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Page 8 of 16 Liu et al. Hepatoma Res 2020;6:7 I http://dx.doi.org/10.20517/2394-5079.2019.39
[45]
Aside from their natural aversion to alcohol, mice metabolise alcohol five times faster than humans .
As a result, the aforementioned ALD mouse models tend to exhibit less liver injury than seen in human
[21]
disease . The Lieber-DeCarli model induces mild steatosis with little to no inflammation or fibrosis. The
technically demanding Tsukamoto-French model produces severe steatosis but only mild inflammation and
mild fibrosis. Although chronic or binge ethanol feeding regimens cause minor liver changes by themselves,
their combination in the NIAAA model synergistically induces more severe steatosis and inflammation,
[46]
with only mild chicken-wire fibrosis .
Many have studied liver injury patterns of ALD mouse models; however, few have examined
hepatocarcinogenesis specifically. As described above, the mild severity of liver inflammation and fibrosis
induced by standalone mouse models of ALD means HCCs do not develop spontaneously. Therefore, a
“second-hit” usually consisting of a chemical hepatotoxin is required for progression of ALD to cirrhosis
and/or HCC. Indeed, of the hepatotoxins, DEN-induced C57BL/6 tumours have recently been matched
to most resemble alcohol-induced HCC both morphologically and by comparative genomic hybridisation
[47]
[48]
in a study comparing five different HCC models with human data . Ambade et al. established a
model of alcohol-driven HCC in adult C57BL/6 male mice. The four-week-old mice were administered
six doses of DEN (or saline) intraperitoneally (75 mg/kg weekly for three weeks and then 100 mg/kg
weekly for three weeks) followed by the Lieber-DeCarli diet (or calorie-matched control diet) for seven
weeks before sacrifice at 15 weeks. Compared to mice fed with a control diet, alcohol-fed mice had greater
liver inflammation (raised alanine aminotransferase) and fibrosis. The alcohol-fed group also exhibited
numerous liver nodules of hepatic hyperplasia associated with increased AFP expression and cellular
proliferation, which the authors thought represented signs of early hepatocarcinogenesis. There were no
hyperplastic nodules seen in the alcohol-fed saline-injected group or the control-fed DEN-injected group,
thus confirming the need for a second stressor to initiate hepatocarcinogenesis. Early precancerous lesions
[49]
were also described in another model using the combination of DEN and alcohol diet . In this study,
male C57BL/6 mice were injected intraperitoneally with DEN (25 mg/kg) at two weeks of age and then
fed with the Lieber-DeCarli diet at eight weeks of age for 21 days. Over half of DEN-injected alcohol-fed
mice developed precancerous basophilic foci compared to none in the DEN-injected control diet group.
Interestingly, dietary luteolin (a flavonoid with anti-cancer properties) co-administration completely
abrogated the development of precancerous lesions potentially by restoring sirtuin 1 activity and increasing
downstream proliferator-activated receptor gamma coactivator 1 alpha protein expression. In a longer
[50]
model, Brandon-Warner et al. studied DEN-injected alcohol-fed B6C3 mice for 48 weeks and observed
tumours in 94% and 36% of males and females, respectively. While chronic ethanol feeding exacerbated
tumour formation in DEN-injected males, fewer and smaller tumours were observed in females exposed
to ethanol compared to DEN-injected control-fed mice of respective sexes. Further analysis of liver mRNA
revealed elevated SMAD3 in male compared to female mice in response to liver injury from DEN and
alcohol, suggesting that increased TGFβ-SMAD3 signalling may enhance HCC promotion. Indeed, gender
disparity (males > females) in liver cancer both in humans and in DEN-injected mice is well-recognised
and may be related to sex differences in MyD88-dependent IL-6 production mediated by the protective
effect of oestrogen .
[51]

The combination of alcohol and CCl has also been experimented, although predominantly in rats. Weekly
4
[52]
injections of CCl and alcohol administration through drinking water led to HCC after 104 weeks in mice .
4
[53]
The impact of chemical carcinogens on HCC formation appears to be additive. Recently, Xin et al.
combined DEN (100 mg/kg intraperitoneal and 50 mg/kg gavage once each), CCl twice weekly and 9%
4
alcohol as drinking water together in adult (seven-week-old) BALB/c mice. Multifocal HCC was noted only
five months (150 days) after DEN injection. Tumours were moderate to highly differentiated and secreted
AFP, resembling human HCC. Furthermore, there was no evidence of toxicity in this model as these mice
survived until sacrifice.