Abstract
Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.
Footnotes
This work was supported by the Research Participation Program at the National Center for Toxicological Research 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 (to X.Y.); in part by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK081406] (to D.W.R.); by the Arkansas Children’s Hospital Research Institute and the Arkansas Biosciences Institute, the major research component of the Tobacco Settlement Proceeds Act of 2000; and by a grant from the National Institutes of Health [Grant R01-DK079008] (to J.A.H.).
↵1 Part owners of Acetaminophen Toxicity Diagnostics, LLC, a company working to develop a medical device for diagnosis of acetaminophen-induced liver injury.
↵2 Completed work while at the Division of Systems Biology, National Center for Toxicological Research.
The opinions expressed in this manuscript do not reflect the official positions or policies of the U.S. Food and Drug Administration.
- Received August 23, 2012.
- Accepted October 1, 2012.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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