Digestive Diseases and Sciences

, Volume 58, Issue 7, pp 1923–1933 | Cite as

Chronic Ingestion of Ethanol Induces Hepatocellular Carcinoma in Mice Without Additional Hepatic Insult

  • Mutsumi Tsuchishima
  • Joseph George
  • Hisakazu Shiroeda
  • Tomiyasu Arisawa
  • Tsutomu Takegami
  • Mikihiro Tsutsumi
Original Article



Chronic intake of alcohol increases the risk of gastrointestinal and hepatic carcinogenesis. The present study was focused to investigate the incidence and mechanism of pathogenesis of hepatocellular carcinoma (HCC) during chronic ingestion of alcohol without any additional hepatic injury.


Ethanol was administered to Institute for Cancer Research male mice through drinking water for 70 weeks at concentrations of 5 % (first week), 10 % (next 8 weeks), and 15 % thereafter. The animals were killed at 60 and 70 weeks, the livers were examined for hepatic tumors, and evaluated for foci of cellular alteration (FCA). Immunohistochemical staining was performed in the liver sections for cytochrome P4502E1 (CYP2E1), 4-hydroxy-nonenal (4-HNE), and proto-oncogene, c-Myc.


At the 60th week, 40 % of the mice in the ethanol group had visible white nodules (5–10 mm) in the liver, but not in the control mice. At the 70th week, several larger nodules (5–22 mm) were present in the livers of 50 % mice in the ethanol group. In the control group, one mouse developed a single nodule. All nodules were histologically trabecular HCC composed of eosinophilic and vacuolated cells. In the livers of both control and ethanol group, several foci with cellular alteration were present, which were significantly higher in ethanol group. Staining for CYP2E1, 4-HNE and c-Myc depicted marked upregulation of all these molecules in the FCA.


Our data demonstrated that upregulation of CYP2E1 and subsequent production of reactive oxygen species along with the persistent expression of c-Myc play a significant role in the pathogenesis of HCC during chronic ingestion of ethanol.


Hepatocellular carcinoma HCC Ethanol Carcinogenesis Cellular alteration 



This work was supported by a grant for specially promoted research from Kanazawa Medical University, Japan (SR 2012-04).

Conflict of interest



  1. 1.
    Chun JM, Kwon HJ, Sohn J, et al. Prognostic factors after early recurrence in patients who underwent curative resection for hepatocellular carcinoma. J Surg Oncol. 2011;103:148–151.CrossRefGoogle Scholar
  2. 2.
    Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108.CrossRefGoogle Scholar
  3. 3.
    Cornellà H, Alsinet C, Villanueva A. Molecular pathogenesis of hepatocellular carcinoma. Alcohol Clin Exp Res. 2011;35:821–825.CrossRefGoogle Scholar
  4. 4.
    French SW, Oliva J, French BA, Li J, Bardag-Gorce F. Alcohol, nutrition and liver cancer: role of Toll-like receptor signaling. World J Gastroenterol. 2010;16:1344–1348.CrossRefGoogle Scholar
  5. 5.
    Chemin I, Zoulim F. Hepatitis B virus-induced hepatocellular carcinoma. Cancer Lett. 2009;286:52–59.CrossRefGoogle Scholar
  6. 6.
    Vezali E, Aghemo A, Colombo M. A review of the treatment of chronic hepatitis C virus infection in cirrhosis. Clin Ther. 2010;32:2117–2138.CrossRefGoogle Scholar
  7. 7.
    Rehm J, Taylor B, Mohapatra S, et al. Alcohol as a risk factor for liver cirrhosis: a systematic review and meta-analysis. Drug Alcohol Rev. 2010;29:437–445.CrossRefGoogle Scholar
  8. 8.
    Lefton HB, Rosa A, Cohen M. Diagnosis and epidemiology of cirrhosis. Med Clin N Am. 2009;93:787–799.CrossRefGoogle Scholar
  9. 9.
    Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002;31:339–346.CrossRefGoogle Scholar
  10. 10.
    Wong N, Lai P, Pang E, et al. Genomic aberrations in human hepatocellular carcinomas of differing etiologies. Clin Cancer Res. 2000;6:4000–4009.Google Scholar
  11. 11.
    Wogan GN. Aflatoxins as risk factors for hepatocellular carcinoma in humans. Cancer Res. 1992;52:2114s–2118s.Google Scholar
  12. 12.
    Montalto G, Cervello M, Giannitrapani L, Dantona F, Terranova A, Castagnetta LA. Epidemiology, risk factors, and natural history of hepatocellular carcinoma. Ann N Y Acad Sci. 2002;963:13–20.CrossRefGoogle Scholar
  13. 13.
    Pan H, Fu X, Huang W. Molecular mechanism of liver cancer. Anticancer Agents Med Chem. 2011;11:493–499.CrossRefGoogle Scholar
  14. 14.
    Moradpour D, Blum HE. Pathogenesis of hepatocellular carcinoma. Eur J Gastroenterol Hepatol. 2005;17:477–4783.CrossRefGoogle Scholar
  15. 15.
    Pöschl G, Seitz HK. Alcohol and cancer. Alcohol Alcohol. 2004;39:155–165.CrossRefGoogle Scholar
  16. 16.
    Seitz HK, Stickel F. Molecular mechanisms of alcohol-mediated carcinogenesis. Nat Rev Cancer. 2007;7:599–612.CrossRefGoogle Scholar
  17. 17.
    Wang Y, Millonig G, Nair J, et al. Ethanol-induced cytochrome P4502E1 causes carcinogenic etheno-DNA lesions in alcoholic liver disease. Hepatology. 2009;50:453–461.CrossRefGoogle Scholar
  18. 18.
    Quertemont E. Genetic polymorphism in ethanol metabolism: acetaldehyde contribution to alcohol abuse and alcoholism. Mol Psychiatry. 2004;9:570–581.CrossRefGoogle Scholar
  19. 19.
    Tsutsumi M, George J, Ishizawa K, Fukumura A, Takase S. Effect of chronic dietary ethanol in the promotion of N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats. J Gastroenterol Hepatol. 2006;21:805–813.CrossRefGoogle Scholar
  20. 20.
    Lieber CS, Garro A, Leo MA, Mak KM, Worner T. Alcohol and cancer. Hepatology. 1986;6:1005–1019.CrossRefGoogle Scholar
  21. 21.
    Anttila S, Raunio H, Hakkola J. Cytochrome p450-mediated pulmonary metabolism of carcinogens: regulation and cross-talk in lung carcinogenesis. Am J Respir Cell Mol Biol. 2011;44:583–590.CrossRefGoogle Scholar
  22. 22.
    Su AI, Cooke MP, Ching KA, et al. Large-scale analysis of the human and mouse transcriptomes. Proc Natl Acad Sci USA. 2002;99:4465–4470.CrossRefGoogle Scholar
  23. 23.
    Rice MC, O’Brien SJ. Genetic variance of laboratory outbred Swiss mice. Nature. 1980;283:157–161.CrossRefGoogle Scholar
  24. 24.
    McClain CJ, Hill DB, Song Z, Deaciuc I, Barve S. Monocyte activation in alcoholic liver disease. Alcohol. 2002;27:53–561.CrossRefGoogle Scholar
  25. 25.
    Fattovich G, Stroffolini T, Zagni I, Donato F. Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology. 2004;127:S35–S50.CrossRefGoogle Scholar
  26. 26.
    Miller AM, Horiguchi N, Jeong WI, Radaeva S, Gao B. Molecular mechanisms of alcoholic liver disease: innate immunity and cytokines. Alcohol Clin Exp Res. 2011;35:787–793.CrossRefGoogle Scholar
  27. 27.
    Yam JW, Wong CM, Ng IO. Molecular and functional genetics of hepatocellular carcinoma. Front Biosci (Schol Ed). 2010;2:117–134.CrossRefGoogle Scholar
  28. 28.
    Beland FA, Benson RW, Mellick PW, et al. Effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in B6C3F1 mice. Food Chem Toxicol. 2005;43:1–19.CrossRefGoogle Scholar
  29. 29.
    Wu D, Cederbaum AI. Oxidative stress and alcoholic liver disease. Semin Liver Dis. 2009;29:141–154.CrossRefGoogle Scholar
  30. 30.
    Campbell JS, Hughes SD, Gilbertson DG, et al. Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma. Proc Natl Acad Sci USA. 2005;102:3389–3394.CrossRefGoogle Scholar
  31. 31.
    Cederbaum AI, Lu Y, Wu D. Role of oxidative stress in alcohol-induced liver injury. Arch Toxicol. 2009;83:519–548.CrossRefGoogle Scholar
  32. 32.
    Amidi F, French BA, Chung D, Halsted CH, Medici V, French SW. M-30 and 4HNE are sequestered in different aggresomes in the same hepatocytes. Exp Mol Pathol. 2007;83:296–300.CrossRefGoogle Scholar
  33. 33.
    Maronpot RR, Haseman JK, Boorman GA, Eustis SE, Rao GN, Huff JE. Liver lesions in B6C3F1 mice: the national toxicology program, experience and position. Arch Toxicol Suppl. 1987;10:10–26.CrossRefGoogle Scholar
  34. 34.
    Baldrick P, Reeve L. Carcinogenicity evaluation: comparison of tumor data from dual control groups in the CD-1 mouse. Toxicol Pathol. 2007;35:562–569.CrossRefGoogle Scholar
  35. 35.
    Engelhardt JA, Gries CL, Long GG. Incidence of spontaneous neoplastic and nonneoplastic lesions in Charles River CD-1 mice varies with breeding origin. Toxicol Pathol. 1993;21:538–541.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Mutsumi Tsuchishima
    • 1
  • Joseph George
    • 1
  • Hisakazu Shiroeda
    • 1
  • Tomiyasu Arisawa
    • 1
  • Tsutomu Takegami
    • 2
  • Mikihiro Tsutsumi
    • 1
  1. 1.Department of GastroenterologyKanazawa Medical UniversityUchinada, IshikawaJapan
  2. 2.Medical Research InstituteKanazawa Medical UniversityUchinada, IshikawaJapan

Personalised recommendations