Role Of Cytochrome P450 2E1 Research Articles

The CYP2E1 inhibitor Q11 ameliorates LPS-induced sepsis in mice by suppressing oxidative stress and NLRP3 activation

Sepsis is an infection-induced, multi-organ system failure with a pathophysiology related to inflammation and oxidative stress. Increasing evidence indicates that cytochrome P450 2E1 (CYP2E1) is involved in the incidence and development of inflammatory diseases. However, a role for CYP2E1 in lipopolysaccharide (LPS)-induced sepsis has not been completely explored. Here we use Cyp2e1 knockout (cyp2e1-/-) mice to determine if CYP2E1 could be a therapeutic target for sepsis. We also evaluated the ability of Q11, a new specific CYP2E1 inhibitor, to prevent and ameliorate LPS-induced sepsis in mice and in LPS-treated J774A.1 and RAW264.7 cells. Cyp2e1 deletion significantly reduced hypothermia, multi-organ dysfunction and histological abnormalities in LPS-treated mice; consistent with this finding, the CYP2E1 inhibitor Q11 significantly prolonged the survival time of septic mice and ameliorated multi-organ injury induced by LPS. CYP2E1 activity in liver correlated with indicators of multi-organ injury, such as the level of lactate dehydrogenase (LDH) and blood urea nitrogen (BUN) (P < 0.05). Q11 significantly suppressed the expression of NLRP3 in tissues after LPS injection; in vitro studies revealed that activation of NLRP3 signaling and increase of ROS was attenuated by Q11 in LPS-stimulated macrophages, which was reflected by reduced expression of caspase-1 and formation of ASC specks. Overall, our results indicate that Q11 improves the survival of mice with LPS-induced sepsis and attenuates sepsis-induced multiple-organ injury, suggesting that CYP2E1 could be a therapeutic target for sepsis.

Toxic effect of carpet dust on the biochemical indices and histological structure of the lung in rats: the potential role of cytochrome P450 2E1 and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways

Background: Carpet dust exposure in the carpet industry causes various respiratory hazards that lead to permanent loss of lung function. This study investigated the potentially toxic effects of knotted and tufted carpet dust on rat lungs and the possible involvement of cytochrome P450 2E1 (CYP2E1) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways in the induced toxicity, as well as histological changes in the lung induced by carpet dust. Methods: This study divided 48 adult rats into six groups: group I was the control group, group II (vehicle group) received phosphate buffer saline (50 µL/rat), groups III and IV received knotted dust (2.5 and 5 mg/kg, respectively), and groups V and VI received tufted dust (2.5 and 5 mg/kg, respectively). All treatments were intranasally administered once a day for 7 days. Results: Both dust types significantly decreased the lung content of GSH compared with the control. Significantly elevated malondialdehyde (MDA) and nitric oxide (NO) lung contents were observed with an increased CYP2E1, interleukin (IL)-6, nuclear factor kappa B (NF-κβ), and ERK/MAPK. The histological lung structure was moderately affected with a moderately increased number of CD68-positive macrophages in the lung parenchyma of knotted dust-exposed rats, whereas tufted dust exposure severely affected the lung tissue with significantly increased CD68-positive macrophages. Conclusions: Carpet dust exposure could induce oxidative stress and inflammatory response in the lung tissue via induction of CYP2E1 that stimulates ERK/MAPK signalling pathway proteins, resulting in elevated MDA, NO and IL-6 levels in the lung tissue with suppressed GSH content. Tufted dust could possess a more toxic response than knotted ones.

Cytochrome P450 2E1 gene knockout or inhibition prevents obesity induced by high-fat diet via regulating energy expenditure

Cytochrome P450 2E1 (CYP2E1), an important member of the CYP metabolic enzyme family in the liver, regulates the disposal of drugs and the biotransformation of endogenous substances. Although previous studies have found that CYP2E1 is related to energy metabolism, the role of CYP2E1 in energy homeostasis remains unclear. Herein this study shows that the deletion of Cyp2e1 gene in rats can prevent obesity, fatty liver and insulin resistance induced by high-fat diet. Mechanism studies uncover that Cyp2e1 deficiency not only increases the expression of thermogenic genes in brown adipose tissue (BAT) and subcutaneous adipose tissue (SAT), but also promotes fatty acid metabolism in the liver and BAT. In particular, Cyp2e1 deficiency elevates energy expenditure through an increase of liver-generated acylcarnitines, which promote BAT thermogenesis and increase β-oxidation. Interestingly, disulfiram as a CYP2E1 inhibitor can also prevent obesity induced by high-fat diet in normal rats. In general, this study explains the relationship between CYP2E1 and energy metabolism, and provides a new perspective for the prevention and treatment of obesity.

A New CYP2E1 Inhibitor, 12-Imidazolyl-1-dodecanol, Represents a Potential Treatment for Hepatocellular Carcinoma.

Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 μM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD.

Ethanol-Induced Oxidative Stress Modifies Inflammation and Angiogenesis Biomarkers in Retinal Pigment Epithelial Cells (ARPE-19): Role of CYP2E1 and its Inhibition by Antioxidants.

The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.

Susceptibility to the acute toxicity of acrylonitrile in streptozotocin-induced diabetic rats: protective effect of phenethyl isothiocyanate, a phytochemical CYP2E1 inhibitor

Diabetes mellitus is a significant global public health issue. The diabetic state not only precipitates chronic disease but also has the potential to change the toxicity of drugs and chemicals. Acrylonitrile (AN) is a potent neurotoxin widely used in industrial products. This study used a streptozotocin (STZ)-induced diabetic rat model to examine the role of cytochrome P450 2E1 (CYP2E1) in acute AN toxicity. The protective effect of phenethyl isothiocyanate (PEITC), a phytochemical inhibitor of CYP2E1, was also investigated. A higher incidence of convulsions and loss of the righting reflex, and decreased rates of survival, as well as elevated CYP2E1 activity, were observed in diabetic rats treated with AN when compared to those in non-diabetic rats, suggesting that diabetes confers susceptibility to the acute toxicity of AN. Pretreatment with PEITC (20–80 mg/kg) followed by AN injection alleviated the acute toxicity of AN in diabetic rats as evidenced by the decreased incidence of convulsions and loss of righting reflex, and increased rates of survival. PEITC pretreatment at 40 and 80 mg/kg decreased hepatic CYP2E1 activity in AN-exposed diabetic rats. PEITC pretreatment (20 mg/kg) increased the glutathione (GSH) content and glutathione S-transferase (GST) activity and further decreased ROS levels in AN-exposed diabetic rats. Collectively, STZ-induced diabetic rats were more sensitive to AN-induced acute toxicity mainly due to CYP2E1 induction, and PEITC pretreatment significantly alleviated the acute toxicity of AN in STZ-induced diabetic rats. PEITC might be considered as a potential effective chemo-preventive agent against AN-induced acute toxicity in individuals with an underlying diabetic condition.

Non‐Additive Effects of Changing the Composition of the Cytochrome P450 Ensemble in Human Liver Microsomes: the Focus on CYP2E1 and Alcohol‐Drug Interactions

Recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a straight correlation between the composition of human cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of physical interactions between multiple P450 species co‐localized in the microsomal membrane. Due to complex effects of these interactions any change in the content of a particular P450 enzyme may affect drug metabolism in a complex, hard‐to‐predict manner. Of particular practical importance are the functional effects of the changes in the composition of P450 ensemble induced by alcohol consumption. Although the multi‐fold increase in the content of cytochrome P450 2E1 (CYP2E1) in liver observed in both alcoholics and moderate alcohol consumers is well documented, the role of CYP2E1 in the instances of alcohol‐drug interactions is commonly considered insignificant due to a minor role of this enzyme in drug metabolism. However, the involvement of CYP2E1 in alcohol‐drug interactions may stretch far beyond its immediate effects on the metabolism of its substrates.The aim of this study is to probe the effect of increasing concentrations of CYP2E1 in HLM on the metabolism of drug substrates of other P450 species. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into HLM that allows creating the microsomal preparations with altered composition of the P450 ensemble. Incorporation of CYP2E1 into HLM results in an increase in the rate of CYP2E1‐dependent metabolism of chlorzoxazone, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Studies with a series of substrates of drug‐metabolizing P450 species showed that CYP2E1 incorporation also results in pronounced alterations in the metabolism of substrates of several other P450 enzymes. The most important effect is observed on CYP2C19. The changes in the effective KM and Vmax values with such probe substrates as 7‐etoxy‐4‐cyanocoumarin (CEC, the substrate of CYP2C19 and CYP1A2), 7‐dimethylamino‐4‐trifluoromethylcoumarin (coumarin‐152, the substrate of CYP2B6, CYP2C19 and CYP2C9) and N‐butyl 7‐methoxy‐4‐(aminomethyl)‐coumarin (BMAMC, the substrate of CYP1A2, CYP2C19 and CYP2D6) reveal a substantial decrease in the involvement of CYP2C19 in their metabolism. Furthermore, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5. These observations suggest that ethanol‐dependent induction CYP2E1 may be involved in pronounced pharmacokinetic interactions of alcohol with such drug substrates of CYP2C19 and CYP3A enzymes as benzodiazepines (diazepam), tricyclic antidepressants (amitriptylline, imipramine) and anticonvulsants (phenytoin, carbamazepine).Support or Funding InformationThis research was supported by grant R21‐AA024548 from NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Possible Mechanisms of Ethanol-Mediated Colorectal Carcinogenesis: The Role of Cytochrome P4502E1, Etheno-DNA Adducts, and the Anti-Apoptotic Protein Mcl-1.

Chronic alcohol consumption is a risk factor for colorectal cancer. The mechanisms by which ethanol (EtOH) exerts its carcinogenic effect on the colorectal mucosa are not clear and may include oxidative stress with the action of reactive oxygen species (ROS) generated through EtOH metabolism via cytochrome P4502E1 (CYP2E1) leading to carcinogenic etheno-DNA adducts. ROS may also induce apoptosis. However, the effect of chronic EtOH consumption on CYP2E1, etheno-DNA adducts as well as anti-apoptotic proteins in the colorectal mucosa of heavy drinkers without colorectal inflammation is still not known. Rectal biopsies from 32 alcoholics (>60g EtOH/d) and from 12 controls (<20g EtOH/d) were histologically examined, and immunohistochemistry for CYP2E1 and etheno-DNA adducts was performed. Apoptosis (cleaved PARP) as well as anti-apoptotic proteins including Bcl-xL , Bcl-2, and Mcl-1 were immunohistochemically determined. No significant difference in mucosal CYP2E1 or etheno-DNA adducts was observed between alcoholics and control patients. However, CYP2E1 and etheno-DNA adducts correlated significantly when both groups were combined (p<0.001). In addition, although apoptosis was found not to be significantly affected by EtOH, the anti-apoptotic protein Mcl-1, but neither Bcl-xL nor Bcl-2, was found to be significantly increased in heavy drinkers as compared to controls (p=0.014). Although colorectal CYP2E1 was not found to be significantly increased in alcoholics, CYP2E1 correlated overall with the level of etheno-DNA adducts in the colorectal mucosa, which identifies CYP2E1 as an important factor in colorectal carcinogenesis. Most importantly, however, is the up-regulation of the anti-apoptotic protein Mcl-1 in heavy drinkers counteracting apoptosis and possibly stimulating cancer development.

Protective effects of garlic oil against 1,3-dichloro-2-propanol-induced hepatotoxicity: role of CYP2E1 and MAPKs

1,3-Dichloro-2-propanol (1,3-DCP) is a well-known food contaminant that can be detected during the production of a wide range of foods. We investigated whether garlic oil (GO) has protective effects against 1,3-DCP-induced hepatotixicy and oxidative damage in rats. GO significantly attenuated the 1,3-DCP-induced increase in serum aminotransferase activities. This effect was accompanied by attenuation of the histopahological alterations in liver caused by 1,3-DCP. In 1,3-DCP-treated rats, GO prevented formation of hepatic malondialdehyde and depletion of reduced glutathione content, and promoted hepatic antioxidant enzymes activities. 1,3-DCP caused apoptosis in the liver with increases phosphorylation of mitogen-activated protein kinases (MAPKs); GO significantly attenuated these alterations and suppressed the expression of hepatic cytochrome P450 2E1 (CYP2E1). These results suggest that the protective effects of GO against 1,3-DCP-induced hepatotoxicity involve the ability to induce antioxidant enzymes, block metabolic activation of 1,3-DCP by suppressing CYP2E1 expression, and induce antiapoptotic activity by inhibiting phosphorylation of MAPKs.

Alcoholic liver disease: a synopsis of the Charles Lieber's Memorial Symposia 2009-2012.

This paper is based upon the 'Charles Lieber Satellite Symposia' organized by Manuela G. Neuman at each of the 2009-2012 Research Society on Alcoholism (RSA) Annual Meetings. The presentations represent a broad spectrum dealing with alcoholic liver disease (ALD). In addition, a literature search (2008-2013) in the discussed area was performed in order to obtain updated data. The presentations are focused on genetic polymorphisms of ethanol metabolizing enzymes and the role of cytochrome P4502E1 (CYP2E1) in ALD. In addition, alcohol-mediated hepatocarcinogenesis, immune response to alcohol and fibrogenesis in alcoholic hepatitis as well as its co-morbidities with chronic viral hepatitis infections in the presence or absence of human deficiency virus are discussed. Finally, emphasis was led on alcohol and drug interactions as well as liver transplantation for end-stage ALD.

Protective Effects of Diallyl Sulfide against Thioacetamide-Induced Toxicity: A Possible Role of Cytochrome P450 2E1.

Effects of diallyl sulfide (DAS) on thioacetamide-induced hepatotoxicity and immunotoxicity were investigated. When male Sprague-Dawley rats were treated orally with 100, 200 and 400 mg/kg of DAS in corn oil for three consecutive days, the activity of cytochrome P450 (CYP) 2E1-selective p-nitrophenol hydroxylase was dose-dependently suppressed. In addition, the activities of CYP 2B-selective benzyloxyresorufin O-debenzylase and pentoxyresorufin O-depentylase were significantly induced by the treatment with DAS. Western immunoblotting analyses also indicated the suppression of CYP 2E1 protein and/or the induction of CYP 2B protein by DAS. To investigate a possible role of metabolic activation by CYP enzymes in thioacetamide-induced hepatotoxicity, rats were pre-treated with 400 mg/kg of DAS for 3 days, followed by a single intraperitoneal treatment with 100 and 200 mg/kg of thioacetamide in saline for 24 hr. The activities of serum alanine aminotransferase and aspartate aminotransferase significantly elevated by thioacetamide were protected in DAS-pretreated animals. Likewise, the suppressed antibody response to sheep erythrocytes by thioacetamide was protected by DAS pretreatment in female BALB/c mice. Taken together, our present results indicated that thioacetamide might be activated to its toxic metabolite(s) by CYP 2E1, not by CYP 2B, in rats and mice.

Critical role of cytochrome P450 2E1 (CYP2E1) in the development of high fat-induced non-alcoholic steatohepatitis

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) activity contributes to oxidative stress. However, CYP2E1 may have an important role in the pathogenesis of high-fat mediated non-alcoholic steatohepatitis (NASH). Thus, the role of CYP2E1 in high-fat mediated NASH development was evaluated. Male wild type (WT) and Cyp2e1-null mice were fed a low-fat diet (LFD, 10% energy-derived) or a high-fat diet (HFD, 60% energy-derived) for 10 weeks. Liver histology and tissue homogenates were examined for various parameters of oxidative stress and inflammation. Liver histology showed that only WT mice fed a HFD developed NASH despite the presence of increased steatosis in both WT and Cyp2e1-null mice fed HFD. Markers of oxidative stress such as elevated CYP2E1 activity and protein amounts, lipid peroxidation, protein carbonylation, nitration, and glycation with increased phospho-JNK were all markedly elevated only in the livers of HFD-fed WT mice. Furthermore, while the levels of inflammation markers osteopontin and F4/80 were higher in HFD-fed WT mice, TNFα and MCP-1 levels were lower compared to the corresponding LFD-fed WT. Finally, only HFD-fed WT mice exhibited increased insulin resistance and impaired glucose tolerance. These data suggest that CYP2E1 is critically important in NASH development by promoting oxidative/nitrosative stress, protein modifications, inflammation, and insulin resistance.

Ethanol‐Induced Oxidative Stress via the CYP2E1 Pathway Disrupts Adiponectin Secretion from Adipocytes

Adipose tissue is an important target for ethanol action. One important effect of ethanol is to reduce the secretion of adiponectin from adipocytes; this decrease is associated with lowered circulating adiponectin in rodent models of chronic ethanol feeding. Adiponectin is an insulin-sensitizing, anti-inflammatory adipokine; decreased adiponectin activity may contribute to tissue injury in response to chronic ethanol. Here, we investigated the role of cytochrome P450 2E1 (CYP2E1) and oxidative stress in the mechanism for impaired adiponectin secretion from adipocytes in response to ethanol. Male Wistar rats were fed a liquid diet containing ethanol as 36% of calories or pair-fed a control diet for 4 weeks. 3T3-L1 adipocyte cultures, expressing CYP2E1 or not, were exposed to ethanol or 4-hydroxynonenal (4-HNE). Chronic ethanol feeding to rats suppressed the secretion of adiponectin from isolated epididymal adipocytes. Ethanol feeding induced the expression of CYP2E1 in adipocytes and increased markers of oxidative stress, including 4-HNE and protein carbonyls. Because adiponectin is posttranslationally processed in the endoplasmic reticulum and Golgi, we investigated the impact of ethanol on the redox status of high-density microsomes. Chronic ethanol decreased the ratio of reduced glutathione to oxidized glutathione (4.6:1, pair-fed; 2.9:1, ethanol-fed) in high-density microsomes isolated from rat epididymal adipose tissue. We next utilized the 3T3-L1 adipocyte-like cell model to interrogate the mechanisms for impaired adiponectin secretion. Culture of 3T3-L1 adipocytes overexpressing exogenous CYP2E1, but not those overexpressing antisense CYP2E1, with ethanol increased oxidative stress and impaired adiponectin secretion from intracellular pools. Consistent with a role of oxidative stress in impaired adiponectin secretion, challenge of 3T3-L1 adipocytes with 4-HNE also reduced adiponectin mRNA expression and secretion, without affecting intracellular adiponectin concentration. These data demonstrate that CYP2E1-dependent reactive oxygen species production in response to ethanol disrupts adiponectin secretion from adipocytes.

Role of CYP2E1 in Ethanol-Induced Oxidant Stress, Fatty Liver and Hepatotoxicity

Background/Aims: Several pathways contribute to mechanisms by which ethanol induces oxidant stress. While some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. There is a need to develop oral models of significant ethanol-induced liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. Methods: We evaluated chronic ethanol-induced liver injury, steatosis and oxidant stress in wild-type (WT) mice, CYP2E1 knockout (KO) mice and in humanized CYP2E1 knockin (KI) mice, where the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and CYP2E1 KO and KI mice (both provided by Dr. F. Gonzalez, NCI) were fed a high-fat Lieber-DeCarli liquid diet for 3 weeks; pair-fed controls received dextrose. Results: Ethanol produced fatty liver and oxidant stress in WT mice, but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress were blunted in the KO mice (no liver injury) but restored in the KI mice. Significant liver injury was produced in the ethanol-fed KI mice with elevated transaminases and necrosis. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK was observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in WT and KI mice was associated with lower levels of lipolytic PPAR-α. No such changes were found in the ethanol-fed KO mice. Conclusions: These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. Restoring CYP2E1 in the CYP2E1 KO mice restores ethanol-induced fatty liver and oxidant stress.

The role of cytochrome P-4502E1 in the development of hepatic fat, fibrosis and DNA lesions in patients with of alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD)

The role of cytochrome P-4502E1 in the development of hepatic fat, fibrosis and DNA lesions in patients with of alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD)

ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes

Molecular mechanisms underlying alcoholic liver disease (ALD) are still not fully understood. Activating transcription factor-4 (ATF4) is the master coordinator of the integrated stress response (ISR), an adaptive pathway triggered by multiple stressors. which can promote cell death and induce metabolic dysregulation if the stress is intense or prolonged. The aim of this study was to assess the effect of alcohol on the ISR signaling pathway in human liver cells and to define the role of cytochrome P450 2E1 (CYP2E1) in this response. Primary cultured human hepatocytes and human HepG2 cells over-expressing CYP2E1 by adenoviral infection were exposed to ethanol (25-100mM) for 8-48h. Ethanol treatment of both liver cells up-regulated ATF4 as well as the pro-survival and the pro-apoptotic transcriptional program of the ISR. Indeed, in CYP2E1-expressing HepG2 cells exposed to ethanol, the expression of ISR target genes (HMOX-1, GCLC, AsnS, IGFBP-1, GADD34,CHOP, ATF3, CHAC1) was induced. Up-regulation of ATF4 and the ISR transcriptional program was decreased by addition of the anti-oxidant glutathione. Several mechanisms mediated ATF4 protein induction, including, at early times, the phosphorylation of eIF2α which controls ATF4 translation, and, at later times, increased mRNA level and increased stability of the protein. A decrease in cell survival was also observed. This study demonstrates that both CYP2E1 and ethanol induce ATF4 and the integrated stress response, a pathway which coordinates signals from multiple stresses, as well as established risk factors for ALD, and can display detrimental cellular effects upon prolonged activation.

Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice

A major pathway for chronic ethanol-induced liver injury is ethanol-induced oxidant stress. Several pathways contribute to mechanisms by which ethanol induces oxidant stress. Although some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. Most previous studies were conducted in the intragastric infusion model of ethanol administration. There is a need to develop oral models of significant liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. We evaluated chronic ethanol-induced liver injury, steatosis, and oxidant stress in wild-type (WT) mice, CYP2E1 knock out (KO) mice, and humanized CYP2E1 knock-in (KI) mice, in which the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and the CYP2E1 KO and KI mice (both provided by Dr. F. Gonzalez, National Cancer Institute) were fed a high-fat Lieber–DeCarli ethanol liquid diet for 3 weeks; pair-fed controls received dextrose. Ethanol produced fatty liver and oxidant stress in WT mice but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress were blunted in the KO mice (no liver injury) but restored in the KI mice. Significant liver injury was produced in the ethanol-fed KI mice, with elevated transaminases, necrosis, and increased levels of collagen type 1 and smooth muscle actin. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK and decreased levels of Bcl-2 and Bcl-XL were observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in the WT and the KI mice was associated with lower levels of PPARα and acyl-CoA oxidase. No such changes were found in the ethanol-fed KO mice. These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. It is the absence of CYP2E1 in the KO mice that is responsible for the blunting of steatosis and oxidant stress because restoring the CYP2E1 restores the fatty liver and oxidant stress. Moreover, it is the human CYP2E1 that restores these effects of ethanol, which suggests that results for fatty liver and oxidant stress from rodent models of ethanol intake and mouse CYP2E1 can be extrapolated to human models of ethanol intake and to human CYP2E1.

The role of cytochrome P-4502E1 in the progression of alcoholic and non-alcoholic fatty liver

Aims: Although the pathogenesis of alcoholic liver disease (ALD) is still unclear, there is increasing evidence of the involvement of cytochrome P-4502E1 (CYP2E1) in its progression, since the inhibition of CYP2E1 in animal experiments prevents ALD. The purpose of this study was to further investigate the role of CYP2E1 in hepatic steatosis, fibrosis and carcinogenesis in human livers and various animal models.

Molecular basis for a functional role of cytochrome P450 2E1 in non‐alcoholic steatohepatitis

See article in J. Gastroenterol. Hepatol. 2010; 25: 1136–1143