Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease

AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma(HCC) that develops in livers with alcoholic liver disease(ALD).METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine(DEN) followed by 7 wk of 4% Lieber-De Carli diet. Serum alanine aminotransferase(ALT), alpha fetoprotein(AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6 G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/i NOS(M1) and Arg-1/IL-10/CD163/CD206(M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen(PCNA).RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed- DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicatingadvanced liver disease. Molecular characterization showed high estlevels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M 2 macrophages were edominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to al l other groups. This correlated with increased serum alphafetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice.CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC.