Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. Conclusion: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. (HEPATOLOGY 2010;52:2096-2108 P rohibitin (PHB) proteins are highly conserved and ubiquitously expressed proteins that have diverse cellular functions.1,2 Two PHB proteins, PHB1 and PHB2, encoded by genes located on different chromosomes, form a large multimeric complex (PHB complex) that is found largely in the inner mitochondrial membrane where it exerts a chaperone-like function to stabilize newly synthesized mitochondrial

Background & Aims-The tumor suppressor PTEN inhibits AKT2 signaling; both are aberrantly expressed in liver tumors. We investigated how PTEN and AKT2 regulate liver carcinogenesis. Loss of PTEN leads to spontaneous development of liver tumors from progenitor

The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.

There is clinical evidence that chronic liver diseases in which MDBs (Mallory Denk Bodies) form progress to hepatocellular carcinoma. The present study provides evidence that links MDB formation induced by chronic drug injury, with preneoplasia and later to the formation of tumors, which develop long after drug withdrawal. Evidence indicated that this link was due to an epigenetic cellular memory induced by chronic drug ingestion. Microarray analysis showed that the expressions of many markers of preneoplasia (UBD, Alpha Fetoprotein, KLF6 and glutathione-S-transferase mu2) were increased together when the drug DDC was refed. These changes were suppressed by S-adenosylmethionine feeding, indicating that the drug was affecting DNA and histones methylation in an epigenetic manner. The link between MDB formation and neoplasia formation was likely due to the over expression of UBD (also called FAT10), which is up regulated in 90% of human hepatocellular carcinomas. Immunohistochemical staining of drug-primed mouse livers showed that FAT10 positive liver cells persisted up to 4 months after drug withdrawal and they were still found in the livers of mice, 14 months after drug withdrawal. The refeeding of DDC increased the percent of FAT10 hepatocytes.

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Although alcohol abuse is the major cause of chronic pancreatitis, the pathogenesis of alcoholic chronic pancreatitis (ACP) remains obscure. A critical obstacle to understanding the mechanism of ACP is lack of animal models. Our objective was to develop one such model. Rats were pair-fed for 8 wk ethanol or control Lieber-DeCarli liquid diet. For the last 2 wk, they received cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. After 1 wk on CsA, one episode of acute pancreatitis was induced by four 20 microg/kg injections of cerulein (Cer); controls received saline. Pancreas was analyzed 1 wk after the acute pancreatitis. CsA or Cer treatments alone did not result in pancreatic injury in either control (C)- or ethanol (E)-fed rats. We found, however, that alcohol dramatically aggravated pathological effect of the combined CsA+Cer treatment on pancreas, resulting in massive loss of acinar cells, persistent inflammatory infiltration, and fibrosis. Macrophages were prominent in the inflammatory infiltrate. Compared with control-fed C+CsA+Cer rats, their ethanol-fed E+CsA+Cer counterparts showed marked increases in pancreatic NF-kappaB activation and cytokine/chemokine mRNA expression, collagen and fibronectin, the expression and activities of matrix metalloproteinase-2 and -9, and activation of pancreatic stellate cells. Thus we have developed a model of alcohol-mediated postacute pancreatitis that reproduces three key responses of human ACP: loss of parenchyma, sustained inflammation, and fibrosis. The results indicate that alcohol impairs recovery from acute pancreatitis, suggesting a mechanism by which alcohol sensitizes pancreas to chronic injury.

This paper is based upon the “Charles Lieber Satellite Symposia” organized by Manuela G. Neuman at the Research Society on Alcoholism (RSA) Annual Meetings, 2013 and 2014. The present review includes pre-clinical, translational and clinical research that characterize alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH). In addition, a literature search in the discussed area was performed. Strong clinical and experimental evidence lead to recognition of the key toxic role of alcohol in the pathogenesis of ALD. The liver biopsy can confirm the etiology of NASH or alcoholic steatohepatitis (ASH) and assess structural alterations of cells, their organelles, as well as inflammatory activity. Three histological stages of ALD are simple steatosis, ASH, and chronic hepatitis with hepatic fibrosis or cirrhosis. These latter stages may also be associated with a number of cellular and histological changes, including the presence of Mallory's hyaline, megamitochondria, or perivenular and perisinusoidal fibrosis. Genetic polymorphisms of ethanol metabolizing enzymes such as cytochrome p450 (CYP) 2E1 activation may change the severity of ASH and NASH. Alcohol mediated hepatocarcinogenesis, immune response to alcohol in ASH, as well as the role of other risk factors such as its comorbidities with chronic viral hepatitis in the presence or absence of human deficiency virus are discussed. Dysregulation of hepatic methylation, as result of ethanol exposure, in hepatocytes transfected with hepatitis C virus (HCV), illustrates an impaired interferon signaling. The hepatotoxic effects of ethanol undermine the contribution of malnutrition to the liver injury. Dietary interventions such as micro and macronutrients, as well as changes to the microbiota are suggested. The clinical aspects of NASH, as part of metabolic syndrome in the aging population, are offered. The integrative symposia investigate different aspects of alcohol-induced liver damage and possible repair. We aim to (1) determine the immuno-pathology of alcohol-induced liver damage, (2) examine the role of genetics in the development of ASH, (3) propose diagnostic markers of ASH and NASH, (4) examine age differences, (5) develop common research tools to study alcohol-induced effects in clinical and pre-clinical studies, and (6) focus on factors that aggravate severity of organ-damage. The intention of these symposia is to advance the international profile of the biological research on alcoholism. We also wish to further our mission of leading the forum to progress the science and practice of translational research in alcoholism.

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