title: Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo.
abstract: 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.
authors.affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Wang
authors.forename: Defeng
authors.initials: D
authors.lastname: Wu
authors.forename: Lili
authors.initials: L
authors.lastname: Yang
authors.forename: Lixia
authors.initials: L
authors.lastname: Gan
authors.forename: Arthur I
authors.initials: AI
authors.lastname: Cederbaum
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism / Q000503:physiopathology; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000008:administration & dosage*; D005978:Glutathione / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D000860:Hypoxia / Q000139:chemically induced / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D008099:Liver / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017382:Reactive Oxygen Species; D000431:Ethanol; D011393:Procollagen-Proline Dioxygenase; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D019392:Cytochrome P-450 CYP2E1; D005978:Glutathione
keywords: 2-ME; 2-methoxyestradiol; 3-NT; 3-nitrotyrosine; 4-HNE; 4-hydroxynonenal; AA; ALT; BSO; CYP2E1; Ethanol; Free radicals; GSH; HE; HIF; HIF prolyl hydroxylase 2; HIF-1α; HPH-2; Hepatotoxicity; IHC; KI; KO; LDHA; MDA; Oxidative stress; PNP; ROS; TBARS; WT; alanine aminotransferase; arachidonic acid; cytochrome P450 2E1; hematoxylin–eosin; hypoxia-inducible factor; immunohistochemistry; knock-in; knockout; l-buthionine sulfoximine; lactate dehydrogenase A; malondialdehyde; p-nitrophenol; reactive oxygen species; reduced glutathione; thiobarbituric acid-reactive substances; wild type