Background & Aims Alcohol abuse is a major cause of liver injury. The pathologic features of alcoholic liver disease develop over prolonged periods, yet the cellular defense mechanisms against the detrimental effects of alcohol are not well understood. We investigated whether macroautophagy, an evolutionarily conserved cellular mechanism that is commonly activated in response to stress, could protect liver cells from ethanol toxicity. Methods Mice were acutely given ethanol by gavage. The effects of ethanol on primary hepatocytes and hepatic cell lines were studied in vitro. Results Ethanol-induced macroautophagy in the livers of mice and cultured cells required ethanol metabolism, generated reactive oxygen species, and inhibited mTOR signaling. Suppression of macroautophagy with pharmacological agents or small interfering RNAs significantly increased hepatocyte apoptosis and liver injury; macroautophagy therefore protected cells from the toxic effects of ethanol. Macroautophagy induced by ethanol seemed to be selective for cells with damaged mitochondria and accumulated lipid droplets, but not long-lived proteins, which could account for its protective effects. Increasing macroautophagy pharmacologically reduced hepatotoxicity and steatosis associated with acute ethanol exposure. Conclusions Macroautophagy protects against ethanol-induced toxicity in livers of mice. Reagents that modify macroautophagy might be developed as therapeutics for patients with alcoholic liver disease.

Patients with alcoholic liver disease frequently exhibit iron overload in association with increased hepatic fibrosis. Even moderate alcohol consumption elevates body iron stores; however, the underlying molecular mechanisms are unknown. Hepcidin, a circulatory peptide synthesized in the liver, is a key mediator of iron metabolism. Ethanol metabolism significantly down-regulated both in vitro and in vivo hepcidin mRNA and protein expression. 4-Methylpyrazole, a specific inhibitor of the alcohol-metabolizing enzymes, abolished the effects of ethanol on hepcidin. However, ethanol did not alter the expression of transferrin receptor1 and ferritin or the activation of iron regulatory RNA-binding proteins, IRP1 and IRP2. Mice maintained on 10 -20% ethanol for 7 days displayed down-regulation of liver hepcidin expression without changes in liver triglycerides or histology. This was accompanied by elevated duodenal divalent metal transporter1 and ferroportin protein expression. Injection of hepcidin peptide negated the effect of ethanol on duodenal iron transporters. Ethanol down-regulated hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein ␣ (C/EBP␣) but not ␤. Interestingly, the antioxidants vitamin E and N-acetylcysteine abolished both the alcohol-mediated down-regulation of C/EBP␣ binding activity and hepcidin expression in the liver and the up-regulation of duodenal divalent metal transporter 1. Collectively, these findings indicate that alcohol metabolism-mediated oxidative stress regulates hepcidin transcription via C/EBP␣, which in turn leads to increased duodenal iron transport.

The inhibition of apoptosis is a critical event in the development of colorectal malignancies, although the mechanism(s) remain incompletely understood. The anti-apoptotic proto-oncogene, AKT, has been implicated in the molecular pathogenesis of a variety of human malignancies; however, no data exist on the role of AKT in colon carcinogenesis. We therefore evaluated the presence of AKT in human and experimental colon neoplasms by immunohistochemistry. Normal colonic mucosa and hyperplastic polyps exhibited no significant AKT expression, in marked contrast to the dramatic AKT immunoreactivity seen in colorectal cancers (57% positive) and in both human colorectal cancer cell lines examined. Importantly, AKT was also detected in 57% of the adenomas examined, implicating overexpression of this proto-oncogene as an early event during colon carcinogenesis. Moreover, in the rodent-carcinogen model, azoxymethane (AOM)-treatment induced AKT expression in premalignant rat colonocytes. Tumors that evolve via different genetic pathways displayed a lower incidence of AKT overexpression. Indeed, only 22% of mismatch repair defective tumors and 42% of AOM-induced rodent tumors upregulated AKT. Staining with an antibody specific for AKT 2 duplicated findings with the AKT 1&2 antibody, suggesting that AKT 2 was the predominant isoform involved in colon carcinogenesis. Furthermore, utilizing an antibody that specifically recognizes the serine-473 phosphorylated form of AKT, we observed that activated AKT was detectable in the neoplastic but not normal epithelium. In summary, our immunohistochemical analysis indicates AKT overexpression occurs frequently during human colon carcinogenesis, but is less common in colon cancers with microsatellite instability. The early inhibition of apoptosis during sporadic colon carcinogenesis may be related, at least partly, to the overexpression of AKT.

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T he vast majority of ingested ethanol is oxidized to acetaldehyde by the hepatocytes of the liver. 1,2 It is thought that the metabolism of ethanol by hepatocytes is the reason that the liver is a target for the detrimental effects of chronic alcohol abuse. 3 Ethanol oxidation by hepatocytes results in many metabolic changes, some of which have been shown to be detrimental to cells. It has been proposed that chronic ethanol abuse promotes continual hepatocyte destruction that, in turn, stimulates abnormal hepatocyte regeneration and fibrotic scarring, which over many years results in alcoholic liver disease. Thus, alcoholic liver disease results from an eventual inability of hepatocytes to appropriately respond and regenerate in response to the toxic effects of the metabolic changes that occur during ethanol oxidation. 4 Liver regeneration is the mechanism by which cells that have been lost as a result of hepatotoxicity are replaced. It is well documented that ethanol metabolism impairs the regenerative capacity of the liver. 3,5 Therefore, it appears that ethanol oxidation not only results in hepatotoxicity, but also impairs the ability of the liver to respond to this toxic assault. The mechanism(s) by which chronic ethanol metabolism affects replication is not well understood. This is partially because the biochemical events that take place during ethanol oxidation occur simultaneously, making it difficult to attribute specific impairments to specific biochemical events.In an attempt to dissect these biochemical events, we have developed a cell-culture system with cells of hepatic origin that stably expresses alcohol dehydrogenase and efficiently metabolizes ethanol. Using these cells, we in-

Background This study investigated the role of ethanol-inducible CYP2E1 in enhancing CYP2E1 and other P450 proteins in extracellular vesicles (EVs) from alcohol-exposed rodents and human alcoholics and their effects on oxidative hepatocyte injury Methods Female Fischer rats and wild-type (WT) or Cyp2e1-null mice were exposed to three oral doses of binge ethanol or dextrose-control at 12-h intervals. Plasma EV and hepatic proteins from alcohol-exposed rodents, alcoholics and their respective controls were isolated and characterized. Results The number of EVs and EV CYP2E1, CYP2A, CYP1A1/2, CYP4B proteins were markedly elevated in both alcoholics and alcohol-exposed rats and mice. The number of EVs and EV P450 proteins were significantly reduced in ethanol-exposed rats fed a diet containing polyunsaturated fatty acids. The increased number of EVs and EV CYP2E1 and other P450 isoforms in alcohol-exposed WT were significantly reduced in the corresponding Cyp2e1-null mice. EV CYP2E1 amounts depended on increased oxidative and ER stress, because their levels were decreased by co-treatment with an anti-oxidant N-acetylcysteine or a CYP2E1 inhibitor chlormethiazole, but increased by ER stress inducer thapsigargin, which was blocked by 4-phenylbutyric acid. Furthermore, cell death rates were elevated when primary hepatocytes or human hepatoma cells were exposed to EVs from alcohol-exposed rodents and alcoholics, demonstrating that EVs from alcohol-exposed rats and alcoholics are functional and can promote cell death by activating the apoptosis signaling pathway, including phospho-JNK, proapoptotic Bax and activated caspase-3. Conclusion These results demonstrate an important role of CYP2E1 in elevating EV CYP2E1 and other P450 isoforms through increased oxidative and ER stress. Elevated EV-CYP2E1 detected after withdrawal from alcohol or exposure to a CYP2E1 inducer pyrazole can be a potential biomarker for liver injury.

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