Abstract
In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by initial hepatic metabolism to N-acetyl-p-benzoquinone imine (NAPQI). After low doses NAPQI is efficiently detoxified by GSH. However, in overdose GSH is depleted, NAPQI covalently binds to proteins as APAP adducts, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. Manganese superoxide dismutase (MnSOD) inactivation by protein nitration has been reported to occur during other oxidant stress-mediated diseases. MnSOD is a critical mitochondrial antioxidant enzyme that prevents peroxynitrite formation within the mitochondria. To examine the role of MnSOD in APAP toxicity, mice were treated with 300 mg/kg APAP. GSH was significantly reduced by 65% at 0.5 h and remained reduced from 1 to 4 h. Serum alanine aminotransferase did not significantly increase until 4 h and was 2290 IU/liter at 6 h. MnSOD activity was significantly reduced by 50% at 1 and 2 h. At 1 h, GSH was significantly depleted by 62 and 80% at nontoxic doses of 50 and 100 mg/kg, respectively. No further GSH depletion occurred with hepatotoxic doses of 200 and 300 mg/kg APAP. A dose response decrease in MnSOD activity was observed for APAP at 100, 200, and 300 mg/kg. Immunoprecipitation of MnSOD from livers of APAP-treated mice followed by Western blot analysis revealed nitrated MnSOD. APAP-MnSOD adducts were not detected. Treatment of recombinant MnSOD with NAPQI did not produce APAP protein adducts. The data indicate that MnSOD inactivation by nitration is an early event in APAP-induced hepatic toxicity.
Footnotes
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grants R01-DK079008, R01-DK059872, R01-DK75936]. D.W.R. and L.P.J. received partial support from an Arkansas Biosciences Institute grant.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.176321.
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ABBREVIATIONS:
- APAP
- acetaminophen
- APAP-Cys
- 3-(cystein-S-yl)-APAP
- ALT
- alanine aminotransferase
- BSA
- bovine serum albumin
- HPLC-EC
- high-pressure liquid chromatography with electrochemical detection
- SOD
- superoxide dismutase
- MnSOD
- manganese SOD
- MPT
- mitochondrial permeability transition
- NAPQI
- N-acetyl-p-benzoquinone imine
- OA
- ovalbumin
- PAGE
- polyacrylamide gel electrophoresis
- PBS
- phosphate-buffered saline
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase.
- Received October 20, 2010.
- Accepted December 29, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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