Pyrazole induced oxidative liver injury independent of CYP2E1/2A5 induction due to Nrf2 deficiency

doi:10.1016/j.tox.2008.07.058

Toxicology

Volume 252, Issues 1–3, 30 October 2008, Pages 9-16

https://doi.org/10.1016/j.tox.2008.07.058 Get rights and content

Abstract

Pyrazole can induce CYP2E1 and 2A5, which produce reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates important antioxidant enzymes to remove ROS. In this study, we applied Nrf2 knockout mice to test the hypothesis that pyrazole will cause hepatotoxicity and elevate oxidative stress to a greater extent in Nrf2 knockout mice compared to wild type mice. Pyrazole induced severe oxidative liver damage in Nrf2 knockout mice but not in wild type mice. Activities and levels of CYP2E1 and 2A5 were elevated by pyrazole in the wild type mice but not in the Nrf2 knockout mice. However, expression or activity of Nrf2-regulated antioxidant enzymes, such as γ-glutamylcysteine synthetase (GCS), heme oxygenase-1 (HO-1) and glutathione-S-transferase (GST), were upregulated in the pyrazole-treated wild type mice, but to a lesser extent or not at all in the pyrazole-treated Nrf2 knockout mice. Treatment with antioxidants such as vitamin C or S-adenosyl-l-methionine (SAM) or an inhibitor of iNOS prevented the pyrazole-induced oxidative liver damage, thus validating the role of oxidative/nitrosative stress in the pyrazole induced liver injury to the Nrf2 knockout mice. In summary, even though ROS-producing CYP2E1/2A5 were not elevated by pyrazole, impaired antioxidant capacity resulting from Nrf2 deficiency appear to be sufficient to promote pyrazole-induced oxidative liver injury.

Section snippets

Reagents

Pyrazole, lipopolyssachride (LPS), N(omega)-nitro-l-arginine methyl ester (l-NAME), S-adenosyl-methionine (SAM), 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrophenol (PNP), H₂O₂, 7-ethoxycoumarin, coumarin, Ac-DEVD-AMC, were purchased from Sigma (St. Louis, MO); thiobarbituric acid (TBA), o-phthalaldehyde, and vitamin C were from Fisher (Pittsburgh, PA).

Antibodies

Anti-3-nitrotyrosine (3-NT) adducts Ig G was from Upstate (Lake Placid, NY); Ig G for Nrf2 was from Santa Cruz Biotechnology (Santa Cruz, CA); Ig

Pyrazole induces necrotic hepatotoxicity in Nrf2 knockout mice but not in wild type mice

Serum ALT and AST, two markers for hepatic necrosis, increased dramatically after treatment with pyrazole in the Nrf2 knockout mice, but no changes in both serum ALT and AST were observed in the wild type mice (Fig. 1). Pathology evaluation showed that many necrotic areas around the central veins were found in the liver sections from the pyrazole-treated Nrf2 knockout mice, but no pathological change was observed in the pyrazole-treated wild type mice (Fig. 2). TUNEL staining, an assay for

Discussion

In this study, we found that pyrazole treatment alone induced hepatotoxicity in Nrf2 knockout mice but not in wild type mice. Similarly, elevated oxidative stress was detected in pyrazole-treated Nrf2 knockout mice but not in wild type mice. Vc, SAM and l-NAME protected against the pyrazole-induced liver injury in Nrf2 knockout mice. These results suggest that pyrazole-induced liver injury in Nrf2 knockout mice is due to oxidative stress.

Oxidative stress reflects a balance between production of

Conflicts of interest statement

The authors claim that the manuscript entitled “Pyrazole induced oxidative liver injury in Nrf2 knockout mice independent of cytochrome P450s” by Yongke Lu, Pengfei Gong, and Arthur Cederbaum has no conflict of interest.

Acknowledgement

We thank Drs. Jerome Lasker (Hackensack Biomedical Research Institute, Hackensack, NJ) and Risto Juvonen (Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland) for generously providing antibodies against CYP2E1 and 2A5, respectively.

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^☆: These studies were supported by United State Public Health Service Grant AA06610 from the National Institute on Alcohol Abuse and Alcoholism.

¹: Equal contribution to this study.

View full text

Pyrazole induced oxidative liver injury independent of CYP2E1/2A5 induction due to Nrf2 deficiency☆

Abstract

Section snippets

Reagents

Antibodies

Pyrazole induces necrotic hepatotoxicity in Nrf2 knockout mice but not in wild type mice

Discussion

Conflicts of interest statement

Acknowledgement

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2

Toxicol. Lett.

Regulation of CYP2A5 gene by the transcription factor nuclear factor (erythroid-derived 2)-like 2

Drug Metab. Dispos.

Nrf2, a Cap‘n’Collar transcription factor, regulates induction of the heme oxygenase-1 gene

J. Biol. Chem.

Ethanol withdrawal induced CYP2E1 degradation in vivo, blocked by proteasomal inhibitor PS-341

Free Radic. Biol. Med.

Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats

Arch. Biochem. Biophys.

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-l-methionine

Biochem. Pharmacol.

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Biochim. Biophys. Acta

Effects of the nitric oxide synthase inhibitors NG-nitro-l-arginine methyl ester and aminoethyl-isothiourea on the liver microcirculation in rat endotoxemia

J. Hepatol.

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

Biochem. Pharmacol.

Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary rat hepatocyte cell cultures

Biochem. Biophys. Res. Commun.

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

Toxicology

Endoplasmic reticulum stress due to altered cellular redox status positively regulates murine hepatic CYP2A5 expression

J. Pharmacol. Exp. Ther.

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity

Free Radic. Biol. Med.

Nrf2 is increased by CYP2E1 in rodent liver and HepG2 cells and protects against oxidative stress caused by CYP2E1

Hepatology

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

J. Biol. Chem.

Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species

Arch. Biochem. Biophys.

Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice

Eur. J. Biochem.

Molecular mechanism of nrf2 activation by oxidative stress

Antioxid. Redox Signal.

Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione

Mol. Med.

Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway

Mol. Cell Biol.