Inhibition of CYP2E1 leads to decreased malondialdehyde–acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure

AimEthanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde–acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated.Main methodsChronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor — diallyl sulfide or ADH inhibitor — pyrazole or ADH and CYP2E1 inhibitor — 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis.Key findingsInhibition of CYP2E1 with 10 μM diallyl sulfide or ADH with 2 mM pyrazole or ADH and CYP2E1 with 5 mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury.SignificanceCYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.     

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.     

Gene-environmental interactions between alcohol-drinking behavior and ALDH2 and CYP2E1 polymorphisms and their impact on micronuclei frequency in human lymphocytes

Ethanol is converted to acetaldehyde by alcohol dehydrogenase (ADH), cytochrome p4502E1 (CYP2E1) and catalase. This metabolite is then detoxified by aldehyde dehydrogenase 2 (ALDH2), a key enzyme in the elimination of acetaldehyde, via further oxidation to acetic acid. The toxic effects of acetaldehyde are well documented and may be partially mediated by genotoxic damage. In the present study, we investigated the effects of alcohol-drinking behavior and genetic polymorphisms in two different genes (ALDH2 and CYP2E1) on the micronuclei (MN) frequency in 248 healthy Japanese men. Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient type) was significantly associated with an increased MN frequency in subjects drinking more than three times/wk, while habitual drinkers with wild-type CYP2E1 also had a significantly increased MN frequency. Furthermore, when the subjects were divided into eight groups according to their drinking frequency and genotypes of ALDH2 and CYP2E1, we found that habitual drinkers with homozygous CYP2E1*1/*1 and heterozygous ALDH2*1/*2 or homozygous ALDH2*2/*2 showed the highest mean MN frequency. In the present study, we found clear associations among ALDH2 and CYP2E1 gene polymorphisms, alcohol-drinking behavior and genotoxic effects in a healthy Japanese population. Therefore, analysis of the polymorphisms of alcohol-metabolizing enzymes may lead to elucidation of the mechanism(s) for individual susceptibilities to the toxicity of ethanol metabolites.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.     

Induction of nicotine-metabolizing CYP2B1 by ethanol and ethanol-metabolizing CYP2E1 by nicotine: summary and implications

Alcohol and tobacco are frequently co-abused. Increased alcohol use and alcoholism are associated with smoking, and vice versa. Functional and/or metabolic cross-tolerance may contribute to this occurrence. This review summarizes recent studies published from our laboratory focusing on metabolic aspects of tolerance, which demonstrate that in rat, subchronic, behaviourally relevant doses of ethanol induce hepatic nicotine-metabolizing cytochrome P450 (CYP) 2B1, and that subchronically administered nicotine, at behaviourally relevant doses, induces hepatic ethanol-metabolizing CYP2E1. Increased CYP2B1 protein, mRNA and CYP2B1-mediated nicotine metabolism was observed following ethanol treatments. CYP2E1 protein and activity were induced by nicotine, but no changes were seen in levels of CYP2E1 mRNA. These data indicate that metabolic cross-tolerance may occur between nicotine and ethanol, suggesting that nicotine use may increase the elimination of ethanol, and ethanol use may increase the elimination of nicotine. Other implications, such as altered pharmacology and toxicology of drugs metabolized by these enzymes, as well as changes in pro-carcinogen and pro-toxin activation are also discussed.     

Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells

It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12?±?3 and 6?±?1 nmol/h/mg of total protein to 191?±?9 and 57?±?9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect – enzyme overexpression.     

CYP2E1 and oxidative liver injury by alcohol

Ethanol-induced oxidative stress seems to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway seems to be the induction of cytochrome P450 2E1 (CYP2E1) by ethanol. CYP2E1 metabolizes and activates many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide and, in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This review article summarizes some of the biochemical and toxicological properties of CYP2E1 and briefly describes the use of cell lines developed to constitutively express CYP2E1 and CYP2E1 knockout mice in assessing the actions of CYP2E1. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help us to understand the actions of CYP2E1 and its role in alcoholic liver injury.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390-401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H(2)O(2) [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414-419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390–401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H₂O₂ [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414–419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

In vitro model using mouse hepatocytes for study of alcohol stress

In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5 to approximately 5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.     

In Vitro Model using Mouse Hepatocytes for Study of Alcohol Stress

In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5～5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.     

The role of cytochrome P450 2E1 on ethanol-mediated oxidative stress and HIV replication in human monocyte-derived macrophages

BackgroundAlcohol consumption is considered to be a major health problem among people living with HIV/AIDS. Our previous reports have shown that ethanol reduced intracellular concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1 cell line. Ethanol also increased HIV-1 replication despite the presence of elvitegravir. Our previous finding has also shown that the levels of cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in ethanol-enhanced oxidative stress and HIV-1 replication is still unclear.MethodsThis study examined the chronic effects (14 days) of ethanol on HIV viral load, oxidative DNA damage, expression of CYP2E1, expression of antioxidant enzymes (AOEs), expression of reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM). Further, to evaluate the role of CYP2E1 in mediating ethanol-induced viral replication, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-infected U1 cell line following ethanol treatment.ResultsChronic ethanol exposure demonstrated an increase in oxidative DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM. Our results showed that ethanol chronic exposure increased HIV-1 replication by ~3-fold in HIV-1-infected MDM. This ethanol-enhanced HIV-1 replication was associated with an increased oxidative DNA damage, an increased expression of CYP2E1, and a decreased expression of antioxidant enzyme PRDX6. In HIV-1-infected U1 cell line, we observed a decreased viral replication (~30%) and a decreased DNA damage (~100%) after repression of CYP2E1 by siRNA, upon ethanol exposure. We also observed a decreased viral replication (~25%) after inhibition of CYP2E1 by using selective CYP2E1 inhibitor.ConclusionsThe data suggest that chronic ethanol exposure increases HIV-1 replication in MDM, at least in part, through CYP2E1-mediated oxidative stress. These results are clinically relevant to potentially find effective treatment strategies for HIV-1-infected alcohol users.     

CYP2E1 enhances ethanol-induced lipid accumulation but impairs autophagy in HepG2 E47 cells

The regulation and function of autophagy and lipid metabolism have recently been reported to be reciprocally related. Macroautophagy mediates the breakdown of lipids stored in lipid droplets. An inhibition of autophagy leads to the development of a fatty liver. We evaluated the ability of CYP2E1 to modulate the effects of ethanol on lipid accumulation and autophagy in vitro. The E47 HepG2 cell which expresses CYP2E1 was treated with ethanol at 50, 100 and 150 mM for 4 or 5 days. Ethanol-induced lipid accumulation and an increase of triglycerides (TG) in E47 cells to a greater extent than in control C34 cells which do not express CYP2E1. In contrast, autophagy (LC3 II/LC3 I ratio) was significantly induced by ethanol in C34 cells to a greater extent than in E47 cells. P62 was significantly increased in E47 cells after ethanol treatment. Thus, there is a reciprocal relationship between the effects of ethanol on lipid accumulation and autophagy in the CYP2E1-expressing cells. Inhibition of autophagy by 3-methyladenine (3MA), increased lipid accumulation and TG levels in C34 cells which display elevated autophagy, but enhanced lipid accumulation and TG level to a lesser extent in E47 cells which displayed lower autophagy. Ethanol induced CYP2E1 activity and oxidative stress in E47 cells compared with C34 cells. These experiments suggest that the expression of CYP2E1 may impair autophagy formation which contributes to lipid accumulation in the liver. We hypothesize that CYP2E1-induced oxidative stress promotes the accumulation of lipid droplets by ethanol and this may be responsible for the suppression of autophagy in the liver.     

CYP2E1: biochemistry, toxicology, regulation and function in ethanol-induced liver injury

Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. CYP2E1 is of interest because of its ability to metabolize and activate many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This Review Article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines developed to constitutively express the human CYP2E1 in assessing the actions of CYP2E1. Regulation of CYP2E1 is quite complex and will be briefly reviewed. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help to understand the actions of CYP2E1 and its role in alcoholic liver injury.     

Application of the reverse dept polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy

Using the reverse 13C----1H DEPT polarization-transfer pulse sequence the metabolism of 13C ethanol in vitro and in vivo has been monitored by 1H-NMR spectroscopy. Using yeast alcohol dehydrogenase, acetaldehyde, the hydrated form of acetaldehyde and acetate were identified as metabolites of [2-13C]-ethanol. The ratio of hydrated to free acetaldehyde was dependent upon the protein concentration of the reaction mixture. Binding of acetaldehyde in an irreversible Schiffs base resulted in optimal enzyme activity. Hepatocytes from rats fasted for 20 h, metabolised [1-13C] and [2-13C]ethanol in a linear fashion, but no [13C]acetaldehyde was detected. Metabolic integrity of the hepatocytes was confirmed with [2-13C]acetate. The addition of disulfiram (50 micron) to hepatocyte suspensions which had been incubated with [1-13C]ethanol, resulted in the resynthesis of [13C]ethanol. The amount of [13C]ethanol resynthesized under these conditions represents intracellular acetaldehyde whose concentration was in the range of 400-800 mumol/g wet weight of hepatocytes when 50 mM ethanol had been originally incubated with the hepatocyte suspension. These studies show how NMR-polarization transfer pulse sequences can be used to monitor the metabolism of 13C-ethanol in vivo, and provide a unique tool to measure in vivo concentrations of acetaldehyde. The studies also suggest that cytoplasmic aldehyde dehydrogenase may play a major role in hepatic ethanol metabolism.     

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.     

Regulation of cytochrome P450 2e1 expression by ethanol: role of oxidative stress-mediated pkc/jnk/sp1 pathway

CYP2E1 metabolizes ethanol leading to production of reactive oxygen species (ROS) and acetaldehyde, which are known to cause not only liver damage but also toxicity to other organs. However, the signaling pathways involved in CYP2E1 regulation by ethanol are not clear, especially in extra-hepatic cells. This study was designed to examine the role of CYP2E1 in ethanol-mediated oxidative stress and cytotoxicity, as well as signaling pathways by which ethanol regulates CYP2E1 in extra-hepatic cells. In this study, we used astrocytic and monocytic cell lines, because they are important cells in central nervous system . Our results showed that 100 mM ethanol significantly induced oxidative stress, apoptosis, and cell death at 24 h in the SVGA astrocytic cell line, which was rescued by a CYP2E1 selective inhibitor, diallyl sulfide (DAS), CYP2E1 siRNA, and antioxidants (vitamins C and E). Further, we showed that DAS and vitamin C abrogated ethanol-mediated (50 mℳ) induction of CYP2E1 at 6 h, as well as production of ROS at 2 h, suggesting the role of oxidative stress in ethanol-mediated induction of CYP2E1. We then investigated the role of the protein kinase C/c-Jun N-terminal kinase/specificity protein1 (PKC/JNK/SP1) pathway in oxidative stress-mediated CYP2E1 induction. Our results showed that staurosporine, a non-specific inhibitor of PKC, as well as specific PKCζ inhibitor and PKCζ siRNA, abolished ethanol-induced CYP2E1 expression. In addition, inhibitors of JNK (SP600125) and SP1 (mithramycin A) completely abrogated induction of CYP2E1 by ethanol in SVGA astrocytes. Subsequently, we showed that CYP2E1 is also responsible for ethanol-mediated oxidative stress and apoptotic cell death in U937 monocytic cell lines. Finally, our results showed that PKC/JNK/SP1 pathway is also involved in regulation of CYP2E1 in U937 cells. This study has clinical implications with respect to alcohol-associated neuroinflammatory toxicity among alcohol users.