Arthur I. Cederbaum

Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT), CYP2E1 knockout (KO) or CYP2E1 humanized transgenic knockin (KI), mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA), an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These results suggest that autophagy is protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. We speculate that autophagy-dependent processes such as mitophagy and lipophagy help to minimize ethanol-induced CYP2E1-dependent oxidative stress and therefore the subsequent liver injury and steatosis. Attempts to stimulate autophagy may be helpful in lowering ethanol and CYP2E1-dependent liver toxicity.

The mechanisms by which alcohol causes cell injury are not clear. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol-elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other; however, associations and interactions between them, especially in vivo, have not been extensively evaluated. We have shown that increased oxidative stress from induction of CYP2E1 in vivo sensitizes hepatocytes to LPS and TNFα toxicity and that oxidative stress, activation of p38 and JNK MAP kinases, and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNFα potentiated hepatotoxicity. This Review will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved including activation of the mitogen-activated kinase kinase kinase ASK-1 as a result of CYP2E1-derived reactive oxygen intermediates promoting dissociation of the inhibitory thioredoxin from ASK-1. This activation of ASK-1 is followed by activation of the mitogen-activated kinase kinases MKK3/MKK6 and MKK4/MMK7 and subsequently p38 and JNK MAP kinases. Synergistic toxicity occurs between CYP2E1 and the JNK1 but not the JNK2 isoform as JNK1 knockout mice are completely protected against CYP2E1 plus TNFα toxicity, elevated oxidative stress, and mitochondrial dysfunction. We hypothesize that similar interactions occur as a result of ethanol induction of CYP2E1 and TNFα.

The transcription factor Nrf2 regulates the expression of important cytoprotective enzymes. Induction of cytochrome P450 2E1(CYP2E1) is one of the central pathways by which ethanol generates oxidative stress. CYP2E1 can be induced by ethanol and several low molecular weight chemicals such as pyrazole. The chapter discusses biochemical and toxicological effects of CYP2E1 and the effects of Nrf2 in modulating these actions of CYP2E1.Besides ethanol, CYP2E1 metabolizes and activates many other important toxicological compounds. One approach to try to understand basic effects and actions of CYP2E1 was to establish HepG2 cell lines that constitutively express human CYP2E1. Ethanol, polyunsaturated fatty acids and iron were toxic to the HepG2 cells which express CYP2E1 (E47 cells) but not control C34HepG2 cells which do not express CYP2E1.Toxicity was associated with enhanced oxidant stress and could be prevented by antioxidants and potentiated if glutathione (GSH) was removed. The E47 cells had higher GSH levels and a Twofold increase in catalase, cytosolic and microsomal glutathione transferase, and heme oxygenase-1 (HO-1) than control HepG2 cells due to activation of their respective genes. These activations were prevented by antioxidants, suggesting that reactive oxygen species (ROS) generated by CYP2E1 were responsible for the up-regulation of these antioxidant genes. This upregulation of antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. Increases in Nrf2 protein and mRNA were observed in livers of chronic alcohol-fed mice or rats and of pyrazole-treated rats or mice, conditions known to elevate CYP2E1. E47 cells showed increased Nrf2 mRNA and protein expression compared with control HepG2 C34 cells. Upregulation of antioxidant genes in E47 cells is dependent on Nrf2 and is prevented by siRNA-Nrf2. Blocking Nrf2 by siRNA-Nrf2 decreases GSH and increases ROS and lipid peroxidation, resulting in decreased mitochondrial membrane potential and loss of cell viability of E47 cells but not C34 cells. Nrf2 is activated and levels of Nrf2 protein and mRNA are increased when CYP2E1 is elevated. These results suggest that Nrf2 plays a key role in the adaptive response against increased oxidative stress caused by CYP2E1 in the HepG2 cells.