Dilinoleoylphosphatidylcholine is responsible for the beneficial effects of polyenylphosphatidylcholine on ethanol-induced mitochondrial injury in rats

Chronic ethanol consumption depletes phosphatidylcholines (PC) in membranes and hepatic mitochondria are an early target of this toxicity. Our previous studies showed that soybean-derived polyenylphosphatidylcholine (PPC), attenuated mitochondrial liver injury. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed whether it is responsible for the protection of PPC. Forty-two male rats were fed the following liquid diets for 8 weeks: Control; Control with DLPC (1.5 g/1000 Calories (Cal); Alcohol (36% of Cal); Alcohol with DLPC (1.5 g/1000 Cal) and Alcohol with PPC (3 g/1000 Cal). As expected, ethanol feeding diminished the capacity of hepatic mitochondria to oxidize glutamate and palmitoyl-1-carnitine, and also decreased the activity of mitochondrial cytochrome oxidase. These effects were equally prevented by either PPC or DLPC. In conclusion, DLPC fully reproduced PPC's protective action and may be effective in the prevention or delay of more severe liver damage.     

Dilinoleoylphosphatidylcholine decreases acetaldehyde-induced TNF-alpha generation in Kupffer cells of ethanol-fed rats

We previously reported that dilinoleoylphosphatidylcholine (DLPC) decreases lipopolysaccharide-induced TNF-alpha generation by Kupffer cells of ethanol-fed rats by blocking p38, ERK1/2, and NF-kappaB activation. Here we show that DLPC also decreases TNF-alpha induction by acetaldehyde, a toxic metabolite released by ethanol oxidation. Acetaldehyde induces TNF-alpha generation with a maximal effect at 200 microM and activates p38 and ERK1/2; the latter in turn activates NF-kappaB. This effect is augmented in Kupffer cells of ethanol-fed rats, with upregulation of cytochrome P4502E1 by ethanol. DLPC decreases TNF-alpha generation by blocking p38, ERK1/2, and NF-kappaB activation. Likewise, SB203580, which abolishes p38 activation, and PD098059, which abrogates ERK1/2 and NF-kappaB activation, diminish TNF-alpha generation. Since increased TNF-alpha generation plays a pathogenic role in alcoholic liver disease, the DLPC action on Kupffer cells may explain, in part, its beneficial effects on liver cell injury after ethanol consumption.     

Inhibition of ethanol-induced liver disease in the intragastric feeding rat model by chlormethiazole

The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.     

Effect of dietary epigallocatechin-3-gallate on cytochrome P450 2E1-dependent alcoholic liver damage: enhancement of fatty acid oxidation

This study was designed to determine whether dietary epigallocatechin-3-gallate (EGCG), the most abundant catechin polyphenol in green tea, can protect the liver from cytochrome P450 2E1 (CYP2E1)-dependent alcoholic liver damage. Compared with an ethanol group, when EGCG was present in the ethanol diet, the formation of a fatty liver was significantly reduced and the serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were much lower. Ethanol treatment significantly elevated hepatic CYP2E1 expression while simultaneously reducing hepatic phospho-acetyl CoA carboxylase (p-ACC) and carnitine palmitoyl-transferase 1 (CPT-1) levels. While EGCG markedly reversed the effect of ethanol on hepatic p-ACC and CPT-1 levels, it had no effect on the ethanol-induced elevation in CYP2E1 expression. EGCG prevents ethanol-induced hepatotoxicity and inhibits the development of a fatty liver. These effects were associated with improvements in p-ACC and CPT-1 levels. The use of EGCG might be useful in treating patients with an alcoholic fatty liver.     

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.     

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.     

Differential in vivo effects of α‐naphthoflavone and β‐naphthoflavone on CYP1A1 and CYP2E1 in rat liver,lung, heart,and kidney

Male Sprague–Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist β‐naphthoflavone (βNF), or the relatively weak AHR agonist α‐naphthoflavone (αNF). Animals treated with βNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with αNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by αNF treatment to levels comparable to that seen with βNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to βNF treatment, whereas αNF was without effect. Large increases of CYP1A1‐dependent 7‐ethoxyresorufin O‐deethylation (EROD) activity occurred with microsomes prepared from the tissues of βNF‐treated animals. Comparatively small changes were associated with αNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with βNF treatment. CYP2E1‐dependent p‐nitrophenol hydroxylase (PNP) activity was also increased by βNF treatment in microsomes prepared from kidney (3.1‐fold), whereas αNF was without effect. In contrast, αNF or βNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7‐pentoxyresorufin O‐deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that βNF and αNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR‐mediated toxicity. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 29–40, 1999     

The CYP inhibitor 1-aminobenzotriazole does not prevent oxidative stress associated with alcohol-induced liver injury in rats and mice

Cytochrome P450 (CYP) 2E1 is induced by ethanol and is postulated to be a source of reactive oxygen species during alcoholic liver disease. However, there was no difference in liver pathology and radical formation between wild-type and CYP2E1 knockout mice fed ethanol. Other CYP isoforms may contribute these effects if CYP2E1 is inhibited or absent. The purpose of this study was, therefore, to determine if blocking most of the P450 isoforms with 1-aminobenzotriazole (ABT; 100 mg/kg i.g.), has any effect on liver damage and oxidative stress due to alcohol in rats and mice. Male C57BL/6 mice and Wistar rats were fed either high-fat control or ethanol-containing enteral diet for 4 weeks. ABT had a significant inhibitory effect on many P450 isoforms independent of concomitant alcohol administration. However, ABT did not protect against liver damage due to alcohol in either species. Indices of oxidative stress and inflammation were also similar in livers from vehicle-treated and ABT-treated animals fed ethanol. In summary, suppression of P450 activity with ABT had no apparent effect on oxidative stress caused by alcohol in both rats and mice. These data support the hypothesis that oxidative stress and liver damage can occur independently of CYP activities in both rats and mice during early alcohol-induced liver injury.     

Respective roles of the microsomal ethanol oxidizing system and catalase in ethanol metabolism by deermice lacking alcohol dehydrogenase

To evaluate the roles of MEOS (microsomal ethanol oxidizing system) and catalase in non-alcohol dehydrogenase (ADH) ethanol metabolism, MEOS and catalase activities in vitro and ethanol oxidation rates in hepatocytes from ADH-negative deermice were measured after treatment with catalase inhibitors and/or a stimulator of H2O2 generation. Inhibition of ethanol peroxidation by 3-amino-1,2,4-triazole (aminotriazole) was found to be greater than 85% up to 3 h and 80% at 6 h in liver homogenates. Urate (1 mM) which stimulates H2O2 production in living systems, increased ethanol oxidation fourfold in control but not in cells from aminotriazole-treated animals, documenting effective inhibition of catalase-mediated ethanol peroxidation by aminotriazole. While aminotriazole slightly depressed (15%) basal ethanol oxidation in hepatocytes, in vitro experiments showed a similar decrease in MEOS activity after aminotriazole pretreatment. Azide (0.1 mM), a potent inhibitor of catalase in vitro, also did not affect ethanol oxidation in control cells. By contrast, 1-butanol, a competitive inhibitor of MEOS, but neither a substrate nor an inhibitor of catalase, decreased ethanol oxidation rates in a dose-dependent manner. These results show that, in deermice lacking ADH, catalase plays little if any role in hepatic ethanol oxidation, and that MEOS mediates non-ADH metabolism.     

Microsomal ethanol-oxidizing system in Euglena gracilis. Similarities between Euglena and mammalian cell systems.

1. ADH activity of Euglena grown with 50 mM ethanol decreased, but MEOS activity increased with a corresponding increase in the total amount of cytochrome P-450. 2. Phenobarbital treatment increased the total amount of cytochrome P-450. 3. CO and KCN, cytochrome P-450 ligands, diminished acetaldehyde formed from ethanol oxidation by MEOS. 4. The amounts of NAD(P)H cytochrome c reductases and cytochrome b5 type, components of microsomal monooxygenase reaction, have been spectrophotometrically measured. 5. NAD(P)H cytochrome c reductases activities were induced by phenobarbital. 6. DMSO, an inhibitor of rabbit MEOS, inhibited O2 consumption (11-20%) by Euglena grown with an ethanol, but not a lactate medium. 7. These studies indicate the presence of cytochrome P-450-dependent MEOS in Euglena similar to that in the mammalian hepatic cell.     

Effect of acetaldehyde on ethanol- and aldehyde-metabolising systems of the liver and brain of rats

Lately the mechanism of craving for alcohol has been related to the local level of brain acetaldehyde occurring in ethanol consumption and depending on the activities of the brain and liver ethanol and acetaldehyde-metabolizing systems. In this connection, we studied the effect of chronic acetaldehyde intoxication on the activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), the microsomal ethanol oxidizing system (MEOS) and liver and brain catalase as well as ethanol and acetaldehyde levels in the blood. The results showed that the chronic acetaldehyde intoxication did not alter significantly the activities of liver ADH, MEOS and catalase as well as liver and brain ALDH. In parallel with this, the systemic acetaldehyde administration led to shortened time of ethanol narcosis and activation of catalase in the cerebellum and left hemisphere, which may indicate involvement of this enzyme into metabolic tolerance development.     

D2-receptor-linked signaling pathways regulate the expression of hepatic CYP2E1

This study investigated the role of catecholamine-related signaling pathways in the regulation of hepatic cytochrome P450 (CYP2E1). Central and peripheral catecholamine depletion with reserpine down-regulated CYP2E1. On the other hand, selective peripheral catecholamine depletion with guanethidine increased CYP2E1 apoprotein levels. Enrichment of peripheral catecholamines with adrenaline suppressed p-nitrophenol hydroxylase activity (PNP). PNP activity was also markedly suppressed by l-DOPA. Stimulation of D(2)-receptors with bromocriptine up-regulated CYP2E1, as assessed by enzyme activity and protein levels, whereas blockade of D(2)-dopaminergic receptors with sulpiride down-regulated this isozyme. These findings indicate that central and peripheral catecholamines have different effects on CYP2E1. Central catecholamines appear related to the up-regulation, whereas the role of peripheral catecholamines is clearly related to the type and location of adrenoceptors involved. D(2)-receptor-linked signaling pathways have an up-regulating effect on CYP2E1, while D(1)-receptor pathways may down-regulate this isozyme. It is worth noting that the widespread environmental pollutant benzo(alpha)pyrene (B(alpha)P) altered the modulating effect of catecholaminergic systems on CYP2E1 regulation. In particular, whereas stimulation or blockade of adrenoceptors had no effect on constitutive PNP activity, exposure to B(alpha)P modified the impact of central and peripheral catecholamines and alpha(2)-adrenoceptors on CYP2E1 expression. It appears that under the influence of B(alpha)P, alpha(2)-adrenergic receptor-linked signaling pathways increased CYP2E1 apoprotein levels. Given that a wide range of xenobiotics and clinically used drugs are activated by CYP2E1 to toxic metabolites, including the production of reactive oxygen species (ROS), it is possible that therapies challenging dopaminergic receptor- and/or alpha(2)-adrenoceptor-linked signaling pathways may alter the expression of CYP2E1, thus affecting the progress and development of several pathologies.     

p-Nitrophenol and glutathione response in medaka (Oryzias latipes) exposed to MX,a drinking water carcinogen

When chlorine is introduced into public drinking water for disinfection, it can react with organic compounds in surface waters to form toxic by-products such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX). We investigated the effect of exposure to MX on cytochrome P450 2E1 (CYP2E1)-like activity and total glutathione (GSH) in the liver of the small fish model, medaka (Oryzias latipes). The multi-site carcinogen methylazoxymethanol acetate (MAMAc) was the positive control compound. Both medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p-nitrophenol (PNP), suggesting CYP2E1-like activity in the medaka. Male medaka exposed for 96 h to the CYP2E1 inducers ethanol and acetone under fasted conditions showed significant increases in PNP-hydroxylation activity. Furthermore, total reduced hepatic GSH was reduced in fish fasted for 96 h, indicating that normal feeding is a factor in maintaining xenobiotic defenses. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, however MX exposure did not alter hepatic GSH levels. These data strengthen the role of the medaka as a suitable species for examining cytochrome P450 and GSH detoxification processes and the role these systems play in chemical carcinogenesis.     

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.     

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.     

Effects of binge ethanol on lipid homeostasis and oxidative stress in a rat model of nonalcoholic fatty liver disease

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.     

Alcohol steatosis and cytotoxicity: The role of cytochrome P4502E1 and autophagy

The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.     

Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to lipopolysaccharide

Kupffer cells become activated in response to elevated levels of LPS during ethanol feeding, but the role of ethanol in the molecular processes of activation remains unclear. Because cytochrome P4502E1 (CYP2E1) is upregulated in Kupffer cells after ethanol, we hypothesized that this effect primes Kupffer cells, sensitizing them to increase TNF-alpha production in response to LPS. However, cultured Kupffer cells rapidly lose their CYP2E1. This difficulty was overcome by transfecting CYP2E1 to RAW 264.7 macrophages. Macrophages with stable increased CYP2E1 expression (E2) displayed increased levels of CD14/Toll-like receptor 4, NADPH oxidase and H2O2, accompanied by activation of ERK1/2, p38, and NF-kappaB. These increases primed E2 cells, sensitizing them to LPS stimuli, with amplification of LPS signaling, resulting in increased TNF-alpha production. Diphenyleneiodonium, a NADPH oxidase inhibitor, and diallyl sulfide, a CYP2E1 inhibitor, decreased approximately equally H2O2 levels in E2 cells, suggesting that NADPH oxidase and CYP2E1 contribute equally to H2O2 generation. Because CYP2E1 expression also enhanced the levels of the membrane localized NADPH oxidase subunits p47phox and p67phox, thereby contributing to the oxidase activation, it may augment H2O2 generation via this mechanism. H2O2, derived in part from NADPH and CYP2E1, activated ERK1/2 and p38. ERK1/2 stimulated TNF-alpha production via activation of NF-kappaB, whereas p38 promoted TNF-alpha production by stabilizing TNF-alpha mRNA. Oxidant generation after CYP2E1 overexpression appears to be central to macrophage priming and their sensitization to LPS. Accordingly, CYP2E1 priming could explain the sensitization of Kupffer cells to LPS activation by ethanol, a critical early step in alcoholic liver disease.