Hepatology International

, Volume 8, Supplement 2, pp 421–430 | Cite as

Epigenetic histone modifications in a clinically relevant rat model of chronic ethanol-binge-mediated liver injury

  • Annayya R. Aroor
  • Ricardo J. Restrepo
  • Kusum K. Kharbanda
  • Shivendra D. ShuklaEmail author
Supplement Issue: ALPD



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.


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.


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.


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.


Alcoholic steatohepatitis Binge ethanol Epigenetics Histone modifications Necrosis 



This work was supported by NIH grant AA16347 (SDS) and in part by MERIT Review grant BX001155 (KKK) from the Department of Veterans Affairs, Office of Research and Development (Biomedical Laboratory Research & Development).

Compliance with ethical requirements and Conflict of interest

In this study the protocol for the use of rats was approved by the University of Missouri Animal Care and Use Committee (approval no. 7178). This committee follows the guidelines set by the National Institutes of Health (USA) for the use and care of laboratory animals. Annayya R. Aroor, Ricardo J. Restrepo, Kususm K. Kharbanda, and Shivendra D. Shukla declare that they have no conflict of interest.


  1. 1.
    Ceccanti M, Attili A, Balducci G, Attilia F, Giacomelli S, Rotondo C, et al. Acute alcoholic hepatitis. J Clin Gastroenterol. 2006;40:833–841PubMedCrossRefGoogle Scholar
  2. 2.
    Zakhari S, Li TK. Determinants of alcohol use and abuse: impact of quantity and frequency patterns on liver disease. Hepatology. 2007;46:2032–2039PubMedCrossRefGoogle Scholar
  3. 3.
    Mathurin P, Beuzin F, Louvet A, Carrié-Ganne N, Balian A, Trinchet JC, et al. Fibrosis progression occurs in a subgroup of heavy drinkers with typical histological features. Aliment Pharmacol Ther. 2007;25:1047–1054PubMedCrossRefGoogle Scholar
  4. 4.
    Courtney KE, Polich J. Binge drinking in young adults: data, definitions, and determinants. Psychol Bull. 2009;135:142–156PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Grucza RA, Norberg KE, Bierut LJ. Binge drinking among youths and young adults in the United States: 1979–2006. J Am Acad Child Adolesc Psychiatry. 2009;48:692–702PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Rehm J, Mathers C, Popova S, Thavorncharoensap M, Teerawattananon Y, Patra J. Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders. Lancet. 2009;373:2223–2233PubMedCrossRefGoogle Scholar
  7. 7.
    Mathurin P, Deltenre P. Effect of binge drinking on the liver: an alarming public health issue? Gut. 2009;58:613–617PubMedCrossRefGoogle Scholar
  8. 8.
    Bobak M, Room R, Pikhart H, Kubinova R, Malyutina S, Pajak A, et al. Contribution of drinking patterns to differences in rates of alcohol related problems between three urban populations. J Epidemiol Community Health. 2004;58:238–242PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Crosse KI, Anania FK. Alcoholic hepatitis. Current treatment options. Gastroenterology. 2002;5:417–423Google Scholar
  10. 10.
    Delcuve GP, Rastegar M, Davie JR. Epigenetic regulation. J Cell Physiol. 2009;219:243–250PubMedCrossRefGoogle Scholar
  11. 11.
    Patel DJ, Wang Z. Readout of epigenetic modifications. Annu Rev Biochem. 2013;82:81–118PubMedCrossRefGoogle Scholar
  12. 12.
    Hübner MR, Eckersley-Maslin MA, Spector DL. Chromatin organization and transcriptional regulation. Curr Opin Genet Dev. 2013;23:89–95PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Greer EL, Shi Y. Histone methylation: a dynamic mark in health, disease and inheritance. Nat Rev Genet. 2012;13:343–357PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Oliver SS, Denu JM. Dynamic interplay between histone H3 modifications and protein interpreters: emerging evidence for a “histone language”. ChemBioChem. 2011;12:299–307PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Shukla SD, Aroor AR. Epigenetic effects of ethanol on liver and gastrointestinal injury. World J Gastroenterol. 2006;12:5265–5271PubMedPubMedCentralGoogle Scholar
  16. 16.
    Shukla SD, Pruett SB, Szabo G, Arteel GE. Binge ethanol and liver: new molecular developments. Alcohol Clin Exp Res. 2013;37:550–557PubMedCrossRefGoogle Scholar
  17. 17.
    Pal-Bhadra M, Bhadra U, Jackson DE, Mamatha L, Park PH, Shukla SD. Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- & down-regulation of genes by ethanol in hepatocytes. Life Sci. 2007;81:979–987PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Kim JS, Shukla SD. Acute in vivo effect of ethanol (binge drinking) on histone H3 modifications in rat tissues. Alcohol Alcohol. 2006;41:126–132PubMedCrossRefGoogle Scholar
  19. 19.
    Kirpich I, Zhang J, Gobejishvili L, Kharebava G, Barker D, Ghare S, et al. Binge ethanol-induced HDAC3 down-regulates Cpt1α expression leading to hepatic steatosis and injury. Alcohol Clin Exp Res. 2013;37:1920–1929PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Kirpich I, Ghare S, Zhang J, Gobejishvili L, Kharebava G, Barve, SJ et al. Binge alcohol-induced microvesicular liver steatosis and injury are associated with down-regulation of hepatic Hdac 1, 7, 9, 10, 11 and up-regulation of Hdac 3. Alcohol Clin Exp Res. 2012;36:1578–1586PubMedCrossRefGoogle Scholar
  21. 21.
    James TT, Aroor AR, Lim RW, Shukla SD. Histone H3 phosphorylation (Ser10, Ser28) and phosphoacetylation (K9S10) are differentially associated with gene expression in liver of rats treated in vivo with acute ethanol. J Pharmacol Exp Ther. 2012;340:237–247PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Aroor AR, James TT, Jackson DE, Shukla SD. Differential changes in MAP kinases, histone modifications, and liver injury in rats acutely treated with ethanol. Alcohol Clin Exp Res. 2010;34:1543–1551PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Li J, Bardag-Gorce F, Oliva J, Dedes J, French BA, French SW. Gene expression modifications in the liver caused by binge drinking and S-adenosylmethionine feeding. The role of epigenetic changes. Genes Nutr. 2010;5:169–179PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Esfandiari F, Medici V, Wong DH, Jose S, Dolatshahi M, Quinlivan E, et al. Epigenetic regulation of hepatic endoplasmic reticulum stress pathways in the ethanol-fed cystathionine beta synthase-deficient mouse. Hepatology. 2010;51:932–941PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Park PH, Lim RW, Shukla SD. Gene-selective histone H3 acetylation in the absence of increase in global histone acetylation in liver of rats chronically fed alcohol. Alcohol Alcohol. 2012;47:233–239PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Shukla SD, Velazquez J, French SW, Lu SC, Ticku MK, Zakhari S. Emerging role of epigenetics in the actions of alcohol. Alcohol Clin Exp Res. 2008;32:1525–1534PubMedCrossRefGoogle Scholar
  27. 27.
    You M, Cao Q, Liang X, Ajmo JM, Ness GC. Mammalian sirtuin 1 is involved in the protective action of dietary saturated fat against alcoholic fatty liver in mice. J Nutr. 2008;138:497–501PubMedGoogle Scholar
  28. 28.
    Aroor AR, Roy LJ, Restrepo RJ, Mooney BP, Shukla SD. A proteomic analysis of liver after ethanol binge in chronically ethanol treated rats. Proteome Sci. 2012;10:29PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Aroor AR, Jackson DE, Shukla SD. Elevated activation of ERK1 and ERK2 accompany enhanced liver injury following alcohol binge in chronically ethanol-fed rats. Alcohol Clin Exp Res. 2011;35:2128–2138PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Kharbanda KK, Rogers DD II, Mailliard ME, Siford GL, Barak AJ, Beckenhauer HC, et al. Role of elevated S-adenosylhomocysteine in rat hepatocyte apoptosis: protection by betaine. Biochem Pharmacol. 2005;70:1883–1890PubMedCrossRefGoogle Scholar
  31. 31.
    Peinnequin A, Mouret C, Birot O, Alonso A, Mathieu J, Clarençon D, et al. Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green. BMC Immunol. 2004;5(5):3PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Lee YJ, Shukla SD. Histone H3 phosphorylation at serine 10 and serine 28 is mediated by p38 MAPK in rat hepatocytes exposed to ethanol and acetaldehyde. Eur J Pharmacol. 2007;573:29–38PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Choudhury M, Pandey RS, Clemens DL, Davis JW, Lim RW, Shukla SD. Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells. Alcohol. 2011;45:311–324PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Park PH, Lim RW, Shukla SD. Involvement of histone acetyltransferase (HAT) in ethanol-induced acetylation of histone H3 in hepatocytes: potential mechanism for gene expression. Am J Physiol Gastrointest Liver Physiol. 2005;289:G1124–1136PubMedCrossRefGoogle Scholar
  35. 35.
    Kharbanda KK. Methionine metabolic pathway in alcoholic liver injury. Curr Opin Clin Nutr Metab Care. 2013;16:89–95PubMedCrossRefGoogle Scholar
  36. 36.
    Halsted CH, Medici V. Vitamin-dependent methionine metabolism and alcoholic liver disease. Adv Nutr. 2011;2:421–427PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Iimuro Y, Bradford BU, Yamashina S, Rusyn I, Nakagami M, Enomoto N, et al. The glutathione precursor L-2-oxothiazolidine-4-carboxylic acid protects against liver injury due to chronic enteral ethanol exposure in the rat. Hepatology. 2000;31:391–398PubMedCrossRefGoogle Scholar
  38. 38.
    Kharbanda KK, Todero SL, King AL, Osna NA, McVicker BL, Tuma DJ, et al. Betaine treatment attenuates chronic ethanol-induced hepatic steatosis and alterations to the mitochondrial respiratory chain proteome. Int J Hepatol. 2012;2012:962183PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Peng Z, Borea PA, Varani K, Wilder T, Yee H, Chiriboga L, et al. Adenosine signaling contributes to ethanol-induced fatty liver in mice. J Clin Invest. 2009;119:582–594PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Peng Z, Fernandez P, Wilder T, Yee H, Chiriboga L, Chan ES, et al. Ecto-5′-nucleotidase (CD73)-mediated extracellular adenosine production plays a critical role in hepatic fibrosis. FASEB J. 2008;22:2263–2272PubMedCrossRefGoogle Scholar
  41. 41.
    McGehee RE Jr, Ronis MJ, Badger TM. Regulation of the hepatic CYP 2E1 gene during chronic alcohol exposure: lack of an ethanol response element in the proximal 5′-flanking sequence. DNA Cell Biol. 1997;16:725–736PubMedGoogle Scholar
  42. 42.
    Balusikova K, Kovar J. Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells. In Vitro Cell Dev Biol Anim. 2013;49:619–625PubMedCrossRefGoogle Scholar
  43. 43.
    Yang H, Nie Y, Li Y, Wan YJ. Histone modification-mediated CYP2E1 gene expression and apoptosis of HepG2 cells. Exp Biol Med (Maywood). 2010;235:32–39CrossRefGoogle Scholar
  44. 44.
    Rossetto D, Avvakumov N, Côté J. Histone phosphorylation: a chromatin modification involved in diverse nuclear events. Epigenetics. 2012;7:1098–1108PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Szerlong HJ, Prenni JE, Nyborg JK, Hansen JC. Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome–nucleosome interactions. J Biol Chem. 2010;285:31954–31964PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Edmondson DG, Davie JK, Zhou J, Mirnikjoo B, Tatchell K, Dent SY. Site-specific loss of acetylation upon phosphorylation of histone H3. J Biol Chem. 2002;277:29496–29502PubMedCrossRefGoogle Scholar
  47. 47.
    Duan Q, Chen H, Costa M, Dai W. Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis. J Biol Chem. 2008;283:33585–33590PubMedCrossRefPubMedCentralGoogle Scholar
  48. 48.
    El Gazzar M, Yoza BK, Chen X, Hu J, Hawkins GA, McCall CE. G9a and HP1 couple histone and DNA methylation to TNFα transcription silencing during endotoxin tolerance. J Biol Chem. 2008;283:32198–208PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    El Gazzar M, Yoza BK, Hu JY, Cousart SL, McCall CE. Epigenetic silencing of tumor necrosis factor-α during endotoxin tolerance. J Biol Chem. 2007;282:26857–26864PubMedCrossRefGoogle Scholar
  50. 50.
    Wang B, Chen J, Santiago FS, Janes M, Kavurma MM, Chong BH, et al. Phosphorylation and acetylation of histone H3 and autoregulation by early growth response 1 mediate interleukin 1β induction of early growth response 1 transcription. Arterioscler Thromb Vasc Biol. 2010;30:536–545PubMedCrossRefGoogle Scholar
  51. 51.
    Meissner Joachim D, Freund Robert, Krone Dorothee, Umeda Patrick K, Chang Kin-Chow, Gros Gerolf, et al. Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/β gene expression via acetylation of nuclear factor of activated T cells c1. Nucl Acids Res. 2011;39:5907–5925PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Derdak Z, Villegas KA, Wands JR. Early growth response-1 transcription factor promotes hepatic fibrosis and steatosis in long-term ethanol-fed Long-Evans rats. Liver Int. 2012;32:761–770PubMedCrossRefGoogle Scholar
  53. 53.
    Pritchard MT, Nagy LE. Ethanol-induced liver injury: potential roles for egr-1. Alcohol Clin Exp Res. 2005;29(11 Suppl):146S–50SPubMedCrossRefGoogle Scholar
  54. 54.
    Kim ND, Moon JO, Slitt AL, Copple BL. Early growth response factor-1 is critical for cholestatic liver injury. Toxicol Sci. 2006;90:586–595PubMedCrossRefGoogle Scholar
  55. 55.
    Beier JI, Arteel GE. Alcoholic liver disease and the potential role of plasminogen activator inhibitor-1 and fibrin metabolism. Exp Biol Med (Maywood). 2012;237:1–9PubMedCrossRefGoogle Scholar
  56. 56.
    Kahata K, Hayashi M, Asaka M, Hellman U, Kitagawa H, Yanagisawa J, et al. Regulation of transforming growth factor-beta and bone morphogenetic protein signaling by transcriptional coactivator GCN5. Genes Cells. 2004;9:143–151PubMedCrossRefGoogle Scholar
  57. 57.
    Tur G, Georgieva EI, Gagete A, López-Rodas G, Rodríguez JL, Franco L. Factor binding and chromatin modification in the promoter of murine Egr1 gene upon induction. Cell Mol Life Sci. 2010;67:4065–4077PubMedCrossRefGoogle Scholar
  58. 58.
    Kikuchi H, Kuribayashi F, Kiwaki N, Takami Y, Nakayama T. GCN5 regulates the superoxide-generating system in leukocytes via controlling gp91-phox gene expression. J Immunol. 2011;186:3015–3022PubMedCrossRefGoogle Scholar
  59. 59.
    Xiong S, Salazar G, San Martin A, Ahmad M, Patrushev N, Hilenski L, et al. PGC-1α serine 570 phosphorylation and GCN5-mediated acetylation by angiotensin II drive catalase down-regulation and vascular hypertrophy. J Biol Chem. 2010;285:2474–2487PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Asian Pacific Association for the Study of the Liver 2014

Authors and Affiliations

  • Annayya R. Aroor
    • 1
  • Ricardo J. Restrepo
    • 1
  • Kusum K. Kharbanda
    • 2
  • Shivendra D. Shukla
    • 1
    Email author
  1. 1.Department of Medical Pharmacology and PhysiologyUniversity of Missouri School of MedicineColumbiaUSA
  2. 2.Veterans Affairs Nebraska-Western Iowa Health Care SystemOmahaUSA

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