DNA-DAMAGE INDUCED BY TUMOR-NECROSIS-FACTOR-ALPHA IN L929 CELLS IS MEDIATED BY MITOCHONDRIAL OXYGEN RADICAL FORMATION

Treatment of L929 cells with tumour necrosis factor-alpha (TNF-alpha) plus actinomycin D induced DNA damage (indicated by the appearance of a sub-G(1) peak due to extracellular leakage of low molecular weight D NA following DNA fragmentation) before significant cell lysis occurred . The DNA damage occurred in parallel with a decrease of the intracell ular total glutathione content and an increase of intracellular reacti ve oxygen intermediates (ROI), as indicated by increased dihydrorhodam ine 123 oxidation. Because the inhibition of mitochondrial respiration suppressed the increase of dihydrorhodamine 123 oxidation and DNA dam age as well as the decrease in the total glutathione content, it was s uggested that increased mitochondrial formation of ROI was responsible for DNA damage after TNF treatment. Deferoxamine (a ferric iron chela tor) and dithiothreitol (a sulfhydryl reagent) both prevented DNA dama ge and cell killing, indicate that hydroxyl radicals generated from O- 2(-) and H2O2 produced by the mitochondria in a process catalysed by i ron contributed to DNA damage and that this pathway may be involved in TNF-alpha-induced cytotoxicity. An inhibitor of poly(ADP)-ribose poly merase (3-aminobenzamide), worsened DNA damage, but was protective aga inst cell lysis, suggesting that DNA repair subsequent to injury was m ore important than DNA damage per se in development of TNF-alpha cytot oxicity.