HCC is one of the most common causes of mortality from solid organ malignancy. Developing an animal model of alcohol-induced liver neoplasia would be an invaluable tool for studying the role of alcohol consumption in HCC and possibly other alcohol-related cancers. Since most rodents do not voluntarily consume alcohol (Lieber et al., 1989
; Ponnappa and Rubin, 2000
; Siegmund et al., 2003
), the effects of alcohol have been assessed in rodents receiving forced, chronic alcohol exposure via
alcohol-containing liquid diets as the sole source of fluid, or continuous housing in alcohol vapor inhalation chambers (Becker and Veatch, 2002
; Siegmund et al., 2003
). Force feeding alcohol in a liquid diet, such as the Lieber-DeCarli diet, has become standard protocol for assuring high alcohol intake in rodents. With this diet, the only source of both nutrients and water is a liquid diet that can be supplemented with alcohol (Lieber et al., 1989
). Although the animals have an aversion to alcohol, they must consume this diet or starve (Lieber et al., 1989
; Ponnappa and Rubin, 2000
). The Lieber and DeCarli diet also provides isocaloric maltodextrin in control-fed animals, minimizing nutrient deficiencies that are characteristic of human alcoholism. When given over a period of 4 to 6 weeks, this diet does not cause liver necrosis, inflammation, or fibrosis, but can result in fatty liver (DeCarli and Lieber, 1967
; Ponnappa and Rubin, 2000
). The important limitations of forced alcohol exposure models are that they do not mimic the human condition of alcoholism, characterized by a preference for alcohol and voluntary consumption of alcohol which is accompanied by nutrient deficiencies. These significant differences make translation of the findings from forced alcohol exposure in rodents to human alcoholics difficult.
Another variable that should be considered when relating animal models of alcohol consumption to human drinkers is that the effect of alcohol on hepatic tumorigenesis may be influenced, in part, by pattern of alcohol intake. Blood alcohol concentration (BAC) resulting from voluntary alcohol drinking is characterized by moderate/high BAC associated with each alcohol drinking bout (“intermittent”). This pattern of drinking may allow periods of liver recovery and/or regeneration after alcohol-induced hepatocyte necrosis. This differs from the BAC produced by forced alcohol vapor inhalation (continuous elevation of BAC). In addition to route of administration, whether alcohol is given simultaneously or separately from food may also affect hepatic tumorigenesis. Nonetheless, the results of prior studies with forced alcohol exposure are important and serve as the basis for the present study.
The P rat model employed in our study meets all five criteria of an animal model of alcoholism. One of the strengths of the model is that it mimics human behavior because the rat voluntarily drinks large amounts of alcohol, comparable to amounts consumed by alcoholic patients. We showed that gross neoplastic lesions developed almost exclusively in the livers of P rats that had been drinking alcohol for 18 months. Tumor incidence, multiplicity, and size (determined either grossly or by ultrasound) were increased in P rats drinking alcohol for 18 months compared to the water drinkers. To our knowledge, this is the first time an animal model of chronic alcohol intake/alcoholism has demonstrated formation of liver neoplasia. Histopathologically, we have classified the neoplastic lesions as well-differentiated HCC, but they also bear similarity to adenoma or dysplastic foci.
While these data demonstrate hepatic carcinogenesis in a rat model of voluntary alcohol consumption, no evidence of alcohol-induced hepatic cirrhosis occurred in P rats drinking alcohol. In the clinical setting, HCC associated with alcohol abuse most commonly arises in the setting of cirrhosis, and the risk for HCC in decompensated alcohol-induced cirrhosis approaches 1% per year (Morgan et al., 2004
). Importantly, HCC may also occur in persons with alcohol-induced liver disease who do not have cirrhosis (Donato et al., 2002
; Morgan et al., 2004
). Of interest, the iP rats, which consume approximately half as much alcohol as P rats, did not develop gross neoplastic liver lesions. This result supports studies which suggest that chronic alcohol use is a risk factor for HCC in humans, whereas moderate use (1–3 drinks/day) is not (Yu and Yuan, 2004
). As such, it will be of considerable interest to expand these studies to address the incidence and degree of HCC that arises in this model when underlying hepatic cirrhosis is induced.
Because the rats in this study were 6 months of age when they began consuming alcohol, the existence of a low level of background preneoplastic lesions associated with age is expected (Schulte-Hermann et al., 1983
). Livers from water-drinking rats had a background level of GSTp+ lesions that increased in number and size in alcohol-drinking iP and P rats. This suggests that alcohol may promote the growth of preneoplastic focal lesions from pre-existing initiated cells that arise in the liver as the rat ages. This is supported by our results from a related study in which P rats were given free access to water or alcohol beginning earlier at 6 weeks of age (Supplementary Figure 1
). The number of GSTp+ lesions was significantly higher in P rats enrolled in the study at 6 months of age () compared to 6 weeks of age after drinking water for 18 months (corresponding to 24 vs. 19.5 months of age respectively). Similarly, GSTp+ lesion size was significantly greater in P rats enrolled in the study at 6 months () versus
6 weeks of age after drinking water for 12 or 18 months. Thus, in the absence of alcohol, number and size of background preneoplastic hepatic lesions increases with age. In P rats enrolled at 6 weeks of age, tumor incidence was 50% (data not shown) after drinking alcohol for 18 months compared to 83% with enrollment at 6 months of age. Taken together, our results suggest that hepatic tumorigenesis is influenced by age at which alcohol consumption is initiated.
In the liver, alcohol is metabolized to acetaldehyde by ADH, with acetaldehyde subsequently being converted to acetate by ALDH (Crabb and Liangpunsakul, 2007
; McKillop and Schrum, 2009
). Both reactions result in the production of NADH, leading to reactive oxygen species (ROS) production and oxidative stress (Crabb and Liangpunsakul, 2007
). ROS can cause peroxidation damage to lipids, proteins and DNA (Wu and Cederbaum, 2003
). Alcohol metabolism and alcohol-induced oxidative stress also generate reactive aldehydes (malondialdehyde [MDA] and 4-hydroxy-2-nonenal [HNE]) in addition to acetaldehyde, all of which can form potentially harmful adducts (Niemela, 2001
). While ADH/ALDH are involved in the oxidation of most alcohol consumed, cytochrome P450IIE1 (CYP2E1) becomes more important for metabolism during chronic alcohol consumption (Lu and Cederbaum, 2008
). CYP2E1 activity is linked to alcohol-associated toxicity through the production of ROS and further oxidative stress. In H4IIE HCC cells, it has been reported that ethanol, at doses similar to blood ethanol content observed following moderate ethanol consumption (10–25 mmol/L), significantly increases CYP2E1 expression and activity without affecting ADH or ALDH levels in vitro
(Brandon-Warner et al., 2009
In the present study, we evaluated whether alcohol preference was determined by differences in the basal levels of alcohol metabolizing enzymes. The protein levels of CYP2E1, ADH and ALDH were similar in the Wistar background control rats, alcohol-naïve P rats at 6 months of age, and P rats drinking water for 6, 12 and 18 months (corresponding to 12, 18 and 24 months of age respectively). This suggests that differences in basal liver enzyme levels do not contribute to alcohol preference or predispose the rats to alcohol drinking, nor does expression of these enzymes change with age in the P rat model. In contrast, long-term alcohol drinking induced CYP2E1 protein levels, decreased ALDH levels, but did not alter ADH levels in the P rats. The alcohol-induced changes in CYP2E1 protein levels were confirmed by increased CYP2E1 mRNA expression as well as enzymatic activity. The combined effect of higher CYP2E1 and lower ALDH level/activity would be predicted to result in the accumulation of acetaldehyde, ROS and oxidative stress. This was supported by the detection of elevated MDA levels, indicative of ROS formation and subsequent lipid peroxidation, in the livers of alcohol-drinking P rats. The timing and extent of these intracellular changes coincided with the detection of preneoplastic and neoplastic changes in the liver after 12 months and especially after 18 months of alcohol drinking. Although our results provide evidence that these events may be associated, causal association remains to be proven experimentally in this model.
Finally, we report that staining of phosphorylated MAPK/ERK increased in sinusoidal lining cells within the livers of rats consuming alcohol for 18 months. We further determined that P-ERK-positive cells within the sinusoids appear to be predominantly endothelial, not Kupffer, cells based upon staining serial slides with P-ERK and CD68. Studies to explore the significance of this finding are ongoing in our laboratory.
In summary, we report that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. This novel animal model of alcohol-induced liver neoplasia will likely be an invaluable tool for further elucidating the mechanisms which support this specific etiology and may further our understanding of the general pathogenic mechanisms of alcohol-induced cancers.