Short-term Treatment with Alcohols Causes Hepatic Steatosis and Enhances Acetaminophen Hepatotoxicity in Cyp2e1(-/-) Mice

doi:10.1006/taap.2000.9023

Toxicology and Applied Pharmacology

Volume 168, Issue 2, 15 October 2000, Pages 114-122

https://doi.org/10.1006/taap.2000.9023 Get rights and content

Abstract

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.

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The zebrafish Danio rerio embryo test with metabolic activation (mDarT) was developed to assess the teratogenic effects of proteratogens. In this study induced rat liver microsomes (RLM) were used as a mammalian metabolic activation system (MAS), since they contain various cytochrome P450 (CYP) isoforms at high concentrations. Acetaminophen (APAP) is considered not to be teratogenic in vivo, however, in vitro teratogenic effects were observed, e.g. in rat whole embryo culture. The CYP2E1 activation of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) mainly occurs, when the glucuronidation and sulfatation pathways are saturated. In vivo the soft electrophile NAPQI is usually inactivated by hepatic reduced glutathione (GSH), a soft nucleophile. In this study, we investigated the teratogenic and lethal effects of APAP after CYP activation in zebrafish embryos. In the test groups with APAP and metabolic activation 11.7 ± 7.6% (2 mM), 25.0 ± 8.7% (4 mM) and 50.0 ± 21.8% (6 mM) affected embryos were seen, reaching statistical significance at 4 mM APAP. When embryos were exposed to 6 mM APAP, MAS and 3 mM GSH the percentage of affected embryos decreased to 6.7 ± 5.8%. In contrast teratogenic and lethal effects of metabolically activated cyclophosphamide (CPA) could not be prevented by GSH addition, because the CPA metabolites are strong electrophiles, which preferentially bind to hard nucleophiles like DNA and RNA.
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The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long–Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5–25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations ≥ 75 μg/ml and ≥ 750 μg/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 μg/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [¹⁴C]-leucine incorporation. At the level present in a modulating concentration (50 μg/ml) of the extract, ginkgolide A (0.55 μg/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.
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The pathophysiology of binge drinking of ethanol and its potentiation of acetaminophen (APAP) toxicity has received very little attention. To evaluate if ethanol binging sensitizes hepatic sinusoidal endothelial cells (SEC) and liver to APAP toxicity.
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¹: To whom correspondence should be addressed at Research 151, Veterans Administration Medical Center, White River Junction, VT 05009. Fax: (802) 296-6308; E-mail: [email protected].

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Regular ArticleShort-term Treatment with Alcohols Causes Hepatic Steatosis and Enhances Acetaminophen Hepatotoxicity in Cyp2e1(-/-) Mice

Abstract

Hepatology

Arch. Biochem. Biophys.

J. Hepatol.

J. Hepatol.

Alcohol

Biochim. Biophys. Acta

Int. J. Biochem.

Toxicol. Appl. Pharmacol.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Hepatology

Exp. Mol. Pathol.

Alcohol

Biochem. Pharmacol.

Arch. Biochem. Biophys.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Hepatology

Hydroxyethyl radicals in ethanol hepatotoxicity

Front. Biosci.

Alcohol and lipids

Recent Dev. Alcohol

Effects of ethanol, dexamethasone and RU 486 on expression of cytochromes P450 2B, 2E, 3A and glutathione transferase pi in a rat hepatoma cell line (Fao)

Pharmacogenetics

Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efflux, and response to incubation with ethanol

J. Clin. Invest.

Hydroxyethyl radicals in ethanol hepatotoxicity

Front. Biosci.

Regular Article
Short-term Treatment with Alcohols Causes Hepatic Steatosis and Enhances Acetaminophen Hepatotoxicity in Cyp2e1(-/-) Mice