The ability of acute and chronic ethanol treatment to increase production of reactive oxygen species and enhance peroxidation of lipids, proteins, and DNA has been demonstrated in a variety of systems, cells, and species, including humans. The mechanism(s) by which alcohol causes cell injury is (are) still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces ‘oxidative stress’ [1
]. Our laboratory has focused on ethanol induction of CYP2E1 [3
]. It is likely that several, indeed many, systems contribute to the ability of ethanol to induce a state of oxidative stress.
CYP2E1 is of interest because of its ability to metabolize and activate many toxicologically important substrates including ethanol, carbon tetrachloride, acetaminophen, and N-nitrosodimethylamine to more toxic products [8
]. Major interest in CYP2E1 reflects the ability of this enzyme to oxidize ethanol, to generate reactive products from ethanol oxidation, e.g. acetaldehyde and the 1-hydroxyethyl radical, to activate various agents (CCl4
, acetaminophen, benzene, halothane, halogenated alkanes, and alcohols) to reactive products, to generate reactive oxygen radical species and to be induced by ethanol. In the intragastric infusion model of ethanol administration, the ethanol-induced liver pathology has been shown to correlate with CYP2E1 levels and lipid peroxidation [14
]. Inhibitors of CYP2E1 prevented the elevation of lipid peroxidation and the ethanol-induced liver pathology [19
]. How CYP2E1 may be mediating liver injury beyond oxidative stress is still not clear from these studies.
Understanding the biochemical and toxicological properties of CYP2E1 is important for many reasons, even besides its role in contributing to alcohol-induced liver injury since CYP2E1 is induced under a variety of pathophysiological conditions such as fasting, diabetes, obesity and high-fat diet [21
] and by drugs [8
]. Non-alcohol-induced steatohepatitis (NASH) causes steatosis, liver cell injury, inflammation and variable necrosis. NASH is associated with obesity, type 2 diabetes and hyperlipidemia, conditions in which CYP2E1 is induced [25
Thurman and colleagues [27
] have suggested that CYP2E1 may not play a role in alcohol liver injury based on studies with gadolinium chloride or CYP2E1 knockout (KO) mice or a broad-spectrum P450 inhibitor aminobenzotriazole. Induction by ethanol of other CYPs or of the reductase in the CYP2E1 KO mice might have served as alternative sources of oxidative stress, especially in the absence of CYP2E1 [30
]. Clearly, further studies are necessary to resolve the above discrepancies. As mentioned earlier, it is likely that several mechanisms contribute to alcohol-induced liver injury, and that ethanol-induced oxidant stress is likely to arise from several sources, including CYP2E1, mitochondria and activated Kupffer cells. Bradford et al. [31
] reported that CYP2E1, but not NADPH oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver, and concluded that CYP2E1 is required for the induction of oxidative stress to DNA and may play a key role in ethanol-associated hepatocarcinogenesis.
Almost all the above studies assessing the role of CYP2E1 in alcoholic liver disease involve the intragastric infusion model where significant liver injury occurs [14
]. Although much valuable information has been gained from this model, it is complex, requires special expertise, is stressful, and is not accessible to most researchers. There is a need to develop oral models of ethanol treatment which result in the production of significant liver injury. Rodents administered Lieber-DeCarli alcohol-containing liquid diets [32
] developed steatosis and oxidative stress in the liver, but significant liver injury beyond steatosis generally does not occur. A modified low-carbohydrate ethanol liquid diet caused mild liver injury in rats [33
]. Nanji's group [37
] recently reported the development of alcoholic liver disease in a voluntary feeding regimen consisting of fish oil and ethanol in female rats. In all these oral models, ALT levels were elevated about 2-fold by the chronic ethanol feeding. Although CYP2E1 was elevated by ethanol in all these models, whether CYP2E1 played a role in the elevated ALT and oxidative stress or the steatosis was not directly evaluated.
Fatty liver is a uniform and early response of the liver to ethanol consumption. Previously, fatty liver was considered benign, however, it is now known that fatty liver can increase sensitivity to hepatotoxins such as lipopolysaccharide (LPS) and high levels of fatty acids promote lipotoxicity [38
]. Hence, there is a need to understand the mechanisms responsible for fatty liver production by ethanol. Early hypotheses for mechanisms responsible for fatty liver production by ethanol included redox state changes (elevated NADH) when ethanol was metabolized by alcohol dehydrogenase, elevated formation of acetyl-CoA from ethanol/acetaldehyde oxidation and impairment of β-oxidation of fatty acids [41
]. Recent studies by Crabb, You and colleagues [42
] have identified new mechanisms which regulate the synthesis and the oxidation of fatty acids as being central to how ethanol produces fatty liver. Ethanol has been shown to elevate levels of SREBP-1c, a master transcriptional regulator of lipogenic enzymes, but to lower levels of PPAR-α, a master transcriptional regulator of lipolytic enzymes. Decreased activation of AMPK by ethanol plays a role in these effects. Is there a role for CYP2E1 in ethanol-induced fatty liver? In the IG infusion model, Kono et al. [28
] reported that after feeding ethanol for 4 weeks, there was no difference in ethanol-induced steatosis between CYP2E1 KO mice and wild-type (WT) mice. Similarly, in this model, Wan et al. [46
] reported that ethanol infusion for 21 days promoted fat accumulation in CYP2E1 KO mice but not in the WT mice. No explanation or mechanism was provided for these unusual observations. In contrast, the CYP2E1 inhibitor chlormethiazole (CMZ) was reported to blunt ethanol-induced fatty liver in the IG model [20
]. We are not aware of any studies providing direct evidence for or against a role for CYP2E1 in fatty liver produced in oral models of ethanol consumption. Studies described below were designed to further evaluate a possible role of CYP2E1 in ethanol-induced fatty liver and to assess downstream factors which may play a role in the molecular mechanisms by which CYP2E1 promotes development of fatty liver by ethanol.