Cadmium-induced apoptosis of hepatocytes is not associated with death receptor-related caspase-dependent pathways in the rat

Cadmium (Cd) is a heavy metal of considerable environmental and occupational concern. The liver is the major target organ of Cd toxicity that follows from repeated exposure to Cd. The aim of this study was to investigate the mechanism of cell death of Cd-induced hepatotoxicity in a rat model. Eighteen adult male Sprague–Dawley (SD) rats were injected daily with a dose of Cd acetate (30 μM/kg body weight, subcutaneously). After 1, 2 and 7 days rats were euthanized and blood and liver tissues were sampled for analysis. Biochemical analyses of the level of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were undertaken. Histopathological and Western blot analyses for liver cellular damage and cell death were also performed. The results for the Cd-treated group of animals were compared to those from 12 control rats. The serum AST/ALT levels increased significantly 24 h after CD exposure. From the Western blot analyses, activation of Bid, independent of caspase-8 was seen and Bax induced the release of cytochrome c into the cytosol from mitochondria in a dose-dependent manner. The level of Bcl-2 was decreased. Eventually, caspase-9 and caspase-3 were activated, and poly(ADP-ribose) polymerase (PARP) was cleaved in a dose-dependent manner. A histopathological analysis and DNA fragmentation test showed apoptotic cell death of the hepatocytes increased over time. These results suggest that Cd-induced liver cell apoptosis in the rat, over a period of 7 days, may not be related to the death-receptor pathway. Moreover, apoptosis is dose-dependent and associated with the decrement of Bcl-2.     

Aspartame-induced apoptosis in PC12 cells

Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR.Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.     

Cadmium-induced apoptosis of hepatocytes is not associated with death receptor-related caspase-dependent pathways in the rat

Cadmium (Cd) is a heavy metal of considerable environmental and occupational concern. The liver is the major target organ of Cd toxicity that follows from repeated exposure to Cd. The aim of this study was to investigate the mechanism of cell death of Cd-induced hepatotoxicity in a rat model. Eighteen adult male Sprague–Dawley (SD) rats were injected daily with a dose of Cd acetate (30 μM/kg body weight, subcutaneously). After 1, 2 and 7 days rats were euthanized and blood and liver tissues were sampled for analysis. Biochemical analyses of the level of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were undertaken. Histopathological and Western blot analyses for liver cellular damage and cell death were also performed. The results for the Cd-treated group of animals were compared to those from 12 control rats. The serum AST/ALT levels increased significantly 24 h after CD exposure. From the Western blot analyses, activation of Bid, independent of caspase-8 was seen and Bax induced the release of cytochrome c into the cytosol from mitochondria in a dose-dependent manner. The level of Bcl-2 was decreased. Eventually, caspase-9 and caspase-3 were activated, and poly(ADP-ribose) polymerase (PARP) was cleaved in a dose-dependent manner. A histopathological analysis and DNA fragmentation test showed apoptotic cell death of the hepatocytes increased over time. These results suggest that Cd-induced liver cell apoptosis in the rat, over a period of 7 days, may not be related to the death-receptor pathway. Moreover, apoptosis is dose-dependent and associated with the decrement of Bcl-2.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses apoptosis and leads to hyperphosphorylation of p53 in rat hepatocytes

Inhibition of apoptosis of preneoplastic cells is thought to represent a major mechanism of action of tumor promoters. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent promoter of liver carcinogenesis in rodents, suppressed apoptosis in rat hepatocytes pretreated in vitro with an apoptogenic dose of UV light. This effect, which was also observable in DNA fragmentation analysis, coincided with a pronounced inhibition of the p53 increase usually seen after UV irradiation of rat hepatocytes. Interestingly, TCDD also led to a very minor but consistent enhancement of DNA fragmentation and to a slight increase in p53. Furthermore, TCDD resulted in a dose-dependent increase in p53 phosphorylation in intact cells. The concentration-response curves of the effects of TCDD on p53 phosphorylation and aromatic hydrocarbon receptor (AhR)-dependent induction of cytochrome P450 1A1 activity were almost superimposable, suggesting that TCDD induces p53 phosphorylation via an AhR-linked kinase activity. In an extract prepared from rat liver homogenate, 1 nM TCDD also stimulated p53 phosphorylation. Since the tyrosine kinase c-src was previously shown by others to be activated upon binding of TCDD to the AhR, extracts were pretreated with anti-src-antibodies. This treatment almost completely abrogated the effect of TCDD on p53 phosphorylation suggesting a key role for AhR-associated c-src. This mode of action may result in the observed suppression of the p53 response to apoptogenic UV irradiation, and may contribute to the inhibition of apoptosis.     

Induction of mitochondria-dependent apoptosis in HepG2 human hepatocellular carcinoma cells by timosaponin A-III

Timosaponin A-III (TSA-III), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. However, the molecular mechanism underlying the anticancer activity of TSA-III has not been fully elucidated. In this study, the apoptotic effects of TSA-III were investigated in HepG2 cells. Treatment with TSA-III significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis in HepG2 cells. This induction was associated with increased fluorescence intensity of Annexin V-FITC, activation of caspases, and altered expression of inhibitor of apoptosis protein (IAP) family members. In addition, TSA-III mediated mitochondrial dysfunction with the release of HtrA2/Omi, Smac/Diablo, and cytochrome c. These findings suggest that TSA-III induces mitochondria-mediated and caspase-dependent apoptosis in HepG2 cells by altering expression of the IAP family. Thus, TSA-III could possibly be used to treat other types of cancer with similar pathologic mechanisms.     

Allyl alcohol cytotoxicity in isolated rat hepatocytes: mechanism of cell death does not involve an early rise in cytosolic free calcium

 We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes. Received: 16 February 1994 / Accepted: 25 May 1994     

Mouse strain-dependent caspase activation during acetaminophen hepatotoxicity does not result in apoptosis or modulation of inflammation

The mechanisms of acetaminophen (APAP)-mediated hepatic oncotic necrosis have been extensively characterized. However, it was recently demonstrated that fed CD-1 mice have a transient caspase activation which initiates apoptosis. To evaluate these findings in more detail, outbred (Swiss Webster, SW) and inbred (C57BL/6) mice were treated with APAP with or without pan-caspase inhibitor and compared to the apoptosis model of galactosamine (GalN)/endotoxin (ET). Fasted or fed APAP-treated C57BL/6 mice showed no evidence of caspase-3 processing or activity. Interestingly, a minor, temporary increase in caspase-3 processing and activity (150% above baseline) was observed after APAP treatment only in fed SW mice. The degree of caspase-3 activation in SW mice after APAP was minor compared to that observed in GalN/ET-treated mice (1600% above baseline). The pancaspase inhibitor attenuated caspase activation and resulted in increased APAP-induced injury (plasma ALT, necrosis scoring). The caspase inhibitor did not affect apoptosis because regardless of treatment only < 0.5% of hepatocytes showed consistent apoptotic morphology after APAP. In contrast, > 20% apoptotic cells were observed in GalN/ET-treated mice. Presence of the caspase inhibitor altered hepatic glutathione levels in SW mice, which could explain the exacerbation of injury. Additionally, the infiltration of hepatic neutrophils was not altered by the fed state of either mouse strain. Conclusion: Minor caspase-3 activation without apoptotic cell death can be observed only in fed mice of some outbred strains. These findings suggest that although the severity of APAP-induced liver injury varies between fed and fasted animals, the mechanism of cell death does not fundamentally change.     

Oncotic necrosis and caspase-dependent apoptosis during galactosamine-induced liver injury in rats

The mode of cell death during galactosamine (Gal)-induced liver injury was originally thought to be oncotic necrosis but recently it was suggested to be apoptosis. Thus, the objective was to assess whether apoptosis and oncosis are sequential or independent events in the pathophysiology. In addition, the role of caspases in Gal-induced apoptotic signaling was investigated. A dose of 500 mg/kg Gal caused a time-dependent increase in plasma alanine transaminase (ALT) levels (24 h: 430 +/- 122 U/L) in female Sprague-Dawley rats. This was accompanied by processing of procaspase-3 and significant increases in hepatic and plasma caspase-3 activities. Using morphology and TUNEL staining, apoptotic and oncotic cells were quantitated. The number of apoptotic hepatocytes increased from 0.14% in controls to 5.4 +/- 1.0% 24 h after Gal treatment. In addition, the number of cells with oncotic morphology increased from 0 to 6.9% of total hepatocytes. Treatment with the pan-caspase inhibitor IDN-7314 (10 mg/kg) or pretreatment with uridine (1 g/kg), reduced all parameters of apoptosis to baseline. However, IDN-7314 administration did not affect plasma ALT activities and the number of oncotic cells at 6 h and only modestly reduced these parameters at 24 h. Uridine, on the other hand, prevented the increase of plasma ALT levels and reduced the number of apoptotic and oncotic cells by >80%. In conclusion, galactosamine-induced hepatocellular apoptosis in rats is caspase dependent. Although some of the apoptotic cells may undergo secondary necrosis, a significant number of hepatocytes die through oncotic necrosis as an independent mechanism of cell death.     

Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman™ Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.     

Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes

Vimang is an aqueous extract from stem bark of Mangifera indica L. (Mango) with pharmacological properties. It is a mixture of polyphenols (as main components), terpenoids, steroids, fatty acids and microelements. In the present work we studied the cytotoxic effects of Vimang on rat hepatocytes, possible interactions of the extract with drug-metabolizing enzymes and its effects on GSH levels and lipid peroxidation. No cytotoxic effects were observed after 24 h exposure to Vimang of up to 1000 μg/mL, while a moderate cytotoxicity was observed after 48 and 72 h of exposure at higher concentrations (500 and 1000 μg/mL). The effect of the extract (50–400 μg/mL) on several P450 isozymes was evaluated. Exposure of hepatocytes to Vimang at concentrations of up to 100 μg/mL produced a significant reduction (60%) in 7-methoxyresorufin-O-demethylase (MROD; CYP1A2) activity, an increase (50%) in 7-penthoxyresorufin-O-depentylase (PROD; CYP2B1) activity, while no significant effect was observed with other isozymes. To our knowledge, this is the first report regarding the modulation of the activity of the P450 system by an extract of Mangifera indica L. The antioxidant properties of Vimang were also evaluated in t-butyl-hydroperoxide-treated hepatocytes. A 36-h pre-treatment of cells with Vimang (25–200 μg/mL) strongly inhibited the decrease of GSH levels and lipid peroxidation induced by t-butyl-hydroperoxide dose- and time-dependently.     

Tetrandrine-induced apoptosis in rat primary hepatocytes is initiated from mitochondria: caspases and endonuclease G (Endo G) pathway

Tetrandrine, a bisbenylisoquinoline alkaloid isolated from the dried root of Stephenia tetrandra (S Moore), possesses a remarkable pharmacological profile. However, the mechanisms of tetrandrine hepatotoxicity remain to be elucidated. In this study, we first proved apoptosis and mitochondrial dysfunction induced by tetrandrine in Sprague-Dawley rat liver in vivo. By further assuming apoptosis as an important mechanism in tetrandrine-induced hepatotoxicity, we focused on mitochondria-initiated apoptosis in primary hepatocytes isolated from Sprague-Dawley male rats. Tetrandrine treatment led to significant release of cytochrome c and downregulation of Bcl-X(L) accompanied by caspase 3 activation, and ultimately, DNA fragmentation. Caspase 3 activation was markedly inhibited by cyclosporin A (CsA) and Ac-DEVD-CHO. Furthermore, Endo G, a caspase-independent apoptotic protein, was detected for its expression and DNase activity. CsA blocked the release both of Endo G and cytochrome c significantly. Additionally, the generation of reactive oxygen species (ROS) increased in a time-dependent manner corresponding with a fall in intracellular GSH content after 10 microM tetrandrine treatment in 4h. Tetrandrine also induced mitochondrial dysfunction indicated by transition of mitochondrial transmembrane potential and decrease of intracellular ATP level. The findings indicated that the caspase-dependent mitochondrial apoptosis pathway was primarily involved in tetrandrine-induced apoptosis in rat primary hepatocytes. In addition, a caspase-independent pathway indicated by Endo G also contributed to apoptosis caused by tetrandrine. Meanwhile, ROS was proved an important inducer in this apoptosis process.     

N-acetylcysteine protects against fluoride-induced oxidative damage in primary rat hepatocytes

Fluoride induces the overproduction of free radicals, which might in turn affect various biochemical parameters. Therefore, the aim of this study was to elucidate the role of N-acetylcysteine (NAC) in decreasing fluoride-induced oxidative stress. The fluoride intoxicated (0.002; 0.082; 0.164 mmol/l) rat hepatocytes was pre-treated (60 min) and simultaneously treated with NAC (1 mmol/l). The resulting levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and intracellular reduced glutathione (GSH) were measured along with the total antioxidant status (TAS) to determine whether NAC treatment reduced cell damage and/or the antioxidant state. These results suggest that NAC pre-treatment provides protection against fluoride-induced oxidative stress in hepatocytes.     

Rifampicin exacerbates isoniazid-induced toxicity in human but not in rat hepatocytes in tissue-like cultures

BACKGROUND AND PURPOSE: Rifampicin has been extensively reported to exacerbate the hepatotoxicity of isoniazid in patients with tuberculosis. However, this was controversially claimed by previous reports using rat models. This study evaluated the effect of rifampicin on isoniazid-induced hepatocyte toxicity by using human and rat hepatocytes in tissue-like culture.EXPERIMENTAL APPROACH: Hepatocytes in tissue-like gel entrapment were used to examine isoniazid toxicity, as shown by cell viability, intracellular glutathione content and albumin secretion. For demonstration of the differential effects of rifampicin on human and rat hepatocytes, induction by rifampicin of cytochrome P450 (CYP) 2E1, a major enzyme associated with isoniazid hepatotoxicity, was detected by 4-nitrocatechol formation and RT-PCR analysis.KEY RESULTS: Rifampicin (12 microM) enhanced isoniazid-induced toxicity in human hepatocytes but not in rat hepatocytes. Enhanced CYP 2E1 enzymic activity and mRNA expression were similarly detected in human hepatocytes but not in rat hepatocytes. Both rat and human hepatocytes in gel entrapment were more sensitive to isoniazid treatment compared with the corresponding hepatocytes in a monolayer culture.CONCLUSIONS AND IMPLICATIONS: The difference in induction of CYP 2E1 by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatocyte toxicity by rifampicin, with more significant toxicity in gel entrapment than in monolayer cultures. Thus, human hepatocytes in tissue-like cultures (gel entrapment) could be an effective model for hepatotoxicity research in vitro, closer to the in vivo situation.     

Time-course of cadmium-induced acute hepatotoxicity in the rat liver: the role of apoptosis

Exposure to toxic metals and pollutants is a major environmental problem. Cadmium is a metal causing acute hepatic injury but the mechanism of this phenomenon is poorly understood. In the present study, we investigated the mechanism and time-course of cadmium-induced liver injury in rats, with emphasis being placed on apoptosis in parenchymal and nonparenchymal liver cells. Cadmium (3.5 mg/kg body weight) was injected intraperitoneally and the rats were killed 0, 9, 12, 16, 24, 48 and 60 h later. The extent of liver injury was evaluated for necrosis, apoptosis, peliosis, mitoses and inflammatory infiltration in hematoxylin–eosin-stained liver sections, and by assaying serum enzyme activities. The number of cells that died via apoptosis was quantified by TUNEL assay. The identification of nonparenchymal liver cells and activated Kupffer cells was performed histochemically. Liver regeneration was evaluated by assaying the activity of liver thymidine kinase and by the rate of ³H-thymidine incorporation into DNA. Both cadmium-induced necrotic cell death and parenchymal cell apoptosis showed a biphasic elevation at 12 and 48 h and peaked at 48 and 12 h, respectively. Nonparenchymal cell apoptosis peaked at 48 h. Peliosis hepatis, another characteristic form of liver injury, was first observed at 16 h and, at all time points, closely correlated with the apoptotic index of nonparenchymal liver cells, where the lesion was also maximial at 48 h. Kupffer cell activation and neutrophil infiltration were minimal for all time points examined. Based on thymidine kinase activity, liver regeneration was found to discern a classic biphasic peak pattern at 12 and 48 h. It was very interesting to observe that cadmium-induced liver injury did not involve inflammation at any time point. Apoptosis seems to be a major mechanism for the removal of damaged cells, and constitutes the major type of cell death in nonparenchymal liver cells. Apoptosis of nonparenchymal cells is the basis of the pathogenesis of peliosis hepatis. The first peaks of necrosis and parenchymal cell apoptosis seem to evolve as a result of direct cadmium effects whereas the latter ones result from ischemia.     

Transfection of adult primary rat hepatocytes in culture

The use of adult primary hepatocytes in culture is of importance for the understanding of hepatic processes at the cellular and molecular levels, and the possibility to employ transient transfection of reporter constructs is invaluable for mechanistic studies on hepatic gene regulation. Although frequently used, there is a lack of reports addressing optimization and characterization of transfection of primary rodent hepatocytes. Here, we have shown that the efficiency of biochemical transfection reagents varies significantly and that Lipofectamine2000 was a superior transfection reagent for adult primary rat hepatocytes when using luciferase reporter vectors. The efficiency increased when the cells were allowed ample time to adapt to the in vitro milieu. Cotransfection of a second reporter gene indicated a risk for promoter competition, and we found that relating reporter activity to total cellular protein content gave consistent and reliable results. Differentiation of the cells, achieved by including biomatrix from the Engelbreth-Holm-Swarm mouse sarcoma in the culture system, was to a larger extent required for hormonal/drug responses of transfected constructs than for responses of endogenous genes and assured responses of transfected constructs. Dexamethasone (Dex) is most often included in hepatocyte culture media, but we could not demonstrate a general beneficial effect of Dex on expression of luciferease reporter contructs. Using the established protocol, we have demonstrated responses of transfected constructs to growth hormone, glucocorticoid and LXR stimuli.     

Possible involvement of active oxygen species in selenite toxicity in isolated rat hepatocytes

Mechanisms of selenite cytotoxicity were examined using isolated rat hepatocytes. When selenite was added to a suspension of rat hepatocytes, intracellular reduced glutathione (GSH) was decreased and the oxygen consumption rate was increased. Subsequently, thiobarbituric acid-reactive substances (TBA-RS) and lactate dehydrogenase (LDH) leakage were increased. A ferric iron chelator, desferrioxamine (DF), and a synthetic Superoxide dismutase (SOD) mimic, desferrioxamine manganese (DFMn), reduced the selenite toxicity. These results suggest that Superoxide anion and its reactive metabolites such as the hydroxyl radical may be involved in the cytotoxicity of selenite.     

The peroxisome proliferator nafenopin does not suppress hepatocyte apoptosis in guinea-pig liver in vivo nor in human hepatocytes in vitro

In rats and mice, nafenopin is a nongenotoxic hepatocarcinogen, which induces hepatic DNA synthesis and enzyme induction both in vivo and in hepatocyte cultures in vitro. However, humans and guinea-pigs are considered to be non-responsive to the liver growth effects of peroxisome proliferators (PPs). The ability to stimulate cell replication coupled with the ability to suppress apoptosis is thought to underpin the carcinogenicity of nongenotoxic carcinogens such as PPs. Previous studies in this laboratory have shown that in rats in vivo and in vitro nafenopin suppressed spontaneous hepatocyte apoptosis and that induced by the physiological negative growth regulator transforming growth factors β1 (TGFβ1). In addition nafenopin suppressed apoptosis in cultured hepatocytes from guinea-pig and hamster. The effects of PPs on apoptosis in human hepatocyte cultures is not known. To correlate these previous in vitro findings to the known species differences in hepatocarcinogenicity of PPs we have investigated the effects of nafenopin on guinea-pig liver growth in vivo. Also, we have examined the effects of nafenopin on apoptosis in cultures of human hepatocytes, a valuable model for human risk assessment. Nafenopin did not inhibit either spontaneous or TGFβ1 induced apoptosis in human hepatocytes in vitro. Administration of nafenopin to guinea-pigs in vivo produced none of the changes seen previously in responsive species, such as rats and mice. There was no change in liver/body weight ratio, peroxisomal volume of hepatocytes or DNA synthesis as determined by incorporation of bromodeoxyuridine and there was no suppression of apoptosis. The lack of response to nafenopin in guinea-pigs in vivo and human hepatocytes in vitro provides further evidence that these species may be refractory to the liver growth effects of PPs despite the ability of guinea-pigs and humans to respond to PPs by alterations in lipid metabolism. The data presented add to our overall understanding of species differences in response to the PP class of rodent nongenotoxic carcinogens. Received: 9 June 1998 / Accepted: 21 September 1998     

Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes

AIM: To investigate the mitochondria-initiated apoptosis pathway involved in Carbon tetrachloride (CCl4) hepatotoxicity in vitro. METHODS: Several cytotoxicity endpoints, including WST-8 metabolism, lactate dehydrogenase leakage and morphological changes, were examined. The 5,5'-dithio-bis(2-nitrobenzoic acid) reaction was used to measure reduced glutathione level, and the malondialdehyde level was determined using the thiobarbituric acid assay. The release of cytochrome c and Bcl-X(L) was detected by Western blot. Caspase-3 activity was measured using the fluorogenic substrate Ac-DEVD-AMC. DNA fragmentation was used to evaluate cell apoptosis. RESULTS: A time- and dose-dependent decrease in cellular glutathione content was observed, along with a concomitant increase in malondialdehyde levels following the application of CCl4. Caspase 3 activity was stimulated at all doses of CCl4, with the most significant activation at 3 mmol/L. Cytochrome c was released obviously after CCl4 treatment. A time-dependent decrease in Bcl-X(L) expression was observed. DNA fragmentation results revealed apoptosis and necrosis following CCl4 treatment. CONCLUSION: Oxidative damage is one of the essential mechanisms of CCl4 hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.     

Glutathione dependent detoxication in adult rat hepatocytes under various culture conditions

In order to obtain more information concerning the effects of culture and medium conditions on the glutathione dependent detoxication system in hepatocyte cultures, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were studied in both pure cultures of adult rat hepatocytes and their co-cultures with rat epithelial cells. Cells were isolated either with an oxygen saturated Krebs Henseleit buffer (KHB) or with a non-gassed Hepes buffer. As medium conditions, additions of 10% fetal calf serum (FCS), 25 mM nicotinamide, 0.1 M selenium and 2% dimethylsulphoxide, respectively, to the culture medium were examined. It was found that co-cultures of rat hepatocytes can cope better with oxidative stress than pure cultures do. This conclusion was reached from the following observations. When oxygenated KHB was used as isolating buffer, GR and GPx activities increased during the first days of pure culture and then slowly decreased. This was observed for all the medium conditions studied and no significant differences between the different media could be observed. For co-cultures, however, after some initial variations GR and GPx activities reached stabilized levels which were not only significantly lower than those observed for pure cultures, but were also maintained throughout the whole culture period. Supplementation of the medium had no effect on these findings with the exception of high GPx activities when Se was added to the co-culture medium. When Hepes buffer with a low oxygen content was used in cell isolation, pure cultures showed significantly lower GR and GPx activities than those first mentioned. Both approached the values measured for cocultures. The shape of the enzymatic activity curve as a function of culture time remained essentially unchanged. In co-cultures no significant differences could be observed in GPx activities when both perfusion techniques were involved, with the exception of the measurements done during the first 2 days of co-culture. Using the non-gassed Hepes buffer instead of the oxygenated KHB had no statistical effect on the GSH content of the hepatocytes in either of the two culture systems.