Abstract
The role of protein glutathionylation in acetaminophen (APAP)-induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dose-dependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepatocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.
Footnotes
X.Y. was supported by the Research Participation Program at the National Center for Toxicological Research, which is administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. D.W.R. was supported, in part, by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK081406], the Arkansas Children's Hospital Research Institute, and the Arkansas Biosciences Institute, the major research component of the Tobacco Settlement Proceeds Act of 2000. J.A.H. was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK079008].
D.W.R. and J.A.H. are part owners of Acetaminophen Toxicity Diagnostics, LLC, a company working to develop a medical device for diagnosis of acetaminophen-induced liver injury.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- APAP
- acetaminophen
- NAPQI
- N-acetyl-p-benzoquinoneimine
- ALT
- alanine aminotransferase
- AST
- aspartate aminotransferase
- MPT
- mitochondrial permeability transition
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- PAGE
- polyacrylamide gel electrophoresis
- RT
- room temperature
- TTBS
- Tris-buffered saline containing 1% Tween 20.
- Received September 14, 2011.
- Accepted October 31, 2011.
- U.S. Government work not protected by U.S. copyright
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