CYP2E1-dependent toxicity and oxidative stress in HepG2 cells.

Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. To study the biochemical and toxicological actions of CYP2E1, our laboratory established HepG2 cell lines which constitutively overexpress CYP2E1 and characterized these cells with respect to ethanol toxicity. Addition of ethanol or an unsaturated fatty acid such as arachidonic acid or iron was toxic to the CYP2E1-expressing cells but not control cells. This toxicity was associated with elevated lipid peroxidation and could be prevented by antioxidants and inhibitors of CYP2E1. Apoptosis occurred in the CYP2E1-expressing cells exposed to ethanol, arachidonic acid, or iron. Removal of GSH caused a loss of viability in the CYP2E1-expressing cells even in the absence of added toxin or pro-oxidant. This was associated with mitochondrial damage and decreased mitochondrial membrane potential. Surprisingly, CYP2E1-expressing cells had elevated GSH levels, due to transcriptional activation of gamma glutamyl cysteine synthetase. Similarly, levels of catalase, alpha-, and microsomal glutathione transferase were also increased, suggesting that upregulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. While it is likely that several mechanisms contribute to alcohol-induced liver injury, the linkage between CYP2E1-dependent oxidative stress, mitochondrial injury, and GSH homeostasis may contribute to the toxic action of ethanol on the liver. HepG2 cell lines overexpressing CYP2E1 may be a valuable model to characterize the biochemical and toxicological properties of CYP2E1.

Synergistic toxicity of iron and arachidonic acid in HepG2 cells overexpressing CYP2E1.

Priming of the liver for ethanol-induced injury, by nutrients such as polyunsaturated fat and iron, plays a key role in alcoholic liver disease. The objective of this work was to evaluate the effect of the combination of Fe-nitrilotriacetic acid (Fe-NTA) and arachidonic acid (AA) on the viability of HepG2 cells (E47 cells) transfected to express human CYP2E1. Cells were plated, preloaded with arachidonic acid, washed, and exposed to Fe-NTA for variable periods. Fe-NTA (10 microM) or AA (5 microM) alone showed low toxicity to E47 cells (18 and 8%, respectively, at 24 h), whereas the combination produced synergistic injury (62% toxicity at 24 h). Exposure of cells not expressing any cytochrome P450 (P450), or HepG2-C3A4 cells (expressing CYP3A4) to 10 microM Fe-NTA plus 5 microM AA produced lower toxicity (14 and 32%, respectively), demonstrating a role for P450, and in particular CYP2E1, in the development of toxicity by exposure to Fe + AA. Lipid peroxidation was induced in the E47 cells exposed to Fe plus arachidonic acid and the synergistic toxicity was prevented by antioxidants, which also decreased lipid peroxidation. Damage to mitochondria plays a role in the CYP2E1-dependent toxicity of Fe + AA, because the mitochondrial transmembrane potential decreased early in the process, and cyclosporin A prevented the toxicity. Toxicity in E47 cells exposed to Fe + AA is mainly necrotic in nature. Hepatocytes from pyrazole-treated rats, with high levels of CYP2E1, were more sensitive to Fe + AA toxicity than were saline control hepatocytyes. The results presented suggest that low concentrations of Fe and AA can act as priming or sensitizing factors for CYP2E1-induced injury in HepG2 cells, and such interactions may play a role in alcohol-induced liver injury.

An ESR and HPLC-EC assay for the detection of alkyl radicals.

The correlation of lipid peroxidation with release of alkanes (RH) is considered a noninvasive method for the in vivo evaluation of oxidative stress. The formation of RH is believed to reflect a lipid hydroperoxide (LOOH)-dependent generation of alkoxyl radicals (LO*) that undergo beta-scission with release of alkyl radicals (R*). Alternatively, R* could be spin-trapped with a nitrone before the formation of RH and analyzed by ESR. Extracts from the liver and lung of CCl(4)- and asbestos-treated rats that were previously loaded with nitrones exhibited ESR spectra suggesting the formation of iso-propyl, n-butyl, ethyl, and pentyl radical-derived nitroxides. In biological systems, various nitroxides with indistinguishable ESR spectra could be formed. Hence, experiments with N-tert-butyl-alpha-phenylnitrone (PBN) for spin trapping of R* were carried out in which the nitroxides formed were separated and analyzed by HPLC with electrochemical detection (EC). The C(1-5) homologous series of PBN nitroxides and hydroxylamines were synthesized, characterized by ESR, GC-MS, and HPLC-EC, and used as HPLC standards. For in vivo generation and spin trapping of R*, rats were loaded with CCl(4) and PBN. The HPLC-EC chromatograms of liver extracts from CCl(4)-treated rats demonstrated the formation of both the nitroxide and hydroxylamine forms of PBN/*CCl(3), as well as the formation of a series of unidentified PBN nitroxides and hydroxylamines. However, formation of PBN adducts with retention times similar to these of the PBN/C(2-5) derivatives was not observed. In conclusion, we could not correlate the production of PBN-detectable alkyl radicals with the reported CCl(4)-dependent production of C(1-5) alkanes. We speculate that the major reason for this is the low steady-state concentrations of R* produced because only a small fraction of LO* undergo beta-scission to release R*.

Oxidation of the ketoxime acetoxime to nitric oxide by oxygen radical-generating systems.

Ketoximes undergo a cytochrome P450-catalyzed oxidation to nitric oxide and ketones in liver microsomes. In addition, nitric oxide synthase (NOS) can catalyze the oxidative denitration of the >C=N-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (acetoxime) and to assess the ability of NOS to catalyze the generation of nitric oxide/nitrogen monoxide (*NO) from acetoxime. Acetoxime was oxidized to NO2- (and NO3-) by microsomes enriched with several P450 isoforms, including CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermediate in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO2- from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acetoxime with production of NO2- and NO3-, whereas oxidation by superoxide or by H2O2 was inefficient. The results presented suggest a role for hydroxyl radical-like oxidants in the oxidation of acetoxime to nitric oxide. O-Acetylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5'-dimethyl-1-pyrroline-N-oxide/. OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime were inactive. These results suggest that the. OH-dependent oxidation of acetoxime initially proceeds via abstraction of a hydrogen atom from its hydroxyl group, as opposed to the oxidation of its >C=N- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO2- from acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a substrate or as an inhibitor of iNOS activity. These results indicate that reactive oxygen species play a key role in the oxidation of acetoxime to. NO by liver microsomes by a mechanism involving H abstraction from the OH moiety by hydroxyl radical-like oxidants and suggest the possibility that acetoxime may be an effective producer of. NO primarily in the liver by a pathway independent of NOS.

Ethanol inhibits the JAK-STAT signaling pathway in freshly isolated rat hepatocytes but not in cultured hepatocytes or HepG2 cells: evidence for a lack of involvement of ethanol metabolism.

OBJECTIVES: To understand the molecular mechanism underlying alcoholic liver injury, effects of acute ethanol on the Janus kinase-signal transducer and activator transcription factor (JAK-STAT) signaling in hepatic cells were studied. DESIGNS AND METHODS: Effects of acute ethanol on the JAK-STAT signaling in freshly isolated, cultured rat hepatocytes, and HepG2 cells were explored. RESULTS: Acute ethanol exposure inhibited IL-6- or IFN-activated STAT in freshly isolated hepatocytes but not in cultured hepatocytes, HepG2 cells, or HepG2 cells transfected with alcohol dehydrogenase (ADH) or cytochrome P450(2E1). The inhibitory action of ethanol in freshly isolated hepatocytes was not antagonized by the ADH inhibitor 4-methylpyrazole (4-MP). Acute exposure of hepatocytes to acetaldehyde or hydrogen peroxide did not suppress STAT activation. Further studies indicated that the loss of response to the inhibitory effect of ethanol was not due to hepatocyte proliferation and collagen contact. CONCLUSIONS: Freshly isolated hepatocytes are more susceptible to the inhibitory action of ethanol on the JAK-STAT signaling than cultured hepatocytes or HepG2 cells, which may be implicated in pathogenesis and progression of alcoholic liver disease.

Preparation and properties of S-nitroso-L-cysteine ethyl ester, an intracellular nitrosating agent.

In this report, a protocol for the preparation of the hydrochloride of S-nitroso-L-cysteine ethyl ester (SNCEE.HCl; 2) is presented. The synthesis of 2 has been targeted because S-nitroso-L-cysteine (SNC; 2b), which is extensively used for trans-S-nitrosation of thiol-containing proteins, has a limited ability of crossing cellular membranes. The nitrosothiol 2 was prepared via direct S-nitrosation of the hydrochloride of L-cysteine ethyl ester (CEE.HCl; 1a) with ethyl nitrite. 2 is relatively stable in crystal form and when neutralized to SNCEE (2a) in aqueous solutions treated with chelators of metal ions. Traces of metal ions, however, triggered the decomposition of 2a to nitric oxide and a S-centered radical, which were detected by ESR spectrometry. In contrast to 2b, 2a is a lipophilic compound that was taken up by human neutrophils. The latter process was paralleled by inhibition of the NADPH oxidase-dependent generation of superoxide anion radicals, presumably via reaction(s) of intracellular trans-S-nitrosation. Intracellular accumulation of S-nitrosothiols was observed with 2a but not with 2b. It is expected that the use of 2a will be advantageous when intracellular reactions of trans-S-nitrosation are to be studied.

Alcoholic liver disease and apoptosis.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol A. Casey and Amin Nanji. The presentations were (1) Mechanisms of apoptosis in alcoholic liver disease, by Amin A. Nanji; (2) Impaired receptor-mediated endocytosis: Its role in alcoholic apoptosis, by Carol A. Casey; (3) Toxicity of ethanol in HepG2 cells that express CYP2E1, by Arthur I. Cederbaum; (4) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis, by M. Adachi; and (5) Apoptosis in alcoholic hepatitis, by T. Takahashi.

Mitochondrial catalase and oxidative injury.

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.

Removal of glutathione produces apoptosis and necrosis in HepG2 cells overexpressing CYP2E1.

BACKGROUND: Previous studies have shown that addition of ethanol, iron, or arachidonic acid to HepG2 cells expressing CYP2E1 produced a loss in cell viability and caused apoptosis. These effects were enhanced when cellular reduced glutathione (GSH) levels were lowered by treatment with buthionine sulfoximine (BSO). Overexpression of CYP2E1 in HepG2 cells could produce toxicity even in the absence of added toxin after BSO treatment. Studies were carried out to characterize this CYP2E1-and BSO-dependent toxicity. METHODS: HepG2 cells expressing CYP2E1 were treated with BSO for 1 to 4 days, and various parameters associated with apoptosis and cell viability were assayed. RESULTS: Treatment of cells expressing CYP2E1 (E47 cells) with BSO resulted in apoptosis as well as necrosis. The apoptosis and necrosis were independent of each other. No toxicity was found with control HepG2 cells or HepG2 cells expressing CYP3A4 instead of CYP2E1 under these conditions. The antioxidant trolox partially prevented the apoptosis and necrosis, whereas diallylsulfide, a CYP2E1 inhibitor, was fully protective. The activity of caspase 3, but not caspases 1, 8, or 9, was increased in the BSO-treated E47 cells, and an inhibitor of caspase 3 prevented apoptosis. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity, because mitochondrial membrane potential was decreased and cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition, prevented the apoptosis and the necrosis. The fall in membrane potential was prevented by trolox and diallylsulfide, suggesting damage to the mitochondria by CYP2E1-derived reactive oxygen species. CONCLUSIONS: These results indicate the critical role of GSH in protecting against CYP2E1-mediated oxidative stress and that mitochondria may be a target for CYP2E1-derived reactive oxygen species, and suggest that interactions between CYP2E1, mitochondria, and altered GSH homeostasis may play a role in alcohol-induced liver injury.

Induction of catalase, alpha, and microsomal glutathione S-transferase in CYP2E1 overexpressing HepG2 cells and protection against short-term oxidative stress.

Induction of cytochrome P450 2E1 (CYP2E1) and the formation of reactive oxygen species (ROS) appear to be one of the mechanisms by which ethanol is hepatotoxic. Glutathione peroxidase and catalase detoxify H(2)O(2). Glutathione S-transferases (GST) provide protection from membrane lipid peroxidation, have GSH peroxidase activity, and reduce lipid hydroperoxides. Previous studies showed an up-regulation of GSH synthesis in CYP2E1 expressing HepG2 cells; this finding prompted an evaluation of the levels of other antioxidant exzymes. In CYP2E1 expressing cells, the alpha and microsomal GST messenger RNA (mRNA) are increased by 4- and 2-fold, respectively, and catalase protein and mRNA is increased by 2-fold. The increase in alpha and microsomal GST mRNA correlates with increased total enzymatic activity and is caused by increased transcription as evidenced by run-on transcription assays. In HepG2 cells transfected to express a different cytochrome P450, CYP3A4, there was an increase in alpha GST. However, in contrast to the CYP2E1 expressing cells, neither microsomal GST nor catalase were induced, suggesting some specificity for CYP2E1. In agreement with an increased antioxidant defense system, the sensitivity to added prooxidants such as menadione, antimycin A, H(2)O(2), and 4-hydroxynonenal was lower in the CYP2E1 expressing cells as compared with control cells. In conclusion, overexpression of CYP2E1 in HepG2 cells, besides elevating total GSH levels, also induces expression of catalase and alpha and microsomal GST. This induction confers resistance to the cells against several prooxidants and is suggested to reflect an adaptive response by the cells against CYP2E1-mediated oxidative stress.

Spin trapping agents (Tempol and POBN) protect HepG2 cells overexpressing CYP2E1 against arachidonic acid toxicity.

Polyunsaturated fatty acids such as arachidonic acid were previously shown to be toxic to HepG2 cells expressing CYP2E1 by a mechanism involving oxidative stress and lipid peroxidation. This study investigated the effects of the spin trapping agents Tempol and POBN on the arachidonic acid toxicity. Arachidonic acid caused toxicity and induced lipid peroxidation and mitochondrial membrane damage in cells overexpressing CYP2E1 but had little or no effect in control cells not expressing CYP2E1. The toxicity appeared to be both apoptotic and necrotic in nature. 4-Hydroxy-[2,2,6,6-tetramethylpiperidine-1-oxyl] (Tempol) and alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) protected against the decrease in cell viability and the apoptosis and necrosis. These spin traps prevented the enhanced lipid peroxidation and the loss of mitochondrial membrane potential. Tempol and POBN had little or no effect on cellular viability or on CYP2E1 activity at concentrations which were protective. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently damages the mitochondrial membrane leading to a loss in cell viability when the cells are enriched with arachidonic acid. Tempol and POBN, which scavenge various radical intermediates, prevent in this way the enhanced lipid peroxidation, mitochondrial dysfunction, and the cell toxicity. Since oxidative stress appears to play a key role in ethanol hepatotoxicity, it may be of interest to evaluate whether such spin trapping agents are useful candidates for the prevention or improvement of ethanol-induced liver injury.

Sodium salicylate increases CYP2E1 levels and enhances arachidonic acid toxicity in HepG2 cells and cultured rat hepatocytes.

Sodium salicylate and acetylsalicylic acid are drugs used as anti-inflammatory agents. Salicylate prevents nuclear factor-kappa B activation and can cause apoptosis. However, salicylate, a substrate of CYP2E1, is also an antioxidant and can scavenge reactive oxygen species. Experiments were carried out to evaluate whether salicylate can modulate CYP2E1-dependent toxicity. Addition of a polyunsaturated fatty acid such as arachidonic acid (AA) to HepG2 cells resulted in loss of cell viability, especially in cells expressing CYP2E1 (E47 cells). Toxicity was enhanced by the addition of 1 to 10 mM salicylate to the E47 cells but not to control HepG2 cells or HepG2 cells expressing CYP3A4. Salicylate alone was not toxic, and the enhanced toxicity by AA in the presence of salicylate was prevented by diallyl sulfide, a CYP2E1 inhibitor, and by the antioxidant (+/-)6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. Salicylate potentiated AA-induced lipid peroxidation in the E47 cells, a reaction blocked by diallyl sulfide. CYP2E1 levels were elevated by salicylate at concentrations (<5 mM), which did not increase CYP2E1 mRNA levels. This increase was associated with a decrease of CYP2E1 turnover by salicylate in the presence of cycloheximide. Salicylate also potentiated AA toxicity in hepatocytes isolated from pyrazole treated rats with high levels of CYP2E1 and from saline controls. In view of the potential role of CYP2E1 in contributing to alcohol-induced oxidative stress and liver injury, the potentiation of CYP2E1-dependent toxicity and the elevation of CYP2E1 levels by salicylate may be of clinical significance and merit caution in the use of salicylate and salicylate precursors such as acetylsalicylic acid with certain other drugs.

CYP2E1-dependent toxicity and up-regulation of antioxidant genes.

Induction of cytochrome P450 2E1 (CYP2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. Glutathione (GSH) is critical in preserving the proper cellular redox balance and for its role as a cellular protectant. The goal of the present study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2-E47 cells) that overexpress CYP2E1. Toxicity in the E47 cells was markedly enhanced after GSH depletion by buthionine sulfoximine (BSO) treatment. The antioxidant trolox partially prevented the apoptosis and necrosis, while diallylsulfide, a CYP2E1 inhibitor, was fully protective. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity. CYP2E1-overexpressing cells showed increases in total GSH levels, GSH synthetic rate and in gamma-glutamylcysteine synthetase (GCS) mRNA. This GCS increase was due to transcriptional activation of the GCS gene and could be blocked by certain antioxidants. Activity, protein and mRNA levels for other antioxidants such as catalase, alpha- and microsomal glutathione transferases were also increased in the E47 cells. Up-regulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. These oxidants are diffusable and were able to elevate collagen type I protein in a co-culture system consisting of the E47 cells + rat hepatic stellate cells. Such interactions between CYP2E1, mitochondria and altered GSH homeostasis, and elevation of collagen levels, may play a role in alcohol-induced liver injury.

Adenovirus-mediated overexpression of catalase in the cytosolic or mitochondrial compartment protects against cytochrome P450 2E1-dependent toxicity in HepG2 cells.

Cytochrome P450 2E1 (CYP2E1) is an effective producer of reactive oxygen species such as superoxide radical and hydrogen peroxide, which may contribute to the development of alcohol liver disease or cytotoxicity. To investigate the protective role of catalase against CYP2E1-dependent cytotoxicity, E47 cells, a transfected HepG2 cell line overexpressing CYP2E1, were infected with adenoviral vectors containing human catalase cDNA (AdCat) and catalase cDNA with a mitochondrial leader sequence (AdmCat). Forty-eight hours after infection with AdCat or AdmCat at a multiplicity of infection of 100, intracellular catalase protein was increased >2-fold compared with uninfected E47 cells and E47 cells infected with empty adenoviral vector (AdNull) as determined by Western blotting and catalase activity measurements. Overexpression of catalase in the cytosol (AdCat) and in mitochondria (AdmCat) was confirmed by confocal microscopy. Cell death caused by arachidonic acid plus iron was considerably suppressed in both AdCat- and AdmCat-infected E47 cells as determined by assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide absorbance, lactate dehydrogenase release, and morphology changes. AdCat- and AdmCat-infected cells were also more resistant to the loss of mitochondrial membrane potential and to the increase in lipid peroxidation induced by arachidonic acid and iron. This study indicates that catalase in the cytosol and catalase in mitochondria are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by oxidants that promote lipid peroxidation and suggests the possibility that such agents may be useful in protecting against the development of alcohol liver injury.

CYP2E1 degradation by in vitro reconstituted systems: role of the molecular chaperone hsp90.

One major mode of regulation of cytochrome P450 2E1 (CYP2E1) is at the posttranscriptional level, since many low-molecular-weight compounds stabilize the enzyme against proteolysis by the proteasome complex. In an in vitro system containing human liver microsomes, degradation of CYP2E1 in the microsomes required addition of the human liver cytosol fraction in a reaction sensitive to inhibitors of the proteasome complex. It is not clear how CYP2E1 in the microsomal membrane becomes accessible to the cytosolic proteasome. Since molecular chaperones play a role in protein folding and degradation, the possible role of heat shock proteins in CYP2E1 degradation by this reconstituted system was evaluated. Degradation of CYP2E1 required ATP; ATP-gammaS, a nonhydrolyzable analogue of ATP, did not catalyze CYP2E1 degradation by the cytosol fraction, indicating that ATP hydrolysis is required. Geldanamycin, a specific inhibitor of hsp90, inhibited the degradation of microsomal CYP2E1 by the cytosol fraction. Control experiments indicated that geldanamycin was not a substrate/ligand of CYP2E1 nor did it prevent microsomal lipid peroxidation, a process which increases CYP2E1 turnover. Inhibition by geldanamycin was prevented by molybdate. Both of these compounds have been shown to promote alterations in hsp90 structure and to modulate hsp90-protein interactions. The proteasome activity in the cytosol, as assayed by the cleavage of a fluorogenic peptide, was enhanced when ATP was added and inhibited by 30-40% by geldanamycin, effects that are similar, although less pronounced, to the degradation of CYP2E1 by the cytosol. Purified 20S proteasome could catalyze degradation of CYP2E1; however, in an assay using equal peptidase activity, the cytosol fraction was much more effective than the 20S proteasome in promoting CYP2E1 degradation. Immunodepletion of hsp90 from the cytosol resulted in prevention of the degradation of CYP2E1, a reaction that was reversed by the addition of pure hsp90 to this cytosol. These results suggest that in addition to the proteasome, the cytosol fraction contains other factors that modulate the efficiency of CYP2E1 degradation. The sensitivity to geldanamycin and molybdate and the immunodepletion experiments suggest that hsp90 is one of these factors that interact with CYP2E1 and/or with the proteasome to promote the degradation of this microsomal P450.

Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxygenase-2.

The ability of ethanol and arachidonic acid (AA), as inducers of oxidative stress and key factors in alcoholic liver disease, to up-regulate alpha 2 collagen type I (COL1A2) gene expression was studied in a hepatic stellate cell line overexpressing the ethanol-inducible cytochrome P450 2E1 (CYP2E1) (E5 cells). A time- and dose-dependent induction in COL1A2 mRNA by ethanol or AA was observed that was prevented by diallylsulfide, a CYP2E1 inhibitor. Nuclear run-on experiments showed transcriptional activation of the COL1A2 gene by ethanol and AA. Catalase abrogated the increase in COL1A2 mRNA suggesting an H(2)O(2)-dependent mechanism. Cyclooxygenase-2 (COX-2) levels and production of prostaglandin E(2) upon addition of AA were elevated in the E5 cells. Incubation with NS-398, a COX-2 inhibitor, blocked the effect of AA, but not of ethanol, on COL1A2 expression suggesting that CYP2E1 activates COX-2 expression, and the oxidation of AA by COX-2 is responsible for the increase in COL1A2. Activity of a reporter construct driven by -378 base pairs of the proximal promoter region of the COL1A2 gene increased in E5 but not control cells and was further increased by ethanol or AA. These experiments link CYP2E1-dependent oxidative stress to induction of COX-2 and the actions of ethanol and AA on activation of collagen gene expression in hepatic stellate cells.

CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase.

Induction of CYP2E1 (cytochrome P450 2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. CYP2E1 is a loosely coupled enzyme; formation of reactive oxygen species occurs even in the absence of added substrate. GSH is critical for preserving the proper cellular redox balance and for its role as a cellular protectant. Since cells must maintain optimal GSH levels to cope with a variety of stresses, the goal of this study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2) that overexpress CYP2E1. This study was prompted by the finding that toxicity in CYP2E1-overexpressing cells was markedly enhanced after GSH depletion by buthionine sulfoximine treatment. CYP2E1-overexpressing cells showed a 40-50% increase in intracellular H(2)O(2); a 30% increase in total GSH levels; a 50% increase in the GSH synthesis rate; and a 2-fold increase in gamma-glutamylcysteine synthetase heavy subunit (GCS-HS) mRNA, the rate-limiting enzyme in GSH synthesis. This GCS-HS mRNA increase was due to increased synthesis since nuclear run-on assays showed increased transcription in CYP2E1-expressing cells, and the GCS-HS mRNA decay after actinomycin D treatment was similar in CYP2E1-expressing cells and empty vector-transfected cells. The facts that treatment with GSH ethyl ester almost completely prevented the increase in GCS-HS mRNA and decreased H(2)O(2) levels and that transient transfection with catalase (but not manganese-superoxide dismutase) produced a decrease in GCS-HS mRNA only in CYP2E1-expressing cells suggest a possible role for H(2)O(2) in the induction of GCS-HS gene transcription. In contrast to results with HepG2 cells expressing CYP2E1, no increase in GCS-HS mRNA was found with a HepG2 cell line engineered to express human cytochrome P450 3A4. In summary, CYP2E1 overexpression in HepG2 cells up-regulates the levels of reduced GSH by transcriptional activation of GCS-HS; this may reflect an adaptive mechanism to remove CYP2E1-derived oxidants such as H(2)O(2).

Overexpression of catalase in the mitochondrial or cytosolic compartment increases sensitivity of HepG2 cells to tumor necrosis factor-alpha-induced apoptosis.

The sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-alpha (TNF-alpha) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-alpha-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-alpha, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-alpha to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-alpha-induced apoptosis and toxicity produced by catalase overexpression. Addition of H(2)O(2) prevented TNF-alpha-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-alpha plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-alpha did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-alpha signaling was not altered by the overexpression of catalase, as activation of nuclear factor kappaB and AP-1 by TNF-alpha was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-alpha-induced apoptosis and activation of caspases by removal of H(2)O(2).

Ethanol and arachidonic acid produce toxicity in hepatocytes from pyrazole-treated rats with high levels of CYP2E1.

Ethanol and polyunsaturated fatty acids such as arachidonic acid were shown to be toxic and cause apoptosis in HepG2 cells which express CYP2E1 but not in control HepG2 cell lines. The goal of the current study was to extend the observations made with the HepG2 cells to non-transformed, intact hepatocytes. Rats were treated with pyrazole to increase CYP2E1 levels, hepatocytes were isolated and placed into culture and treated for varying time points with ethanol or arachidonic acid. Comparisons were made to hepatocytes from saline-treated rats, with low CYP2E1 content. Incubation with ethanol (100 mM) or especially arachidonic acid (60 microM) resulted in loss of viability of hepatocytes from the pyrazole-treated rats, without any effect on the hepatocytes from the saline-treated rats. The toxicity appeared to be apoptotic in nature and was prevented by diallyldisulfide, an inhibitor of CYP2E1. Toxicity was reduced by trolox, an antioxidant. The treatment with ethanol or arachidonic acid resulted in release of cytochrome c into the cytosol fraction, and activation of caspase 3 (but not caspase 1) in hepatocytes from the pyrazole-treated rats but not hepatocytes from the saline-treated rats. The activation of caspase 3 was prevented by diallyldisulfide, by trolox, and by DEVD-fmk. The latter also prevented the toxicity produced by ethanol or arachidonic acid. These results extend previous observations found with HepG2 cells expressing CYP2E1 to intact hepatocytes and suggest that release of cytochrome c and activation of caspase 3 play a role in the overall pathway by which CYP2E1 contributes towards the hepatotoxic actions of ethanol and polyunsaturated fatty acids.