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 microM AA or 100 microM 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.     

Metallothionein 2A induction by zinc protects HEPG2 cells against CYP2E1-dependent toxicity

Zinc has been shown to have antioxidant actions, which may be due, in part, to induction of metallothionein (MT). Such induction can protect tissues against various forms of oxidative injury because MT can function as an antioxidant. The objective of this study was to investigate if zinc or MT induction by zinc could afford protection against CYP2E1-dependent toxicity. HepG2 cells overexpressing CYP2E1 (E47cells) were treated with 60 microM arachidonic acid (AA), which is known to be toxic to these cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. E47 cells were preincubated overnight in the absence or presence of metals such as zinc or cadmium that can induce MT. The culture medium containing the metals was removed, AA was added, and cell viability determined after 24 h incubation. Preincubation overnight with 150 microM zinc sulfate or 5 microM cadmium chloride induced a 20- to 30-fold increase of MT2A mRNA; high levels of MT2A mRNA were maintained during the subsequent challenge period with AA, even after the zinc was removed. MT protein levels were increased about 4- to 5-fold during the overnight preincubation with zinc and a 20- to 30-fold increase was observed 24 h after zinc removal during the AA challenge. The treatment with zinc was associated with significant protection against the loss of cell viability caused by AA in E47 cells. The zinc pretreatment protected about 50% against the DNA fragmentation, cell necrosis, the enhanced lipid peroxidation and increased generation of reactive oxygen species, and the loss of mitochondrial membrane potential induced by AA treatment in E47 cells. CYP2E1 catalytic activity and components of the cell antioxidant defense system such as glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxidase (GPX), catalase, Cu,Zn superoxide dismutase (SOD), and MnSOD were not altered under these conditions. Zinc preincubation also protected the E47 cells against BSO-dependent toxicity. When E47 cells were coincubated with zinc plus AA for 24 h (i.e., zinc was not removed, nor was there a preincubation period prior to challenge with AA), AA toxicity was increased. Thus, zinc had a direct pro-oxidant effect in this model and an indirect antioxidant effect, perhaps via induction of MT. MT may have potential clinical utility for the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Role of p38 MAPK in CYP2E1-dependent arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-kappaB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-kappaB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-kappaB activation.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

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.     

Resistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1

Cancer cell survival is known to be related to the ability to counteract oxidative stress, and glutathione (GSH) depletion has been proposed as a mechanism to sensitize cells to anticancer therapy. However, we observed that GI-ME-N cells, a neuroblastoma cell line without MYCN amplification, are able to survive even if GSH-depleted by l-buthionine-(S,R)-sulfoximine (BSO). Here, we show that in GI-ME-N cells, BSO activates Nrf2 and up-regulates heme oxygenase-1 (HO-1). Silencing of Nrf2 restrained HO-1 induction by BSO. Inhibition of HO-1 and silencing of Nrf2 or HO-1 sensitized GI-ME-N cells to BSO, leading to reactive oxygen/nitrogen species overproduction and decreasing viability. Moreover, targeting the Nrf2/HO-1 axis sensitized GI-ME-N cells to etoposide more than GSH depletion. Therefore, we have provided evidence that in GI-ME-N cells, the Nrf2/HO-1 axis plays a crucial role as a protective factor against cellular stress, and we suggest that the inhibition of Nfr2/HO-1 signaling should be considered as a central target in the clinical battle against neuroblastoma.     

Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HEPG2 cells.

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca2+ and activation of Ca2+-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca2+ were inhibited by omission of Ca2+ from the extracellular medium, and toxicity was restored by reincorporation of Ca2+. An inhibitor of Ca2+ release from intracellular stores did not prevent the toxicity or the increase in Ca2+, reflecting a role for the influx of extracellular Ca2+ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca2+ in E47 cells exposed to iron-AA were inhibited by alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca2+ and activation of Ca2+-dependent proteases.     

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.     

Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver, and liver injury. This study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild-type (WT), CYP2E1-knock-in (KI), and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair-fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolyl hydroxylase 2, which promotes HIF-1α degradation, were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol-fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were colocalized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells, which express CYP2E1, with ethanol plus arachidonic acid (AA) or ethanol plus buthionine sulfoximine (BSO), which depletes glutathione, caused loss of cell viability to a greater extent than in HepG2 C34 cells, which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than in C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress, and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis, and liver injury.     

Antioxidant and pro-oxidant effects of a manganese porphyrin complex against CYP2E1-dependent toxicity

Superoxide dismutases (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP) on CYP2E1-dependent toxicity. The synergistic toxicity of iron and arachidonic acid has been associated with oxidative stress and lipid peroxidation in HepG2 cells that overexpress CYP2E1. Iron plus arachidonic acid caused loss of viability, increased lipid peroxidation and reactive oxygen species generation, and mitochondrial membrane injury in these cells. MnTMPyP partially protected against the decrease in cell viability, the enhanced lipid peroxidation and oxygen radical production, and the loss of mitochondrial membrane potential. The effect of MnTMPyP on arachidonic acid (absence of iron) toxicity was also evaluated. Arachidonic acid also caused toxicity, lipid peroxidation and reduction of the mitochondrial membrane potential. However, in this model, all of these alterations were actually enhanced by MnTMPyP. MnTMPyP also enhanced toxicity in CYP2E1-expressing HepG2 cells depleted of reduced glutathione (GSH). MnCl(2) had little or no effect on the toxicity by arachidonic acid, and MnTMPyP itself did not peroxidize arachidonic acid. MnTMPyP, an SOD mimetic that also scavenges hydrogen peroxide and peroxynitrite, thus showed an antioxidant and protective effect against iron plus arachidonic acid toxicity, but a pro-oxidant and cytotoxic effect against arachidonic acid toxicity in CYP2E1-expressing cells. These different actions may relate to the ability of MnTMPyP to either scavenge or produce free radicals in cells depending upon the prevailing MnTMPyP oxidation-reduction pathways. MnTMPyP and related manganese porphyrin compounds may have potential clinical utility against diseases associated with the overproduction of reactive oxygen species such as ethanol-induced liver injury but it is clear that further investigation of all the pathways of manganese porphyrin oxidation-reduction are necessary.     

Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways

The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HT's strengthening of the antiapoptotic actions of the endogenous antioxidant system.     

Serum deprivation-induced HepG2 cell death is potentiated by CYP2E1

Induction of oxidative stress plays a key role in serum deprivation-induced apoptosis. CYP2E1 plays an important role in toxicity of many chemicals and ethanol and produces oxidant stress. We investigated whether CYP2E1 expression can sensitize HepG2 cells to toxicity as a consequence of serum deprivation. The models used were HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells which do not express CYP2E1. E47 cells showed greater growth inhibition and enhanced cell death after serum deprivation, as compared to the C34 cells. DNA ladder and flow cytometry assays indicated that apoptosis occurred at earlier times after serum deprivation in E47 than C34 cells. Serum withdrawal-induced E47 cell death could be rescued by antioxidants, the mitochondrial permeability transition inhibitor cyclosporine A, z-DEVD-fmk, and a CYP2E1 inhibitor 4-methylpyrazole. Increased production of reactive oxygen species (ROS) and lipid peroxidation occurred in E47 cells after serum deprivation, and there was a corresponding decline in the E47 cell mitochondrial membrane potential and reduced glutathione (GSH) levels. We propose that the mechanism of this serum withdrawal plus CYP2E1 toxicity involves increased production of intracellular ROS, lipid peroxidation, and decline of GSH levels, which results in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of serum deprivation-induced cell death by CYP2E1 may contribute to the sensitivity of the liver to alcohol-induced ischemia and growth factor deprivation.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.