RT Journal Article
SR Electronic
T1 Peroxynitrite-Induced Mitochondrial and Endonuclease-Mediated Nuclear DNA Damage in Acetaminophen Hepatotoxicity
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 879
OP 887
DO 10.1124/jpet.105.088898
VO 315
IS 2
A1 Cathleen Cover
A1 Abdellah Mansouri
A1 Tamara R. Knight
A1 Mary Lynn Bajt
A1 John J. Lemasters
A1 Dominique Pessayre
A1 Hartmut Jaeschke
YR 2005
UL http://jpet.aspetjournals.org/content/315/2/879.abstract
AB Intracellular sources of peroxynitrite formation and potential targets for this powerful oxidant and nitrating agent have not been identified after acetaminophen (AAP) overdose. Therefore, we tested the hypothesis that peroxynitrite generated in mitochondria may be responsible for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage. C3Heb/FeJ mice were treated with 300 mg/kg AAP and monitored for up to 12 h. Loss of mtDNA (assayed by slot blot hybridization) and substantial nDNA fragmentation (evaluated by anti-histone enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and agarose gel electrophoresis) were observed as early as 3 h after AAP overdose. Analysis of nitrotyrosine protein adducts in subcellular fractions established that peroxynitrite was generated predominantly in mitochondria beginning at 1 h after AAP injection. Delayed treatment with a bolus dose of glutathione (GSH) accelerated the recovery of mitochondrial glutathione, which then effectively scavenged peroxynitrite. However, mtDNA loss was only partially prevented. Despite the absence of nitrotyrosine adducts in the nucleus after AAP overdose, nDNA damage was almost completely eliminated with GSH administration. A direct comparison of nDNA damage after AAP overdose with nDNA fragmentation during tumor necrosis factor receptor-mediated apoptosis showed similar DNA ladders on agarose gels but quantitatively different results in three other assays. We conclude that peroxynitrite may be partially responsible for mtDNA loss but is not directly involved in nDNA damage. In contrast, nDNA fragmentation after AAP overdose is not caused by caspase-activated DNase but most likely by other intracellular DNase(s), whose activation is dependent on the mitochondrial oxidant stress and peroxynitrite formation. The American Society for Pharmacology and Experimental Therapeutics