicity. These results suggest that the cytocidal hepatotoxicity of EN in primary culture depends to some degree on metabolic activation by a cytochrome P450 species.     

Enalapril hepatotoxicity in the rat. Effects of modulators of cytochrome P450 and glutathione.

The effects of modulators of cytochrome P450 and reduced glutathione (GSH) on the hepatotoxicity of enalapril maleate (EN) were investigated in Fischer 344 rats. Twenty-four hours following the administration of EN (1.5 to 1.8 g/kg), increased serum transaminases (ALT and AST) and hepatic necrosis were observed. Pretreatment of the animals with pregnenolone-16 alpha-carbonitrile, a selective inducer of the cytochrome P450IIIA gene subfamily, enhanced EN-induced hepatotoxicity, whereas pretreatment with the cytochrome P450 inhibitor, cobalt protoporphyrin, reduced the liver injury. Depletion of hepatic non-protein sulfhydryls (NPSHs), an indicator of GSH, by combined treatment with buthionine sulfoximine (BSO) and diethyl maleate (DEM) produced marked elevations in serum transaminases by 6 hr after EN treatment. Administered on its own, EN decreased hepatic NPSH content and when combined with the BSO/DEM pretreatment, the liver was nearly completely devoid of NPSHs. Protection from EN-induced hepatotoxicity was observed in animals administered L-2-oxothiazolidine-4-carboxylic acid, a cysteine precursor. Together, these observations suggest the involvement of cytochrome P450 in EN bioactivation and GSH in detoxification. The results corroborate previous in vitro observations pertaining to the mechanism of EN-induced cytotoxicity towards primary cultures of rat hepatocytes. Although the doses of EN used in this study were far in excess of therapeutic doses, under certain circumstances, this metabolism-mediated toxicologic mechanism could form the basis for idiosyncratic liver injury in patients receiving EN therapy.     

Influence of modulators of epoxide metabolism on the cytotoxicity of trans-anethole in freshly isolated rat hepatocytes.

The effect of modulating epoxide metabolism by inhibiting microsomal and cytosolic epoxide hydrolases and depleting glutathione, on the cytotoxicity of trans-anethole has been examined in freshly isolated rat hepatocytes in suspension. Hepatocytes derived from female Sprague-Dawley CD rats by collagenase perfusion were incubated in suspension and sampled at intervals over a 6-hr period. Cytotoxicity was assessed by the leakage of lactate dehydrogenase into the culture medium and in the cells after lysis. Glutathione was determined by fluorimetry. Anethole showed a dose-dependent cytotoxicity at concentrations ranging from 5 x 10(-4) to 5 x 10(-3) M, with concentrations of 10(-3) M and above causing greater than 63% leakage of lactate dehydrogenase in 6 hr. Microsomal epoxide hydrolase was inhibited by trichloropropene oxide (10(-4) M) and cyclohexene oxide (10(-3) M), and cytosolic epoxide hydrolase by 4-fluorochalcone oxide (5 x 10(-4) M). Cellular glutathione was depleted by diethyl maleate (5 x 10(-4) M), and its synthesis inhibited by 2.5 x 10(-3) M-L-buthionine (S,R)-sulphoximine. Suspensions treated with a sub-cytotoxic concentration of anethole (5 x 10(-4) M) showed a rapid increase in cytotoxicity when 4-fluorochalcone oxide was present (complete loss of viability within 2 hr), while pretreatment of hepatocytes with diethyl maleate in combination with buthionine sulphoximine, to deplete glutathione, slowly increased the cytotoxic response at later times (after 4 hr of incubation). The association of the effects of 4-fluorochalcone oxide with the inhibition of cytosolic epoxide hydrolase is strengthened by the inability of chalcone oxide, a close structural analogue of 4-fluorochalcone oxide, which has no effect on epoxide hydrolase or glutathione conjugation, to influence the effects of anethole on hepatocytes. These data are discussed in terms of the role of anethole epoxide in the cytotoxicity of trans-anethole.     

Effect of glutathione depletion and oxidative stress on the in vitro cytotoxicity of velnacrine maleate

Velnacrine maleate (Mentane^®) is an aminoacridine drug developed for the treatment of Alzheimer's disease. Although velnacrine maleate has not been observed to cause prominent cytotoxicity in in vitro hepatocyte cultures, this drug was associated with elevated serum levels of hepatic enzymes in clinical trials. The purpose of the present study was to manipulate cultures of rat hepatocytes in an attempt to elicit a cytotoxic response from this drug and to better understand the in vitro mechanisms of action. Cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) leakage, neutral red (NR) uptake, and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. Preliminary studies with fluorescent probes did not indicate a role for calcium influx or the formation of reactive oxygen species in the cytotoxicity of velnacrine maleate. However, depletion of cellular glutathione (GSH) by diamide (DA) pretreatment resulted in a cytotoxic response at concentrations of velnacrine maleate (1 and 10 μg/ml) which were ∼ 25-fold lower than those in the absence of DA. Similarly, pretreatment with velnacrine maleate enhanced the cytotoxicity of DA. Pre-exposure of cells to a mixture of DA and t-butyl hydroperoxide (t-BHP) at non-toxic concentrations resulted in significant cytotoxicity of the hepatocyte cultures by velnacrine maleate. Results from these studies indicate that oxidative stress and GSH depletion may enhance Alzheimer patients' susceptibility to the hepatotoxic potential of aminoacridine drugs.     

Cytotoxicity of halogenated alkanes in primary cultures of rat hepatocytes from normal, partial hepatectomized, and preneoplastic/neoplastic liver

Six halogenated hydrocarbons, chloroform, 1,2-dibromoethane (1,2-DBE), 1,1-dichloroethane (1,1-DCE), 1,2-dichloroethane (1,2-DCE), 1,1,1-trichloroethane (1,1,1-TCE), and 1,1,2-trichloroethane (1,1,2-TCE), were evaluated for their cytotoxicity in primary cultures of rat hepatocytes isolated from normal, partially hepatectomized, and preneoplastic/neoplastic rat livers. Preneoplastic/neoplastic lesions of phenotypically altered foci and hepatocyte nodules were induced by either (1) initiation by diethylnitrosamine (DENA) followed by 2 weeks of 0.02% 2-acetylaminofluorene (2-AAF) in the diet and a single gavage dose of carbon tetrachloride 1 week after the start of the 2-AAF diet or (2) initiation by DENA followed by promotion with 500 ppm sodium phenobarbital in the drinking water for 24 weeks. The hepatocytes containing preneoplastic/neoplastic cells isolated from animals treated with either protocol, compared to hepatocytes isolated from normal liver, were resistant to the cytotoxicity of aflatoxin B1 (AFB1). None of the six halogenated alkanes exhibited any difference in their cytotoxicity toward hepatocytes isolated from normal liver or from liver containing preneoplastic/neoplastic lesions induced by either procedure. Hepatocytes isolated from partially hepatectomized animals were resistant to the cytotoxicity of AFB1 and chloroform but not to the cytotoxicity of 1,2-DBE or 1,2-DCE. The ranking of relative cytotoxicity in hepatocytes from untreated rats was 1,2-DBE much greater than 1,2-DCE greater than 1,1,2-TCE greater than 1,1,1-TCE greater than chloroform greater than 1,1-DCE. Treatment with SKF-525A protected the hepatocytes from the cytotoxicity of AFB1 while increasing the cytotoxicity of all six halogenated alkanes. Treatment with diethyl maleate increased the cytotoxicity of AFB1 and all six halogenated alkanes. These observations suggest that preneoplastic/neoplastic rat hepatocytes are not resistant to the cytotoxicity of the six halogenated alkanes because their toxicity might be mediated by a cytochrome P-450 species which is not inhibited by SKF-525A and is not decreased in preneoplastic/neoplastic lesions.     

The role of conjugation in hepatotoxicity of troglitazone in human and porcine hepatocyte cultures.

In primary human and porcine hepatocyte cultures, we investigated the relationship between metabolism and cytotoxicity of troglitazone. Treatment of human hepatocytes for 2 h with 10, 20, 25, 35, and 50 microM troglitazone in protein-free medium resulted in concentration-dependent decreases in total protein synthesis. Decreases at 10 and 20 microM were reversible by 24 h, however protein synthesis did not recover at concentrations >/=25 microM. Troglitazone at 50 microM caused cellular death. In porcine hepatocytes, 100 microM troglitazone was lethal, whereas at 50 microM, protein synthesis completely recovered by 24 h. Recovery in protein synthesis was associated with metabolism of parent drug, whereas toxicity correlated (r(2) = 0.82) with accumulation of unmetabolized troglitazone. By 1 h, in human hepatocytes, troglitazone was metabolized to similar amounts of sulfate and quinone metabolites with little glucuronide detected. In contrast, porcine hepatocytes metabolized troglitazone to the similar amounts of glucuronide and the quinone metabolites with little sulfate detected. Exposure of human hepatocytes to a combination of 10 microM troglitazone and 10 microM 2,4-dichloro-4-nitrophenol resulted in a 70% decrease in protein synthesis, associated with 90% inhibition in the formation of troglitazone sulfate, a 4-fold increase in unmetabolized troglitazone, and no effect on formation of the quinone metabolite. Treatment with a combination of acetaminophen or phenobarbital with 20 microM troglitazone resulted in sustained decrease in protein synthesis associated with inhibition of sulfation and accumulation of troglitazone. These results suggest that inhibition of troglitazone sulfation may result in increased hepatotoxicity due to exposure to parent drug, or increased metabolism by alternate pathways.     

Intracellular glutathione in human hepatocytes incubated with S-adenosyl-L-methionine and GSH-depleting drugs.

The present study was undertaken to investigate (a) whether S-adenosyl-L-methionine (SAMe) added to culture medium can increase intracellular glutathione (GSH) levels in human hepatocytes and (b) whether SAMe can prevent the GSH depletion found in human hepatocytes incubated with GSH-depleting drugs (paracetamol, opiates, ethanol). Incubation of hepatocytes with increasing concentrations of SAMe resulted in a dose-dependent elevation of intracellular GSH content, which reached its maximum (35% increase) at 30 microM after 20 h. SAMe, as the only sulfur source in the medium, was efficient in repleting GSH-depleted hepatocytes following treatment with diethyl maleate. Incubation of human hepatocytes with SAMe attenuated the GSH depletion of cells incubated with toxic concentrations of paracetamol (2 mM), heroin (0.5 mM) and methadone (0.2 mM). A decrease in GSH due to exposure of hepatocytes to 50 mM ethanol was prevented when SAMe was simultaneously added to ethanol, and human hepatocytes maintained their GSH levels like non ethanol-treated cells. The experimental results of our work give the first direct evidence of the ability of exogenously administered SAMe to increase intracellular GSH levels in human hepatocytes and to prevent the GSH depletion caused by paracetamol, opiates and ethanol.     

Reactive oxygen species and non-peroxidative mechanisms of cocaine-induced cytotoxicity in rat hepatocyte cultures

Primary short-term cultures of hepatocytes derived from phenobarbital-induced male Sprague-Dawley rats were used to investigate the mechanisms of cocaine-induced cytotoxicity. Exposure of cells to cocaine resulted in a time and concentration-dependent release of lactate dehydrogenase (LDH) into the culture medium which became evident after 7 h of incubation. Over the course of 24 h incubation with cocaine (0.3 mM) there was no significant lipid peroxidation (measured as the formation of thiobarbituric acid-reactive substances. TBA-RS). The addition of the ferric iron chelator, deferoxamine (DFO), prevented in part cocaine-induced LDH release. Alternatively, addition of the antioxidant, alpha-tocopherol polyethylene glycol 1000 succinate (TPGS), did not protect against hepatocyte injury. Depletion of the intracellular glutathione (GSH) with diethyl maleate (DEM) to below critical levels for antioxidative protection markedly accelerated the onset and increased the extent of cocaine-induced LDH release, concomitant with massive production of lipid peroxidation. During the first four hours of incubation DFO and TPGS protected against cocaine-induced cytotoxicity in GSH-depleted cells. However, at later stages (24 h), the protective effect was lost even in the absence of lipid peroxidation. These results suggest that reactive oxygen species are involved in cocaine-mediated hepatocyte injury. However, lipid peroxidation can be dissociated from other, non-peroxidative, iron-dependent mechanisms of oxidative cell injury.     

Depletion of ATP but not of GSH affects viability of rat hepatocytes.

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after a depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.     

The concentration and temporal relationships of acetaminophen-induced changes in intracellular and extracellular total glutathione in freshly isolated hepatocytes from untreated and 3-methylcholanthrene pretreated Sprague-Dawley and Fischer rats.

Fischer rats are more sensitive to acetaminophen-induced hepatotoxicity than Sprague-Dawley rats, however, the mechanisms for this enhanced sensitivity remain unclear. The susceptibility to hepatotoxicity is determined largely by the balance between acetaminophen toxification and detoxification. Since glutathione plays a critical role in the detoxification process, it would be of interest to compare the effects of acetaminophen on hepatic glutathione homeostasis in the Sprague-Dawley and Fischer rat, and relate these effects to cytotoxicity. To this end, we measured the sequential changes of intracellular and extracellular total glutathione in freshly isolated hepatocytes from untreated and 3-methylcholanthrene pretreated Fischer and Sprague-Dawley rats, both in the absence (basal) and presence of acetaminophen. In the basal state, the intracellular total glutathione content was significantly (P less than 0.01) increased in hepatocytes from untreated Fischer rats. Nevertheless, the sequential release of total glutathione into the medium and the sequential depletion of intracellular total glutathione were quantitatively similar in hepatocytes from untreated Fischer and Sprague-Dawley rats. Following exposure to acetaminophen, there was a striking dose and time associated depletion of intracellular total glutathione in untreated hepatocytes from both rat strains, and quantitatively the depletion was similar in untreated hepatocytes from both rat strains. This degree of depletion of intracellular total glutathione was not associated with acetaminophen-induced cytotoxicity in Sprague-Dawley hepatocytes, whereas significant (P less than 0.05) cytotoxicity was demonstrated in Fischer hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocyte toxicity of mechlorethamine and other alkylating anticancer drugs. Role of lipid peroxidation.

The alkylating anticancer drugs, mechlorethamine (HN2), chlorambucil, cyclophosphamide, carmustine and lomustine readily induced cytotoxicity in isolated rat hepatocytes. Hepatocyte glutathione (GSH) was depleted rapidly following addition of the drugs. Lipid peroxidation ensued following GSH depletion and before cytotoxicity occurred. Furthermore, cytotoxicity was delayed by the antioxidants butylated hydroxyanisole (BHA) and alpha-tocopherol, the ferric iron chelator desferoxamine or the radical trap 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) even when added 10 min later. HN2 was much less toxic to hepatocytes under nitrogen and caused much less lipid peroxidation than under aerobic conditions. Cytotoxicity induced by HN2 was also prevented by choline, suggesting that a choline carrier is responsible for HN2 uptake in the hepatocytes. Various sulfur compounds acted as antidotes for HN2 cytotoxicity. Thiosulfate was still effective when added 30 min after HN2. Depletion of GSH in the hepatocytes markedly increased their susceptibility to HN2. However, BHA, desferoxamine or TEMPO protected these hepatocytes from HN2. This suggests that antioxidants could prove useful in preventing the increased risk of hepatotoxicity if GSH-depleting agents are used to overcome tumor resistance to nitrogen mustards.     

Relationship between metabolism and cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

The relationship between the metabolism and the cytotoxicity of ortho-phenylphenol (OPP) was investigated using isolated rat hepatocytes. Addition of OPP (0.5-1.0 mM) to the hepatocytes caused a dose-dependent toxicity; 1.0 mM OPP caused acute cell death. Pretreatment of hepatocytes with SKF-525A (50 microM, a non-toxic level) enhanced the cytotoxicity of OPP (0.5-1.0 mM). This was accompanied by inhibition of OPP metabolism. Conversely, OPP at low concentrations (0.5 or 0.75 mM) was converted sequentially to phenyl-hydroquinol (PHQ) and then to glutathione (GSH) conjugate in the cells. The concentrations of both metabolites, especially PHQ-GSH conjugate, were very low in hepatocytes exposed to 1.0 mM OPP alone as well as with SKF-525A. The cytotoxicity induced by 0.5 mM OPP was enhanced by the addition of diethylmaleate (1.25 mM) which continuously depletes cellular GSH. In contrast, additions to hepatocytes of 5 mM of dithiothreitol, cysteine, N-acetyl-L-cysteine or ascorbic acid significantly inhibited the cytotoxicity induced by 0.5 mM PHQ; GSH, protein thiols and ATP losses were also prevented. Further, these compounds depressed the rate of PHQ loss in hepatocyte suspensions. These results indicate that the acute cytotoxicity caused by the high dose (1.0 mM) of OPP is associated with direct action by the parent compound; at low doses (0.5-0.75 mM) of OPP, the prolonged depletion of GSH in hepatocytes enhances the cytotoxicity induced by PHQ.     

Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity.

Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip). Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS. Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM). These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from na?ve rats. These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects.     

In vitro evaluation for corneal damages by anti-glaucoma combination eye drops using human corneal epithelial cell (HCE-T)

The combination of anti-glaucoma eye drops is frequently used in clinical treatment, and it is known that the combination can cause corneal damage. Recently, an anti-glaucoma combination eye drops is developed, and the treatment by the combination eye drops is expected to enhance quality of life. However, effects of the combination eye drops on corneal epithelial cell damage have not been clarified. In this study, we investigated the corneal epithelial cell damage of commercially available anti-glaucoma combination eye drops, such as Xalacom? (latanoprost/timolol maleate combination eye drops), Duotrav? (travoprost/timolol maleate combination eye drops) and Cosopt? (dorzolamide hydrochloride/timolol maleate combination eye drops) using the human corneal epithelial cell (HCE-T). The cytotoxicity in Xalacom? was higher than that in Xalatan? (eye drops containing latanoprost) and Timoptol? (eye drops containing timolol maleate), and the benzalkonium chloride (BAC) and timolol maleate were related to cytotoxicity in Xalacom?. The cytotoxicity in Duotrav? and Cosopt? was lower than that in Timoptol?. The Duotrav? is preserved with a non-BAC system (POLYQUAD, polidronium chloride). Therefore, it was suggested that the POLYQUAD related to the low cytotoxicity in Duotrav?. On the other hand, the D-mannitol reduced the cytotoxicity by BAC in this study. This result suggested that the cytotoxicity in Cosopt? was reduced by D-mannitol. The Duotrav? and Cosopt? may be less damaging to the ocular surface of glaucoma patients receiving long-term eye drop therapy in compared with the combination of anti-glaucoma eye drops.     

Integrated disinfection by-products (DBP) mixtures research: gene expression alterations in primary rat hepatocyte cultures exposed to DBP mixtures formed by chlorination and ozonation/postchlorination

Large-scale differential gene expression analysis was used to examine the biological effects of disinfected surface waters on cultured rat hepatocytes. Source water from East Fork Lake (Harsha Lake), a reservoir on the Little Miami River in Ohio, was spiked with iodide and bromide and disinfected by chlorination or ozonation/postchlorination. The chlorinated and ozonated/postchlorinated waters were concentrated, respectively, 136- and 124-fold (full strength) by reverse-osmosis membrane techniques. Volatile disinfection by-products (DBP) lost during concentration were restored to the extent possible. Primary rat hepatocytes were exposed to either full-strength or 1:10 or 1:20 dilutions of the concentrates for 24 h and assayed for cytotoxicity and gene expression alterations. The full-strength concentrates were cytotoxic, whereas the diluted samples exhibited no detectable cytotoxicity. Differential gene expression analysis provided evidence for the underlying causes of the severe cytotoxicity observed in rat hepatocytes treated with the full-strength ozonation/postchlorination concentrate (e.g., cell cycle arrest, metabolic stasis, oxidative stress). Many gene expression responses were shared among the hepatocyte cultures treated with dilutions of the ozonation/ postchlorination and chlorination concentrates. The shift in the character of the response between the full-strength concentrates and the diluted samples indicated a threshold for toxicity. A small subset of gene expression changes was identified that was observed in the response of hepatocytes to peroxisome proliferators, phthalate esters, and haloacetic acids, suggesting a peroxisome proliferative response.     

Methotrexate induced mitochondrial injury and cytochrome c release in rat liver hepatocytes

Methotrexate (MTX) is a folic acid antagonist that is widely used to treat a variety of diseases. One of the most serious side effects of MTX therapy is hepatotoxicity. The potential molecular cytotoxic mechanisms of MTX toward isolated rat hepatocytes were investigated using Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques. A concentration and time dependent increase in cytotoxicity and reactive oxygen species (ROS) formation and a decrease in mitochondrial membrane potential (MMP) were observed with MTX. Furthermore, a significant increase in MTX (300?μM)-induced cytotoxicity and ROS formation were observed when glutathione (GSH)-depleted hepatocytes were used whereas addition of N-acetylcysteine (a GSH precursor) decreased cytotoxicity. Catalase inactivation also increased MTX-induced cytotoxicity, while the direct addition of catalase to the hepatocytes decreased cytotoxicity. MTX treatment in isolated rat mitochondria caused swelling and significantly decreased adenosine triphosphate (ATP) and GSH content, and cytochrome c release. Potent antioxidants such as mesna, resveratrol and Trolox decreased MTX-induced cytotoxicity and ROS formation and increased MMP. This study suggests that MTX-induced cytotoxicity caused by ROS formation and GSH oxidation leads to oxidative stress and mitochondrial injury in rat hepatocytes.     

Toxicity of nitrobenzene compounds towards isolated hepatocytes: dependence on reduction potential

1. The cytotoxicity of p-substituted nitrobenzenes towards isolated hepatocytes under aerobic or hypoxic conditions has been determined. The nitrobenzene concentration required to cause 50% cytoxicity in 2 h was a function of the one-electron reduction potential of the nitrobenzene, with the more cytotoxic compounds having the strongest electron-withdrawing substituents. 2. The effectiveness of the nitrobenzenes at causing cytotoxicity under aerobic but not hypoxic conditions was markedly increased if hepatocyte catalase was inhibited with azide. 3. Nitrobenzenes at cytotoxic concentrations induced cyanide-resistant respiration in isolated hepatocytes. Their effectiveness correlated with their cytotoxicity. 4. The rate of oxygen activation of these nitrobenzenes by ascorbate was also a function of the one-electron reduction potential. The nitro compounds with the strongest electron-withdrawing substituents were the most rapidly reduced. 5. Most nitrobenzenes were more cytotoxic under aerobic than hypoxic conditions. Ascorbate enhanced hypoxic, but not aerobic, cytotoxicity. 6. It was concluded that the cytotoxicity of different nitrobenzenes is related to their ease of reduction to nitro radical anions and nitrosobenzenes. Aerobic cytotoxicity is probably initiated by redox cycling and oxygen activation by the nitro radical anions whereas hypoxic cytotoxicity is probably initiated by the alkylation of macromolecules by nitrosobenzene metabolites.     

Influence of CLOCK on cytotoxicity induced by diethylnitrosamine in mouse primary hepatocytes

The Clock gene is a core clock factor that plays an essential role in generating circadian rhythms. In the present study, it was investigated whether the Clock gene affects the response to diethylnitrosamine (DEN)-induced cytotoxicity using mouse primary hepatocytes. DEN-induced cytotoxicity, after 24h exposure, was caused by apoptosis in hepatocytes isolated from wild-type mouse. On the other hand, Clock mutant mouse (Clk/Clk) hepatocytes showed resistance to apoptosis. Because apoptosis is an important pathway for suppressing carcinogenesis after genomic DNA damage, the mechanisms that underlie resistance to DEN-induced apoptosis were examined in Clk/Clk mouse hepatocytes. The mRNA levels of metabolic enzymes bioactivating DEN and apoptosis-inducing factors before DEN exposure were lower in Clk/Clk cells than in wild-type cells. The accumulation of p53 and Ser15 phosphorylated p53 after 8h DEN exposure was seen in wild-type cells but not in Clk/Clk cells. Caspase-3/7 activity was elevated during 24h DEN exposure in wild-type cells but not in Clk/Clk cells. In addition, resistance to DEN-induced apoptosis in Clk/Clk cells affected the cell viability. These studies suggested that the lower expression levels of metabolic enzymes bioactivating DEN and apoptosis inducing factors affected the resistance to DEN-induced apoptosis in Clk/Clk cells, and the Clock gene plays an important role in cytotoxicity induced by DEN.     

Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A‐mediated ortho‐benzoquinone metabolite formation and glutathione depletion

Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)‐mediated reactive ortho‐benzoquinone metabolites formation and glutathione (GSH) depletion in LUT‐induced cytotoxicity in primary rat hepatocytes. A reactive ortho‐benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT‐induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT‐induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L‐buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'‐dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A‐mediated reactive metabolite formation plays a critical role in LUT‐induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT‐mediated cytotoxicity in primary rat hepatocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

Depletion of ATP but not of GSH affects viability of rat hepatocytes

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.