1 873 111 CHRONIC ALCOHOL EXPOSURE INHIBITS BIOTIN UPTAKE BY PANCREATIC ACINAR CELLS: POSSIBLE INVOLVEMENT OF EPIGENETIC MECHANISMS. CHRONIC EXPOSURE TO ALCOHOL AFFECTS DIFFERENT PHYSIOLOGICAL ASPECTS OF PANCREATIC ACINAR CELLS (PAC), BUT ITS EFFECT ON THE UPTAKE PROCESS OF BIOTIN IS NOT KNOWN. WE ADDRESSED THIS ISSUE USING MOUSE-DERIVED PANCREATIC ACINAR 266-6 CELLS CHRONICALLY EXPOSED TO ALCOHOL AND WILD-TYPE AND TRANSGENIC MICE (CARRYING THE HUMAN SLC5A6 5'-PROMOTER) FED ALCOHOL CHRONICALLY. FIRST WE ESTABLISHED THAT BIOTIN UPTAKE BY PAC IS NA(+) DEPENDENT AND CARRIER MEDIATED AND INVOLVES SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER (SMVT). CHRONIC EXPOSURE OF 266-6 CELLS TO ALCOHOL LED TO A SIGNIFICANT INHIBITION IN BIOTIN UPTAKE, EXPRESSION OF SMVT PROTEIN, AND MRNA AS WELL AS IN THE ACTIVITY OF THE SLC5A6 PROMOTER. SIMILARLY, CHRONIC ALCOHOL FEEDING OF WILD-TYPE AND TRANSGENIC MICE CARRYING THE SLC5A6 PROMOTER LED TO A SIGNIFICANT INHIBITION IN BIOTIN UPTAKE BY PAC, AS WELL AS IN THE EXPRESSION OF SMVT PROTEIN AND MRNA AND THE ACTIVITY OF THE SLC5A6 PROMOTERS EXPRESSED IN THE TRANSGENIC MICE. WE ALSO FOUND THAT CHRONIC ALCOHOL FEEDING OF MICE IS ASSOCIATED WITH A SIGNIFICANT INCREASE IN THE METHYLATION STATUS OF CPG ISLANDS PREDICTED TO BE IN THE MOUSE SLC5A6 PROMOTERS AND A DECREASE IN THE LEVEL OF EXPRESSION OF TRANSCRIPTION FACTOR KLF-4, WHICH PLAYS AN IMPORTANT ROLE IN REGULATING SLC5A6 PROMOTER ACTIVITY. THESE RESULTS DEMONSTRATE, FOR THE FIRST TIME, THAT CHRONIC ALCOHOL EXPOSURE NEGATIVELY IMPACTS BIOTIN UPTAKE IN PAC AND THAT THIS EFFECT IS EXERTED (AT LEAST IN PART) AT THE LEVEL OF TRANSCRIPTION OF THE SLC5A6 GENE AND MAY INVOLVE EPIGENETIC/MOLECULAR MECHANISMS. 2014 2 1787 55 EFFECT OF CHRONIC ALCOHOL EXPOSURE ON GUT VITAMIN B7 UPTAKE: INVOLVEMENT OF EPIGENETIC MECHANISMS AND EFFECT OF ALCOHOL METABOLITES. VITAMIN B7 (BIOTIN) IS ESSENTIAL FOR NORMAL HEALTH AND ITS DEFICIENCY/SUBOPTIMAL LEVELS OCCUR IN A VARIETY OF CONDITIONS INCLUDING CHRONIC ALCOHOLISM. MAMMALS, INCLUDING HUMANS, OBTAIN BIOTIN FROM DIET AND GUT-MICROBIOTA VIA ABSORPTION ALONG THE INTESTINAL TRACT. THE ABSORPTION PROCESS IS CARRIER MEDIATED AND INVOLVES THE SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER (SMVT; SLC5A6). WE HAVE PREVIOUSLY SHOWN THAT CHRONIC ALCOHOL EXPOSURE SIGNIFICANTLY INHIBITS INTESTINAL/COLONIC BIOTIN UPTAKE VIA SUPPRESSION OF SLC5A6 TRANSCRIPTION IN ANIMAL AND CELL LINE MODELS. HOWEVER, LITTLE IS KNOWN ABOUT THE TRANSCRIPTIONAL/EPIGENETIC FACTORS THAT MEDIATE THIS SUPPRESSION. IN ADDITION, THE EFFECT OF ALCOHOL METABOLITES (GENERATED VIA ALCOHOL METABOLISM BY GUT MICROBIOTA AND HOST TISSUES) ON BIOTIN UPTAKE IS STILL UNKNOWN. TO ADDRESS THESE QUESTIONS, WE FIRST DEMONSTRATED THAT CHRONIC ALCOHOL EXPOSURE INHIBITS SMALL INTESTINAL AND COLONIC BIOTIN UPTAKE AND SMVT EXPRESSION IN HUMAN DIFFERENTIATED ENTEROID AND COLONOID MONOLAYERS. WE THEN SHOWED THAT CHRONIC ALCOHOL EXPOSURES OF BOTH, CACO-2 CELLS AND MICE, ARE ASSOCIATED WITH A SIGNIFICANT SUPPRESSION IN EXPRESSION OF THE NUCLEAR FACTOR KLF-4 (NEEDED FOR SLC5A6 PROMOTER ACTIVITY), AS WELL AS WITH EPIGENETIC ALTERATIONS (HISTONE MODIFICATIONS). WE ALSO FOUND THAT CHRONIC EXPOSURE OF NCM460 HUMAN COLONIC EPITHELIAL CELLS AS WELL AS HUMAN DIFFERENTIATED COLONOID MONOLAYERS, TO ALCOHOL METABOLITES (ACETALDEHYDE, ETHYL PALMITATE, ETHYL OLEATE) SIGNIFICANTLY INHIBITED BIOTIN UPTAKE AND SMVT EXPRESSION. THESE FINDINGS SHED LIGHT ONTO THE MOLECULAR/EPIGENETIC MECHANISMS THAT MEDIATE THE INHIBITORY EFFECT OF CHRONIC ALCOHOL EXPOSURE ON INTESTINAL BIOTIN UPTAKE. THEY FURTHER SHOW THAT ALCOHOL METABOLITES ARE ALSO CAPABLE OF INHIBITING BIOTIN UPTAKE IN THE GUT.NEW & NOTEWORTHY USING COMPLEMENTARY MODELS, INCLUDING HUMAN DIFFERENTIATED ENTEROID AND COLONOID MONOLAYERS, THIS STUDY SHOWS THE INVOLVEMENT OF MOLECULAR AND EPIGENETIC MECHANISMS IN MEDIATING THE INHIBITORY EFFECT OF CHRONIC ALCOHOL EXPOSURE ON BIOTIN UPTAKE ALONG THE INTESTINAL TRACT. THE STUDY ALSO SHOWS THAT ALCOHOL METABOLITES (GENERATED BY GUT MICROBIOTA AND HOST TISSUES) CAUSE INHIBITION IN GUT BIOTIN UPTAKE. 2021 3 871 75 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 4 1406 35 DIETARY HISTONE DEACETYLASE INHIBITORS: FROM CELLS TO MICE TO MAN. SULFORAPHANE (SFN) IS AN ISOTHIOCYANATE FOUND IN CRUCIFEROUS VEGETABLES, SUCH AS BROCCOLI AND BROCCOLI SPROUTS. THIS ANTICARCINOGEN WAS FIRST IDENTIFIED AS A POTENT INDUCER OF PHASE 2 DETOXIFICATION ENZYMES, BUT EVIDENCE IS MOUNTING THAT SFN ALSO ACTS THROUGH EPIGENETIC MECHANISMS. SFN HAS BEEN SHOWN TO INHIBIT HISTONE DEACETYLASE (HDAC) ACTIVITY IN HUMAN COLON AND PROSTATE CANCER LINES, WITH AN INCREASE IN GLOBAL AND LOCAL HISTONE ACETYLATION STATUS, SUCH AS ON THE PROMOTER REGIONS OF P21 AND BAX GENES. SFN ALSO INHIBITED THE GROWTH OF PROSTATE CANCER XENOGRAFTS AND SPONTANEOUS INTESTINAL POLYPS IN MOUSE MODELS, WITH EVIDENCE FOR ALTERED HISTONE ACETYLATION AND HDAC ACTIVITIES IN VIVO. IN HUMAN SUBJECTS, A SINGLE INGESTION OF 68 G BROCCOLI SPROUTS INHIBITED HDAC ACTIVITY IN CIRCULATING PERIPHERAL BLOOD MONONUCLEAR CELLS 3-6 H AFTER CONSUMPTION, WITH CONCOMITANT INDUCTION OF HISTONE H3 AND H4 ACETYLATION. THESE FINDINGS PROVIDE EVIDENCE THAT ONE MECHANISM OF CANCER CHEMOPREVENTION BY SFN IS VIA EPIGENETIC CHANGES ASSOCIATED WITH INHIBITION OF HDAC ACTIVITY. OTHER DIETARY AGENTS SUCH AS BUTYRATE, BIOTIN, LIPOIC ACID, GARLIC ORGANOSULFUR COMPOUNDS, AND METABOLITES OF VITAMIN E HAVE STRUCTURAL FEATURES COMPATIBLE WITH HDAC INHIBITION. THE ABILITY OF DIETARY COMPOUNDS TO DE-REPRESS EPIGENETICALLY SILENCED GENES IN CANCER CELLS, AND TO ACTIVATE THESE GENES IN NORMAL CELLS, HAS IMPORTANT IMPLICATIONS FOR CANCER PREVENTION AND THERAPY. IN A BROADER CONTEXT, THERE IS GROWING INTEREST IN DIETARY HDAC INHIBITORS AND THEIR IMPACT ON EPIGENETIC MECHANISMS AFFECTING OTHER CHRONIC CONDITIONS, SUCH AS CARDIOVASCULAR DISEASE, NEURODEGENERATION AND AGING. 2007 5 5307 48 PROXIMITY UTILIZING BIOTINYLATION OF NUCLEAR PROTEINS IN VIVO. INTRODUCTION: THE HUMAN GENOME CONSISTS OF ROUGHLY 30,000 GENES CODING FOR OVER 500,000 DIFFERENT PROTEINS, OF WHICH MORE THAN 10,000 PROTEINS CAN BE PRODUCED BY THE CELL AT ANY GIVEN TIME (THE CELLULAR "PROTEOME"). IT HAS BEEN ESTIMATED THAT OVER 80% OF PROTEINS DO NOT OPERATE ALONE, BUT IN COMPLEXES. THESE PROTEIN-PROTEIN INTERACTIONS (PPI) ARE REGULATED BY SEVERAL MECHANISMS. FOR EXAMPLE, POST-TRANSLATIONAL MODIFICATIONS (METHYLATION, ACETYLATION, PHOSPHORYLATION, OR UBIQUITINATION) OR METAL-BINDING CAN LEAD TO CONFORMATIONAL CHANGES THAT ALTER THE AFFINITY AND KINETIC PARAMETERS OF THE INTERACTION. MANY PPIS ARE PART OF LARGER CELLULAR NETWORKS OF INTERACTIONS OR INTERACTOMES. INDEED, THESE INTERACTIONS ARE AT THE CORE OF THE ENTIRE INTERACTOMICS SYSTEM OF ANY LIVING CELL, AND SO, ABERRANT PPIS ARE THE BASIS OF MULTIPLE DISEASES, SUCH AS NEURODEGENERATIVE DISEASES AND CANCER. THE OBJECTIVE OF THIS STUDY WAS TO DEVELOP A METHOD OF MONITORING PROTEIN-PROTEIN INTERACTIONS AND PROXIMITY DEPENDENCE IN VIVO. METHODS: THE BIOTIN LIGASE BIRA WAS FUSED TO THE PROTEIN OF INTEREST, AND THE BIOTIN ACCEPTOR PEPTIDE (BAP) WAS FUSED TO AN INTERACTING PARTNER TO MAKE THE DETECTION OF ITS BIOTINYLATION POSSIBLE BY WESTERN BLOT OR MASS SPECTROMETRY. RESULTS: USING SEVERAL EXPERIMENTAL SYSTEMS (BIRA.A + BAP.B), WE SHOWED THAT THE BIOTINYLATION IS INTERACTION/PROXIMITY DEPENDENT. HERE, A AND B ARE THE NEXT NUCLEAR PROTEINS USED IN THE EXPERIMENTS - 3 PARALOGUES OF HETEROCHROMATIN PROTEIN HP1ALPHA (CBX5), HP1BETA (CBX1), HP1GAMMA (CBX3), WILD TYPE AND TRANSCRIPTION MUTANT FACTOR KAP1, TRANSLESION DNA POLYMERASE POLH AND E3, UBIQUITIN LIGASE RAD18, PROLIFERATIVE CELL NUCLEAR ANTIGEN (PCNA), UBIQUITIN UB, SUMO-2/3, DIFFERENT TYPES AND ISOFORMS OF HISTONES H2A, H2AZ, H3.1, H3.3, CENPA, H2A.BBD, AND MACROH2A. THE VARIANT OF THIS APPROACH IS TERMED PUB-NCHIP (PROXIMITY UTILIZING BIOTINYLATION WITH NATIVE CHROMATIN IMMUNO-PRECIPITATION) AND IS DESIGNED TO PURIFY AND STUDY THE PROTEIN COMPOSITION OF CHROMATIN IN PROXIMITY TO THE NUCLEAR PROTEIN OF INTEREST. USING THE RAD18 PROTEIN AS A MODEL, WE DEMONSTRATED THAT THE RAD18-PROXIMAL CHROMATIN IS ENRICHED IN SOME H4 ACETYLATED SPECIES. MOREOVER, THE RAD18-PROXIMAL CHROMATIN CONTAINING A REPLACEMENT HISTONE H2AZ HAS A DIFFERENT PATTERN OF H4 ACETYLATION. CONCLUSION: PROGRESS IN THE LAST DECADE IN CANCER DRUG THERAPY HAS LED US TO THE CONCLUSION THAT THE NUCLEUS OF EUKARYOTIC CELLS IS AN ACTIVE SITE FOR MANY CELLULAR PROCESSES IMPORTANT TO THE DEVELOPMENT OF CANCER. THESE PROCESSES INCLUDE CHANGES IN GENETIC AND EPIGENETIC LANDSCAPE (E. G. METHYLATION OF DNA, MODIFICATION OF HISTONES) AND THE EXPRESSION LEVELS OF TRANSCRIPTION FACTORS, WHICH REGULATES GENE PRODUCTS (E.G. HYPOXIA-INDUCIBLE FACTOR 1ALPHA (HIF-1ALPHA) IN CHRONIC ANEMIA, ETC.) WHERE PROTEIN-PROTEIN INTERACTIONS PLAY IMPORTANT ROLE. UNDERSTANDING THE NATURE OF PROTEIN-PROTEIN INTERACTIONS MAY IMPROVE DESIGN STRATEGIES FOR SMALL-MOLECULE PPI MODULATORS. PPI ASSAY TECHNOLOGIES THAT CLOSELY REFLECT PHYSIOLOGICAL CONDITIONS HOLD THE KEY TO DEVELOPING SPECIFIC ANTI-CANCER DRUGS. 2014 6 5492 21 REVIEW OF ARSENIC TOXICITY, SPECIATION AND POLYADENYLATION OF CANONICAL HISTONES. ARSENIC CONTAMINATION IMPACTS HUNDREDS OF MILLIONS OF PEOPLE IN THE WORLD. ARSENIC IS A WELL-ESTABLISHED HUMAN CARCINOGEN AND HAS BEEN SHOWN TO CAUSE SKIN, LUNG, BLADDER, LIVER, PROSTATE AND KIDNEY CANCERS, IN HUMANS. MECHANISMS THAT UNDERLIE ARSENIC-MEDIATED CARCINOGENESIS, INCLUDING EPIGENETIC ALTERATIONS, REMAIN LARGELY UNKNOWN. HUMAN EXPOSURE TO ARSENIC IS REVIEWED, AND THE MECHANISMS OF ITS ACUTE AND CHRONIC TOXICITY AND MECHANISMS OF ITS CARCINOGENESIS IN HUMANS ARE DISCUSSED. ARSENIC IS ONE OF THE FEW METALS THAT IS METABOLIZED IN VIVO, AND ARSENIC METHYLATION AND HOW THIS RESULTS IN A SHORTER HALF-LIFE IN VIVO ARE DISCUSSED. A REVIEW OF RECENT FINDINGS THAT ARSENIC CAUSES LOSS IN THE CELLULAR LEVELS OF STEM LOOP BINDING PROTEIN (SLBP) RESULTING IN POLYADENYLATION OF CANONICAL HISTONES (H3.1) AS A DEFAULT, INCREASING LEVELS OF H3.1 PROTEIN OUTSIDE OF S-PHASE. MALIGNANT CELL TRANSFORMATION IS INDUCED BY KNOCKDOWN OF SLBP AND BY OVEREXPRESSION OF POLYADENYLATED H3.1. ARSENIC INDUCED POLYADENYLATION OF H3.1 CAUSES ENHANCED LEVELS OF H3.1 PROTEIN DISPLACING H3.3 PROTEIN FROM ITS CELLULAR BINDING SITES, SINCE THE TWO PROTEINS DIFFER BY ONLY 5 AMINO ACIDS. KNOCKDOWN OF H3.3 ALONE CAN INDUCE CARCINOGENESIS, AND THEREFORE DISPLACEMENT OF FUNCTIONAL H3.3 PROTEIN BY INCREASED H3.1 PROTEIN, IS LIKELY A MECHANISM OF ARSENIC CARCINOGENESIS. 2019 7 1999 27 EPIGENETIC AND NEURONAL ACTIVITY MARKERS SUGGEST THE RECRUITMENT OF THE PREFRONTAL CORTEX AND HIPPOCAMPUS IN THE THREE-HIT MODEL OF DEPRESSION IN MALE PACAP HETEROZYGOUS MICE. DEPRESSION AND ITS INCREASING PREVALENCE CHALLENGE PATIENTS, THE HEALTHCARE SYSTEM, AND THE ECONOMY. WE RECENTLY CREATED A MOUSE MODEL BASED ON THE THREE-HIT CONCEPT OF DEPRESSION. AS GENETIC PREDISPOSITION (FIRST HIT), WE APPLIED PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE HETEROZYGOUS MICE ON CD1 BACKGROUND. MATERNAL DEPRIVATION MODELED THE EPIGENETIC FACTOR (SECOND HIT), AND THE CHRONIC VARIABLE MILD STRESS WAS THE ENVIRONMENTAL FACTOR (THIRD HIT). FLUOXETINE TREATMENT WAS APPLIED TO TEST THE PREDICTIVE VALIDITY OF OUR MODEL. WE AIMED TO EXAMINE THE DYNAMICS OF THE EPIGENETIC MARKER ACETYL-LYSINE 9 H3 HISTONE (H3K9AC) AND THE NEURONAL ACTIVITY MARKER FOSB IN THE PREFRONTAL CORTEX (PFC) AND HIPPOCAMPUS. FLUOXETINE DECREASED H3K9AC IN PFC IN NON-DEPRIVED ANIMALS, BUT A HISTORY OF MATERNAL DEPRIVATION ABOLISHED THE EFFECT OF STRESS AND SSRI TREATMENT ON H3K9AC IMMUNOREACTIVITY. IN THE HIPPOCAMPUS, STRESS DECREASED, WHILE SSRI INCREASED H3K9AC IMMUNOSIGNAL, UNLIKE IN THE DEPRIVED MICE, WHERE THE OPPOSITE EFFECT WAS DETECTED. FOSB IN STRESS WAS STIMULATED BY FLUOXETINE IN THE PFC, WHILE IT WAS INHIBITED IN THE HIPPOCAMPUS. THE FOSB IMMUNOREACTIVITY WAS ALMOST COMPLETELY ABOLISHED IN THE HIPPOCAMPUS OF THE DEPRIVED MICE. THIS STUDY SHOWED THAT FOSB AND H3K9AC WERE MODULATED IN A TERRITORY-SPECIFIC MANNER BY EARLY LIFE ADVERSITIES AND LATER LIFE STRESS INTERACTING WITH THE EFFECT OF FLUOXETINE THERAPY SUPPORTING THE RELIABILITY OF OUR MODEL. 2022 8 3331 33 HISTONE DEACETYLASE INHIBITOR SUBERANILOHYDROXAMIC ACID TREATMENT REVERSES HYPOSENSITIVITY TO GAMMA-AMINOBUTYRIC ACID IN THE VENTRAL TEGMENTAL AREA DURING ETHANOL WITHDRAWAL. BACKGROUND: THE VENTRAL TEGMENTAL AREA (VTA) IS IMPORTANT FOR ALCOHOL-RELATED REWARD AND REINFORCEMENT. MOUSE VTA NEURONS ARE HYPOSENSITIVE TO GAMMA-AMINOBUTYRIC ACID (GABA) DURING ETHANOL (ETOH) WITHDRAWAL, AND GABA RESPONSIVENESS IS NORMALIZED BY IN VITRO TREATMENT WITH HISTONE DEACETYLASE INHIBITORS (HDACI). THE PRESENT STUDY EXAMINED THE EFFECT OF A SYSTEMICALLY ADMINISTERED HDACI, SUBERANILOHYDROXAMIC ACID (SAHA) ON GABA SENSITIVITY, AND RELATED MOLECULAR CHANGES IN VTA NEURONS DURING WITHDRAWAL AFTER CHRONIC ETOH INTAKE IN RATS. METHODS: SPRAGUE DAWLEY MALE ADULT RATS WERE FED WITH LIEBER-DECARLI DIET (9% ETOH OR CONTROL DIET) FOR 16 DAYS. EXPERIMENTAL GROUPS INCLUDED CONTROL DIET-FED AND ETOH DIET-FED (0- OR 24-HOUR WITHDRAWAL) RATS TREATED WITH EITHER SAHA OR VEHICLE INJECTION. SINGLE-UNIT RECORDINGS WERE USED TO MEASURE THE RESPONSE OF VTA NEURONS TO GABA. IMMUNOHISTOCHEMISTRY WAS PERFORMED TO EXAMINE LEVELS OF HDAC2, ACETYLATED HISTONE H3 LYSINE 9 (ACH3K9), AND GABA(A) RECEPTOR ALPHA1 AND ALPHA5 SUBUNITS IN THE VTA; QUANTITATIVE POLYMERASE CHAIN REACTION WAS PERFORMED TO EXAMINE THE MRNA LEVELS OF HDAC2 AND GABA(A) RECEPTOR SUBUNITS. RESULTS: VTA NEURONS FROM THE WITHDRAWAL GROUP EXHIBITED GABA HYPOSENSITIVITY. IN VIVO SAHA TREATMENT 2 HOURS BEFORE SACRIFICE NORMALIZED THE SENSITIVITY OF VTA NEURONS TO GABA. ETOH WITHDRAWAL WAS ASSOCIATED WITH INCREASED HDAC2 AND DECREASED ACH3K9 PROTEIN LEVELS; SAHA TREATMENT NORMALIZED ACH3K9 LEVELS. INTERESTINGLY, NO SIGNIFICANT CHANGE WAS OBSERVED IN THE MRNA LEVELS OF HDAC2. THE MRNA LEVELS, BUT NOT PROTEIN LEVELS, OF GABA(A) RECEPTOR ALPHA1 AND ALPHA5 SUBUNITS WERE INCREASED DURING WITHDRAWAL. CONCLUSIONS: WITHDRAWAL FROM CHRONIC ETOH EXPOSURE RESULTS IN A DECREASE IN GABA-MEDIATED INHIBITION, AND THIS GABA HYPOSENSITIVITY IS NORMALIZED BY IN VIVO SAHA TREATMENT. DISRUPTION OF SIGNALING IN THE VTA PRODUCED BY ALTERATION OF GABA NEUROTRANSMISSION COULD BE 1 NEUROADAPTIVE PHYSIOLOGICAL PROCESS LEADING TO CRAVING AND RELAPSE. THESE RESULTS SUGGEST THAT HDACI PHARMACOTHERAPY WITH AGENTS LIKE SAHA MIGHT BE AN EFFECTIVE TREATMENT FOR ALCOHOLISM. 2018 9 312 34 ALCOHOL FEEDING IN MICE PROMOTES COLONIC HYPERPERMEABILITY AND CHANGES IN COLONIC ORGANOID STEM CELL FATE. BACKGROUND: ALCOHOL INCREASES INTESTINAL PERMEABILITY TO PROINFLAMMATORY MICROBIAL PRODUCTS THAT PROMOTE LIVER DISEASE, EVEN AFTER A PERIOD OF SOBRIETY. WE SOUGHT TO TEST THE HYPOTHESIS THAT ALCOHOL AFFECTS INTESTINAL STEM CELLS USING AN IN VIVO MODEL AND EX VIVO ORGANOIDS GENERATED FROM JEJUNUM AND COLON FROM MICE FED CHRONIC ALCOHOL. METHODS: MICE WERE FED A CONTROL OR AN ALCOHOL DIET. INTESTINAL PERMEABILITY, LIVER STEATOSIS-INFLAMMATION, AND STOOL SHORT-CHAIN FATTY ACIDS (SCFAS) WERE MEASURED. JEJUNUM AND COLONIC ORGANOIDS AND TISSUE WERE STAINED FOR STEM CELL, CELL LINEAGE, AND APICAL JUNCTION MARKERS WITH ASSESSMENT OF MRNA BY PCR AND RNA-SEQ. CHIP-PCR ANALYSIS WAS CARRIED OUT FOR NOTCH1 USING AN ANTIBODY SPECIFIC FOR ACETYLATED HISTONE 3. RESULTS: ALCOHOL-FED MICE EXHIBITED COLONIC (BUT NOT SMALL INTESTINAL) HYPERPERMEABILITY, STEATOHEPATITIS, AND DECREASED BUTYRATE/TOTAL SCFA RATIO IN STOOL. STEM CELL, CELL LINEAGE, AND APICAL JUNCTION MARKER STAINING IN TISSUE OR ORGANOIDS FROM JEJUNUM TISSUE WERE NOT IMPACTED BY ALCOHOL. ONLY CHROMOGRANIN A (CHGA) WAS INCREASED IN JEJUNUM ORGANOIDS BY QPCR. HOWEVER, COLONIC TISSUE AND ORGANOID STAINING EXHIBITED AN ALCOHOL-INDUCED SIGNIFICANT DECREASE IN CYTOKERATIN 20+ (KRT20+) ABSORPTIVE LINEAGE ENTEROCYTES, A DECREASE IN OCCLUDIN AND E-CADHERIN APICAL JUNCTION PROTEINS, AN INCREASE IN CHGA, AND AN INCREASE IN THE LGR5 STEM CELL MARKER. QPCR REVEALED AN ALCOHOL-INDUCED DECREASE IN COLONIC ORGANOID AND TISSUE NOTCH1, HES1, AND KRT20 AND INCREASED CHGA, SUPPORTING AN ALTERATION IN STEM CELL FATE DUE TO DECREASED NOTCH1 EXPRESSION. COLONIC TISSUE CHIP-PCR REVEALED ALCOHOL FEEDING SUPPRESSED NOTCH1 MRNA EXPRESSION (VIA DEACETYLATION OF HISTONE H3) AND DECREASED NOTCH1 TISSUE STAINING. CONCLUSIONS: DATA SUPPORT A MODEL FOR ALCOHOL-INDUCED COLONIC HYPERPERMEABILITY VIA EPIGENETIC EFFECTS ON NOTCH1, AND THUS HES1, SUPPRESSION THROUGH A MECHANISM INVOLVING HISTONE H3 DEACETYLATION AT THE NOTCH1 LOCUS. THIS DECREASED ENTEROCYTE AND INCREASED ENTEROENDOCRINE CELL COLONIC STEM CELL FATE AND DECREASED APICAL JUNCTIONAL PROTEINS LEADING TO HYPERPERMEABILITY. 2017 10 1848 46 EFFECTS OF VENLAFAXINE ON THE EXPRESSION LEVEL AND METHYLATION STATUS OF GENES INVOLVED IN OXIDATIVE STRESS IN RATS EXPOSED TO A CHRONIC MILD STRESS. RECENT HUMAN AND ANIMAL STUDIES INDICATE THAT OXIDATIVE AND NITROSATIVE STRESS MAY PLAY A ROLE IN THE AETIOLOGY AND PATHOGENESIS OF DEPRESSION. THIS STUDY INVESTIGATES THE EFFECT OF CHRONIC ADMINISTRATION OF THE SEROTONIN-NOREPINEPHRINE REUPTAKE INHIBITOR, VENLAFAXINE, ON THE EXPRESSION AND METHYLATION STATUS OF SOD1, SOD2, GPX1, GPX4, CAT, NOS1 AND NOS2 IN THE BRAIN AND BLOOD OF RATS EXPOSED TO A CHRONIC MILD STRESS (CMS) MODEL OF DEPRESSION. SEPARATE GROUPS OF ANIMALS WERE EXPOSED TO CMS FOR 2 OR 7 WEEKS; THE SECOND GROUP RECEIVED SALINE OR VENLAFAXINE (10 MG/KG/D, IP) FOR 5 WEEKS. AFTER COMPLETION OF BOTH STRESS CONDITIONS AND DRUG ADMINISTRATION, THE MRNA AND PROTEIN EXPRESSION OF SELECTED GENES AND THE METHYLATION STATUS OF THEIR PROMOTERS WERE MEASURED IN PERIPHERAL MONONUCLEAR BLOOD CELLS (PBMCS) AND IN BRAIN STRUCTURES (HIPPOCAMPUS, AMYGDALA, HYPOTHALAMUS, MIDBRAIN, CORTEX, BASAL GANGLIA) WITH THE USE OF TAQMAN GENE EXPRESSION ASSAY, WESTERN BLOT AND METHYLATION-SENSITIVE HIGH-RESOLUTION MELTING TECHNIQUES. CMS CAUSED A DECREASE IN SUCROSE CONSUMPTION, AND THIS EFFECT WAS NORMALIZED BY FLUOXETINE. IN PBMCS, SOD1, SOD2 AND NOS2 MRNA EXPRESSION CHANGED ONLY AFTER VENLAFAXINE ADMINISTRATION. IN BRAIN, CAT, GPX1, GPX4 AND NOS1 GENE EXPRESSION CHANGED FOLLOWING CMS OR VENLAFAXINE EXPOSURE, MOST PROMINENTLY IN THE HIPPOCAMPUS, MIDBRAIN AND BASAL GANGLIA. CMS INCREASED THE METHYLATION OF THE GPX1 PROMOTER IN PBMCS, THE SECOND GPX4 PROMOTER IN MIDBRAIN AND BASAL GANGLIA, AND SOD1 AND SOD2 IN HIPPOCAMPUS. THE CMS ANIMALS TREATED WITH VENLAFAXINE DISPLAYED A SIGNIFICANTLY HIGHER CAT LEVEL IN MIDBRAIN AND CEREBRAL CORTEX. CMS CAUSED AN ELEVATION OF GPX4 IN THE HIPPOCAMPUS, WHICH WAS LOWERED IN CEREBRAL CORTEX BY VENLAFAXINE. THE RESULTS INDICATE THAT CMS AND VENLAFAXINE ADMINISTRATION AFFECT THE METHYLATION OF PROMOTERS OF GENES INVOLVED IN OXIDATIVE AND NITROSATIVE STRESS. THEY ALSO INDICATE THAT PERIPHERAL AND CENTRAL TISSUE DIFFER IN THEIR RESPONSE TO STRESS OR ANTIDEPRESSANT TREATMENTS. IT IS POSSIBLE THAT THAT APART FROM DNA METHYLATION, A CRUCIAL ROLE OF EXPRESSION LEVEL OF GENES MAY BE PLAYED BY OTHER FORMS OF EPIGENETIC REGULATION, SUCH AS HISTONE MODIFICATION OR MICRORNA INTERFERENCE. THESE FINDINGS PROVIDE STRONG EVIDENCE FOR THESIS THAT ANALYSIS OF THE LEVEL OF MRNA AND PROTEIN EXPRESSION AS WELL AS THE STATUS OF PROMOTER METHYLATION CAN HELP IN UNDERSTANDING THE PATHOMECHANISMS OF MENTAL DISEASES, INCLUDING DEPRESSION, AND THE MECHANISMS OF ACTION OF DRUGS EFFECTIVE IN THEIR THERAPY. 2020 11 3727 58 INHIBITION OF PANCREATIC ACINAR MITOCHONDRIAL THIAMIN PYROPHOSPHATE UPTAKE BY THE CIGARETTE SMOKE COMPONENT 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE. THIAMIN IS ESSENTIAL FOR NORMAL METABOLISM IN PANCREATIC ACINAR CELLS (PAC) AND IS OBTAINED FROM THEIR MICROENVIRONMENT THROUGH SPECIFIC PLASMA-MEMBRANE TRANSPORTERS, CONVERTED TO THIAMIN PYROPHOSPHATE (TPP) IN THE CYTOPLASM, FOLLOWED BY UPTAKE OF TPP BY MITOCHONDRIA THROUGH THE MITOCHONDRIAL TPP (MTPP) TRANSPORTER (MTPPT; PRODUCT OF SLC25A19 GENE). TPP IS ESSENTIAL FOR NORMAL MITOCHONDRIAL FUNCTION. WE EXAMINED THE EFFECT OF LONG-TERM/CHRONIC EXPOSURE OF PAC IN VITRO (PANCREATIC ACINAR 266-6 CELLS) AND IN VIVO (WILD-TYPE OR TRANSGENIC MICE CARRYING THE SLC25A19 PROMOTER) OF THE CIGARETTE SMOKE TOXIN, 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE (NNK), ON THE MTPP UPTAKE PROCESS. OUR IN VITRO AND IN VIVO FINDINGS DEMONSTRATE THAT NNK NEGATIVELY AFFECTS MTPP UPTAKE AND REDUCED EXPRESSION OF MTPPT PROTEIN, MTPPT MRNA, AND HETEROGENOUS NUCLEAR RNA, AS WELL AS SLC25A19 PROMOTER ACTIVITY. THE EFFECT OF NNK ON SLC25A19 TRANSCRIPTION WAS NEITHER MEDIATED BY CHANGES IN EXPRESSION OF TRANSCRIPTIONAL FACTOR NFY-1 (KNOWN TO DRIVE SLC25A19 TRANSCRIPTION), NOR DUE TO CHANGES IN METHYLATION PROFILE OF THE SLC25A19 PROMOTER. RATHER, IT APPEARS TO BE DUE TO CHANGES IN HISTONE MODIFICATIONS THAT INVOLVE SIGNIFICANT DECREASES IN HISTONE H3K4-TRIMETHYLATION AND H3K9-ACETYLATION (ACTIVATION MARKERS). THE EFFECT OF NNK ON MTPPT FUNCTION IS MEDIATED THROUGH THE NONNEURONAL ALPHA7-NICOTINIC ACETYLCHOLINE RECEPTOR (ALPHA7-NACHR), AS INDICATED BY BOTH IN VITRO (USING THE NACHR ANTAGONIST MECAMYLAMINE) AND IN VIVO (USING AN ALPHA7-NACHR(-/-) MOUSE MODEL) STUDIES. THESE FINDINGS DEMONSTRATE THAT CHRONIC EXPOSURE OF PAC TO NNK NEGATIVELY IMPACTS PAC MTPP UPTAKE. THIS EFFECT APPEARS TO BE EXERTED AT THE LEVEL OF SLC25A19 TRANSCRIPTION, INVOLVE EPIGENETIC MECHANISM(S), AND IS MEDIATED THROUGH THE ALPHA7-NACHR. 2016 12 5651 26 SEX DIFFERENCES IN THE EPIGENETIC REGULATION OF CHRONIC VISCERAL PAIN FOLLOWING UNPREDICTABLE EARLY LIFE STRESS. BACKGROUND: WE PREVIOUSLY REPORTED THAT EARLY LIFE STRESS (ELS) DYSREGULATED GLUCOCORTICOID RECEPTOR (GR) AND CORTICOTROPHIN-RELEASING HORMONE (CRH) EXPRESSION IN THE CENTRAL NUCLEUS OF THE AMYGDALA (CEA). EPIGENETIC MODIFICATIONS SERVE AS MEMORIES OF ADVERSE EVENTS THAT OCCURRED DURING EARLY LIFE. THEREFORE, WE HYPOTHESIZED THAT EPIGENETIC MECHANISMS ALTER GR AND CRH EXPRESSION IN THE CEA AND UNDERLIE CHRONIC VISCERAL PAIN AFTER ELS. METHODS: NEONATAL RATS WERE EXPOSED TO UNPREDICTABLE, PREDICTABLE ELS, OR ODOR ONLY (NO STRESS CONTROL) FROM POSTNATAL DAYS 8 TO 12. IN ADULTHOOD, VISCERAL SENSITIVITY WAS ASSESSED OR THE CEA WAS ISOLATED FOR WESTERN BLOT OR CHIP-QPCR TO STUDY HISTONE MODIFICATIONS AT THE GR AND CRH PROMOTERS. FEMALE ADULT RATS UNDERWENT STEREOTAXIC IMPLANTATION OF INDWELLING CANNULAS FOR MICROINJECTIONS OF GARCINOL (HAT INHIBITOR) INTO THE CEA. AFTER 7 DAYS OF MICROINJECTIONS, VISCERAL SENSITIVITY WAS ASSESSED OR THE CEA WAS ISOLATED FOR CHIP-QPCR ASSAYS. RESULTS: UNPREDICTABLE ELS INCREASED VISCERAL SENSITIVITY IN ADULT FEMALE RATS, BUT NOT IN MALE COUNTERPARTS. ELS INCREASED HISTONE 3 LYSINE 9 (H3K9) ACETYLATION IN THE CEA AND H3K9 ACETYLATION LEVELS AT THE GR PROMOTER IN THE CEA OF ADULT FEMALE RATS. AFTER UNPREDICTABLE ELS, H3K9 ACETYLATION WAS INCREASED AND GR BINDING WAS DECREASED AT THE CRH PROMOTER. ADMINISTRATION OF GARCINOL IN THE CEA OF ADULT FEMALES, THAT UNDERWENT UNPREDICTABLE ELS, NORMALIZED H3K9 ACETYLATION AND RESTORED GR BINDING AT THE CRH PROMOTER. CONCLUSION: DYSREGULATED HISTONE ACETYLATION AND GR BINDING AT THE CRH PROMOTER IN THE CEA ARE AN IMPORTANT MECHANISM FOR "MEMORIZING" ELS EVENTS MEDIATING VISCERAL PAIN IN ADULTHOOD. 2020 13 6175 30 THE HISTONE DEACETYLASE INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID (SAHA) ALLEVIATES DEPRESSION-LIKE BEHAVIOR AND NORMALIZES EPIGENETIC CHANGES IN THE HIPPOCAMPUS DURING ETHANOL WITHDRAWAL. WITHDRAWAL FROM CHRONIC ALCOHOL DRINKING CAN CAUSE DEPRESSION, LEADING TO AN INABILITY TO FUNCTION IN DAILY LIFE AND AN INCREASED RISK FOR RELAPSE TO HARMFUL DRINKING. UNDERSTANDING THE CAUSES OF ALCOHOL WITHDRAWAL-RELATED DEPRESSION MAY LEAD TO NEW THERAPEUTIC TARGETS FOR TREATMENT. EPIGENETIC FACTORS HAVE RECENTLY EMERGED AS IMPORTANT CONTRIBUTORS TO BOTH DEPRESSION AND ALCOHOL USE DISORDER (AUD). SPECIFICALLY, ACETYLATION OF THE N-TERMINAL TAILS OF HISTONE PROTEINS THAT PACKAGE DNA INTO NUCLEOSOMES IS ALTERED IN STRESS-INDUCED MODELS OF DEPRESSION AND DURING ALCOHOL WITHDRAWAL. THE GOAL OF THIS STUDY WAS TO EXAMINE DEPRESSION-LIKE BEHAVIOR DURING ALCOHOL WITHDRAWAL AND ASSOCIATED CHANGES IN HISTONE ACETYLATION AND EXPRESSION OF HISTONE DEACETYLASE 2 (HDAC2) IN THE HIPPOCAMPUS, A BRAIN REGION CRITICAL FOR MOOD REGULATION AND DEPRESSION. MALE SPRAGUE-DAWLEY RATS WERE TREATED WITH THE LIEBER-DECARLI ETHANOL LIQUID DIET FOR 15 DAYS AND THEN UNDERWENT WITHDRAWAL. RATS WERE TREATED WITH THE HDAC INHIBITOR, SUBEROYLANILIDE HYDROXAMIC ACID (SAHA), DURING WITHDRAWAL AND WERE TESTED FOR DEPRESSION-LIKE BEHAVIOR. IN A SEPARATE GROUP OF RATS, THE HIPPOCAMPUS WAS ANALYZED FOR MRNA AND PROTEIN EXPRESSION OF HDAC2 AND LEVELS OF HISTONE H3 LYSINE 9 ACETYLATION (H3K9AC) DURING CHRONIC ETHANOL EXPOSURE AND WITHDRAWAL. RATS UNDERGOING ETHANOL WITHDRAWAL EXHIBITED DEPRESSION-LIKE BEHAVIOR AND HAD INCREASED HDAC2 AND DECREASED H3K9AC LEVELS IN SPECIFIC STRUCTURES OF THE HIPPOCAMPUS. TREATMENT WITH SAHA DURING WITHDRAWAL AMELIORATED DEPRESSION-LIKE BEHAVIOR AND NORMALIZED CHANGES IN HIPPOCAMPAL HDAC2 AND H3K9AC LEVELS. THESE RESULTS DEMONSTRATE THAT ETHANOL WITHDRAWAL CAUSES AN ALTERED EPIGENETIC STATE IN THE HIPPOCAMPUS. TREATMENT WITH AN HDAC INHIBITOR CAN CORRECT THIS STATE AND ALLEVIATE DEPRESSION-LIKE SYMPTOMS DEVELOPED DURING WITHDRAWAL. TARGETING HISTONE ACETYLATION MAY BE A NOVEL STRATEGY TO REDUCE ETHANOL WITHDRAWAL-INDUCED DEPRESSION. 2019 14 2826 40 FLUOXETINE EPIGENETICALLY ALTERS THE CAMKIIALPHA PROMOTER IN NUCLEUS ACCUMBENS TO REGULATE DELTAFOSB BINDING AND ANTIDEPRESSANT EFFECTS. CHRONIC SOCIAL DEFEAT STRESS IN MICE PRODUCES A SUSCEPTIBLE PHENOTYPE CHARACTERIZED BY SEVERAL BEHAVIORAL ABNORMALITIES CONSISTENT WITH HUMAN DEPRESSION THAT ARE REVERSED BY CHRONIC BUT NOT ACUTE EXPOSURE TO ANTIDEPRESSANT MEDICATIONS. RECENT WORK IN ADDICTION MODELS DEMONSTRATES THAT THE TRANSCRIPTION FACTOR DELTAFOSB AND PROTEIN KINASE CALMODULIN-DEPENDENT PROTEIN KINASE II (CAMKII) ARE CO-REGULATED IN NUCLEUS ACCUMBENS (NAC), A BRAIN REWARD REGION IMPLICATED IN BOTH ADDICTION AND DEPRESSION MODELS INCLUDING SOCIAL DEFEAT. PREVIOUS WORK HAS ALSO DEMONSTRATED THAT DELTAFOSB IS INDUCED IN NAC AFTER CHRONIC SOCIAL DEFEAT STRESS OR AFTER CHRONIC ANTIDEPRESSANT TREATMENT, WHEREIN IT MEDIATES A PRO-RESILIENCE OR ANTIDEPRESSANT-LIKE PHENOTYPE. HERE, USING CHROMATIN IMMUNOPRECIPITATION ASSAYS, WE FOUND THAT DELTAFOSB BINDS THE CAMKIIALPHA GENE PROMOTER IN NAC AND THAT THIS BINDING INCREASES AFTER MICE ARE EXPOSED TO CHRONIC SOCIAL DEFEAT STRESS. PARADOXICALLY, CHRONIC EXPOSURE TO THE ANTIDEPRESSANT FLUOXETINE REDUCES BINDING OF DELTAFOSB TO THE CAMKIIALPHA PROMOTER AND REDUCES CAMKII EXPRESSION IN NAC, DESPITE THE FACT THAT DELTAFOSB IS INDUCED UNDER THESE CONDITIONS. THESE DATA SUGGEST A NOVEL EPIGENETIC MECHANISM OF ANTIDEPRESSANT ACTION, WHEREBY FLUOXETINE INDUCES SOME CHROMATIN CHANGE AT THE CAMKIIALPHA PROMOTER, WHICH BLOCKS THE DELTAFOSB BINDING. INDEED, CHRONIC FLUOXETINE REDUCES ACETYLATION AND INCREASES LYSINE-9 DIMETHYLATION OF HISTONE H3 AT THE CAMKIIALPHA PROMOTER IN NAC, EFFECTS ALSO SEEN IN DEPRESSED HUMANS EXPOSED TO ANTIDEPRESSANTS. OVEREXPRESSION OF CAMKII IN NAC BLOCKS FLUOXETINE'S ANTIDEPRESSANT EFFECTS IN THE CHRONIC SOCIAL DEFEAT PARADIGM, WHEREAS INHIBITION OF CAMKII ACTIVITY IN NAC MIMICS FLUOXETINE EXPOSURE. THESE FINDINGS SUGGEST THAT EPIGENETIC SUPPRESSION OF CAMKIIALPHA EXPRESSION IN NAC IS BEHAVIORALLY RELEVANT AND OFFER A NOVEL PATHWAY FOR POSSIBLE THERAPEUTIC INTERVENTION IN DEPRESSION AND RELATED SYNDROMES. 2014 15 1800 23 EFFECT OF HISTONE DEACETYLASE INHIBITOR ON ETHANOL WITHDRAWAL-INDUCED HYPERALGESIA IN RATS. BACKGROUND: INCREASED PAIN SENSITIVITY IS OBSERVED FOLLOWING ALCOHOL WITHDRAWAL, AND ATTEMPTS TO ALLEVIATE THIS HYPERALGESIA CAN CONTRIBUTE TO THE CYCLE OF ADDICTION. THE AIM OF THIS STUDY WAS TO DETERMINE IF ALCOHOL WITHDRAWAL-INDUCED HYPERALGESIA WAS OBSERVED IN A CHRONIC ETHANOL EXPOSURE MODEL AND IF THIS PAIN WAS AFFECTED BY HISTONE DEACETYLASE INHIBITORS, THUS REVEALING AN EPIGENETIC MECHANISM. METHODS: ADULT MALE SPRAGUE DAWLEY RATS RECEIVED LIEBER-DECARLI LIQUID CONTROL OR ETHANOL (9% V/V) DIET FOR 15 DAYS. MECHANICAL SENSITIVITY WAS MEASURED WITH VON FREY HAIR STIMULATION OF THE HINDPAW DURING ETHANOL ADMINISTRATION AND 24- AND 72-HOUR WITHDRAWAL. RESULTS: ETHANOL WITHDRAWAL PRODUCED SEVERE AND SUSTAINED MECHANICAL HYPERALGESIA, AN EFFECT NOT OBSERVED IN THE CONTROL OR ETHANOL-MAINTAINED GROUPS. FURTHERMORE, THIS HYPERALGESIA WAS ATTENUATED BY THE HISTONE DEACETYLASE INHIBITOR, SUBEROYLANILIDE HYDROXAMIC ACID TREATMENT. CONCLUSIONS: HEIGHTENED PAIN SENSITIVITY WAS OBSERVED FOLLOWING WITHDRAWAL FROM CHRONIC ETHANOL EXPOSURE, AND HISTONE DEACETYLASE INHIBITORS COULD BE NOVEL TREATMENTS FOR THIS ALCOHOL WITHDRAWAL-INDUCED HYPERALGESIA. 2019 16 2120 29 EPIGENETIC HISTONE MODIFICATIONS IN A CLINICALLY RELEVANT RAT MODEL OF CHRONIC ETHANOL-BINGE-MEDIATED LIVER INJURY. PURPOSE: ETHANOL BINGE AUGMENTS LIVER INJURY AFTER CHRONIC ETHANOL CONSUMPTION IN HUMANS, BUT THE MECHANISM BEHIND THE ENHANCED LIVER INJURY BY ETHANOL BINGE IS NOT KNOWN. IN THIS STUDY WE USED A CLINICALLY RELEVANT RAT MODEL IN WHICH LIVER INJURY IS AMPLIFIED BY BINGE AFTER CHRONIC ETHANOL TREATMENT AND INVESTIGATED THE IMPORTANCE OF HISTONE MODIFICATIONS. METHODS: EIGHT-WEEK-OLD SPRAGUE-DAWLEY RATS WERE FED ETHANOL IN A LIQUID DIET FOR 4 WEEKS. CONTROL RATS WERE FED AN ISOCALORIC LIQUID DIET. THIS WAS FOLLOWED BY THREE BINGE ADMINISTRATIONS OF ETHANOL (INTRAGASTRIC 5 G/KG BODY WEIGHT, 12 H APART). IN THE CONTROL, ETHANOL WAS REPLACED BY WATER. FOUR HOURS AFTER THE LAST BINGE ADMINISTRATION, LIVER SAMPLES WERE ANALYZED FOR HISTONE MODIFICATIONS AND PARAMETERS OF LIVER INJURY. RESULTS: CHRONIC ETHANOL ADMINISTRATION ALONE CAUSED AN INCREASE IN HISTONE H3 SER10 AND SER28 (H3S10 OR S28) PHOSPHORYLATION, AND BINGE ETHANOL REDUCED THEIR LEVELS. LEVELS OF DUALLY MODIFIED PHOSPHOACETYLATED HISTONE H3 (H3ACK9/PS10) INCREASED AFTER ACUTE BINGE ETHANOL AND REMAINED SAME AFTER CHRONIC ETHANOL BINGE. IN CONTRAST, HISTONE H3 LYSINE-9 ACETYLATION (H3ACK9) WAS NOT INCREASED AFTER CHRONIC ETHANOL BUT INCREASED SIGNIFICANTLY AFTER ACUTE BINGE AND CHRONIC ETHANOL BINGE. INCREASE IN HISTONE ACETYLATION WAS ACCOMPANIED BY INCREASED PHOSPHO-ERK1/2 IN THE NUCLEAR EXTRACTS. INCREASED ACETYLATION AFTER CHRONIC ETHANOL BINGE WAS ALSO ACCOMPANIED BY INCREASED PROTEIN LEVELS OF GCN5 HISTONE ACETYL TRANSFERASE AND A MODEST INCREASE IN HDAC3 IN THE NUCLEUS. HISTONE LYSINE-9 DIMETHYLATION WAS SIGNIFICANTLY INCREASED AFTER CHRONIC ETHANOL BINGE. CHRONIC ETHANOL BINGE ALSO RESULTED IN A DECREASE IN THE SAM:SAH RATIO WITH A RELATIVE DECREASE OF SAM LEVELS AND A CORRESPONDING INCREASE IN SAH LEVELS. CONCLUSIONS: ETHANOL BINGE AFTER CHRONIC ETHANOL ALTERED THE PROFILE OF SITE-SPECIFIC HISTONE MODIFICATIONS AND MAY UNDERLIE THE MECHANISM OF AUGMENTED LIVER INJURY BY CHRONIC-ETHANOL-BINGE-TREATED RATS. 2014 17 2156 41 EPIGENETIC MECHANISMS ARE INVOLVED IN THE REGULATION OF ETHANOL CONSUMPTION IN MICE. BACKGROUND: REPEATED ALCOHOL EXPOSURE IS KNOWN TO INCREASE SUBSEQUENT ETHANOL CONSUMPTION IN MICE. HOWEVER, THE UNDERLYING MECHANISMS HAVE NOT BEEN FULLY ELUCIDATED. ONE POSTULATED MECHANISM INVOLVES EPIGENETIC MODIFICATIONS, INCLUDING HISTONE MODIFICATIONS AND DNA METHYLATION OF RELEVANT GENES SUCH AS NR2B OR BDNF. METHODS: TO INVESTIGATE THE ROLE OF EPIGENETIC MECHANISMS IN THE DEVELOPMENT OF ALCOHOL DRINKING BEHAVIOR, AN ESTABLISHED CHRONIC INTERMITTENT ETHANOL EXPOSURE REINFORCED ETHANOL DRINKING MOUSE MODEL WITH VAPOR INHALATION OVER TWO 9-DAY TREATMENT REGIMENS WAS USED. THE DNA METHYLTRANSFERASE INHIBITOR, 5-AZACYTIDINE OR THE HISTONE DEACETYLASE INHIBITOR, TRICHOSTATIN A WAS ADMINISTERED (INTRAPERITONEALLY) TO C57BL/6 MICE 30 MIN BEFORE DAILY EXPOSURE TO CHRONIC INTERMITTENT ETHANOL. CHANGES IN ETHANOL CONSUMPTION WERE MEASURED USING THE 2-BOTTLE CHOICE TEST. RESULTS: THE RESULTS INDICATED THAT SYSTEMIC ADMINISTRATION OF TRICHOSTATIN A (2.5 MICROG/G) FACILITATED CHRONIC INTERMITTENT ETHANOL-INDUCED ETHANOL DRINKING, BUT SYSTEMIC ADMINISTRATION OF 5-AZACYTIDINE (2 MICROG/G) DID NOT CAUSE THE SAME EFFECT. HOWEVER, WHEN 5-AZACYTIDINE WAS ADMINISTERED BY INTRACEREBROVENTRICULAR INJECTION, IT FACILITATED CHRONIC INTERMITTENT ETHANOL-INDUCED ETHANOL DRINKING. FURTHERMORE, THE INCREASED DRINKING CAUSED BY CHRONIC INTERMITTENT ETHANOL WAS PREVENTED BY INJECTION OF A METHYL DONOR, S-ADENOSYL-L-METHIONINE. TO PROVIDE EVIDENCE THAT CHRONIC INTERMITTENT ETHANOL- OR TRICHOSTATIN A-INDUCED DNA DEMETHYLATION AND HISTONE MODIFICATIONS OF THE NR2B PROMOTER MAY UNDERLIE THE ALTERED ETHANOL CONSUMPTION, WE EXAMINED EPIGENETIC MODIFICATIONS AND NR2B EXPRESSION IN THE PREFRONTAL CORTEX OF THESE MICE. CHRONIC INTERMITTENT ETHANOL OR TRICHOSTATIN A DECREASED DNA METHYLATION AND INCREASED HISTONE ACETYLATION IN THE NR2B GENE PROMOTER, AS WELL AS MRNA LEVELS OF NR2B IN THESE MICE. CONCLUSIONS: TAKEN TOGETHER, THESE RESULTS INDICATE THAT EPIGENETIC MODIFICATIONS ARE INVOLVED IN REGULATING ETHANOL DRINKING BEHAVIOR, PARTIALLY THROUGH ALTERING NR2B EXPRESSION. 2014 18 6177 26 THE HISTONE METHYLTRANSFERASE G9A MEDIATES STRESS-REGULATED ALCOHOL DRINKING. THE EPIGENETIC ENZYME G9A IS A HISTONE METHYLTRANSFERASE THAT DIMETHYLATES LYSINE 9 ON HISTONE H3 (H3K9ME2), AND IN THE ADULT NUCLEUS ACCUMBENS (NAC), G9A REGULATES MULTIPLE BEHAVIORS ASSOCIATED WITH SUBSTANCE USE DISORDER. WE SHOW HERE THAT CHRONIC INTERMITTENT ETHANOL (CIE) EXPOSURE IN MALE MICE REDUCED BOTH G9A AND H3K9ME2 LEVELS IN THE ADULT NAC, BUT NOT DORSAL STRIATUM. VIRAL-MEDIATED REDUCTION OF G9A IN THE NAC HAD NO EFFECTS ON BASELINE VOLITIONAL ETHANOL DRINKING OR ESCALATED ALCOHOL DRINKING PRODUCED BY CIE EXPOSURE; HOWEVER, NAC G9A WAS REQUIRED FOR STRESS-REGULATED CHANGES IN ETHANOL DRINKING, INCLUDING POTENTIATED ALCOHOL DRINKING PRODUCED BY ACTIVATION OF THE KAPPA-OPIOID RECEPTOR. IN ADDITION, WE OBSERVED THAT CHRONIC SYSTEMIC ADMINISTRATION OF A G9A INHIBITOR, UNC0642, ALSO BLOCKED STRESS-POTENTIATED ALCOHOL DRINKING. TOGETHER, OUR FINDINGS SUGGEST THAT CHRONIC ALCOHOL USE, SIMILAR TO OTHER ABUSED SUBSTANCES, PRODUCES A NAC-SELECTIVE REDUCTION IN G9A LEVELS THAT SERVES TO LIMIT STRESS-REGULATED ALCOHOL DRINKING. MOREOVER, OUR FINDINGS SUGGEST THAT PHARMACOLOGICAL INHIBITION OF G9A MIGHT PROVIDE A NOVEL THERAPEUTIC APPROACH TO TREAT STRESS-INDUCED ALCOHOL DRINKING, WHICH IS A MAJOR TRIGGER OF RELAPSE IN INDIVIDUALS SUFFERING FROM AUD. 2022 19 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 20 3841 27 IRON SUPPLEMENTATION REVERSES THE REDUCTION OF HYDROXYMETHYLCYTOSINE IN HEPATIC DNA ASSOCIATED WITH CHRONIC ALCOHOL CONSUMPTION IN RATS. BACKGROUND: ALCOHOL IS KNOWN TO AFFECT TWO EPIGENETIC PHENOMENA, DNA METHYLATION AND DNA HYDROXYMETHYLATION, AND IRON IS A COFACTOR OF TEN-ELEVEN TRANSLOCATION (TET) ENZYMES THAT CATALYZE THE CONVERSION FROM METHYLCYTOSINE TO HYDROXYMETHYLCYTOSINE. IN THE PRESENT STUDY WE AIMED TO DETERMINE THE EFFECTS OF ALCOHOL ON DNA HYDROXYMETHYLATION AND FURTHER EFFECTS OF IRON ON ALCOHOL ASSOCIATED EPIGENETIC CHANGES. METHODS: TWENTY-FOUR MALE SPRAGUE-DAWLEY RATS WERE FED EITHER LIEBER-DECARLI ALCOHOL DIET (36% CALORIES FROM ETHANOL) OR LIEBER-DECARLI CONTROL DIET ALONG WITH OR WITHOUT IRON SUPPLEMENTATION (0.6% CARBONYL IRON) FOR 8 WEEKS. HEPATIC NON-HEME IRON CONCENTRATIONS WERE MEASURED BY COLORIMETRIC ASSAYS. PROTEIN LEVELS OF HEPATIC FERRITIN AND TRANSFERRIN RECEPTOR WERE DETERMINED BY WESTERN BLOTTING. METHYLCYTOSINE, HYDROXYMETHYLCYTOSINE AND UNMODIFIED CYTOSINE IN DNA WERE SIMULTANEOUSLY MEASURED BY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY METHOD. RESULTS: IRON SUPPLEMENTATION SIGNIFICANTLY INCREASED HEPATIC NON-HEME IRON CONTENTS (P < 0.05) BUT ALCOHOL ALONE DID NOT. HOWEVER, BOTH ALCOHOL AND IRON SIGNIFICANTLY INCREASED HEPATIC FERRITIN LEVELS AND DECREASED HEPATIC TRANSFERRIN RECEPTOR LEVELS (P < 0.05). ALCOHOL REDUCED HEPATIC DNA HYDROXYMETHYLATION (0.21% +/- 0.04% VS. 0.33% +/- 0.04%, P = 0.01) COMPARED TO CONTROL, WHILE IRON SUPPLEMENTATION TO ALCOHOL DIET DID NOT CHANGE DNA HYDROXYMETHYLATION. THERE WAS NO SIGNIFICANT DIFFERENCE IN METHYLCYTOSINE LEVELS, WHILE UNMODIFIED CYTOSINE LEVELS WERE SIGNIFICANTLY INCREASED IN ALCOHOL-FED GROUPS COMPARED TO CONTROL (95.61% +/- 0.08% VS. 95.26% +/- 0.12%, P = 0.03), SUGGESTING THAT ALCOHOL FURTHER INCREASES THE CONVERSION FROM HYDROXYMETHYLCYTOSINE TO UNMODIFIED CYTOSINE. CONCLUSIONS: CHRONIC ALCOHOL CONSUMPTION ALTERS GLOBAL DNA HYDROXYMETHYLATION IN THE LIVER BUT IRON SUPPLEMENTATION REVERSES THE EPIGENETIC EFFECT OF ALCOHOL. 2016