Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

doi:10.1016/j.freeradbiomed.2009.11.008

Free Radical Biology and Medicine

Volume 48, Issue 3, 1 February 2010, Pages 391-398

https://doi.org/10.1016/j.freeradbiomed.2009.11.008 Get rights and content

Abstract

The aim of this study was to investigate the mechanism of inhibition of mitochondrial aldehyde dehydrogenase (ALDH2) by carbon tetrachloride (CCl₄). CCl₄ administration caused marked hepatocyte ballooning and necrosis in the pericentral region. CCl₄ also inhibited hepatic ALDH2 activity in a time-dependent manner without altering the protein level, suggesting ALDH2 inhibition through covalent modifications such as phosphorylation by JNK. To demonstrate phosphorylation, the isoelectric point (pI) of ALDH2 in CCl₄-exposed rats was compared to that of untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCl₄-exposed rats were shifted to acidic pI ranges on two-dimensional electrophoresis (2-DE) gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with a concurrent alkaline pI shift of the ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria after CCl₄ exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity, with an acidic pI shift on 2-DE gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser–Pro antibody revealed phosphorylation of a Ser residue(s) of ALDH2. These results collectively indicate a novel underlying mechanism by which CCl₄ exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied by decreased cellular defense capacity and increased lipid peroxidation.

Section snippets

Chemicals and other materials

NAD⁺, propionyl aldehyde, pyrazole, and calf intestine alkaline phosphatase were purchased from Sigma (St. Louis, MO, USA). Specific antibodies to c-Jun, phospho-c-Jun (Ser⁶³), JNK, phospho-JNK, phospho-Ser–Pro, or phospho-Thr–Pro were purchased from Cell Signaling Technology (Beverly, MA, USA). Specific monoclonal antibody to mitochondrial ALDH2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant JNK (Cat. No. 420105) was purchased from Calbiochem (San Diego, CA, USA).

Hepatic damage and oxidative stress after CCl₄ exposure

To assess hepatocellular damage in CCl₄-exposed rat liver, liver histopathology analysis was performed. H&E staining data showed that normal liver histology was observed in untreated controls and rats exposed to CCl₄ for 2 and 4 h. However, marked hepatocyte ballooning and necrosis in the centrilobular region were observed in 16-h CCl₄-exposed rat liver (Fig. 1A). Consistent with the histological data, the level of cytochrome c detected in the cytoplasm [31] was markedly elevated at 16 h after

Discussion

Mitochondrial ALDH2 is involved in the metabolism of carbonyl compounds with a very low K_m for acetaldehyde and other lipid aldehydes, which are highly reactive and toxic. ALDH2 is known to be inhibited by many chemicals such as disulfiram, HNE, MDA, acetaminophen, ecstasy (3,4-methylenedioxymethamphetamine), and CCl₄ [6], [7], [9], [37], [44], [45]. Its activity is also decreased in many pathological conditions such as alcoholic fatty liver, hepatic cancer, aging, and hepatic

Acknowledgments

This research was supported by the Intramural Research Program of National Institute on Alcohol Abuse and Alcoholism. We are grateful to Drs. Klaus Gawrisch and Insong James Lee for support and critical reading of the manuscript, respectively. The authors are also thankful to Dr. Jae-Young Lee, DongKuk University, Seoul, Korea for preparation and analysis of the structural model of ALDH2 protein. Preliminary results of this work were presented at the 13th Carbonyl Meeting in 2006.

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Original ContributionInhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

Abstract

Section snippets

Chemicals and other materials

Hepatic damage and oxidative stress after CCl4 exposure

Discussion

Acknowledgments

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Oral Oncol.

Clin. Chim. Acta

Pharmacol. Ther.

Biochem. Pharmacol.

Gastroenterology

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Hepatol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

Gastroenterology

Arch. Biochem. Biophys.

Chem. Biol. Interact.

J. Hepatol.

J. Biol. Chem.

J. Biol. Chem.

Investigation of the role of polymorphisms at the alcohol and aldehyde dehydrogenase loci in genetic predisposition to alcohol-related end-organ damage

Hepatology

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J. Clin. Invest.

An essential role for mitochondrial aldehyde dehydrogenase in nitroglycerin bioactivation

Proc. Natl. Acad. Sci. USA

Inhibition of rat liver aldehyde dehydrogenase by carbon tetrachloride

J. Pharmacol. Exp. Ther.

Original Contribution
Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

Hepatic damage and oxidative stress after CCl₄ exposure