The DEN and CCl4‐Induced Mouse Model of Fibrosis and Inflammation‐Associated Hepatocellular Carcinoma

Takeki Uehara1, Igor P. Pogribny2, Ivan Rusyn3

1 Strategic Development Department, Shionogi & Co, Osaka, 2 National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, 3 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.30
DOI:  10.1002/0471141755.ph1430s66
Online Posting Date:  September, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Human hepatocellular carcinoma (HCC) develops most often as a complication of fibrosis or cirrhosis. While most human studies of HCC provide crucial insights into the molecular signatures of HCC, seldom do they address the etiology of HCC. Mouse models are essential tools for investigating the pathogenesis of HCC; however, the overwhelming majority of cancer models in rodents do not feature liver fibrosis. Detailed in this unit is a protocol for an experimental mouse model of HCC that arises in association with advanced liver fibrosis. The disease model is induced by a single injection of N‐nitrosodiethylamine (DEN) followed by repeated administration of carbon tetrachloride (CCl4). A dramatic potentiation of liver tumor incidence is observed following administration of DEN and CCl4, with100% of mice developing liver tumors at 5 months of age. This model can be employed for studying the molecular mechanisms of fibrogenesis and HCC development, and in cancer hazard/chemotherapy testing of drug candidates. Curr. Protoc. Pharmacol. 66:14.30.1‐14.30.10. © 2014 by John Wiley & Sons, Inc.

Keywords: liver; fibrosis; cancer; mechanisms; genotoxic

PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • Female pregnant B6C3F1/J mice (Jackson Laboratory) ∼1 week before delivery
  • Diethylnitrosamine (see recipe)
  • Sterile phosphate‐buffered saline (PBS; vehicle)
  • Carbon tetrachloride (CCl 4; see recipe)
  • Olive oil (vehicle)
  • 5‐Bromo‐2′‐deoxyuridine (BrDU; see recipe)
  • Nembutal (Oak Pharmaceutical) for anesthesia or other approved anesthetic
  • Neutral buffered formalin (10%)
  • Paraffin
  • Hematoxylin and eosin
  • Animal scale
  • Disposable plastic syringes (1.0 ml) with needles for i.p. injection
  • Sharp dissecting scissors
  • Heparin‐containing serum gel Z/1.1‐ml centrifuge tubes (Sardstedt)
  • 0.9% NaCl‐moistened filter paper
  • Razor blades
  • Tissue processing/embedding cassettes (any commercial supplier)
  • Screw‐lid containers
  • Light microscope
  • Additional reagents and equipment for the Masson's trichrome procedure (Masson, )
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  AACR Cancer Progress Report Writing Committee, Sawyers, C.L., Abate‐Shen, C., Anderson, K.C., Barker, A., Baselga, J., Berger, N.A., Foti, M., Jemal, A., Lawrence, T.S., Li, C.I., Mardis, E.R., Neumann, P.J., Pardoll, D.M., Prendergast, G.C., Reed, J.C., and Weiner, G.J. 2013. AACR Cancer Progress Report 2013. Clin. Cancer Res. 19:S4‐S98.
  Alkofer, B., Lepennec, V., and Chiche, L. 2011. Hepatocellular cancer in the non‐cirrhotic liver. J. Visc. Surg. 148:3‐11.
  Aravalli, R.N., Cressman, E.N., and Steer, C.J. 2013. Cellular and molecular mechanisms of hepatocellular carcinoma: an update. Arch. Toxicol. 87:227‐247.
  Bannasch, P. 1983. Strain and species differences in susceptibility to liver tumour induction. IARC Sci. Publ. 51:9‐38.
  Becker, F.F. 1983. Thioacetamide hepatocarcinogenesis. J. Natl. Cancer Inst. 71:553‐558.
  Bucher, J.R. 1998. Update on national toxicology program (NTP) assays with genetically altered or “transgenic” mice. Environ. Health Perspect. 106:619‐621.
  Center, M.M. and Jemal, A. 2011. International trends in liver cancer incidence rates. Cancer Epidemiol. Biomarkers Prev. 20:2362‐2368.
  Chappell, G., Kutanzi, K., Uehara, T., Tryndyak, V., Hong, H.H., Hoenerhoff, M., Beland, F.A., Rusyn, I., and Pogribny, I.P. 2014. Genetic and epigenetic changes in fibrosis‐associated hepatocarcinogenesis in mice. Int. J. Cancer 134:2778‐2788.
  Dapito, D.H., Mencin, A., Gwak, G.Y., Pradere, J.P., Jang, M.K., Mederacke, I., Caviglia, J.M., Khiabanian, H., Adeyemi, A., Bataller, R., Lefkowitch, J.H., Bower, M., Friedman, R., Sartor, R.B., Rabadan, R., and Schwabe, R.F. 2012. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 21:504‐516.
  Della Corte, C. and Colombo, M. 2012. Surveillance for hepatocellular carcinoma. Semin. Oncol. 39:384‐398.
  Dragani, T.A., Manenti, G., and Della, P.G. 1986. Enhancing effects of carbon tetrachloride in mouse hepatocarcinogenesis. Cancer Lett. 31:171‐179.
  Druckrey, H., Steinhoff, D., Preussmann, R., and Ivankovic, S. 1964. Induction of cancer by a single dose of methylnitroso‐urea and various dialkylnitrosamines in rats. Z. Krebsforsch. 66:1‐10.
  El Serag, H.B. and Rudolph, K.L. 2007. Hepatocellular carcinoma: Epidemiology and molecular carcinogenesis. Gastroenterology 132:2557‐2576.
  Farazi, P.A. and DePinho, R.A. 2006. Hepatocellular carcinoma pathogenesis: From genes to environment. Nat. Rev. Cancer 6:674‐687.
  Fattovich, G., Stroffolini, T., Zagni, I., and Donato, F. 2004. Hepatocellular carcinoma in cirrhosis: Incidence and risk factors. Gastroenterology 127:S35‐S50.
  Fausto, N. and Campbell, J.S. 2010. Mouse models of hepatocellular carcinoma. Semin. Liver Dis. 30:87‐98.
  Fujii, T., Fuchs, B.C., Yamada, S., Lauwers, G.Y., Kulu, Y., Goodwin, J.M., Lanuti, M., and Tanabe, K.K. 2010. Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor. BMC Gastroenterol. 10:79.
  Goldsworthy, T.L. and Fransson‐Steen, R. 2002. Quantitation of the cancer process in C57BL/6J, B6C3F1 and C3H/HeJ mice. Toxicol. Pathol. 30:97‐105.
  Grivennikov, S.I., Greten, F.R., and Karin, M. 2010. Immunity, inflammation, and cancer. Cell 140:883‐899.
  Guzman, G., Chennuri, R., Voros, A., Boumendjel, R., Locante, A., Patel, R., and Valyi‐Nagy, T. 2011. Nucleometric study of anisonucleosis, diabetes and oxidative damage in liver biopsies of orthotopic liver transplant recipients with chronic hepatitis C virus infection. Pathol. Oncol. Res. 17:191‐199.
  Heindryckx, F., Colle, I., and Van Vlierberghe, H. 2009. Experimental mouse models for hepatocellular carcinoma research. Int. J. Exp. Pathol. 90:367‐386.
  Hoenerhoff, M.J., Hong, H.H., Ton, T.V., Lahousse, S.A., and Sills, R.C. 2009. A review of the molecular mechanisms of chemically induced neoplasia in rat and mouse models in National Toxicology Program bioassays and their relevance to human cancer. Toxicol. Pathol. 37:835‐848.
  Huff, J., Cirvello, J., Haseman, J., and Bucher, J. 1991. Chemicals associated with site‐specific neoplasia in 1394 long‐term carcinogenesis experiments in laboratory rodents. Environ. Health Perspect. 93:247‐270.
  Jacobson‐Kram, D. 2010. Cancer risk assessment approaches at the FDA/CDER: Is the era of the 2‐year bioassay drawing to a close? Toxicol. Pathol. 38:169‐170.
  Jepsen, P., Vilstrup, H., Tarone, R.E., Friis, S., and Sorensen, H.T. 2007. Incidence rates of hepatocellular carcinoma in the U.S. and Denmark: Recent trends. Int. J. Cancer 121:1624‐1626.
  Luedde, T. and Schwabe, R.F. 2011. NF‐kappaB in the liver—linking injury, fibrosis and hepatocellular carcinoma. Nat. Rev. Gastroenterol. Hepatol. 8:108‐118.
  Maeda, S., Kamata, H., Luo, J.L., Leffert, H., and Karin, M. 2005. IKKbeta couples hepatocyte death to cytokine‐driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121:977‐990.
  Masson, P.J. 1929. Some histological methods: Trichrome stainings and their preliminary technique. Tech. Methods 12:75‐90.
  Mitchell, K.A. 2013. Hepatocellular carcinoma: Histologic considerations: Pure, mixed, and motley. J. Clin. Gastroenterol. 47:S20‐S26.
  National Research Council. 2005. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals. The National Academies Press, Washington, D.C.
  National Toxicology Program. 2011. 12th Report on Carcinogens. Department of Health and Human Services, Research Ttiangle Park, North Carolina.
  Oh, J.Y., Jeong, J.S., Kim, Y.J., Nam, K.J., Park, B.H., Kwon, E.Y., Kim, Y.H., and Hwang, T.H. 2002. Ultrasonographic evidence of phenotypic instability during hepatocarcinogenesis in N‐nitrosomorpholine‐treated rats. Exp. Mol. Pathol. 73:67‐73.
  Siegel, R., Naishadham, D., and Jemal, A. 2013. Cancer statistics, 2013. CA Cancer J. Clin. 63:11‐30.
  Stowell, R.E., Lee, C.S., Tsuboi, K.K., and Villasana, A. 1951. Histochemical and microchemical changes in experimental cirrhosis and hepatoma formation in mice by carbon tetrachloride. Cancer Res. 11:345‐354.
  Titford, M. 2005. The long history of hematoxylin. Biotech. Histochem. 80:73‐78.
  Uehara, T., Ainslie, G.R., Kutanzi, K., Pogribny, I.P., Muskhelishvili, L., Izawa, T., Yamate, J., Kosyk, O., Shymonyak, S., Bradford, B.U., Boorman, G.A., Bataller, R., and Rusyn, I. 2013. Molecular mechanisms of fibrosis‐associated promotion of liver carcinogenesis. Toxicol. Sci. 132:53‐63.
  Vesselinovitch, S.D. and Mihailovich, N. 1983. Kinetics of diethylnitrosamine hepatocarcinogenesis in the infant mouse. Cancer Res. 43:4253‐4259.
  Vucur, M., Roderburg, C., Bettermann, K., Tacke, F., Heikenwalder, M., Trautwein, C., and Luedde, T. 2010. Mouse models of hepatocarcinogenesis: What can we learn for the prevention of human hepatocellular carcinoma? Oncotarget 1:373‐378.
  Wells, M.Y. and Williams, E.S. 2009. The transgenic mouse assay as an alternative test method for regulatory carcinogenicity studies—implications for REACH. Regul. Toxicol. Pharmacol. 53:150‐155.
  Zucman‐Rossi, J. 2010. Molecular classification of hepatocellular carcinoma. Dig. Liver Dis. 42:S235‐S241.
PDF or HTML at Wiley Online Library