1 2792 111 FAT10 IS AN EPIGENETIC MARKER FOR LIVER PRENEOPLASIA IN A DRUG-PRIMED MOUSE MODEL OF TUMORIGENESIS. 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. 2008 2 6310 56 THE REGULATION OF NON-CODING RNA EXPRESSION IN THE LIVER OF MICE FED DDC. MALLORY-DENK BODIES (MDBS) ARE FOUND IN THE LIVER OF PATIENTS WITH ALCOHOLIC AND CHRONIC NONALCOHOLIC LIVER DISEASE, AND HEPATOCELLULAR CARCINOMA (HCC). DIETHYL 1,4-DIHYDRO-2,4,6,-TRIMETHYL-3,5-PYRIDINEDICARBOXYLATE (DDC) IS USED AS A MODEL TO INDUCE THE FORMATION OF MDBS IN MOUSE LIVER. PREVIOUS STUDIES IN THIS LABORATORY SHOWED THAT DDC INDUCED EPIGENETIC MODIFICATIONS IN DNA AND HISTONES. THE COMBINATION OF THESE MODIFICATIONS CHANGES THE PHENOTYPE OF THE MDB FORMING HEPATOCYTES, AS INDICATED BY THE MARKER FAT10. THESE EPIGENETIC MODIFICATIONS ARE PARTIALLY PREVENTED BY ADDING TO THE DIET S-ADENOSYLMETHIONINE (SAME) OR BETAINE, BOTH METHYL DONORS. THE EXPRESSION OF THREE IMPRINTED NCRNA GENES WAS FOUND TO CHANGE IN MDB FORMING HEPATOCYTES, WHICH IS THE SUBJECT OF THIS REPORT. NCRNA EXPRESSION WAS QUANTITATED BY REAL-TIME PCR AND RNA FISH IN LIVER SECTIONS. MICROARRAY ANALYSIS SHOWED THAT THE EXPRESSION OF THREE NCRNAS WAS REGULATED BY DDC: UP REGULATION OF H19, ANTISENSE IGF2R (AIR), AND DOWN REGULATION OF GTL2 (ALSO CALLED MEG3). S-ADENOSYLMETHIONINE (SAME) FEEDING PREVENTED THESE CHANGES. BETAINE, ANOTHER METHYL GROUP DONOR, PREVENTED ONLY H19 AND AIR UP REGULATION INDUCED BY DDC, ON MICROARRAYS. THE RESULTS OF THE SAME AND BETAINE GROUPS WERE CONFIRMED BY REAL-TIME PCR, EXCEPT FOR AIR EXPRESSION. AFTER 1 MONTH OF DRUG WITHDRAWAL, THE EXPRESSION OF THE THREE NCRNAS TENDED TOWARD CONTROL LEVELS OF EXPRESSION. LIVER TUMORS THAT DEVELOPED ALSO SHOWED UP REGULATION OF H19 AND AIR. THE RNA FISH APPROACH SHOWED THAT THE MDB FORMING CELLS' PHENOTYPE CHANGED THE LEVEL OF EXPRESSION OF AIR, H19 AND GTL2, COMPARED TO THE SURROUNDING CELLS. FURTHERMORE, OVER EXPRESSION OF H19 AND AIR WAS DEMONSTRATED IN TUMORS FORMED IN MICE WITHDRAWN FOR 9 MONTHS. THE DYSREGULATION OF NCRNA IN MDB FORMING LIVER CELLS HAS BEEN OBSERVED FOR THE FIRST TIME IN DRUG-PRIMED MICE ASSOCIATED WITH LIVER PRENEOPLASTIC FOCI AND TUMORS. 2009 3 6361 59 THE ROLE OF INNATE IMMUNITY IN THE PATHOGENESIS OF PRENEOPLASIA IN DRUG-INDUCED CHRONIC HEPATITIS BASED ON A MOUSE MODEL. INNATE IMMUNITY FACTORS SUCH AS CONVERSION OF THE 26S PROTEASOME TO FORM THE IMMUNOPROTEASOME AND THE TOLL-LIKE RECEPTOR SIGNALING PATHWAYS ARE ACTIVATED IN CHRONIC HEPATITIS INDUCED BY THE CARCINOGENIC DRUG DDC. OVER TIME, PRENEOPLASTIC HEPATOCYTE PHENOTYPES APPEAR IN THE LIVER PARENCHYMA. THESE CHANGED HEPATOCYTES EXPAND IN NUMBER BECAUSE THEY HAVE A GROWTH ADVANTAGE OVER NORMAL HEPATOCYTES WHEN RESPONDING TO CHRONIC LIVER INJURY. THE CHANGED HEPATOCYTES CAN BE IDENTIFIED USING IMMUNOFLUORESCENT ANTIBODIES TO PRENEOPLASTIC CELLS E.G. FAT10/UBD, A2 MACROGLOBULIN, GLUTATHIONE TRANSPEPTIDASE, ALPHA FETOPROTEIN, GLYCIPAN 3, FAS, AND GAMMA GLUTAMYL TRANSPEPTIDASE. THE FORMATION OF THE PRENEOPLASTIC CELLS OCCURS CONCOMITANT WITH ACTIVATION OF THE TOLL-LIKE RECEPTOR SIGNALING PATHWAYS AND THE TRANSFORMATION OF THE 26S PROTEASOME TO FORM THE IMMUNOPROTEASOME. THIS TRANSFORMATION IS IN RESPONSE TO INTERFERON STIMULATING RESPONSE ELEMENT ON THE PROMOTER OF THE FAT10/UBD GENE. NFKAPPAB, ERK, P38 AND JNK ARE ALSO UP REGULATED. SPECIFIC INHIBITORS BLOCK THESE RESPONSES IN VITRO IN A MOUSE TUMOR CELL LINE EXPOSED TO INTERFERON GAMMA. MALLORY-DENK BODIES FORM IN THESE PRENEOPLASTIC CELLS, BECAUSE OF THE DEPLETION OF THE 26S PROTEASOME DUE TO FORMATION OF THE IMMUNOPROTEASOME. THUS, MDB FORMING CELLS ARE ALSO MARKERS OF THE PRENEOPLASTIC HEPATOCYTES. THE UBD POSITIVE PRENEOPLASTIC CELLS REGRESS WHEN THE LIVER INJURY INDUCED CHRONIC HEPATITIS SUBSIDES. WHEN THE DRUG DDC IS REFED TO MICE AND CHRONIC HEPATITIS IS ACTIVATED, THE PRENEOPLASTIC CELL POPULATION EXPANDS AND MALLORY-DENK BODIES RAPIDLY REFORM. THIS RESPONSE IS REMEMBERED BY THE PRENEOPLASTIC CELLS FOR AT LEAST FOUR MONTHS INDICATING THAT AN EPIGENETIC CELLULAR MEMORY HAS FORMED IN THE PRENEOPLASTIC CELLS. THIS PROLIFERATIVE RESPONSE IS PREVENTED BY FEEDING METHYL DONORS SUCH AS S-ADENOSYLMETHIONINE OR BETAINE. DRUG FEEDING REDUCES THE METHYLATION OF H(3) K4, 9, AND 27 AND THIS RESPONSE IS PREVENTED BY FEEDING THE METHYL DONORS. AFTER 8 TO 15MONTHS OF DRUG WITHDRAWAL IN MICE THE PRENEOPLASTIC LIVER CELLS PERSIST AS SINGLE OR SMALL CLUSTERS OF CELLS IN THE LIVER LOBULES. MULTIPLE LIVER TUMORS FORM, SOME OF WHICH ARE HEPATOCELLULAR CARCINOMAS. THE TUMORS IMMUNOSTAIN POSITIVELY FOR THE SAME PRENEOPLASTIC MARKERS AS THE PRENEOPLASTIC CELLS. SIMILAR CELLS ARE IDENTIFIED IN HUMAN CIRRHOSIS AND HEPATOCELLULAR CARCINOMA INDICATING THE RELEVANCE OF THE DRUG MODEL DESCRIBED HERE TO THE PRENEOPLASTIC CHANGES ASSOCIATED WITH HUMAN CHRONIC HEPATITIS AND HEPATOCELLULAR CARCINOMA. 2011 4 894 34 CHRONIC ETHANOL FEEDING ALTERS HEPATOCYTE MEMORY WHICH IS NOT ALTERED BY ACUTE FEEDING. BACKGROUND: GENE EXPRESSION CHANGES IN THE LIVER AFTER ACUTE BINGE DRINKING MAY DIFFER FROM THE CHANGES SEEN IN CHRONIC ETHANOL FEEDING IN THE RAT. THE CHANGES IN GENE EXPRESSION AFTER CHRONIC ETHANOL FEEDING MAY SENSITIZE THE LIVER TO ALCOHOL-INDUCED LIVER DAMAGE, WHICH IS NOT SEEN AFTER ACUTE BINGE DRINKING. METHODS: TO TEST THIS HYPOTHESIS, GENE MICROARRAY ANALYSIS WAS PERFORMED ON THE LIVERS OF RATS (N = 3) FED AN ACUTE BINGE DOSE OF ETHANOL (6 G/KG BODY WT) AND KILLED AT 3 AND 12 HOURS AFTER ETHANOL BY GAVAGE. THE GENE MICROARRAYS WERE COMPARED WITH THOSE MADE ON THE LIVER OF RATS FROM A PREVIOUS STUDY, IN WHICH THE RATS WERE FED ETHANOL BY INTRAGASTRIC TUBE FOR 1 MONTH (36% OF CALORIES DERIVED FROM ETHANOL). RESULTS: MICROARRAY ANALYSIS DATA VARIED BETWEEN THE ACUTE AND CHRONIC MODELS IN SEVERAL IMPORTANT RESPECTS. GROWTH FACTORS INCREASED MAINLY IN THE CHRONIC ALCOHOL FED RAT. CHANGES IN ENZYMES INVOLVED IN OXIDATIVE STRESS WERE NOTED ONLY WITH CHRONIC ETHANOL FEEDING. GENE EXPRESSION OF FAT METABOLISM WAS INCREASED ONLY WITH CHRONIC ETHANOL FEEDING. MOST IMPORTANTLY, EPIGENETIC RELATED ENZYMES AND ACETYLATION AND METHYLATION OF HISTONES CHANGED ONLY AFTER CHRONIC ETHANOL FEEDING. CONCLUSIONS: THE RESULTS SUPPORT THE CONCEPT THAT CHRONIC ETHANOL INGESTION INDUCES ALTERED GENE EXPRESSION AS A RESULT OF CHANGES IN EPIGENETIC MECHANISMS, WHERE ACETYLATION AND METHYLATION OF HISTONES WERE ALTERED. 2009 5 5057 22 PHENOBARBITAL MECHANISTIC DATA AND RISK ASSESSMENT: ENZYME INDUCTION, ENHANCED CELL PROLIFERATION, AND TUMOR PROMOTION. CHRONIC EXPOSURE TO HIGH DOSES OF PHENOBARBITAL (PB) CAUSES HEPATOCELLULAR ADENOMAS IN BOTH MICE AND RATS AND HEPATOCELLULAR CARCINOMAS IN SOME STRAINS OF MICE. LONG-TERM PB THERAPY HAS NOT BEEN FOUND TO CAUSE HUMAN TUMORS. PB IS NOT DNA REACTIVE, AND MOST GENOTOXICITY TESTS HAVE YIELDED NEGATIVE RESULTS. PB HAS BEEN EXTENSIVELY STUDIED AS AN EPIGENETIC, RODENT LIVER TUMOR PROMOTER. AT EXPOSURES CAUSING RODENT LIVER TUMORS, PB HAS MEASURABLE EFFECTS ON HEPATOCYTES: PB INHIBITS CELL-TO-CELL COMMUNICATION; PB INDUCES ENZYMES, INCLUDING P450 CYTOCHROMES; PB STIMULATES PROLIFERATION AND INHIBITS APOPTOSIS OF HEPATOCYTES IN NEOPLASTIC FOCI. THRESHOLD EXPOSURES FOR SOME OF THESE ENDPOINTS COINCIDE WITH THE THRESHOLD EXPOSURE FOR TUMORIGENESIS. 1996 6 871 29 CHRONIC ALCOHOL EXPOSURE AFFECTS PANCREATIC ACINAR MITOCHONDRIAL THIAMIN PYROPHOSPHATE UPTAKE: STUDIES WITH MOUSE 266-6 CELL LINE AND PRIMARY CELLS. THIAMIN IS ESSENTIAL FOR NORMAL METABOLIC ACTIVITY OF ALL MAMMALIAN CELLS, INCLUDING THOSE OF THE PANCREAS. CELLS OBTAIN THIAMIN FROM THEIR SURROUNDINGS AND ENZYMATICALLY CONVERT IT INTO THIAMIN PYROPHOSPHATE (TPP) IN THE CYTOPLASM; TPP IS THEN TAKEN UP BY MITOCHONDRIA VIA A SPECIFIC CARRIER THE MITOCHONDRIAL TPP TRANSPORTER (MTPPT; PRODUCT OF THE SLC25A19 GENE). CHRONIC ALCOHOL EXPOSURE NEGATIVELY IMPACTS THE HEALTH OF PANCREATIC ACINAR CELLS (PAC), BUT ITS EFFECT ON PHYSIOLOGICAL/MOLECULAR PARAMETERS OF MTPPT IS NOT KNOWN. WE ADDRESSED THIS ISSUE USING MOUSE PANCREATIC ACINAR TUMOR CELL LINE 266-6 AND PRIMARY PAC OF WILD-TYPE AND TRANSGENIC MICE CARRYING THE SLC25A19 PROMOTER THAT WERE FED ALCOHOL CHRONICALLY. CHRONIC ALCOHOL EXPOSURE OF 266-6 CELLS (BUT NOT TO ITS NONOXIDATIVE METABOLITES ETHYL PALMITATE AND ETHYL OLEATE) LED TO A SIGNIFICANT INHIBITION IN MITOCHONDRIAL TPP UPTAKE, WHICH WAS ASSOCIATED WITH A DECREASED EXPRESSION OF MTPPT PROTEIN, MRNA, AND ACTIVITY OF THE SLC25A19 PROMOTER. SIMILARLY, CHRONIC ALCOHOL FEEDING OF MICE LED TO A SIGNIFICANT INHIBITION IN EXPRESSION OF MTPPT PROTEIN, MRNA, HETEROGENEOUS NUCLEAR RNA, AS WELL AS IN ACTIVITY OF SLC25A19 PROMOTER IN PAC. WHILE CHRONIC ALCOHOL EXPOSURE DID NOT AFFECT DNA METHYLATION OF THE SLC25A19 PROMOTER, A SIGNIFICANT DECREASE IN HISTONE H3 EUCHROMATIN MARKERS AND AN INCREASE IN H3 HETEROCHROMATIN MARKER WERE OBSERVED. THESE FINDINGS SHOW, FOR THE FIRST TIME, THAT CHRONIC ALCOHOL EXPOSURE NEGATIVELY IMPACTS PANCREATIC MTPPT, AND THAT THIS EFFECT IS EXERTED, AT LEAST IN PART, AT THE LEVEL OF SLC25A19 TRANSCRIPTION AND APPEARS TO INVOLVE EPIGENETIC MECHANISM(S). 2015 7 2907 37 GENE EXPRESSION MODIFICATIONS IN THE LIVER CAUSED BY BINGE DRINKING AND S-ADENOSYLMETHIONINE FEEDING. THE ROLE OF EPIGENETIC CHANGES. CHRONIC ETHANOL INGESTION, ACHIEVED BY FEEDING ETHANOL AT A CONSTANT RATE USING INTRAGASTRIC TUBE FEEDING, ALTERS THE EXPRESSION OF GENES IN THE LIVER. THIS IS DONE BY EPIGENETIC MECHANISMS, WHICH DEPEND ON THE BLOOD ALCOHOL LEVELS AT THE TIME OF KILLING. HOWEVER, ACUTE BOLUS FEEDING OF ETHANOL CHANGES GENE EXPRESSION WITHOUT LASTING EPIGENETIC CHANGES. THIS OCCURS WITH HISTONE 3 METHYLATION AND ACETYLATION MODIFICATIONS. THE GENE EXPRESSION RESPONSE TO AN ACUTE BOLUS OF ETHANOL MIGHT BE MODIFIED BY FEEDING S-ADENOSYLMETHIONINE (SAME), A METHYL DONOR. IN THE PRESENT STUDY, RATS WERE GIVEN A BOLUS OF ETHANOL (6 G/KG BODY WEIGHT (BW), SAME (1 G/KG BW), ETHANOL + SAME, OR ISOCALORIC GLUCOSE. THE GROUP OF RATS (N = 3) WERE KILLED AT 3 AND 12 H POST BOLUS, AND GENE MICROARRAY ANALYSIS WAS PERFORMED ON THEIR LIVER CELLS. SAME REDUCED THE 3 H BLOOD ETHANOL LEVELS AND INCREASED THE ALT LEVELS AT 3 H. VENN DIAGRAMS SHOWED THAT ALCOHOL CHANGED THE EXPRESSION OF 646 GENES AT 3 H POST BOLUS AND 586 GENES AT 12 H. SAME CHANGED THE EXPRESSION OF 1,012 GENES WHEN FED WITH ETHANOL 3 H POST ETHANOL BOLUS AND 554 GENES AT 12 H POST ETHANOL BOLUS. SAME ALONE CHANGED THE EXPRESSION OF 1,751 GENES AT 3 H AND 1,398 AT 12 H. THERE WERE MORE CHANGES IN GENE EXPRESSION AT 3 H THAN AT 12 H POST ETHANOL WHEN ETHANOL ALONE WAS COMPARED TO THE DEXTROSE CONTROL. THE SAME WAS TRUE WHEN SAME WAS COMPARED TO SAME + ETHANOL. ETHANOL UP REGULATED GENE EXPRESSION IN MOST FUNCTIONAL PATHWAYS AT 3 H. HOWEVER, WHEN SAME WAS FED WITH ETHANOL AT 3 H, MOST PATHWAYS WERE DOWN REGULATED. AT 12 H, HOWEVER, WHEN ETHANOL WAS FED, THE PATHWAYS WERE HALF UP REGULATED AND HALF DOWN REGULATED. THE SAME WAS TRUE WHEN SAME + ETHANOL WAS FED. THE EXPRESSION OF EPIGENETICALLY IMPORTANT GENES, SUCH AS BHMT AND FOXN3, WAS UP REGULATED 3 H POST ALCOHOL BOLUS. AT 3 H, SAME DOWN REGULATED THE EXPRESSION OF GENES, SUCH AS BHMT, MAT2A, JUN, TNFRS9, AHCY 1, TGFBR1 AND 2, AND PCAF. AT 12 H, THE INSULIN SIGNALING PATHWAYS WERE HALF DOWN REGULATED BY ETHANOL, WHICH WAS PARTLY PREVENTED BY SAME. THE MAPK PATHWAY WAS UP REGULATED BY ETHANOL, BUT SAME DID NOT PREVENT THIS. IN CONCLUSION, PROFOUND CHANGES IN GENE EXPRESSION EVOLVED BETWEEN 3 H AND 12 POST ETHANOL BOLUS. SAME DOWN REGULATED THESE CHANGES IN GENE EXPRESSION AT 3 H, AND LESS SO AT 12 H. 2010 8 2302 32 EPIGENETIC REGULATION OF CANCER STEM CELL MARKER CD133 BY TRANSFORMING GROWTH FACTOR-BETA. HEPATOCELLULAR CARCINOMA (HCC) IS THE THIRD LEADING CAUSE OF CANCER MORTALITY WORLDWIDE. CD133, A TRANSMEMBRANE GLYCOPROTEIN, IS AN IMPORTANT CELL SURFACE MARKER FOR BOTH STEM CELLS AND CANCER STEM CELLS IN VARIOUS TISSUES INCLUDING LIVER. CD133 EXPRESSION HAS BEEN RECENTLY LINKED TO POOR PROGNOSIS IN HCC PATIENTS. CD133+ LIVER CANCER CELLS ARE CHARACTERIZED BY RESISTANCE TO CHEMOTHERAPY, SELF-RENEWAL, MULTILINEAGE POTENTIAL, INCREASED COLONY FORMATION, AND IN VIVO CANCER INITIATION AT LIMITED DILUTION. RECENT STUDIES DEMONSTRATE THAT CD133 EXPRESSION IS REGULATED BY DNA METHYLATION. IN THIS STUDY, WE EXPLORED THE ROLE OF TRANSFORMING GROWTH FACTOR BETA (TGFBETA), A MULTIFUNCTIONAL CYTOKINE THAT PLAYS A CRITICAL ROLE IN CHRONIC LIVER INJURY, IN THE REGULATION OF CD133 EXPRESSION. TGFBETA1 IS CAPABLE OF UP-REGULATING CD133 EXPRESSION SPECIFICALLY WITHIN THE HUH7 HCC CELL LINE IN A TIME- AND DOSE-DEPENDENT MANNER. MOST IMPORTANT, TGFBETA1-INDUCED CD133+ HUH7 CELLS DEMONSTRATE INCREASED TUMOR INITIATION IN VIVO. FORCED EXPRESSION OF INHIBITORY SMADS, INCLUDING SMAD6 AND SMAD7, ATTENUATED TGFBETA1-INDUCED CD133 EXPRESSION. WITHIN CD133- HUH7 CELLS, TGFBETA1 STIMULATION INHIBITED THE EXPRESSION OF DNA METHYLTRANSFERASES (DNMT) 1 AND DNMT3BETA, WHICH ARE CRITICAL IN THE MAINTENANCE OF REGIONAL DNA METHYLATION, AND GLOBAL DNMT ACTIVITY IN CD133- HUH7 CELLS WAS INHIBITED BY TGFBETA1. DNMT3BETA INHIBITION BY TGFBETA1 WAS PARTIALLY RESCUED WITH OVEREXPRESSION OF INHIBITORY SMADS. LASTLY, TGFBETA1 TREATMENT LED TO SIGNIFICANT DEMETHYLATION IN CD133 PROMOTER-1 IN CD133- HUH7 CELLS. CONCLUSION: TGFBETA1 IS ABLE TO REGULATE CD133 EXPRESSION THROUGH INHIBITION OF DNMT1 AND DNMT3BETA EXPRESSION AND SUBSEQUENT DEMETHYLATION OF PROMOTER-1. TGFBETA1-INDUCED CD133+ HUH7 CELLS ARE TUMORIGENIC. THE MECHANISM BY WHICH TGFBETA INDUCES CD133 EXPRESSION IS PARTIALLY DEPENDENT ON THE SMADS PATHWAY. 2010 9 5609 35 S-ADENOSYLMETHIONINE DECREASES THE PEAK BLOOD ALCOHOL LEVELS 3 H AFTER AN ACUTE BOLUS OF ETHANOL BY INDUCING ALCOHOL METABOLIZING ENZYMES IN THE LIVER. INTRODUCTION: AN ALCOHOL BOLUS CAUSES THE BLOOD ALCOHOL LEVEL (BAL) TO PEAK AT 1-2 H POST INGESTION. THE ETHANOL ELIMINATION RATE IS REGULATED BY ALCOHOL METABOLIZING ENZYMES, PRIMARILY ALCOHOL DEHYDROGENASE (ADH1), ACETALDEHYDE DEHYDROGENASE (ALDH), AND CYTOCHROME P450 (CYP2E1). RECENTLY, S-ADENOSYLMETHIONINE (SAME) WAS FOUND TO REDUCE ACUTE BALS 3 H AFTER AN ALCOHOL BOLUS. THE QUESTION, THEN, WAS: WHAT IS THE MECHANISM INVOLVED IN THIS REDUCTION OF BAL BY FEEDING SAME? TO ANSWER THIS QUESTION, WE INVESTIGATED THE CHANGES IN ETHANOL METABOLIZING ENZYMES AND THE EPIGENETIC CHANGES THAT REGULATE THE EXPRESSION OF THESE ENZYMES DURING ACUTE BINGE DRINKING AND CHRONIC DRINKING. METHODS: RATS WERE FED A BOLUS OF ETHANOL WITH OR WITHOUT SAME, AND WERE SACRIFICED AT 3 H OR 12 H AFTER THE BOLUS. RESULTS: RT-PCR AND WESTERN BLOT ANALYSES SHOWED THAT SAME SIGNIFICANTLY INDUCED ADH1 LEVELS IN THE 3 H LIVER SAMPLES. HOWEVER, SAME DID NOT AFFECT THE CHANGES IN ADH1 PROTEIN LEVELS 12 H POST BOLUS. SINCE SAME IS A METHYL DONOR, IT WAS POSTULATED THAT THE ADH1 GENE EXPRESSION UP REGULATION AT 3 H WAS DUE TO A HISTONE MODIFICATION INDUCED BY METHYLATION FROM METHYL TRANSFERASES. DIMETHYLATED HISTONE 3 LYSINE 4 (H3K4ME2), A MODIFICATION RESPONSIBLE FOR GENE EXPRESSION ACTIVATION, WAS FOUND TO BE SIGNIFICANTLY INCREASED BY SAME AT 3 H POST BOLUS. CONCLUSION: THESE RESULTS CORRELATED WITH THE LOW BAL FOUND AT 3 H POST BOLUS, AND SUPPORT THE CONCEPT THAT SAME INCREASED THE GENE EXPRESSION TO INCREASE THE ELIMINATION RATE OF ETHANOL IN BINGE DRINKING BY INCREASING H3K4ME2. 2010 10 3240 35 HEPATIC LIPID ACCUMULATION ALTERS GLOBAL HISTONE H3 LYSINE 9 AND 4 TRIMETHYLATION IN THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA NETWORK. RECENT DATA SUGGEST THAT THE ETIOLOGY OF SEVERAL METABOLIC DISEASES IS CLOSELY ASSOCIATED WITH TRANSCRIPTOME ALTERATION BY ABERRANT HISTONE METHYLATION. WE PERFORMED DNA MICROARRAY AND CHIP-ON-CHIP ANALYSES TO EXAMINE TRANSCRIPTOME PROFILING AND TRIMETHYLATION ALTERATIONS TO IDENTIFY THE GENOMIC SIGNATURE OF NONALCOHOLIC FATTY LIVER DISEASE (NAFLD), THE MOST COMMON FORM OF CHRONIC LIVER DISEASE. TRANSCRIPTOME ANALYSIS SHOWED THAT STEATOTIC LIVERS IN HIGH-FAT DIET-FED APOLIPOPROTEIN E2 MICE SIGNIFICANTLY ALTERED THE EXPRESSION OF APPROXIMATELY 70% OF TOTAL GENES COMPARED WITH NORMAL DIET-FED CONTROL LIVERS, SUGGESTING THAT HEPATIC LIPID ACCUMULATION INDUCES DRAMATIC ALTERATIONS IN GENE EXPRESSION IN VIVO. ALSO, PATHWAY ANALYSIS SUGGESTED THAT GENES ENCODING CHROMATIN-REMODELING ENZYMES, SUCH AS JUMONJI C-DOMAIN-CONTAINING HISTONE DEMETHYLASES THAT REGULATE HISTONE H3K9 AND H3K4 TRIMETHYLATION (H3K9ME3, H3K4ME3), WERE SIGNIFICANTLY ALTERED IN STEATOTIC LIVERS. THUS, WE FURTHER INVESTIGATED THE GLOBAL H3K9ME3 AND H3K4ME3 STATUS IN LIPID-ACCUMULATED MOUSE PRIMARY HEPATOCYTES BY CHIP-ON-CHIP ANALYSIS. RESULTS SHOWED THAT HEPATIC LIPID ACCUMULATION INDUCED ABERRANT H3K9ME3 AND H3K4ME3 STATUS IN PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA AND HEPATIC LIPID CATABOLISM NETWORK GENES, REDUCING THEIR MRNA EXPRESSION COMPARED WITH NON-TREATED CONTROL HEPATOCYTES. THIS STUDY PROVIDES THE FIRST EVIDENCE THAT EPIGENETIC REGULATION BY H3K9ME3 AND H3K4ME3 IN HEPATOCYTES MAY BE INVOLVED IN HEPATIC STEATOSIS AND THE PATHOGENESIS OF NAFLD. THUS, CONTROL OF H3K9ME3 AND H3K4ME3 REPRESENTS A POTENTIAL NOVEL NAFLD PREVENTION AND TREATMENT STRATEGY. 2012 11 6456 27 THYMOSIN BETA4 PREVENTS OXIDATIVE STRESS, INFLAMMATION, AND FIBROSIS IN ETHANOL- AND LPS-INDUCED LIVER INJURY IN MICE. THYMOSIN BETA 4 (TBETA4), AN ACTIN-SEQUESTERING PROTEIN, IS INVOLVED IN TISSUE DEVELOPMENT AND REGENERATION. IT PREVENTS INFLAMMATION AND FIBROSIS IN SEVERAL TISSUES. WE INVESTIGATED THE ROLE OF TBETA4 IN CHRONIC ETHANOL- AND ACUTE LIPOPOLYSACCHARIDE- (LPS-) INDUCED MOUSE LIVER INJURY. C57BL/6 MICE WERE FED 5% ETHANOL IN LIQUID DIET FOR 4 WEEKS PLUS BINGE ETHANOL (5 G/KG, GAVAGE) WITH OR WITHOUT LPS (2 MG/KG, INTRAPERITONEAL) FOR 6 HOURS. TBETA4 (1 MG/KG, INTRAPERITONEAL) WAS ADMINISTERED FOR 1 WEEK. WE DEMONSTRATED THAT TBETA4 PREVENTED ETHANOL- AND LPS-MEDIATED INCREASE IN LIVER INJURY MARKERS AS WELL AS CHANGES IN LIVER PATHOLOGY. IT ALSO PREVENTED ETHANOL- AND LPS-MEDIATED INCREASE IN OXIDATIVE STRESS BY DECREASING ROS AND LIPID PEROXIDATION AND INCREASING THE ANTIOXIDANTS, REDUCED GLUTATHIONE AND MANGANESE-DEPENDENT SUPEROXIDE DISMUTASE. IT ALSO PREVENTED THE ACTIVATION OF NUCLEAR FACTOR KAPPA B BY BLOCKING THE PHOSPHORYLATION OF THE INHIBITORY PROTEIN, IKAPPAB, THEREBY PREVENTED PROINFLAMMATORY CYTOKINE PRODUCTION. MOREOVER, TBETA4 PREVENTED FIBROGENESIS BY SUPPRESSING THE EPIGENETIC REPRESSOR, METHYL-CPG-BINDING PROTEIN 2, THAT COORDINATELY REVERSED THE EXPRESSION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA AND DOWNREGULATED FIBROGENIC GENES, PLATELET-DERIVED GROWTH FACTOR-BETA RECEPTOR, ALPHA-SMOOTH MUSCLE ACTIN, COLLAGEN 1, AND FIBRONECTIN, RESULTING IN REDUCED FIBROSIS. OUR DATA SUGGEST THAT TBETA4 HAS ANTIOXIDANT, ANTI-INFLAMMATORY, AND ANTIFIBROTIC POTENTIAL DURING ALCOHOLIC LIVER INJURY. 2018 12 2590 36 EPIGENETICS OF PROTEASOME INHIBITION IN THE LIVER OF RATS FED ETHANOL CHRONICALLY. AIM: TO EXAMINE THE EFFECTS OF ETHANOL-INDUCED PROTEASOME INHIBITION, AND THE EFFECTS OF PROTEASOME INHIBITION IN THE REGULATION OF EPIGENETIC MECHANISMS. METHODS: RATS WERE FED ETHANOL FOR 1 MO USING THE TSUKAMOTO-FRENCH MODEL AND WERE COMPARED TO RATS GIVEN THE PROTEASOME INHIBITOR PS-341 (BORTEZOMIB, VELCADE(TM)) BY INTRAPERITONEAL INJECTION. MICROARRAY ANALYSIS AND REAL TIME PCR WERE PERFORMED AND PROTEASOME ACTIVITY ASSAYS AND WESTERN BLOT ANALYSIS WERE PERFORMED USING ISOLATED NUCLEI. RESULTS: CHRONIC ETHANOL FEEDING CAUSED A SIGNIFICANT INHIBITION OF THE UBIQUITIN PROTEASOME PATHWAY IN THE NUCLEUS, WHICH LED TO CHANGES IN THE TURNOVER OF TRANSCRIPTIONAL FACTORS, HISTONE-MODIFYING ENZYMES, AND, THEREFORE, AFFECTED EPIGENETIC MECHANISMS. CHRONIC ETHANOL FEEDING WAS RELATED TO AN INCREASE IN HISTONE ACETYLATION, AND IT IS HYPOTHESIZED THAT THE PROTEASOME PROTEOLYTIC ACTIVITY REGULATED HISTONE MODIFICATIONS BY CONTROLLING THE STABILITY OF HISTONE MODIFYING ENZYMES, AND, THEREFORE, REGULATED THE CHROMATIN STRUCTURE, ALLOWING EASY ACCESS TO CHROMATIN BY RNA POLYMERASE, AND, THUS, PROPER GENE EXPRESSION. PROTEASOME INHIBITION BY PS-341 INCREASED HISTONE ACETYLATION SIMILAR TO CHRONIC ETHANOL FEEDING. IN ADDITION, PROTEASOME INHIBITION CAUSED DRAMATIC CHANGES IN HEPATIC REMETHYLATION REACTIONS AS THERE WAS A SIGNIFICANT DECREASE IN THE ENZYMES RESPONSIBLE FOR THE REGENERATION OF S-ADENOSYLMETHIONINE, AND, IN PARTICULAR, A SIGNIFICANT DECREASE IN THE BETAINE-HOMOCYSTEINE METHYLTRANSFERASE ENZYME. THIS SUGGESTED THAT HYPOMETHYLATION WAS ASSOCIATED WITH PROTEASOME INHIBITION, AS INDICATED BY THE DECREASE IN HISTONE METHYLATION. CONCLUSION: THE ROLE OF PROTEASOME INHIBITION IN REGULATING EPIGENETIC MECHANISMS, AND ITS LINK TO LIVER INJURY IN ALCOHOLIC LIVER DISEASE, IS THUS A PROMISING APPROACH TO STUDY LIVER INJURY DUE TO CHRONIC ETHANOL CONSUMPTION. 2009 13 1646 31 DOES THE HEPATITIS B ANTIGEN HBX PROMOTE THE APPEARANCE OF LIVER CANCER STEM CELLS? HEPATITIS B VIRUS (HBV) IS A MAJOR ETIOLOGIC AGENT OF CHRONIC LIVER DISEASE AND HEPATOCELLULAR CARCINOMA (HCC). HBV-ENCODED X ANTIGEN, HBX, AND PATHWAYS IMPLICATED IN THE SELF-RENEWAL OF STEM CELLS CONTRIBUTE TO HCC, BUT IT IS NOT CLEAR WHETHER HBX EXPRESSION PROMOTES "STEMNESS." THUS, EXPERIMENTS WERE DESIGNED TO TEST THE HYPOTHESIS THAT HBX TRIGGERS MALIGNANT TRANSFORMATION BY PROMOTING PROPERTIES THAT ARE CHARACTERISTIC OF CANCER STEM CELLS (CSC). TO TEST THIS HYPOTHESIS, HEPG2 CELLS WERE STABLY TRANSDUCED WITH HBX AND THEN ASSAYED FOR PHENOTYPIC AND MOLECULAR CHARACTERISTICS OF "STEMNESS." THE RELATIONSHIP BETWEEN HBX AND "STEMNESS"-ASSOCIATED MARKERS WAS ALSO EVALUATED BY IMMUNOHISTOCHEMICAL STAINING OF LIVER AND TUMOR TISSUE SECTIONS FROM HBV-INFECTED PATIENTS. THE RESULTS SHOWED THAT OCT-4, NANOG, KLF-4, BETA-CATENIN, AND EPITHELIAL CELL ADHESION MOLECULE (EPCAM) WERE ACTIVATED BY HBX IN VITRO AND IN VIVO. EPCAM WAS DETECTED IN THE NUCLEI OF HUMAN HCC CELLS FROM INFECTED PATIENTS. HBX PROMOTES "STEMNESS" BY ACTIVATING BETA-CATENIN AND EPIGENETIC UPREGULATION OF MIR-181, BOTH OF WHICH TARGET EPCAM. HBX EXPRESSION WAS ALSO ASSOCIATED WITH DEPRESSED LEVELS OF E-CADHERIN. MOREOVER, HBX STIMULATED CELL MIGRATION, GROWTH IN SOFT AGAR, AND SPHEROID FORMATION. THIS WORK IS THE FIRST TO PROPOSE THAT HBV PROMOTES "STEMNESS" IN THE PATHOGENESIS OF HCC. HBX-ASSOCIATED UPREGULATED EXPRESSION OF MULTIPLE "STEMNESS" MARKERS SUPPORTS THE HYPOTHESIS THAT HBX CONTRIBUTES TO HEPATOCARCINOGENESIS, AT LEAST IN PART, BY PROMOTING CHANGES IN GENE EXPRESSION THAT ARE CHARACTERISTICS OF CSCS. 2011 14 3619 30 IN VIVO ACUTE ON CHRONIC ETHANOL EFFECTS IN LIVER: A MOUSE MODEL EXHIBITING EXACERBATED INJURY, ALTERED METABOLIC AND EPIGENETIC RESPONSES. CHRONIC ALCOHOLICS WHO ALSO BINGE DRINK (I.E., ACUTE ON CHRONIC) ARE PRONE TO AN EXACERBATED LIVER INJURY BUT ITS MECHANISM IS NOT UNDERSTOOD. WE THEREFORE INVESTIGATED THE IN VIVO EFFECTS OF CHRONIC AND BINGE ETHANOL INGESTION AND COMPARED TO CHRONIC ETHANOL FOLLOWED BY THREE REPEAT BINGE ETHANOL ON THE LIVER OF MALE C57/BL6 MICE FED ETHANOL IN LIQUID DIET (4%) FOR FOUR WEEKS FOLLOWED BY BINGE ETHANOL (INTRAGASTRIC ADMINISTRATION, 3.5 G/KG BODY WEIGHT, THREE DOSES, 12H APART). CHRONIC FOLLOWED BY BINGE ETHANOL EXACERBATED FAT ACCUMULATION, NECROSIS, DECREASE IN HEPATIC SAM AND SAM:SAH RATIO, INCREASE IN ADENOSINE LEVELS, AND ELEVATED CYP2E1 LEVELS. HISTONE H3 LYSINE ACETYLATION (H3ACK9), DUALLY MODIFIED PHOSPHOACETYLATED HISTONE H3 (H3ACK9/PS10), AND PHOSPHORYLATED H2AX INCREASED AFTER BINGE WHEREAS PHOSPHORYLATION OF HISTONE H3 SER 10 (H3S10) AND H3 SER 28 (H3S28) INCREASED AFTER CHRONIC ETHANOL-BINGE. HISTONE H3 LYSINE 4 AND 9 DIMETHYLATION INCREASED WITH A MARKED DIMETHYLATION IN H3K9 IN CHRONIC ETHANOL BINGE GROUP. TRIMETHYLATED HISTONE H3 LEVELS DID NOT CHANGE. NUCLEAR LEVELS OF HISTONE ACETYL TRANSFERASE GCN5 AND HISTONE DEACETYLASE HDAC3 WERE ELEVATED WHEREAS PHOSPHO-CREB DECREASED IN A DISTINCTIVE MANNER. TAKEN TOGETHER, ACUTE ON CHRONIC ETHANOL INGESTION CAUSED AMPLIFICATION OF LIVER INJURY AND ELICITED CHARACTERISTIC PROFILES OF HISTONE MODIFICATIONS, METABOLIC ALTERATIONS, AND CHANGES IN NUCLEAR PROTEIN LEVELS. THESE FINDINGS DEMONSTRATE THAT CHRONIC ETHANOL EXPOSURE RENDERS LIVER MORE SUSCEPTIBLE TO REPEAT ACUTE/BINGE ETHANOL INDUCED ACCELERATION OF ALCOHOLIC LIVER DISEASE. 2015 15 3264 34 HEPATITIS VIRUS INFECTION AFFECTS DNA METHYLATION IN MICE WITH HUMANIZED LIVERS. BACKGROUND & AIMS: CELLS OF TUMORS ASSOCIATED WITH CHRONIC INFLAMMATION FREQUENTLY HAVE ALTERED PATTERNS OF DNA METHYLATION, INCLUDING HEPATOCELLULAR CARCINOMAS. CHRONIC HEPATITIS HAS ALSO BEEN ASSOCIATED WITH ABERRANT DNA METHYLATION, BUT LITTLE IS KNOWN ABOUT THEIR RELATIONSHIP. METHODS: PYROSEQUENCING WAS USED TO DETERMINE THE METHYLATION STATUS OF CULTURED HUH7.5.1 HEPATOMA CELLS AFTER HEPATITIS C VIRUS (HCV) INFECTION. WE ALSO STUDIED MICE WITH SEVERE COMBINED IMMUNODEFICIENCY CARRYING THE UROKINASE-TYPE PLASMINOGEN ACTIVATOR TRANSGENE CONTROLLED BY AN ALBUMIN PROMOTER (UROKINASE-TYPE PLASMINOGEN ACTIVATOR/SEVERE COMBINED IMMUNODEFICIENT MICE), IN WHICH UP TO 85% OF HEPATOCYTES WERE REPLACED BY HUMAN HEPATOCYTES (CHIMERIC MICE). MICE WERE GIVEN INTRAVENOUS INJECTIONS OF HEPATITIS B VIRUS (HBV) OR HCV, LIVER TISSUES WERE COLLECTED, AND DNA METHYLATION PROFILES WERE DETERMINED AT DIFFERENT TIME POINTS AFTER INFECTION. WE ALSO COMPARED METHYLATION PATTERNS BETWEEN PAIRED SAMPLES OF HEPATOCELLULAR CARCINOMAS AND ADJACENT NONTUMOR LIVER TISSUES FROM PATIENTS. RESULTS: NO REPRODUCIBLE CHANGES IN DNA METHYLATION WERE OBSERVED AFTER INFECTION OF HUH7.5.1 CELLS WITH HCV. LIVERS FROM HBV- AND HCV-INFECTED MICE HAD GENOME-WIDE, TIME-DEPENDENT CHANGES IN DNA METHYLATION, COMPARED WITH UNINFECTED UROKINASE-TYPE PLASMINOGEN ACTIVATOR/SEVERE COMBINED IMMUNODEFICIENT MICE. THERE WERE CHANGES IN 160 +/- 63 GENES IN HBV-INFECTED AND 237 +/- 110 GENES IN HCV-INFECTED MICE. METHYLATION OF 149 COMMON GENES INCREASED IN HBV- AND HCV-INFECTED MICE; METHYLATION OF SOME OF THESE GENES ALSO INCREASED IN HEPATOCELLULAR CARCINOMA SAMPLES FROM PATIENTS COMPARED WITH NONTUMOR TISSUES. EXPRESSION OF IFNG, WHICH IS EXPRESSED BY NATURAL KILLER CELLS, INCREASED SIGNIFICANTLY IN CHIMERIC LIVERS, IN CONCORDANCE WITH INDUCTION OF DNA METHYLATION, AFTER INFECTION WITH HBV OR HCV. INDUCTION OF IFNG WAS REDUCED AFTER ADMINISTRATION OF AN INHIBITOR OF NATURAL KILLER CELL FUNCTION (ANTI-ASIALO GM1). CONCLUSIONS: IN CHIMERIC MICE WITH HUMANIZED LIVERS, INFECTION WITH HBV AND HCV APPEARS TO ACTIVATE A NATURAL KILL CELL-DEPENDENT INNATE IMMUNE RESPONSE. THIS CONTRIBUTES TO THE INDUCTION AND ACCUMULATION OF ABERRANT DNA METHYLATION IN HUMAN HEPATOCYTES. 2014 16 4903 19 P16 PROMOTER HYPERMETHYLATION IN HUMAN HEPATOCELLULAR CARCINOMA WITH OR WITHOUT HEPATITIS VIRUS INFECTION. BACKGROUND: EPIGENETIC ALTERATION THROUGH METHYLATION IS ONE OF THE MOST IMPORTANT STEPS IN CARCINOGENESIS. HOWEVER, THE RELATION BETWEEN HEPATITIS VIRUS INFECTION AND EPIGENETIC ALTERATIONS IS POORLY UNDERSTOOD. METHODS: SIXTEEN PATIENTS WITHOUT HEPATITIS B VIRUS (HBV) AND HEPATITIS C VIRUS (HCV) AND 35 PATIENTS WITH HBV OR HCV WHO UNDERWENT LIVER RESECTION FOR HEPATOCELLULAR CARCINOMA (HCC) WERE STUDIED. MUTATION OF P53 WAS DETECTED BY DIRECT SEQUENCING. METHYLATION STATUS OF P16 WAS EVALUATED IN TUMOR AND NONCANCEROUS LIVER TISSUES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. RESULTS: IN HCC WITHOUT HBV AND HCV, P53 MUTATIONS WERE DETECTED IN 5 (31%) OF 16 HCCS. METHYLATION OF P16 PROMOTER WAS DETECTED IN 2 (25%) OF 8 MODERATELY DIFFERENTIATED HCCS, 6 (75%) OF 8 POORLY DIFFERENTIATED HCCS, AND NONE OF 16 NONCANCEROUS TISSUE SPECIMENS. IN HCC WITH HBV OR HCV, P53 MUTATIONS WERE DETECTED IN 8 (23%) OF 35 HCCS. METHYLATION OF P16 PROMOTER WAS DETECTED IN 2 (100%) OF 2 WELL-DIFFERENTIATED HCCS, 13 (76%) OF 17 MODERATELY DIFFERENTIATED HCCS, 12 (75%) OF 16 POORLY DIFFERENTIATED HCCS, AND 9 (26%) OF 35 NONCANCEROUS LIVER TISSUE SPECIMENS. CONCLUSIONS: OUR RESULTS SUGGEST THAT HEPATITIS VIRUSES MIGHT INDUCE METHYLATION OF P16 PROMOTER IN LIVER WITH CHRONIC INFLAMMATION, BEFORE APPEARANCE OF HCC. 2004 17 4215 33 METHYL DEFICIENCY, ALTERATIONS IN GLOBAL HISTONE MODIFICATIONS, AND CARCINOGENESIS. THE METHYL-DEFICIENT MODEL OF ENDOGENOUS HEPATOCARCINOGENESIS IN RODENTS IS UNIQUE IN THAT DIETARY OMISSION RATHER THAN THE ADDITION OF CHEMICAL CARCINOGENS LEADS TO TUMOR FORMATION. THUS, THE BIOCHEMICAL AND MOLECULAR EVENTS PREDISPOSING TO CANCER IN THIS MODEL RESULT FROM CHRONIC METABOLIC STRESS AND PROVIDE AN IDEAL MODEL SYSTEM TO STUDY PROGRESSIVE ALTERATIONS THAT OCCUR DURING CARCINOGENESIS. MOREOVER, EPIGENETIC ALTERATIONS IMPOSED BY THIS DIET ARE BELIEVED TO BE 1 OF THE MAIN MECHANISMS RESPONSIBLE FOR MALIGNANT TRANSFORMATION OF RAT LIVER CELLS. IN THIS STUDY WE EXAMINED THE CHANGES IN GLOBAL HISTONE MODIFICATION PATTERNS IN LIVER DURING HEPATOCARCINOGENESIS INDUCED BY METHYL DEFICIENCY. FEEDING ANIMALS THE METHYL-DEFICIENT DIET (MDD) LED TO PROGRESSIVE LOSS OF HISTONE H4 LYSINE 20 TRIMETHYLATION (H4K20ME3), H3 LYSINE 9 TRIMETHYLATION (H3K9ME3), AND HISTONE H3 LYSINE 9 (H3K9AC) AND HISTONE H4 LYSINE 16 (H4K16AC) ACETYLATION. A CONSIDERABLE DECREASE OF H4K20ME3 AND H3K9AC WAS ALSO DETECTED IN LIVER TUMORS INDUCED BY MDD. IN CONTRAST, LIVER TUMORS DISPLAYED AN INCREASE IN H3K9ME3 AND H4K16AC. TO DETERMINE THE POSSIBLE MECHANISM OF ALTERATIONS OF HISTONE MODIFICATIONS, WE ANALYZED THE EXPRESSION OF HISTONE-MODIFYING ENZYMES IN LIVER DURING HEPATOCARCINOGENESIS. THE EXPRESSION OF SUV4-20H2 AND RIZ1 HISTONE METHYLTRANSFERASES (HMTS) STEADILY DECREASED ALONG WITH THE DEVELOPMENT OF LIVER TUMORS AND REACHED ITS LOWEST LEVEL IN TUMOR TISSUE, WHEREAS THE EXPRESSION OF SUV39-H1 HMT AND HISTONE ACETYLTRANSFERASE 1 (HAT1) SUBSTANTIALLY INCREASED IN TUMORS. THESE RESULTS ILLUSTRATE THE COMPLEXITY AND IMPORTANCE OF HISTONE MODIFICATION CHANGES IN THE ETIOLOGY OF HEPATOCARCINOGENESIS INDUCED BY MDD. 2007 18 2433 30 EPIGENETIC SILENCING OF NAD(P)H:QUINONE OXIDOREDUCTASE 1 BY HEPATITIS B VIRUS X PROTEIN INCREASES MITOCHONDRIAL INJURY AND CELLULAR SUSCEPTIBILITY TO OXIDATIVE STRESS IN HEPATOMA CELLS. NAD(P)H:QUINONE OXIDOREDUCTASE 1 (NQO1) IS A PHASE II ENZYME THAT PARTICIPATES IN THE DETOXIFICATION OF DOPAMINE-DERIVED QUINONE MOLECULES AND REACTIVE OXYGEN SPECIES. OUR PRIOR WORK USING A PROTEOMIC APPROACH FOUND THAT NQO1 PROTEIN LEVELS WERE SIGNIFICANTLY DECREASED IN STABLE HEPATITIS B VIRUS (HBV)-PRODUCING HEPATOMA CELLS RELATIVE TO THE EMPTY-VECTOR-TRANSFECTED CONTROLS. HOWEVER, THE MECHANISM AND BIOLOGICAL SIGNIFICANCE OF THE NQO1 SUPPRESSION REMAIN ELUSIVE. IN THIS STUDY WE DEMONSTRATE THAT HBV X PROTEIN (HBX) INDUCES EPIGENETIC SILENCING OF NQO1 IN HEPATOMA CELLS THROUGH PROMOTER HYPERMETHYLATION VIA RECRUITMENT OF DNA METHYLTRANSFERASE DNMT3A TO THE PROMOTER REGION OF THE NQO1 GENE. IN HBV-RELATED HEPATOCELLULAR CARCINOMA (HCC) SPECIMENS, HBX EXPRESSION WAS CORRELATED NEGATIVELY TO NQO1 TRANSCRIPTS BUT POSITIVELY TO NQO1 PROMOTER HYPERMETHYLATION. DOWNREGULATION OF NQO1 BY HBX REDUCED INTRACELLULAR GLUTATHIONE LEVELS, IMPAIRED MITOCHONDRIAL FUNCTION, AND INCREASED SUSCEPTIBILITY OF HEPATOMA CELLS TO OXIDATIVE STRESS-INDUCED CELL INJURY. THESE RESULTS SUGGEST A NOVEL MECHANISM FOR HBV-MEDIATED PATHOGENESIS OF CHRONIC LIVER DISEASES, INCLUDING HCC. 2013 19 3242 33 HEPATIC NCOR1 DELETION EXACERBATES ALCOHOL-INDUCED LIVER INJURY IN MICE BY PROMOTING CCL2-MEDIATED MONOCYTE-DERIVED MACROPHAGE INFILTRATION. NUCLEAR RECEPTOR COREPRESSOR 1 (NCOR1) IS A COREPRESSOR OF THE EPIGENETIC REGULATION OF GENE TRANSCRIPTION THAT HAS IMPORTANT FUNCTIONS IN METABOLISM AND INFLAMMATION, BUT LITTLE IS KNOWN ABOUT ITS ROLE IN ALCOHOL-ASSOCIATED LIVER DISEASE (ALD). IN THIS STUDY, WE DEVELOPED MICE WITH HEPATOCYTE-SPECIFIC NCOR1 KNOCKOUT (NCOR1(HEP-/-)) USING THE ALBUMIN-CRE/LOXP SYSTEM AND INVESTIGATED THE ROLE OF NCOR1 IN THE PATHOGENESIS OF ALD AND THE UNDERLYING MECHANISMS. THE TRADITIONAL ALCOHOL FEEDING MODEL AND NIAAA MODEL OF ALD WERE BOTH ESTABLISHED IN WILD-TYPE AND NCOR1(HEP-/-) MICE. WE SHOWED THAT AFTER ALD WAS ESTABLISHED, NCOR1(HEP-/-) MICE HAD WORSE LIVER INJURY BUT LESS STEATOSIS THAN WILD-TYPE MICE. WE DEMONSTRATED THAT HEPATOCYTE-SPECIFIC LOSS OF NCOR1 ATTENUATED LIVER STEATOSIS BY PROMOTING FATTY ACID OXIDATION BY UPREGULATING BMAL1 (A CIRCADIAN CLOCK COMPONENT THAT HAS BEEN REPORTED TO PROMOTE PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR ALPHA (PPARALPHA)-MEDIATED FATTY BETA-OXIDATION BY UPREGULATING DE NOVO LIPID SYNTHESIS). ON THE OTHER HAND, HEPATOCYTE-SPECIFIC LOSS OF NCOR1 EXACERBATED ALCOHOL-INDUCED LIVER INFLAMMATION AND OXIDATIVE STRESS BY RECRUITING MONOCYTE-DERIVED MACROPHAGES VIA C-C MOTIF CHEMOKINE LIGAND 2 (CCL2). IN THE MOUSE HEPATOCYTE LINE AML12, NCOR1 KNOCKDOWN SIGNIFICANTLY INCREASED ETHANOL-INDUCED CCL2 RELEASE. THESE RESULTS SUGGEST THAT HEPATOCYTE NCOR1 PLAYS DISTINCT ROLES IN CONTROLLING LIVER INFLAMMATION AND STEATOSIS, WHICH PROVIDES NEW INSIGHTS INTO THE DEVELOPMENT OF TREATMENTS FOR STEATOHEPATITIS INDUCED BY CHRONIC ALCOHOL CONSUMPTION. 2022 20 5357 35 REACTIVE OXYGEN SPECIES INDUCE EPIGENETIC INSTABILITY THROUGH THE FORMATION OF 8-HYDROXYDEOXYGUANOSINE IN HUMAN HEPATOCARCINOGENESIS. CHRONIC HEPATITIS C (CHC) TRIGGERS OXIDATIVE STRESS AND CONTRIBUTES TO THE EMERGENCE OF HEPATOCELLULAR CARCINOMA (HCC). WE PREVIOUSLY REPORTED THAT TUMOR SUPPRESSOR GENE (TSG) METHYLATION IS A CRITICAL FACTOR DURING THE EARLY STAGES OF HEPATOCARCINOGENESIS. IN THIS STUDY, WE CLARIFY THE ASSOCIATION BETWEEN OXIDATIVE STRESS AND EPIGENETIC ALTERATIONS DURING HEPATOCARCINOGENESIS. WE EXAMINED DNA OXIDATION AND METHYLATION PROFILES IN 128 LIVER BIOPSY SAMPLES FROM CHC PATIENTS. THE DNA OXIDATION AND METHYLATED TSG NUMBERS WERE QUANTIFIED USING IMMUNOHISTOCHEMICAL ANALYSIS OF 8-HYDROXYDEOXYGUANOSINE (8-OHDG) AND QUANTITATIVE PCR FOR 11 TSGS, RESPECTIVELY. THE QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION-PCR (CHIP-QPCR) ASSAY IN HEPG2 AND FETAL LIVER HC CELLS TREATED WITH H2O2 WAS USED TO QUANTIFY TRIMETHYL-H3K4, ACETYLATED-H4K16 (AN ACTIVE CHROMATIN MARKER), TRIMETHYL-H3K27 (A REPRESSIVE CHROMATIN MARKER) AND 8-OHDG. WE ANALYZED 30 PROMOTERS OF 25 DIFFERENT TSGS BY QPCR. THE HIGH LEVELS OF 8-OHDG WAS THE ONLY VARIABLE THAT WAS SIGNIFICANTLY ASSOCIATED WITH THE INCREASED NUMBER OF METHYLATED TSGS IN CHC (P < 0.0001). THE CHIP-QPCR REVEALED THAT AFTER H2O2 TREATMENT OF THE CELL LINES, THE 8-OHDG-BOUND PROMOTERS SHOWED A MODIFICATION FROM AN ACTIVE CHROMATIN (TRIMETHYL-H3K4 AND ACETYLATED-H4K16 DOMINANT) TO A REPRESSIVE CHROMATIN (TRIMETHYL-H3K27 DOMINANT) STATUS. WE CONCLUDE THAT OXIDATIVE STRESS ALTERS THE CHROMATIN STATUS, WHICH LEADS TO ABNORMAL METHYLATION OF TSGS, AND CONTRIBUTES TO HEPATOCARCINOGENESIS IN CHC PATIENTS. 2013