Sulforaphane induces Nrf2 and protects against CYP2E1-dependent binge alcohol-induced liver steatosis

https://doi.org/10.1016/j.bbagen.2013.09.018Get rights and content

Highlights

  • Activation of Nrf2 by sulforaphane was studied in CYP2E1 KI mice and HepG2 E47 cells which express CYP2E1.

  • The activation of Nrf2 by sulforaphane prevents binge-ethanol induced oxidative stress and steatosis in CYP2E1 KI mice.

  • Sulforaphane prevents ethanol-induced increases in lipid accumulation in HepG2 E47 cells.

  • Binge-ethanol plus sulforaphane increases hepatic autophagy.

  • Sulforaphane may be effective in lowering ethanol/CYP2E1-induced liver injury.

Abstract

Background

The mechanism(s) 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. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins.

Method

The current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro.

Results

The sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase-1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1.

Conclusions

Sulforaphane proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice.

General significance

The possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study.

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.

References (65)

  • H. Motohashi et al.

    Nrf2-Keap1 defines a physiologically important stress response mechanism

    Trends Mol. Med.

    (2004)
  • W.O. Osburn et al.

    Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults

    Mutat. Res.

    (2008)
  • R. Hu et al.

    Gene expression profiles induced by cancer chemopreventive isothiocyanate sulforaphane in the liver of C57BL/6J mice and C57B6/6J/Nrf2(− 1 −) mice

    Cancer Lett.

    (2006)
  • N. Gan et al.

    Sulforaphane protects microcystin-LR-induced toxicity through activation of the Nrf2-mediated defensive response

    Toxicol. Appl. Pharmacol.

    (2010)
  • J. Chen et al.

    Activation of Nrf2 by cadmium and its role in protection against cadmium-induced apoptosis in rat kidney cells

    Toxicol. Appl. Pharmacol.

    (2009)
  • T. Greco et al.

    Sulforaphane inhibits mitochondrial permeability transition and oxidative stress

    Free Radic. Biol. Med.

    (2011)
  • P. Gong et al.

    Transcription factor Nrf2 protects HepG2 cells against CYP2E1 plus arachidonic acid-dependent toxicity

    J. Biol. Chem.

    (2006)
  • T. Zeng et al.

    The activation of HO-1/Nrf-2 contributes to the protective effects of diallyl disulfide (DADS) against ethanol-induced oxidative stress

    Biochim. Biophys. Acta

    (2013)
  • N.E. Saidu et al.

    Diallyl tetrasulfane activates both the elF2α and Nrf2/HO-1 pathways

    Biochim. Biophys. Acta

    (2013)
  • K.C. Wu et al.

    Nrf2 activation prevents cadmium-induced acute liver injury

    Toxicol. Appl. Pharmacol.

    (2012)
  • H. Dong et al.

    Regulation of lipid droplets by autophagy

    Trends Endocrinol. Metab.

    (2011)
  • W.X. Ding et al.

    Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice

    Gastroenterology

    (2010)
  • D. Wu et al.

    Alcohol steatosis and cytotoxicity: the role of cytochrome P4502E1 and autophagy

    Free Radic. Biol. Med.

    (2012)
  • B. Levine et al.

    Autophagy in the pathogenesis of disease

    Cell

    (2008)
  • P. Yao et al.

    Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways

    J. Hepatol.

    (2007)
  • K.C. Wu et al.

    Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

    Toxicol. Appl. Pharmacol.

    (2012)
  • W.J. Gilmore et al.

    Effects of lipopolysaccharide-stimulated inflammation and pyrazole-mediated hepatocellular injury on mouse hepatic CYP2A5 expression

    Toxicology

    (2003)
  • R. Sharma et al.

    Role of 4-hydroxynonenal in chemopreventive activities of sulforaphane

    Free Radic. Biol. Med.

    (2012)
  • M.A. Abdelmegeed et al.

    Critical role of cytochrome P4502E1 (CYP2E1) in the development of high fat-induced non-alcoholic steatohepatitis

    J. Hepatol.

    (2012)
  • Y.K.J. Zhang et al.

    Nrf2 deficiency improves glucose tolerance in mice fed a high fat diet

    Toxicol. Appl. Pharmacol.

    (2012)
  • S. Barcelo et al.

    Production of DNA strand breaks by N-nitrosodimethylamine and 2-amino-3-methylimidazol [4,5-f] quinoline in THLE cells expressing human CYP isoenzymes and inhibition by sulforaphane

    Mutat. Res.

    (1998)
  • R. Nordman et al.

    Implication of free radical mechanisms in ethanol-induced cellular injury

    Free Radic. Biol. Med.

    (1992)
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