Species differences in the hepatotoxicity of paracetamol are due to differences in the rate of conversion to its cytotoxic metabolite

The cytotoxicity of paracetamol and of its putative toxic metabolite, N-acetyl-p-benzo-quinoneimine (NABQI) have been investigated in hepatocytes from hamster, mouse, rat and human liver. Whereas paracetamol readily caused cell blebbing and a loss of viability in hepatocytes from mouse and hamster, human and rat hepatocytes were much more resistant to these effects. In marked contrast, there were no significant differences in the sensitivity of the cells from any species to the toxic effects of NABQI. Glutathione depletion by NABQI and paracetamol correlated very well with the toxic effects of these compounds. It is concluded that species differences in sensitivity to the hepatotoxicity of paracetamol are due almost entirely to differences in the rate of formation of NABQI, and not to any intrinsic differences in sensitivity or in any difference in the fate of NABQI once formed. Further, man appears to be relatively resistant to the hepatotoxic effects of paracetamol, and the results in hepatocytes were confirmed by both in vitro and in vivo analyses.     

The combined use of human neural and liver cell lines and mouse hepatocytes improves the predictability of the neurotoxicity of selected drugs

The cytotoxicity of amitriptyline (0-100microM), selegiline (0-4.5microM), carbamazepine (0-420microM) and paracetamol (0-10mM) was studied in metabolically competent mouse hepatocytes, metabolically incompetent human hepatoblastoma (HepG2) cells, and in neuroblastoma (SH-SY5Y) and astrocytoma (U-373 MG) cells, by using luminescence-based ATP measurement as an endpoint of cell toxicity. The aim was to evaluate the potential of the selected cell cultures to recognize metabolism-induced toxicity of the test compounds, and to predict further hepatic and neural toxicity. In SH-SY5Y cells amitriptyline was severely toxic, while selegiline and paracetamol failed to show any toxic effect, and carbamazepine was only slightly toxic at the highest concentration. In U-373 MG cells the onset of amitriptyline toxicity started earlier than in SH-SY5Y cells. However, the highest amitriptyline concentration resulted in approximately 100% decrease in the viability of the SH-SY5Y cells, whereas the decrease in the viability of the U-373 MG cells was only approximately 30%. Selegiline, carbamazepine and paracetamol were toxic in mouse hepatocytes (but not in HepG2 cells), which suggests that these drugs may show metabolism-dependent (neuro)toxicity. In conclusion, compared to the use of neurons alone, better estimations of neurotoxicity can be made by the combined use of metabolically competent hepatocytes and glial cells (e.g. U-373 MG) together with neuronal cells (e.g. SH-SY5Y).     

Evidence for a direct role of intracellular calcium in paracetamol toxicity

There is considerable evidence that an increase in cytosolic Ca2+ is involved in the cytotoxicity of a variety of agents. However, the direct demonstration of such involvement has proved difficult. In the present study, loading of freshly isolated hamster hepatocytes with the Ca2+ specific chelator Quin 2 (2-[(2-bis[carboxymethyl]amino-5-methyl-phenoxy)methyl]-6-methoxy-8- bis-[carboxymethyl]amino-quinoline) provided significant protection against the loss of viability caused by paracetamol. This was evident both when the cells were co-incubated with Quin 2-AM and paracetamol, and when the cells were incubated with Quin 2-AM after prior exposure to paracetamol and its complete removal from the hepatocytes. These observations provide direct evidence that an increase in intracellular Ca2+ is the cause of cell death in hepatocytes exposed to paracetamol. Further, the fact that Quin 2 is protective even after some time suggests that, for alterations of cytosolic Ca2+ to be detrimental, they must be sustained. The effects of Quin 2 on plasma membrane blebbing of paracetamol-exposed hepatocytes were less pronounced than on cell viability. This is in contrast to the effects of the direct-acting thiol-reducing reducing agent, dithiothreitol, which was equally effective in preventing blebbing and loss of viability. It is concluded that alterations of cytosolic Ca2+ are less directly linked to plasma membrane blebbing than to loss of cell viability.     

The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine

In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.     

Dissociation of cell death from covalent binding of paracetamol by flavones in a hepatocyte system

Paracetamol metabolism and toxicity were studied in isolated rat hepatocytes. Cell damage, due to paracetamol, was shown to be dose dependent and was worse in cells from animals pre-treated with phenobarbitone. Exposure to 10 mM paracetamol for 1 hr caused a loss of intracellular reduced glutathione (GSH) and a later progressive leakage of isocitrate dehydrogenase (ICD). Treatment with (+)catechin, 3-O-methyl(+)catechin and promethazine reduced or prevented the paracetamol-induced ICD leakage. Similarly, studies on covalent binding of paracetamol showed that 3-O-methyl(+)catechin, which “protected” the cells, did so without affecting the amount of material bound covalently to cellular protein. Incubation in tissue culture for 24 hr, after prior treatment with paracetamol ± the protective agent, showed that the protected cells remained viable and attached to tissue culture plates much better than did the “unprotected” cells. These results suggest that the protective effect is much more than just a temporarily delayed cell death. GSH loss and covalent binding of paracetamol metabolites to cell protein are not sufficient causes of cell death, although they may act as starting points in the chain of events leading to cell death.     

Mechanism of chemical-induced toxicity. II. Role of extracellular calcium

Previous studies disagree as to if chemical-induced cell death is caused by the influx and accumulation of extracellular Ca2+. To determine the role of extracellular Ca2+ in toxic cell death, the viability (leakage of intracellular K+ and lactate dehydrogenase) and total Ca2+ content of isolated hepatocytes incubated in the presence or absence of extracellular Ca2+ were determined during a toxic insult with bromobenzene, ethyl methanesulfonate (EMS), Ca2+ ionophore A23187, and adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The present study utilized the dibutyl phthalate separation technique which enabled the analysis of only viable hepatocytes for changes in intracellular Ca2+ and K+ content during toxic cell injury. The three chemical treatments, bromobenzene, EMS, and ADR-BCNU, each caused an accelerated loss of viability in hepatocytes incubated without extracellular Ca2+ as compared to cells incubated with Ca2+. Furthermore, the total Ca2+ content of viable hepatocytes incubated in the presence of extracellular Ca2+ did not increase during chemically induced cell injury as compared to control cells. In fact, a significant decline in total cellular Ca2+ was observed in viable hepatocytes incubated in Ca2+-free medium during toxic cell injury. Treatment with Ca2+ ionophore A23187 was also toxic to hepatocytes incubated in the presence or absence of extracellular Ca2+. At high concentrations of ionophore (20 microM or 4 micrograms/10(6) cells), cell death was accelerated in hepatocytes incubated with Ca2+ as compared to cells incubated in Ca2+-free medium. In contrast, after treatment with lower concentrations of ionophore (10 microM or 2 micrograms/10(6) cells), the rate of cell death was reversed with hepatocytes incubated without extracellular Ca2+ dying first. Thus, depending on the concentration of A23187 and the time of exposure, the presence of extracellular Ca2+ can be shown either to accelerate or protect against cell death. Surprisingly, reversible and irreversible cell injury were not observed in hepatocytes incubated with extracellular Ca2+ and 2 microM A23187 though this treatment resulted in an 800% increase in total intracellular Ca2+ content. We conclude that chemical-induced hepatic cell death is not caused by an increase in total cellular Ca2+ resulting from the influx of extracellular Ca2+.     

Acute effects of halothane and enflurane on drug metabolism and protein synthesis in isolated rat hepatocytes

The metabolism of sulphanilamide, antipyrine and paracetamol was studied in the absence and presence of the anaesthetics halothane and enflurane at three different concentrations (0.5, 1.0 and 2.0 mM) in isolated hepatocytes from the rat. Cell viability and protein synthesis were monitored to evaluate toxic effects. A strong concentration related inhibition of antipyrine oxidation (40-70%) and paracetamol conjugation (20-40%) was caused by both halothane and enflurane. Acetylation of sulphanilamide was not inhibited, however, as a slight augmentation was noticed. A significant dose related decrease of cell viability (3-13%) was caused by both anaesthetics. Dose dependent inhibition of the synthesis of stationary cell proteins (15-60%) and the synthesis/secretion of medium proteins (35-85%) was caused by halothane. Similar but slightly less pronounced effects were caused by enflurane. The present findings show that volatile anaesthetics may have general effects as well as different degrees of specific effects on both membrane bound enzyme and soluble enzyme activities.     

The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes

Microcystin-YM a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa was radiolabeled with 125I, and used to investigate the uptake of the toxin by freshly isolated rat hepatocytes. The uptake was temperature dependent with apparent activation energy of 18 kcal/mole (77 kJ/mole) for the initial rate of uptake. Uptake of non-toxic (10-20 nM) doses of microcystin by hepatocytes continued with time, the intracellular to extracellular distribution ratio for the toxin was 70 at 60 min for 10(6) cells/ml. Uptake of higher doses of microcystin (100 nM and more) stopped when the cells blebbed: a toxic response of hepatocytes to microcystin. Uptake of microcystin by hepatocytes was inhibited 70-80% by the addition of 10 microM sodium deoxycholate or bromsulphthlein, compounds that protect hepatocytes from the toxic effects of microcystin.     

Warifteine and milonine,alkaloids isolated from Cissampelos sympodialis Eichl: cytotoxicity on rat hepatocyte culture and in V79 cells

Two alkaloids were isolated from the leaves of Cissampelos sympodialis; a bisbenzylisoquinoline compound named warifteine and a novel 8,14-dihydromorphinandienone alkaloid named milonine. The cytotoxic effects of these alkaloids were assayed in cultured hepatocytes and V79 fibroblasts. Three independent endpoint assays for cytotoxicity in vitro were used: the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). Milonine was less toxic than warifteine in both cell cultures. The IC50 values determined in the three different viability assays were around 100 and 400 microM after milonine treatment of V79 cells or hepatocytes. IC50 values ranging from 10 to 35 microM were obtained for warifteine in the viability tests evaluated in V79 cells and hepatocytes. Due to the similar cytotoxic effects detected on V79 cells and hepatocytes, probably warifteine and milonine induced toxic effects independent to the cytochrome P450. This hypothesis was corroborated by the results where Cimetidine (1.0 mM), a traditional cytochrome P450 inhibitor, did not protect the cells from the toxic action of warifteine or milonine. In conclusion, these alkaloids merit further investigations as potential novel pharmacological agents although milonine was less toxic than warifteine in the cells models investigated.     

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.     

Metabolism and toxicity of benzophenone in isolated rat hepatocytes and estrogenic activity of its metabolites in MCF-7 cells

The metabolism and cytotoxicity of benzophenone and estrogenic activity of its metabolites have been studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with benzophenone (0.25-1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by loss of intracellular ATP and depletion of adenine nucleotide pools. Benzophenone at a low-toxic level (0.25 mM) in the hepatocyte suspensions was converted to benzhydrol, p-hydroxybenzophenone and its sulfate conjugate, without marked loss of cell viability. The amounts of benzhydrol and sulfate conjugate increased with time. In contrast, addition of 2,6-dichloro-4-nitrophenol (an inhibitor of sulfotransferase; 0.1 mM), nontoxic to hepatocytes during the incubation period, enhanced benzophenone-induced cytotoxicity, and this effect was accompanied by a decrease in the formation of sulfate conjugate and increase in the amount of free p-hydroxybenzophenone. In another experiment, MCF-7 cells, estrogen-responsible breast cancer cells were cultured in estradiol free medium and then exposed to 10 nM-500 microM benzophenone or its metabolites for 6 days. Although at higher concentrations all the compounds were toxic, except for benzophenone and benzhydrol, 10-100 microM p-hydroxybenzophenone significantly increased cell proliferation. These results indicate that benzophenone is enzymaticaly converted to benzhydrol, p-hydroxybenzophenone and its sulphate conjugate in rat hepatocytes. Even if there is less free p-hydroxybenzophenone than benzhydrol and sulfate conjugate in hepatocyte suspensions, p-hydroxybenzophenone itself acts as a weak xeno-estrogen on MCF-7 cells.     

Fasting increases the susceptibility of rat hepatocytes to the cytotoxic effects of N-hydroxy-acetylaminofluorene. Effects on mitochondrial respiration and membrane potential.

Isolated rat hepatocytes were incubated with the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Cells from fasted rats were much more susceptible to the cytotoxic effects of 1 mM N-OH-AAF than cells from fed rats: after approximately 90 min exposure the former were all dead but the latter still viable. Even after 240 min 25% of the "fed" cells were still viable. The loss of viability was preceded by a decrease in mitochondrial membrane potential (MMP) and inhibition of respiration; the mitochondrial respiration as measured in permeabilized cells appeared uncoupled. Addition of 15 mM fructose prevented cell death and the loss of MMP in cells both from fed and fasted rats to a large extent; however, uncoupling was not prevented. After incubation of hepatocytes from fasted rats with 1 mM [3H]N-OH-AAF for 120 min, 12 nmol [3H]N-OH-AAF became bound per mg cell protein. Addition of fructose decreased this to 7 nmol. In cells from fed animals 4 nmol [3H]N-OH-AAF became bound after 120 min, in this case fructose had no effect. Part of the protective effect of fructose might be explained by a decrease in intracellular ATP, which prevents the formation of reactive intermediates of N-OH-AAF resulting in a decrease of covalent binding, in addition, fructose protects via a yet to be determined mechanism.     

o-hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin.

o-Hydroxyphenylacetaldehyde (o-HPA), the product of coumarin 3, 4-epoxide, was synthesized and its contribution to the hepatotoxic effects of coumarin in the rat was determined. The relative toxicity of coumarin and o-HPA were initially assessed in Chinese hamster ovary K1 (CHO K1) cells, a cell line that does not contain cytochrome P450. In CHO K1 cells, o-HPA-mediated toxicity greatly exceeded that of coumarin. CHO K1 cell viability, determined via the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), was decreased by 95 and 6% in cultures containing o-HPA and coumarin (4 mM), respectively. Coumarin and o-HPA were then incubated in metabolically competent primary rat hepatocyte cultures. Cell viability was determined via the reduction of MTT, and lactic dehydrogenase (LDH) release was used as a measure of cytotoxicity. Concentration-dependent decreases in cell viability and increased LDH release were observed using 0.2 to 0.8 mM o-HPA and coumarin, with coumarin being consistently less toxic than o-HPA. Cell viability was decreased by 11 and 50% at 0.5 mM coumarin or o-HPA, respectively. Hepatocyte LDH release increased 5-fold after a 6-h exposure to 0.8 mM o-HPA, corresponding to a greater than 90% loss of cell viability in these cultures. In contrast, 0.8 mM coumarin decreased cell viability by 60%, an effect likely due to the conversion of coumarin to coumarin epoxide and o-HPA. Furthermore, 3-hydroxycoumarin (0.8 mM), which is not a product of coumarin epoxidation, had no effect on cell viability or hepatocellular LDH release. These studies demonstrate that metabolically active rat hepatocytes convert coumarin into toxic metabolites, and strongly suggest that o-HPA and coumarin 3, 4-epoxide mediate the toxicity of coumarin in rodents in vivo.     

Flow cytometric analysis of the effects of tri-n-butyltin chloride on cytosolic free calcium and thiol levels in isolated rainbow trout hepatocytes

The toxic effects of tri-n-butylin chloride (TBT) were investigated on isolated trout hepatocytes by flow cytometry (FCM). We developed a procedure permitting the study of cytosolic free calcium in these cells using the new fluorescent probe Fura Red. In parallel, changes in thiol levels upon exposure to TBT were also followed by FCM with the probe 5-chloromethylfluorescein-diacetate. Cell viability was monitored through FCM analysis using propidium iodide. Treatment of hepatocytes with TBT caused a time- and concentration-dependent loss of viability. The results show that TBT induced a sustained elevation of cytosolic free calcium in isolated trout hepatocytes before loss of viability was detectable. Data for the viable cells remaining after incubation with TBT were selected after appropriate gating with the flow cytometer. When this was performed, the data revealed that changes in cytosolic free calcium were not dependent upon TBT concentration and duration of exposure. Moreover, TBT induced a rapid and important depletion of thiols in cells which survived TBT exposure. The present results suggest that alterations in calcium homeostasis and intracellular thiols are involved in the mechanism of trout hepatocyte injury by TBT. FCM is a powerful tool to study metabolic disturbances caused by toxic agents on cells at an individual level.     

Enalapril cytotoxicity in primary cultures of rat hepatocytes. II. Role of glutathione

The cytotoxicity of enalapril maleate (EN) in primary cultures of rat hepatocytes, at concentrations of 0.5 mM or greater, was measured by the release of lactate dehydrogenase (LDH) into the culture medium. Pretreatment of the hepatocytes with L-buthionine-(S,R)-sulfoximine (BSO) and diethyl maleate (DEM) potentiated the toxicity whereas N-acetyl-L-cysteine (NAC) provided protection. EN produced a dose-dependent reduction in intracellular glutathione (GSH) concentration. This was an early effect, apparent after only 1 h of exposure to the drug, whereas loss of cell viability occurred after 6-18 h. These results suggest that the mechanism of EN cytotoxicity involves a GSH-dependent detoxification pathway.     

Chemical structure and toxicity of diuretics in isolated hepatocytes

The effects of several diuretics, including tienilic acid and indacrinone, on isolated rat heaptocytes were examined. Addition of tienilic acid and indacrinone at 1 mM to a suspension of freshly isolated cells caused dose-dependent loss of cell viability as judged by the LDH-latency test. Survey of 19 structurally related compounds revealed that the extent of cell injury and chemical structure were correlated, and an intense adverse effect was attributed to the 2-thienylcarbonyl moiety. Several other factors influencing cell viability are also disclosed. Further study revealed that tienilic acid and indacrinone were toxic to the primary culture of hepatocytes at a lower dose than that a freshly isolated hepatocytes. Thus, an isolated hepatocyte system can be used to select compounds displaying low hepatotoxicity, as for example is needed when screening diuretics.     

Segregation of discrete GS alpha-mediated responses that accompany homologous or heterologous desensitization in two related somatic hybrids.

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.     

Furosemide toxicity in isolated mouse hepatocyte suspensions

Incubation of freshly isolated mouse hepatocytes with 0.5 or 1.0 mM furosemide caused a depletion of cellular acid soluble sulfhydryls to approximately 20-30% of control over the course of 4.5 h. The depletion was accompanied by a reduction in cell viability (indicated by the lactate dehydrogenase latency test) which was significant (P less than 0.05) for 0.5 mM but not for 1.0 mM furosemide at 4.5 h. Ultrastructurally, 0.5 or 1.0 mM furosemide caused cytoplasmic changes including loss of glycogen, disaggregation of polyribosomes, vesiculation of endoplasmic reticulum, and occasional appearance of lamellar bodies consisting of concentric arrays of paired smooth membranes. These concentrations of furosemide also caused cell surface changes, including loss of microvilli, development of an irregular shape compared to the spherical appearance of untreated hepatocytes, and the development of occasional blebs. The appearance of pale staining hydropic cells was indicative of the final stages of cell death. N-Acetylcysteine (6.0 mM) was effective at preventing the depletion of soluble sulfhydryls, the loss of viability, and the ultrastructural effects of 0.5 or 1.0 mM furosemide, suggesting a role for soluble sulfhydryls in the pathogenesis of furosemide hepatotoxicity.     

Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes

The relation between the metabolism and the cytotoxic effects of bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane) has been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. The incubation of hepatocytes with BPA (0.25–1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by losses of intracellular ATP and total adenine nucleotide pools. BPA at a low-toxic level (0.25 mM) in the hepatocyte suspensions was rapidly converted to its major conjugate, BPA-glucuronide, and other minor products without marked loss of cell viability, although at a toxic level (0.5 mM), more than 65% of the compound presented in an unaltered form 2 h after the incubation. Addition of salicylamide (2 mM), non-toxic to hepatocytes during the incubation period, enhanced BPA-induced cytotoxicity and reduced the loss of BPA and the formation of BPA-glucuronide. The addition of BPA to isolated hepatic mitochondria caused a concentration (0–0.5 mM)-dependent increase in the rate of state 4 oxygen consumption in the presence of an FAD-linked substrate (succinate), indicating an uncoupling effect, whereas the rate of state 3 oxygen consumption was inhibited by BPA. Further, the addition of BPA (0.25 mM) reduced state 3 respiration with NAD⁺-linked substrates (pyruvate plus malate) and/or with the FAD-linked substrate, whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not significantly affected by BPA. A comparative study of the toxic effects of BPA and some bisphenols on cell viability (at 1.0 mM) and mitochondrial respiration (at 0.25 mM) revealed that 4,4′-(1,2-diethyl-1,2-ethenediyl)bisphenol (diethylstilbestrol) was more toxic than BPA, followed by 4,4′-methylenediphenol and 4,4′-biphenol. These results indicate that the onset of cytotoxicity caused by BPA may depend on the intracellular energy status and that mitochondria are important targets of the compound. The toxicity caused by the inhibition of ATP synthesis may be related to the concentration of unmetabolised free BPA remaining in the cell suspensions. In addition, the toxic potency of bisphenols to hepatocytes and mitochondria depends on the relative elongation and/or molecular size of the hydrocarbon bridge between the phenolic groups. Received: 25 October 1999 / Accepted: 31 January 2000     

Cytotoxic effects of N-acetyl-p-benzoquinone imine,a common arylating intermediate of paracetamol and N-hydroxyparacetamol

The cytotoxic effects of N-acetyl-p-benzoquinone imine (NAPQI), a postulated ultimate reactive metabolite of paracetamol (pHAA), was studied in suspensions of isolated rat hepatocytes. Incubation of cells for 10–300 min with 0.1–0.5 mM NAPQI led to concentration dependent cell damage. as determined by increased trypan blue exclusion, lactate dehydrogenase release and glutathione (GSH) depletion. NAPQI and N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of pHAA, caused cytotoxic effects in the same concentration range. In contrast, no toxic effects of pHAA (? 20 mM) could be demonstrated. With the short half-life of NAPQI, less than 0.5% of the NAPQI added is expected to be left in the incubation medium after a 2 min incubated period. Nevertheless, 10–120 min (depending on the concentration of NAPQI) elapsed before the cells responded with increased membrane permeability. Clearly, the initial damage caused by NAPQI must be followed by subsequent cellular steps before toxicity becomes apparent. The addition of N-acetylcysteine, GSH or ascorbate during the NAPQI exposure period fully protected the hepatocytes from NAPQI damage. Lesser effects were demonstrated when these agents were added after the 5 min NAPQI exposure period. The results presented in this study further support the hypothesis that NAPQI is the ultimate reactive formed from pHAA.