Western Blot Analysis for Nitrotyrosine Protein Adducts in Livers of Saline-Treated and Acetaminophen-Treated Mice

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyr...

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Bibliographic Details
Published in:	Toxicological sciences Vol. 53; no. 2; pp. 467 - 473
Main Authors:	Hinson, Jack A., Michael, Sherryll L., Ault, Subrena G., Pumford, Neil R.
Format:	Journal Article
Language:	English
Published:	Cary, NC Oxford University Press 01-02-2000
Subjects:	acetaminophen Acetaminophen - metabolism Acetaminophen - toxicity Adjuvants, Immunologic Analgesics, Non-Narcotic - metabolism Analgesics, Non-Narcotic - toxicity Animals Biological and medical sciences Blotting, Western Cattle Chemical and Drug Induced Liver Injury - metabolism Chemical and Drug Induced Liver Injury - pathology Drug toxicity and drugs side effects treatment Enzyme-Linked Immunosorbent Assay Hemocyanins - immunology hepatotoxicity Immunoenzyme Techniques Liver - drug effects Liver - metabolism Liver - pathology Male Medical sciences Mice Mice, Inbred C57BL Necrosis Nitrates - immunology Nitrates - metabolism nitrotyrosine peroxynitrite Pharmacology. Drug treatments protein adducts Protein Binding Proteins - immunology Proteins - metabolism Rabbits Serum Albumin, Bovine - immunology Sodium Chloride - pharmacology Toxicity: digestive system Tyrosine - analogs & derivatives Tyrosine - immunology Tyrosine - metabolism Western blot Immunohistochemistry Nitration Peroxonitrites Toxicity Antimigrainous agent Rodentia Hepatic disease Protein Vertebrata Paracetamol Mammalia Analgesic Mouse Animal Antipyretic Digestive diseases Mechanism of action
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Summary:	The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.
Bibliography:	local:0530467 istex:FE0ECFA79782A8E3210C23E81BE38CDBB5EA5FF5 ark:/67375/HXZ-K91KNHJS-Q PII:1096-0929 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	1096-6080 1096-0929 1096-0929
DOI:	10.1093/toxsci/53.2.467