Biochimica et Biophysica Acta (BBA) - General Subjects
Sulforaphane induces Nrf2 and protects against CYP2E1-dependent binge alcohol-induced liver steatosis
Introduction
The pathways by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity [1], [2], [3], [4]. 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 [5] and high levels of fatty acids promote lipotoxicity [6], [7]. 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 [8]. Recent studies [9], [10], [11], [12] have identified mechanisms which regulate the synthesis and the oxidation of fatty acids as being central to how ethanol produces fatty liver. It appears that ethanol-induced steatosis may be related to oxidative stress as initially shown by DiLuzio [13] who reported that antioxidants can prevent the ethanol-induced fatty liver, and studies showing that overexpression of the manganese superoxide dismutase can partially reduce the ethanol-induced steatosis [14]. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress [15], [16], [17] and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver [18], [19], [20]. Both chronic and acute (binge) ethanol-induced fatty liver was blunted in CYP2E1 knockout mice but restored in humanized CYP2E1 knockin mice [20], [21]. Inhibition of CYP2E1 or CYP2E1-generated oxidative stress, by blunting ethanol-induced fatty liver, would likely be effective in preventing ethanol toxicity.
The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of insults produced by electrophilic and oxidative chemicals [22], [23], [24]. Nrf2 has been shown to be protective against a variety of drugs which can cause hepatotoxicity, lung injury, neurotoxicity, carcinogenesis and inflammation [25]. Exposure to many chemicals, oxidants and cellular stresses leads to increased production of Nrf2, disassociation of Nrf2 from the Kelch-like ECH associated protein Keap1 and subsequent entry of Nrf2 into the nucleus. In view of the importance of Nrf2 in upregulating many critical protective enzymes, there has been considerable interest in efforts to activate Nrf2 signaling by administration of low molecular weight molecules [26], [27], [28]. Sulforaphane is an isothiocyanate generated from the enzymatic cleavage of glucoraphanin, which is present in considerable amounts in brassica vegetables such as cabbage, kale, and broccoli [29]. It is thought to induce a phase II detoxification response by promoting the release of Nrf2 from the Keap 1-Nrf2 cytoplasmic complex. Sulforaphane has been shown to be protective against oxidative stress [30] via activation of Nrf2 [31], [32], [33], [34]. The current study was designed to evaluate the ability of sulforaphane to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro.
Section snippets
In vitro model
HepG2 E47 cells are HepG2 cells which were transfected with human CYP2E1 cDNA and constitutively express human CYP2E1 [35]. The cells were treated with or without 100 mM ethanol in the absence or presence of 6 μM sulforaphane. Cells treated with ethanol were cultured in a CO2 incubator which was saturated with 100 mM ethanol to minimize ethanol evaporation from the medium. Fresh medium with or without ethanol or sulforaphane was added daily. Lipid droplets were assayed by Oil Red O staining. Cells
Sulforaphane treatment induces Nrf2 in liver of CYP2E1 knockin mice
SV129 CYP2E1 KI mice were gavaged with 30% ethanol, 3 g/kg body weight, or with saline twice a day in the absence or presence of 0.05 g/kg sulforaphane once a day for a total of five days. Compared with 6 g/kg body weight once a day, we separate the dose to 3 g/kg body weight twice a day, which may prevent mice to develop hypothermia. There was no significant weight loss or changes in food intake between groups (data not shown). ALT and AST levels were not significantly elevated by the binge
Discussion
One major factor in ethanol-induced liver injury is the generation of reactive oxygen species and production of an imbalance between this generation and the cellular levels of anti-oxidant defense enzymes [1], [2], [3], [4]. Since Nrf2 is a master transcription factor for regulation of levels of anti-oxidants, there has been increasing interest as to how ethanol alters Nrf2 activation and whether Nrf2 can modulate the effects of ethanol on the liver. Lamle et al. [50] established a central role
Acknowledgements
These studies were supported by USPHS grants RO1 AA018790 and R21 AA021362 from the National Institute on Alcohol Abuse and Alcoholism, NIH.
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