Alkylation by reactive chemicals or metabolites causes toxic cell death or necrosis. Our ability to prevent such cell death is limited by an incomplete understanding of the process. Frequent correlation of necrosis with the loss of Ca2+ regulation has resulted in the Ca2+ Hypothesis of toxic cell death, which defines uncontrolled Ca2+ as lethal. However, this theory remains incomplete. The critical targets of Ca2+ attack are not known. Based on our demonstration that hepatic necrosis correlates with early fragmentation of DNA, we propose DNA as a vital target and state this in our DNA/Ca2+ Hypothesis: Liver Alkylation Produces DNA Damage, in Part by Ca2+-Endonuclease Mediated Fragmentation of DNA. Significant Unrepaired Damage to DNA is a Final Common Pathway for Liver Necrosis In Vivo and Toxic Hepatocyte Death In Vitro. The research plan tests this hypothesis by examining dimethylnitrosamine and acetaminophen toxicity in mouse liver and cultured mouse hepatocytes. 1) We will measure Ca2+-sensitive enzyme activities in nuclei (Ca2+-endonuclease), mitochondria (pyruvate dehydrogenase) and cytosol (phosphorylase a) to correlate toxicity with compartmental Ca2+ changes in vivo. Ca2+ changes and DNA damage measured at 2-4 h will be related to later ALT release for evidence of dose- toxicity relationships. 2) We will test the relationship of Ca2+- endonuclease DNA fragmentation to toxic cell death. Differences in liver protection by Ca2+ channel blockers and calmodulin antagonists will be related to their unique effects on subcellular Ca2+ pools. Apoptosis inhibitors (cyproterone acetate, cyclosporin A, spermine, interleukin- 1Beta, phorbol ester) and activators (cycloheximide, okadaic acid, dibutyryl cAMP will be used to establish whether fragmented DNA arises from cells dying by necrosis vs-apoptosis based on how agents affect DNA fragmentation, events specific to necrosis (ALT release), and events specific to apoptosis (cell shrinkage and nuclear condensation by flow cytometry). 3) DNA fragmentation, 3H alkylation of DNA and nuclear protein, and single -and double-strand breaks (alkaline and neutral sedimentation) will be measured in limited dose-response studies to learn if one form of early nuclear damage accurately predicts toxicity. 4) If unrepaired DNA damage causes toxic cell death, toxicity may relate in part to DNA repair competency. Toxicity in liver and cultured hepatocytes will be correlated with DNA ligase activity for double-strand breaks and polymerase alpha activity for single-strand breaks. We will also determine whether DNA repair inhibitors (doxorubicin, ara-C, aphidicolin, myricetin, ethidium bromide) potentiate necrosis and cytotoxicity. 5) Finally, the breadth of the DNA/Ca2+ Hypothesis will be determined from DNA fragmentation-toxicity correlations for additional alkylating hepatotoxins (N-hydroxyacetylaminofluorene, aflatoxin Beta1, furosemide) as well as non-alkylating hepatotoxins (ethionine, galactosamine). By directly assailing the validity and scope of the DNA/Ca2+ Hypothesis, the proposed studies have the potential to establish DNA as a vital target of deregulated Ca2+ in toxic cell death. Results may further identify DNA repair as a site for novel therapeutic interventions to ameliorate toxic cell death caused by chemicals.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Toxicology Subcommittee 2 (TOX)
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University of New Mexico
Schools of Pharmacy
United States
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