ANIT-induced disruption of biliary function in rat hepatocyte couplets.

alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rats, involving damage to biliary epithelial cells; our study aims to investigate whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatocyte couplets were used to investigate biliary function in vitro by canalicular vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), within the canalicular vacuole between the two cells. After a 2-h exposure to ANIT, there was a concentration-dependent inhibition of cVA (cVA-IC50; 25 microM), but no cytotoxicity (LDH leakage or [ATP] decline) within this ANIT concentration range. There was no loss of cellular [GSH] at low ANIT concentrations, but, at 50 microM ANIT, a small but significant loss of [GSH] had occurred. Diethylmaleate (DEM) partially depleted cellular [GSH], but addition of 10 microM ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANIT to these cells reduced cVA further, but the magnitude of this further reduction was no greater than that caused by ANIT alone, indicating that glutathione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphological change in the canalicular vacuoles but only a small, non-significant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the pericanalicular web. The results are in accord with a disruption of hepatocyte canalicular secretion within two h in vitro, at low, non-cytotoxic concentrations of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect.     

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.     

Role of MRP2 and GSH in intrahepatic cycling of toxins

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. alpha-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR(-) rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 micromol absolute i.v.) to Wistar and Mrp2-deficient TR(-) rats and collected bile. While both arsenite and GSH secretion were absent in TR(-) rats, the total secretion of arsenite into Wistar bile (2.91 micromol) was accompanied by a excess secretion of 24 micromol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR(-) rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.     

Menadione-induced oxidative stress in hepatocytes isolated from fed and fasted rats: the role of NADPH-regenerating pathways

Isolated hepatocytes were prepared from fed and fasted rats and exposed to a range of menadione (2-methyl-1,4-naphthoquinone) concentrations. Menadione (300 microM) caused a rapid decline in the (NADPH)/(NADPH + NADP+) ratio from 0.85 to 0.39 within 15 min, with further decreases over the 90-min incubation period in cells isolated from fed animals. This decrease of NADPH resulted from oxidation to NADP+ since there was no loss of total pyridine nucleotide (NADP+ + NADPH) content. In addition, menadione (100 microM) caused a five-fold stimulation of the hexose monophosphate shunt by 30 min as indicated by the oxidation of [1-14C]glucose. LDH leakage was slightly but significantly elevated (30% of total) following exposure of cells to 300 microM menadione for 2 hr. Menadione caused a concentration-dependent GSH depletion: 100 microM menadione caused no depletion and 200 and 300 microM menadione caused a 75 and 95% decrease, respectively. Intracellular NADPH was significantly reduced within 30 min by 100 and 200 microM menadione but then returned to values equivalent to or greater than control by 60 min. In contrast, a sustained decrease of NADPH was produced by 300 microM menadione (5% of control after 2 hr). A marked potentiation of the oxidative cell injury produced by menadione was observed in hepatocytes prepared from 24-hr-fasted rats. LDH leakage was 50 and 95% when these cells were exposed to 100 and 200 microM menadione, respectively. Menadione (100 and 200 microM) also caused a marked GSH depletion (95% of control) by 90 min. In contrast to cells isolated from fed animals, menadione (100 and 200 microM) caused an 85% depletion of NADPH by 60 min in cells isolated from fasted rats. This potentiation of menadione-induced oxidative injury was not related to the decreased GSH content produced by fasting since menadione toxicity was not potentiated in control cells partially depleted of GSH by diethyl maleate. A further comparison was made between cells isolated from fasted rats and incubated either with or without supplemental glucose in order to determine a possible protective effect by glucose. In this comparison a significant (p less than 0.05) glucose effect was indeed observed in the direction of preventing GSH and NADPH depletion, as well as attenuating LDH leakage, when hepatocytes were exposed to either 50 or 100 microM menadione.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxic effect and role of exogenous antioxidants in carpet dust mediated toxicity in rat hepatocytes in vitro.

Carpet industries bear a great deal of economic and commercial significance in India. In order to safe guard the workers against the health hazards caused by dust in their occupational environment; it necessitates studying the biological importance of these dusts. The present study was designed to investigate the toxicity of carpet dust (knotted and tuffted) on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and cells were incubated with different concentration of carpet dust (100-5000 microg/10(6) cells) with various time (30-180 min) intervals. An exogenous antioxidant vitamin-E also used to find out the role of antioxidants and free radical production in carpet dust mediated toxicity. Cell viability by trypan blue exclusion and leakage of enzyme lactate dehydrogenase (LDH) were determined. Reduced glutathione (GSH), formation of thiobarbituric acid reactive substance (TBARS) were also measured. A significant decrease in the cell viability was observed after 60, 180 min upon incubation with tuffted carpet dust, while knotted carpet dust caused a significant decrease in the viability after 180 min. LDH leakage was parallel to the cell viability. Thiobarbituric acid reactive substance was significantly increased at 30 and 60 min with carpet dust treated hepatocytes. Dust at 1000 and 5000 microg dose level showed significantly increased formation of TBARS at 30 min incubation. However, when hepatocytes were co-incubated with carpet dust and Vit-E (10, 15 microM), a significant decrease in LDH release and TBARS production was observed while 15 microM Vit-E showed an enhanced protection than 10 microM Vit-E treated hepatocytes. The effect of carpet dust on cell viability, LDH leakage, TBARS production, GSH depletion was time and dose-dependent. Moreover, we observed that tuffted carpet dust causes greater effect than knotted one on the above mentioned parameters. Our studies also revealed that Vit-E in culture media diminishes the carpet dust mediated toxicity.     

Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity

The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2′,7′-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.     

Chloroacetonitrile-induced toxicity and oxidative stress in rat gastric epithelial cells.

Chloroacetonitrile (CAN), is a disinfectant by-product of chlorination of drinking water. Epidemiological studies indicate that exposure to CAN via drinking water might present a potential hazard to human health. The objective of the present work was to investigate the cytotoxic effects as well as the oxidative stress induced by CAN in cultured rat gastric epithelial cells (GECs). GECs were exposed in vitro to different concentrations of CAN (5-40 microm) for 60 min. Also, GECs were incubated with CAN (10 microm) for different time intervals extending to 180 min. Cytotoxicity was determined by assessing cell viability and lactate dehydrogenase (LDH) release, glutathione (GSH) level and lipid peroxidation as indicated by malondialdehyde (MDA) production. Exposure of GECs CAN (10 microm) for 60 min caused a 50% decrease in cell viability and induced an eightfold increase of LDH leakage. In the same experiment, CAN caused a decrease in cellular GSH content to approximately 50% and significantly enhanced MDA accumulation (approx. sevenfold). These toxic responses to CAN were dependent on both concentration and duration of exposure to CAN. There was a good correlation between LDH release and GSH depletion (r =0.96, P<0.05). Treatment of GECs with 5 m mN -acetyl- l -cysteine (NAC) prior to exposure to CAN afforded some degree of protection as indicated by a significant decrease in the LDH leakage (32% of total leakage) and lipid peroxidation (54%) as compared to CAN alone-treated cells. Also, pretreatment of GECs with vitamin C (1 m m) or vitamin E (10 microm) significantly inhibited LDH leakage (20 and 36% of total leakage, respectively). Preincubation with 1 m m desferroxiamine (DFO), a ferric iron chelator, or 10 microm phenanthroline (PHE), a ferrous iron chelator, diminished CAN-induced LDH leakage by 16 and 21% of total leakage, respectively and MDA production by 40 and 44%, respectively. In conclusion, our results suggest that CAN has a potential cytotoxic effect in rat GECs; and thiol group-donors, antioxidants and iron chelators can play a critical role against CAN-induced cellular damage.     

Metabolic alterations in hepatocytes promoted by the herbicides paraquat,dinoseb and 2,4-D

The cytotoxic effects of the herbicides paraquat (1,1′-dimethyl-4,4′-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) on freshly isolated rat hepatocytes were investigated. Paraquat and 2,4-D (1–10 mM) caused a dose and time dependent cell death accompanied by depletion of intracellular glutathione (GSH) and mirroring increase of oxidized glutathione (GSSG). Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000 fold lower than paraquat and 2,4-D), exhibited moderate effects upon the level of GSH and GSSG. These limited effects are at variance with significant effects upon the adenine and pyridine nucleotide contents. ATP and NADH levels are rapidly depleted by herbicide metabolism. This depletion is observed in the millimolar range for paraquat and 2,4-D and in the micromolar range for dinoseb. 2,4-D completely depletes cellular ATP, with subsequent cell death, as detected by LDH leakage. Paraquat rapidly depletes NADH, according to the redox cycling of the herbicide metabolism. The most effective compound is dinoseb since it exerts similar effects as described for paraquat and 2,4-D at concentrations 1000 fold lower. Simultaneously with NADH and ATP depletion, the levels of ADP, AMP and NAd+ increase in hepatocytes incubated in the presence of the herbicides. In contrast to NADH, the time course and extent of ATP depletion and fall in energy charge correlate reasonably with the time of onset and rate of cell death. It is concluded that the herbicides, paraquat and 2,4-D are hepatotoxic and initiate the process of cell death by decreasing cellular GSH. As a consequence of this primary disturbance, alteration of adenine and pyridine nucleotides contents is a critical event in the induction of irreversible cell injury.     

Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

Treatment of isolated rat hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and adriamycin (ADR) produced a complete depletion of cellular glutathione accompanied by a significant increase in lactate dehydrogenase (LDH) leakage. Separation of the mitochondrial and cytoplasmic pools of glutathione by digitonin disruption showed that, although BCNU, a specific inhibitor of glutathione, completely depleted the cytoplasmic pool of glutathione, the mitochondrial supply was not entirely expended and LDH leakage was only moderately stimulated. Only after depletion of the mitochondrial supply of glutathione by ADR and BCNU did LDH leakage increase markedly. Measurement of lipid peroxidation, by monitoring malondialdehyde through the thiobarbituric acid procedure, showed that malondialdehyde accumulated more extensively and at a rate mirroring release of LDH from ADR/BCNU treated cells. The time of increase in LDH leakage and malondialdehyde production corresponded to the time of depletion of mitochondrial glutathione to less than 10% of the initial pool size. No such increase in LDH leakage was observed with BCNU or ADU treatment alone or when aminopyrine, an inhibitor of lipid peroxidation, was included. Aminopyrine was found to prevent, in a dose-dependent manner, both LDH leakage and malondialdehyde production stimulated by ADR/BCNU treatment. The protective effect peaked at 5 mM aminopyrine, and higher concentrations produced significant LDH leakage exhibiting LDH release kinetics different than those observed with ADR/BCNU. Although aminopyrine had no effect on the rate or extent of cytoplasmic glutathione depletion by ADR/BCNU treatment, the mitochondrial pool was conserved significantly in those cells protected by aminopyrine. These data suggest that enhanced hepatocyte damage observed after treatment with a combination of ADR and BCNU versus BCNU or ADR alone is due to the extensive depletion of mitochondrial glutathione supported by ADR after glutathione reductase inhibition. Further, enhancement of lipid peroxidation is strongly implicated in the mechanism of adriamycin toxicity.     

Involvement of diclofenac acyl-β-d-glucuronide in diclofenac-induced cytotoxicity in glutathione-depleted isolated murine hepatocytes co-cultured with peritoneal macrophages

Direct hepatotoxic effects of drugs can occur when a parent drug and/or its reactive metabolites induces the formation of reactive oxygen species. Reactive metabolites of diclofenac (DIC) such as DIC acyl-β-d-glucuronide (DIC-AG) bind covalently to proteins, potentially decreasing protein function or inducing an immune response. However, it is unclear whether the macrophages and GSH depletion participate in DIC-induced cytotoxicity. Mouse hepatocytes (Hep) co-cultured with peritoneal macrophages (PMs) were used to clarify the effects of presence of PM with GSH depletion on DIC-induced cytotoxicity in Hep. DIC-AG but not hydroxy-DIC concentrations in medium were significantly increased in Hep co-cultured with PM with GSH depletion. Depletion of GSH resulted in significantly higher LDH leakage. Interestingly, LDH leakage in Hep/PM (1:0.4) with GSH depletion was significantly higher than in Hep/PM (1:0 and 1:0.1) with BSO. It is likely that macrophages with GSH depletion could facilitate DIC-induced cytotoxicity.     

Protective effects of honokiol and magnolol on tertiary butyl hydroperoxide- or D-galactosamine-induced toxicity in rat primary hepatocytes

The aim of this study was to investigate the protective effect of honokiol and magnolol on hepatocyte injury induced by either tertiary butyl hydroperoxide (tBH)- or D-galactosamine (GalN). The cellular leakage of LDH and AST, and cell death by treatment with 1.5 mM tBH for 1 h, were significantly inhibited by treatment with honokiol (40 and 20 microM) or magnolol (40 microM). Treatment with honokiol or magnolol significantly inhibited lipid peroxidation in both cells and media, the generation of intracellular reactive oxygen species (ROIs), and intracellular glutathione (GSH) depletion induced by tBH. The cellular leakage of LDH and AST, and cell death, by 24-hour treatment with 30 mM GalN were significantly inhibited by treatment with honokiol (20, 5 and 1 microM) or magnolol (20, 5 and 1 microM). Treatment with honokiol (20, 5 and 1 microM) or magnolol (20 and 5 microM) significantly inhibited the intracellular GSH depletion induced by GalN. The hepatoprotective effects of honokiol and magnolol on oxidative stress induced by tBH were probably the result of their antioxidant activity. Honokiol and magnolol also had a protective effect against GalN-induced hepatotoxicity, which was used as an alternate model to oxidative stress, acting by inhibiting intracellular GSH depletion.     

The mechanism of prevention of paracetamol-induced hepatotoxicity by 3,5-dialkyl substitution. The roles of glutathione depletion and oxidative stress

Recently, we have reported that 3,5-dialkyl substitution of paracetamol, in contrast to 3-monoalkyl substitution, prevented the paracetamol-induced toxicity in freshly isolated rat hepatocytes without having any effect on its cytochrome P-450 mediated bioactivation to reactive N-acetyl-p-benzoquinone imines (NAPQI). In the present study the mechanism of this prevention of toxicity, with special emphasis on oxidative stress, was studied in more detail in freshly isolated rat hepatocytes, using paracetamol, 3-methyl-, 3,5-dimethyl-paracetamol, synthetic NAPQI and 3,5-dimethyl-NAPQI. 3-Methyl-paracetamol was found to induce glutathione (GSH) depletion, lipid-peroxidation and cytotoxicity in hepatocytes to the same extent as paracetamol. 3,5-Dimethyl-paracetamol, however, even when added in a ten-fold higher concentration when compared to paracetamol, did not induce any of these effects. Similar differences of toxicity were observed between NAPQI and 3,5-dimethyl-NAPQI; 3,5-dimethyl-NAPQI, in contrast to NAPQI, did not reduce protein thiol levels, did not induce GSH depletion, lipid-peroxidation nor cytotoxicity. Only after artificial depletion of GSH levels in the hepatocytes by DEM or BCNU, 3,5-dimethyl-NAPQI was cytotoxic. This effect was accompanied by depletion of protein thiol levels, but not by lipid-peroxidation. Addition of the disulfide reducing agent, dithiothreitol, prevented the artificially created cytotoxicity of 3,5-dimethyl-NAPQI. It is concluded that prevention of paracetamol-induced toxicity by 3,5-dialkyl substitution is primarily due to prevention of irreversible GSH-depletion, presumably caused by the inability of 3,5-dialkyl-NAPQI to conjugate with thiols. As a result, the GSH-dependent cellular defense mechanism against potential oxidative cellular injury by 3,5-dialkyl-NAPQI is left unimpaired. Our observations indicate that a compound, not capable of covalent binding to thiol groups of proteins, can induce toxicity solely as a result of protein thiol oxidation without inducing lipid-peroxidation.     

The effects of fructose on adenosine triphosphate depletion following mitochondrial dysfunction and lethal cell injury in isolated rat hepatocytes

Mitochondrial injury in aerobic mammalian cells is associated with a rapid depletion of adenosine triphosphate (ATP) which occurs prior to the onset of lethal cell injury. In this report, the relationships between ATP depletion and lethal cell injury were examined in rat hepatocytes using oligomycin as a model mitochondrial toxicant and fructose as an alternative carbohydrate source for glycolysis. Oligomycin was more potent in causing lethal cell injury in hepatocytes isolated from fasted animals than cells from fed animals. The onset of cell injury (leakage of lactate dehydrogenase) in cells from fed animals correlated with the depletion of stored glycogen and ATP. The degree and time course profile of oligomycin-induced ATP depletion could be duplicated with 50 mM fructose alone in hepatocytes from fasted animals; however, fructose did not cause lethal cell injury. Oligomycin caused marked accumulation of adenosine monophosphate (AMP) and inorganic phosphate (Pi) and a conservation of adenine nucleotides. In contrast, fructose (50 mM) caused a decrease in Pi, no persistent change in AMP, and a depletion of the adenine nucleotide pool. Fructose, at concentrations greater than 1.0 mM, protected hepatocytes from oligomycin-induced toxicity. Blockade of mitochondrial ATP synthesis with oligomycin resulted in massive ATP depletion. In the presence of oligomycin, 5.0 mM fructose maintained cellular ATP content similar to that of control cells, whereas 50 mM fructose did not, demonstrating the biphasic effect of increasing fructose concentrations on cellular ATP content. Fructose-induced protection of hepatocytes from oligomycin toxicity was due to glycolytic fructose metabolism as hepatocytes incubated with iodoacetate (30 microM), fructose, and oligomycin had reduced viability and ATP content. In conclusion, interruption of mitochondrial ATP synthesis leads to marked ATP depletion and lethal cell injury. Cell injury is clearly not due to ATP depletion alone since increased glycolytic ATP production from either glycogen or fructose can maintain cell integrity in the absence of mitochondrial ATP synthesis and at low cellular ATP levels.     

A 43-kDa protein from the leaves of the herb Cajanus indicus L. modulates chloroform induced hepatotoxicity in vitro

A 43-kDa protein isolated from the leaves of the herb Cajanus indicus L. has been shown to possess a protective role against drug- and toxin- induced hepatotoxicity both in vivo and in vitro. The current study was conducted to evaluate its protective action against chloroform (CHCl3)-induced cytotoxicity in hepatocytes. Cellular viability and biochemical parameters such as glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) release from the cells were measured. In addition, the antioxidant effect of the protein was investigated from the DPPH radical scavenging assay and by determining the levels of the antioxidant enzyme catalase (CAT), cellular reserves of reduced glutathione (GSH), and lipid peroxidation end products (measured as TBARS). Treatment of the cells with CHCl3 decreased cellular viability and increased GPT and LDH. Cells treated with the protein before and immediately after CHCl3 application showed a marked improvement in their viability and reduced leakage of GPT and LDH. The levels of CAT and GSH, which were diminished in cells treated with CHCl3, were restored by protein treatment. CHCl3 induced enhancement of lipid peroxidation in hepatocytes was significantly reduced by protein treatment. Results of the DPPH assay with the protein showed its radical scavenging activity. This data suggests that the protein possesses protective activity against CHCl3-induced cytotoxicity in hepatocytes and protects against CHCl3-induced hepatic damage.     

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.     

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.     

Novel piperidine derivatives: inhibitory properties towards cytochrome P450 isoforms, and cytoprotective and cytotoxic characteristics

The ability of a series of eight piperidine derivatives, substituted at positions 1, 3 and 4, to inhibit P450-dependent metabolism of specific substrates, is reported. Five different P450 isoforms (1A1, 1A2, 2B1, 2E1 and 3A1) in differentially induced rat liver microsomes were used for this purpose. From the results it is concluded that compound 2 was the most potent and moreover, highly selective inhibitor for P4502B1 with an IC(50) of 2.5 μM. Compound 3 appeared to have high selectivity for P4501A1 but not for P4501A2 (IC(50)s 80 and > 1000 μM, respectively). P4502B1 was found to be the most susceptible P450 isoform for inhibition by compounds 2, 3 and 6, while P4502E1 was largely insensitive to the inhibitory properties of all piperidine derivatives. A preliminary SAR study for the cytotoxicity, cytoprotective and P450 inhibitory properties of the piperidine derivatives, was also attempted. Using freshly isolated rat hepatocytes, the toxicity of the compounds was estimated and expressed as lactate dehydrogenase (LDH) leakage, lipid peroxidation (LPO) levels and GSH depletion. Considering the P450 inhibition and cytotoxicity results, compounds 2 and 3 were tested for possible protective activity against paracetamol-induced cytotoxicities. It was found that compound 2 protects completely against LDH leakage and LPO caused by paracetamol in rat hepatocytes isolated from β-naphthoflavone (β-NF) pretreated rats. It is concluded that the piperidine structures studied proved to be potentially valuable lead compounds for the design of potent and selective P450 inhibitors and for non-toxic cytoprotective agents as well.     

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.     

Preventive effects of fructose and N‐acetyl‐L‐cysteine against cytotoxicity induced by the psychoactive compounds N‐methyl‐5‐(2‐aminopropyl)benzofuran and 3,4‐methylenedioxy‐N‐methamphetamine in isolated rat hepatocytes

Psychoactive compounds, N‐methyl‐5‐(2‐aminopropyl)benzofuran (5‐MAPB) and 3,4‐methylenedioxy‐N‐methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N‐acetyl‐l ‐cysteine (NAC) on 5‐MAPB‐ and MDMA‐induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration‐dependent (0–4 mm ) and time‐dependent (0–3 hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5‐MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or NAC at a concentration of 2.5 mm prevented 5‐MAPB?/MDMA‐induced cytotoxicity. In addition, the exposure of hepatocytes to 5‐MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5‐MAPB?/MDMA‐induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5‐MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress.     

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.