Protected HepG2 Cells Research Articles

Silymarin Prevents Palmitate‐Induced Lipotoxicity in HepG2 Cells: Involvement of Maintenance of Akt Kinase Activation

Whereas adipocytes have a unique capacity to store excess free fatty acids in the form of triglyceride in lipid droplets, non-adipose tissues, such as liver, have a limited capacity for storage of lipids. Saturated long-chain fatty acids, such as palmitate, are the major contributors to lipotoxicity. Silymarin is a mixture of flavonolignans, extracted from the milk thistle (Silibum marianum). Its hepatoprotective properties have been studied both in vitro and in vivo; however, its effect on palmitate-induced lipotoxicity has not been investigated. The objective of this study was to investigate (i) whether silymarin could protect HepG2 cells from palmitate-induced cell death in an in vitro model, and (ii) possible mechanisms involved in this hepatoprotective role of silymarin. HepG2 cells were treated with palmitate in the absence or presence of silymarin and supernatants or cell lysates were collected at varying time-points. Cell death was assayed by measuring DNA fragmentation, caspase-3 activity and lactate dehydrogenase release. Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenals. Akt kinase activity was also measured. Incubation with palmitate caused significant death in HepG2 cells. Palmitate incubation did not cause significant changes in reactive oxygen species production or intracellular glutathione content, but markedly inhibited Akt kinase activity. Pre-treatment of HepG2 cells with silymarin prevented palmitate-induced inhibition of Akt kinase activity and attenuated cell death. Our results suggest that silymarin may be an effective agent in protecting hepatocytes from saturated fatty acids-induced cell death. These data also provide a further rationale for exploration of the use of silymarin in the treatment of non-alcoholic steatohepatitis.

Reduced mitochondrial coenzyme Q10 levels in HepG2 cells treated with high-dose simvastatin: A possible role in statin-induced hepatotoxicity?

Lowering of low-density lipoprotein cholesterol is well achieved by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). Statins inhibit the conversion of HMG-CoA to mevalonate, a precursor for cholesterol and coenzyme Q10 (CoQ 10). In HepG2 cells, simvastatin decreased mitochondrial CoQ 10 levels, and at higher concentrations was associated with a moderately higher degree of cell death, increased DNA oxidative damage and a reduction in ATP synthesis. Supplementation of CoQ 10, reduced cell death and DNA oxidative stress, and increased ATP synthesis. It is suggested that CoQ 10 deficiency plays an important role in statin-induced hepatopathy, and that CoQ 10 supplementation protects HepG2 cells from this complication.

Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage

Common sage ( Salvia officinalis L., Lamiaceae) is an aromatic and medicinal plant well known for its antioxidant properties. Some in vivo studies have shown the biological antioxidant effects of sage. However, the intracellular antioxidant mechanisms of action are still poorly understood. In this study, we evaluated the cytoprotective effects of two sage extracts (a water and a methanolic extract) against tert-butyl hydroperoxide ( t-BHP)-induced toxicity in HepG2 cells. The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts, when co-incubated with the toxicant, protected significantly HepG2 cells against cell death. The methanolic extract, with a higher content of phenolic compounds than the water extract, conferred better protection in this in vitro model of oxidative stress with liver cells. Both extracts, tested in a concentration that protects 80% against cell death (IC 80), significantly prevented t-BHP-induced lipid peroxidation and GSH depletion, but not DNA damage assessed by the comet assay. The ability of sage extracts to reduce t-BHP-induced GSH depletion by 62% was probably the most relevant contributor to the observed cytoprotection. A good correlation between the above cellular effects of sage and the effects of their main phenolic compounds was found. When incubated alone for 5 h, sage extracts induced an increase in basal GSH levels of HepG2 cells, which indicates an improvement of the antioxidant potential of the cells. Compounds present in sage extracts other than phenolics may also contribute to this latter effect. Based in these results, it would be of interest to investigate whether sage has protective effects in suitable in vivo models of liver diseases, where it is known that oxidative stress is involved.

Protective effect of the flavonoid myricetin on glucose induced oxidative stress in Hep G2 cells

Myricetin is a naturally occurring flavonol with hydroxyl substitutions at the 3, 5, 7, 3, , 4, and 5, positions and has a hypoglycaemic and hypotrigyceridemic effect in diabetes mellitus. Hyperglicemia in diabetes can induce oxidative stress via several mechanisms. These include glucose autoxidation, the formation of advanced glycation end-products (AGE), and activation of the polyol pathway. Other circulating factors such free fatty acids and leptin, also contribute to increased reactive oxygen species (ROS). The major roles of GSH ( -glutamiylcysteinylglycine) are to maintain the intracellular redox balance and to eliminate ROS in cells. The aims of this study were 1) to investigate the effect of the flavonoid myricetin (Sigma) on the concentration of total glutathione (GSH) in Hep G2 cells and 2) to determine whether this flavonoid could protect thi cells against glucose-induced oxidative stress. Hep G2 cells was supplemented with with 0.5  M and 1.0  M of myricetin for 4 hours or 0.5  M and 1.0  M of myricetin plus 20  M glucose for same time. Concentration of GSH in cells was determined by Cayman, s GSH assay kit with enzymatic recycling method, using glutathione reductase. Exposure the Hep G2 cells to 0.5  M of myricetin for 4 hours at 37 degrees C resulted in significant increased of the GSH level (p 0.05) when compared with control cells. This data suggest that major features of glucose-induced hepatotoxicity are partially mediated by oxidative stress, and that myricetin in low concentration (0.5  M) protect Hep G2 cells against glucose toxicity affecting the glutathione level.

Macelignan protects HepG2 cells against tert‐butylhydroperoxide‐induced oxidative damage

In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.

Cycloheximide Protects HepG2 Cells from Serum Withdrawal-Induced Apoptosis by Decreasing p53 and Phosphorylated p53 Levels

Cycloheximide (CHX), an inhibitor of protein synthesis, has been reported to prevent cell death in a wide variety of cell types and produced by different apoptotic stimuli. However, the mechanisms by which CHX protects cells from apoptosis are still unclear. In this study, we investigated whether p53 plays a role in the protection by CHX against serum withdrawal-induced apoptosis. Deprivation of serum from the culture medium causes apoptosis in HepG2 cells, and CHX dramatically protects cells from death. p53, p21, and Bax protein levels were elevated, and cell cycle arrest was produced after serum withdrawal. CHX abolished this elevation of p53, p21, and Bax as well as the cell cycle arrest induced by serum deprivation. The p53 inhibitor pifithrin-alpha protects HepG2 cells against apoptosis induced by serum withdrawal. HepG2 cells expressing a dominant negative form of mutant p53 and Hep3B cells lacking p53 were resistant to serum withdrawal-induced apoptosis. Lowering of p53 by small interfering RNA protects HepG2 cells from serum withdrawal-induced apoptosis. p53 phosphorylation was induced by serum withdrawal and other chemotherapeutic reagents such as actinomycin D, doxorubicin, and etoposide. CHX decreases the levels of phosphorylated p53 (pp53) even in the presence of a proteasome inhibitor, which maintains the total p53 levels, whereas it does not affect the dephosphorylation of pp53. These results suggest the possibility that kinases that phosphorylate p53 might be affected by CHX administration. In summary, CHX protects HepG2 cells from serum withdrawal-induced apoptosis through inhibiting the synthesis of p53 and the phosphorylation of p53.

Phenolic compounds protect HepG2 cells from oxidative damage: Relevance of glutathione levels

In the present work, the potential hepatoprotective effects of five phenolic compounds against oxidative damages induced by tert-butyl hydroperoxide ( t-BHP) were evaluated in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damage in HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased levels of reduced glutathione (GSH). All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound's lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even greater importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damage induced in HepG2 cells. This could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.

Hypoxia protects HepG2 cells against etoposide-induced apoptosis VIA a HIF-1-independent pathway

Tumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. It also supports the invasiveness and metastatic potential of the tumor. However, few data are available on the transduction pathway set up under hypoxia and leading to this resistance against anti-cancer therapies. HIF-1, the main transcription factor activated by hypoxia, has been recently shown to participate to this process although its role as an anti- or a pro-apoptotic protein is still controversy. In this study, we showed that hypoxia protected HepG2 cells against etoposide-induced apoptosis. The effect of hypoxia on cell death was assayed by measuring different parameters of the apoptotic pathway, like DNA fragmentation, caspase activity and PARP-1 cleavage. The possible implication of HIF-1 in the anti-apoptotic role of hypoxia was investigated using HIF-1α siRNA. Our results indicated that HIF-1 is not involved in the hypoxia-induced anti-apoptotic pathway. Another transcription factor, AP-1, was studied for its potential role in the hypoxia-induced protection against apoptosis. Specific inhibition of AP-1 decreased the protection effect of hypoxia against etoposide-induced apoptosis. Together, all these data underline that hypoxia could mediate its anti-apoptotic role via different transcription factors depending on the cellular context and pro-apoptotic stimuli.

Protective effects of α1-acid glycoprotein and serum amyloid A on concanavalin A-induced liver failure via interleukin-6 induction by ME3738

We examined whether the 22β-methoxyolean-12-ene-3β,24(4β)-diol (ME3738)-mediated selective induction of interleukin-6 increased α1-acid glycoprotein and serum amyloid A expression, and whether these proteins protected against liver injury in vitro and in vivo. ME3738 treatment in male mice increased gene expression of α1-acid glycoprotein subtypes and serum amyloid A 2 genes, and plasma concentration of serum amyloid A. Treatment with α1-acid glycoprotein at 5 mg/animal or serum amyloid A at 0.03 and 0.1 mg/animal prior to concanavalin A administration reduced multifocal necrosis in the liver. Treatment with α1-acid glycoprotein and serum amyloid A, but not α1-antitrypsin, protected Hep G2 cells against cell injury. These results suggest that α1-acid glycoprotein and serum amyloid A, increased by ME3738-induced interleukin-6, might protect against concanavalin A-induced liver injury.

Silymarin Protects HepG2 Cells from Palmitate Induced Cell Death and Interleukin‐8 Production

BACKGROUND Nonalcoholic steatohepatitis (NASH) is part of the spectrum of nonalcoholic fatty liver disease (NAFLD). About 50% of NASH patients develop liver fibrosis, 15–30% develop cirrhosis, and 3% may progress to liver failure. Elevated serum free fatty acid (FFA) levels may play an etiologic role in NASH, and this is postulated to be a critical link between obesity, insulin resistance and the risk of NASH. Silymarin is a flavonoid, extracted from the milk thistle Silibum marianum. The hepatoprotective properties of silymarin have been reported both from in vitro and in vivo studies, but its possible role in preventing hepatotoxicity from FFA, specifically saturated FFA like palmitate, has not been evaluated. OBJECTIVES We investigated the effects of silymarin on palmitate induced cell death and cytokine (IL-8) production in hepatocytes. METHODS HepG2 cells were treated with palmitate with or without silymarin. Cell death was measured by DNA fragmentation ELISA and caspase-3 activity. IL-8 was assayed by ELISA. RESULTS Silymarin protected HepG2 cells from palmitate induced cell death and the protection was independent of its antioxidant property. Palmitate increased IL-8 from HepG2 cells and silymarin supplementation prevented this IL-8 production by palmitate. A caspase-3 inhibitor also attenuated palmitate induced cell death, but did not prevent palmitate induced IL-8, suggesting that prevention of IL-8 production by silymarin was cell death independent. CONCLUSIONS Silymarin may be an effective agent in saturated FFA induced liver injury/inflammation. We postulate that silymarin may have beneficial effects in the treatment of fatty liver disease such as NASH.

Hepatoprotective constituents of the edible brown alga Ecklonia stolonifera on tacrine-induced cytotoxicity in Hep G2 cells.

In this study, ethanolic extracts from 18 seaweed variants were assessed for hepatoprotective activity against tacrine-induced cytotoxicity in Hep G2 cells. Only one of these, Ecklonia stolonifera Okamura (Laminariaceae), a member of the brown algae, exhibited promising hepatoprotective activity. Bioassay-guided fractionation of the active ethyl acetate (EtOAc) soluble fraction obtained from the ethanolic extract of E. stolonifera, resulted in the isolation of several phlorotannins [phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5)]. Compounds 2 and 4 were determined to protect Hep G2 cells against the cytotoxic effects of tacrine, with EC50 values of 62.0 and 79.2 microg/mL, respectively. Silybin, a well characterized hepatoprotective agent, was used as a positive control, and exhibited an EC50 value of 50.0 microg/mL. It has been suggested that the phlorotannins derived from marine brown algae might prove useful sources in the development of novel hepatoprotective agents.

Hypoxia-inducible Factor-1-dependent Overexpression of Myeloid Cell Factor-1 Protects Hypoxic Cells against tert-Butyl Hydroperoxide-induced Apoptosis

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.

Hepatoprotective Effects of Irisolidone on tert-Butyl Hyperoxide-Induced Liver Injury

To clarify the hepatoprotective effects of kakkalide and its metabolite irisolidone by human fecal microflora, their effects on tert-butyl hydroperoxide (t-BHP)-injured HepG2 cells and mice were investigated. Irisolidone protected HepG2 cells against cytotoxicity induced by t-BHP. However, kakkalide did not protect cytotoxicity. When kakkalide 100 mg/kg was orally administered to mice injured by t-BHP, it significantly inhibited the increase in plasma alanine aminotransferase and aspartate aminotransferase activities by 84% and 85% of t-BHP-treated control group, respectively. The inhibitory effect of kakkalide is much more potent than that of silybin, a hepatoprotective agent. However, intraperitoneally administered kakkalide did not exhibit hepatoprotective activity. When irisolidone was intraperitoneally administered to mice, it exhibited potent hepatoprotective activity. Based on these findings, irisolidone can be hepatoprotective and kakkalide may be a prodrug transformed to irisolidone.

N-acetylcysteine does not protect HepG2 cells against acetaminophen-induced apoptosis.

Acetaminophen in large doses is well-known as hepatotoxic, and early therapy with N-acetylcysteine is frequently life-saving. However, in later stages of acetaminophen poisoning, treatment with N-acetylcysteine is not always effective. Although some of the pathways of acetaminophen toxicity and the effect of N-acetylcysteine have been elucidated, in depth information on this process is still lacking. Hepatoma-derived HepG2 cultured cells were exposed to acetaminophen (5 and 10 mM), with or without N-acetylcysteine (5 mM), for 24 and 48 hr. For the assessment of oxidative damage, apoptosis and necrosis, we followed redox status, glutathione content, nuclear fragmentation, phosphatidylserine externalization and ultrastructural changes. Variations in Ca2+ level and number of mitochondrial dense granules were also studied. Acetaminophen treatment of HepG2 cells caused oxidative damage and apoptosis. Significant decrease of cellular redox potential and glutathione content were time- and concentration-dependent. The protective effect of N-acetylcysteine was expressed by an increase of intracellular glutathione and of the level of metabolic reduction of the redox indicator Alamar Blue. The apoptogenic effect of acetaminophen was assessed by flow cytometry of annexin V binding, nuclear hypodiploidity, intracellular Ca2+, as well as by ultrastructural examination. Beyond 24 hr of acetaminophen exposure, necrosis was also noticed. We conclude that acetaminophen-induced oxidative damage in HepG2 cultured cells can be prevented by exposure to N-acetylcysteine. However, apoptosis, either early or late, here demonstrated, is not avoided by exposure to N-acetylcysteine. N-Acetylcysteine did not prevent acetaminophen-induced plasma membrane asymmetry, nuclear damage, alterations of Ca2+ homeostasis and ultrastructural changes.

Hypoxia and CoCl 2 protect HepG2 cells against serum deprivation- and t-BHP-induced apoptosis: a possible anti-apoptotic role for HIF-1

Hypoxia inducible factor-1 (HIF-1) is the main transcriptional factor activated by hypoxia. Besides the well-described role assigned to HIF-1 in the adaptation of cells to hypoxia, different recent data describe a possible role for HIF-1 in the modulation of apoptosis. However, this precise role is not yet clearly understood. In this study, chemical and physiological hypoxia, which were shown to induce HIF-1α stabilization and HIF-1 activation, were shown to inhibit apoptosis induced in HepG2 cells by two different pro-apoptotic conditions, serum deprivation- and t-BHP-induced oxidative stress. Indeed, hypoxia reduced DNA fragmentation, caspase activation, and PARP cleavage induced by these two pro-apoptotic conditions. These results are very interesting because it is a clear demonstration that hypoxia and chemical hypoxia have a direct protective effect on apoptotic cell death induced by two different stimuli. This observation is an important data in understanding how tumor growth can occur in challenging environmental conditions.

Oxidative stress, toxicology, and pharmacology of CYP2E1.

This review describes some of the biochemical and toxicological properties of CYP2E1, especially as it relates to alcohol metabolism and toxicity and the establishment of human hepatoma HepG2 cell lines that overexpress human CYP2E1. Ethanol, polyunsaturated fatty acids, and iron were found to be cytotoxic in HepG2 cells that overexpress CYP2E1. GSH appears to be essential in protecting HepG2 cells against the CYP2E1-dependent cytotoxicity, and GSH levels were elevated owing to a twofold increase in activity and expression of glutamate cysteine ligase. We suggest that this up-regulation of GSH synthesis was an adaptive response to attenuate CYP2E1-dependent oxidative stress and toxicity. Induction of a state of oxidative stress appears to play a central role in the CYP2E1-dependent cytotoxicity. Mitochondrial membrane potential decreased in the CYP2E1-expressing HepG2 cells, and this decrease shared similar characteristics with the developing toxicity. Alcohol-dependent liver injury is likely to be a multifactorial process involving several mechanisms. We believe that the linkage between CYP2E1-dependent oxidative stress, mitochondrial injury, and GSH homeostasis contribute to the toxic actions of ethanol on the liver.

The liver-selective nitric oxide donor O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) protects HepG2 cells against cytochrome P450 2E1-dependent toxicity.

HepG2 cells expressing CYP2E1 (E47 cells) are more susceptible to toxicity by arachidonic acid (AA) or after glutathione depletion with an inhibitor of glutamate-cysteine ligase, l-buthionine-(S,R)-sulfoximine (BSO), compared with control HepG2 cells (C34 cells). The ability of nitric oxide (NO) to protect against CYP2E1-dependent toxicity has not been evaluated. We therefore studied the ability of O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective NO donor, to protect against CYP2E1-dependent toxicity and compared this with protection by chemical NO donors. E47 cells incubated with V-PYRRO/NO produced NO, whereas C34 cells did not. Incubation of E47 cells with 50 microM AA or 100 microM BSO for 2 days resulted in a 50% loss of cell viability. VPYRRO/NO (1 mM) blocked this toxicity of AA and BSO by a mechanism involving NO release via CYP2E1 metabolism of VPYRRO/NO. NO scavengers hemoglobin and 2-(4-carboxophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide blocked the protective effects of V-PYRRO/NO. V-PYRRO/NO inhibited CYP2E1 activity and production of reactive oxygen species, whereas hemoglobin prevented these events. AA and BSO induced lipid peroxidation and decreased mitochondrial membrane potential; both of these effects were blocked by V-PYRRO/NO. Unlike V-PYRRO/NO, the chemical donors spermine/NO and (S)-nitroso-N-acetylpenicillamine release NO directly when added to the medium; however, they could partially protect against the CYP2E1-dependent toxicity. These results suggest that VPYRRO/NO protects HepG2 cells against CYP2E1-dependent toxicity through inhibition of CYP2E1-derived reactive oxygen species production and lipid peroxidation by the generated NO and that this compound may be valuable in protecting against CYP2E1-dependent toxicity via liver P450-specific generation of NO.

Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates.

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury and HO-1 is believed to be an important defense mechanism against such injury. Arachidonic acid (AA) and l-buthionine-( S,R)-sulfoximine (BSO), which lowers GSH levels, cause cytochrome P450 2E1 (CYP2E1)-dependent oxidative injuries in HepG2 cells (E47 cells). Treatment of E47 cells with 50 μM AA or 100 μM BSO for 48 h was recently shown to increase HO-1 mRNA, protein, and activity. The possible functional significance of this increase in protecting against CYP2E1-dependent toxicity was evaluated in the current study. The treatment with AA and BSO caused loss of cell viability (40 and 50%, respectively) in E47 cells. Chromium mesoporphyrin (CrMP), an inhibitor of HO activity, significantly potentiated this cytotoxicity. ROS production, lipid peroxidation, and the decline in mitochondrial membrane potential produced by AA and BSO were also enhanced in the presence of CrMP in E47 cells. Infection with an adenovirus expressing rat HO-1 protected E47 cells from AA toxicity, increasing cell viability and reducing LDH release. HO catalyzes formation of CO, bilirubin, and iron from the oxidation of heme. Bilirubin was not protective whereas iron catalyzed the AA toxicity. The carbon monoxide (CO) scavenger hemoglobin enhanced AA toxicity in E47 cells analogous to CrMP, whereas exposure to exogenous CO partially reduced AA toxicity and the enhanced AA toxicity by CrMP. Addition of exogenous CO to the cells inhibited CYP2E1 catalytic activity, as did overexpression of the rat HO-1 adenovirus. These results suggest that induction of HO-1 protects against CYP2E1-dependent toxicity and this protection may be mediated in part via production of CO and CO inhibition of CYP2E1 activity.

Adenovirus-mediated expression of Cu/Zn- or Mn-superoxide dismutase protects against CYP2E1-dependent toxicity

CYP2E1 induction by ethanol is one mechanism by which ethanol creates oxidative stress in the liver. The superoxide dismutases (SODs) are an important antioxidant enzyme defense system against reactive oxygen species (ROS). To investigate the protective role of SOD against CYP2E1-dependent toxicity, a transfected HepG2 cell line overexpressing CYP2E1 (E47 cells) was infected with adenoviral vectors containing Cu/Zn-SOD complementary DNA (cDNA) (Ad.SOD1) and Mn-SOD cDNA (Ad.SOD2). Forty-eight hours after infection, intracellular levels and activity of Cu/Zn-SOD and Mn-SOD were increased about 2- and 3-fold, respectively. Localization of the overexpressed Cu/Zn-SOD in the cytosol and Mn-SOD in the mitochondria was confirmed by assaying the levels and activity of SOD in the corresponding isolated fractions. Arachidonic acid (AA) plus iron-induced cell death was partially prevented in both Ad.SOD1- and Ad.SOD2-infected E47 cells. Overexpression of Cu/Zn-SOD and Mn-SOD also partially protected E47 cells from the increase in reactive oxygen production and lipid peroxidation and the loss of mitochondrial membrane potential induced by AA and iron. Infection with Cu/Zn-SOD and Mn-SOD also protected the E47 cells against AA toxicity or buthionine sulfoximine (BSO)-dependent toxicity. CYP2E1 levels and catalytic activity were not altered by overexpression of Cu/Zn-SOD or Mn-SOD. Cu/Zn-SOD in the cytosol and Mn-SOD in mitochondria each are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by pro-oxidants. In conclusion, these enzymes may be useful in the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.