"Second hit" models of alcoholic liver disease

Abstract

Alcoholic liver disease (ALD) is a lifestyle disease with its pathogenesis and individual predisposition governed by gene-environment interactions. Based on the "second hit" or "multiple hits" hypothesis, patients are predisposed to progressive ALD when a magic combination of gene and environmental interactions exists. Reproduction of second or multiple hits in animal models serves to test a combination and to gain mechanistic insights into synergism achieved by such combination. Numerous environmental factors have been incorporated into animal models, largely classified into nutritional, xenobiotic/pharmacologic, hemodynamic, and viral groups. A loss or gain of function genetic model has become a popular experimental approach to test the role of a gene as a second hit. Future research will need to test more subtle or natural hits combined with excessive alcohol intake to test multiple hits in the genesis of ALD. Additionally, animal models of comorbidities are urgently needed particularly for synergistic liver disease and oncogenesis caused by alcohol, obesity, and hepatitis virus.

Figure 1

The “second hit” hypothesis of alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) pathogenesis. The second hit is most likely to be multiple hits in progression of ASH and NASH from hepatic steatosis.

Figure 2

Gene–environment interactions that determine the individual susceptibility to alcoholic liver disease.

Figure 3

How diverse “second hits” interplay with known pathogenetic mechanisms of alcoholic liver disease. Shaded boxes and bold arrows depict some of the second hits discussed in this review.

Figure 4

(A) Kupffer cells (KC) were isolated from mice at 2 weeks after a single subcutaneous injection of iron dextran (25 mg/kg) to assess lipopolysaccharide (LPS) or peroxynitrite stimulated tumor necrosis factor-α (TNFα) expression. Note KC from iron dextran-injected mice release 4 to 5 times more TNFα in response to LPS or peroxynitrite. p < 0.05 compared with the cells from mice without iron dextran injection. (B) Plasma alanine aminotransferase (ALT) levels of mice fed intragastrically with alcohol and high fat diet or pair-fed with isocaloric diet for 4 weeks without or with prior iron dextran injection. Note a single subcutaneous injection of iron dextran (75 mg/kg) prior to alcohol feeding significantly aggravates plasma ALT elevation as compared with alcohol-fed mice injected with vehicle (dextran). (C) Liver histological score of mice fed alcohol for 4 weeks without or with iron dextran injection. Note iron dextran injection at 75 mg/kg significantly worsens the liver histologic score compared with alcohol-fed mice with vehicle treatment. (D) Morphometric analysis of inflammatory cells in the liver of the different experimental groups. Note iron dextran injection significantly increases inflammatory cells in the liver as compared with alcohol-fed mice with vehicle treatment. (E) Representative microphotographs of aggravated alcoholic liver injury in iron dextran-injected mice. In the liver of alcohol-fed mice given 75 mg/kg iron dextran, hepatic macrophages stained for iron (Prussian blue reaction) are noted. (F) These iron staining-positive macrophages are also stained positively for active p65. (G) Pericellular liver fibrosis is evident in the livers of alcohol-fed mice with iron dextran treatment as demonstrated by reticulin staining. (H) Numerous activated hepatic stellate cells are present in a focus of liver necrosis and inflammation as shown by α-smooth muscle actin staining. (From Xiong S, She H, Zhang AS, et al. Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis. Am J Physiol Gastrointest Liver Physiol 2008;295(3):G512–G521. Reprinted with permission from The American Physiological Society.)

Figure 4

(A) Kupffer cells (KC) were isolated from mice at 2 weeks after a single subcutaneous injection of iron dextran (25 mg/kg) to assess lipopolysaccharide (LPS) or peroxynitrite stimulated tumor necrosis factor-α (TNFα) expression. Note KC from iron dextran-injected mice release 4 to 5 times more TNFα in response to LPS or peroxynitrite. p < 0.05 compared with the cells from mice without iron dextran injection. (B) Plasma alanine aminotransferase (ALT) levels of mice fed intragastrically with alcohol and high fat diet or pair-fed with isocaloric diet for 4 weeks without or with prior iron dextran injection. Note a single subcutaneous injection of iron dextran (75 mg/kg) prior to alcohol feeding significantly aggravates plasma ALT elevation as compared with alcohol-fed mice injected with vehicle (dextran). (C) Liver histological score of mice fed alcohol for 4 weeks without or with iron dextran injection. Note iron dextran injection at 75 mg/kg significantly worsens the liver histologic score compared with alcohol-fed mice with vehicle treatment. (D) Morphometric analysis of inflammatory cells in the liver of the different experimental groups. Note iron dextran injection significantly increases inflammatory cells in the liver as compared with alcohol-fed mice with vehicle treatment. (E) Representative microphotographs of aggravated alcoholic liver injury in iron dextran-injected mice. In the liver of alcohol-fed mice given 75 mg/kg iron dextran, hepatic macrophages stained for iron (Prussian blue reaction) are noted. (F) These iron staining-positive macrophages are also stained positively for active p65. (G) Pericellular liver fibrosis is evident in the livers of alcohol-fed mice with iron dextran treatment as demonstrated by reticulin staining. (H) Numerous activated hepatic stellate cells are present in a focus of liver necrosis and inflammation as shown by α-smooth muscle actin staining. (From Xiong S, She H, Zhang AS, et al. Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis. Am J Physiol Gastrointest Liver Physiol 2008;295(3):G512–G521. Reprinted with permission from The American Physiological Society.)