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

• Published in: Free radical biology & medicine Vol. 63; pp. 175 - 186
• Main Authors: Wang, Xiaodong, Wu, Defeng, Yang, Lili, Gan, Lixia, Cederbaum, Arthur I.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2013
• Subjects: Animals; arachidonic acid; Cell Survival - drug effects; cell viability; Chemical and Drug Induced Liver Injury - metabolism; Chemical and Drug Induced Liver Injury - physiopathology; CYP2E1; cytochrome P-450; Cytochrome P-450 CYP2E1 - genetics; Cytochrome P-450 CYP2E1 - metabolism; Ethanol; Ethanol - administration & dosage; fatty liver; Free radicals; glucose; glutathione; Glutathione - metabolism; Hep G2 Cells; Hepatotoxicity; HIF-1α; Humans; hypoxia; Hypoxia - chemically induced; Hypoxia - metabolism; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Hypoxia-Inducible Factor-Proline Dioxygenases; liver; Liver - drug effects; Mice; Mice, Knockout; Oxidative stress; Oxidative Stress - drug effects; Oxidative Stress - genetics; procollagen-proline dioxygenase; Procollagen-Proline Dioxygenase - metabolism; Reactive Oxygen Species - metabolism; toxicity; Oxidative stress; 2-ME; IHC; ALT; HIF-1α; MDA; LDHA; PNP; 4-HNE; GSH; WT; Hepatotoxicity; KI; BSO; AA; Free radicals; HIF; KO; Ethanol; TBARS; 3-NT; ROS; HPH-2; CYP2E1; HE; HIF prolyl hydroxylase 2; thiobarbituric acid-reactive substances; alanine aminotransferase; l-buthionine sulfoximine; lactate dehydrogenase A; reduced glutathione; 2-methoxyestradiol; reactive oxygen species; 3-nitrotyrosine; immunohistochemistry; malondialdehyde; arachidonic acid; hematoxylin–eosin; knockout; cytochrome P450 2E1; p-nitrophenol; knock-in; hypoxia-inducible factor; 4-hydroxynonenal; wild type
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2013.05.009

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. •Chronic ethanol administration induces hypoxia and elevates HIF-1α in the liver.•CYP2E1 is elevated by ethanol and promotes the ethanol-induced hypoxia and increase in HIF-1α.•Inhibition of HIF-1α blunted the toxic actions and oxidative stress produced by CYP2E1.•HIF-1α plays an important role in CYP2E1-dependent ethanol-induced hepatotoxicity.