PEROXYNITRITE (ONOO- ) MEDIATES DNA DAMAGE IN CYTOKINE

• Published in: Japanese Journal of Pharmacology 1997, Vol.75 (suppl), p.104-104
• Main Authors: Ruth E. Billings, Susan M. LaRue, David J. Sieg
• Format: Article
• Language: Japanese
• ISSN: 0021-5198

The cytokines TNFα and IFN-γ, in combination with a nontoxic dose of lead (Pb) cause significantly greater cytotoxicity in mouse hepatocytes compared to cytokines alone. This cytotoxic interaction may be mediated by oxidative DNA damage resulting from cytokine-induced oxidative stress and Pb stimulation of mitogenic signals (Sieg and Billings, Toxicol. Appl. Pharmacol. 142, 106-115,1997). In the present study, the role of NO-derived oxidants in these cells was investigated. Primary cultures of mouse hepatocytes were treated with TNFα (0.1 μg/ml) and IFN-γ (100U/ml), DETA-NONOate (25 μM), or SIN-1 (45 μM) in the presence or absence of Pb (50 μM). Cytotoxicity (release of lactate dehydrogenase) and single-stranded DNA damage using the a single cell gel electrophoresis (COMET) assay were measured. All COMET assays were conducted prior to the onset of cytotoxicity, at 12 hr after treatment. In the absence of Pb, none of the treatments were cytotoxic and minimal DNA damage was observed. In the presence of Pb, the cytokines and SIN-1 (which generates ONOO^- ) caused single-stranded DNA fragmentation in 74-82% of the cells. DETA-NONOate, a generator of NO but not ONOO^- , caused DNA fragmentation in only 20% of the cells simultaneously treated with Pb. Inhibition of NOS activity with the arginine analog, L-NAME (500 μM), partially inhibited (48% vs 74%) the number of cytokine + Pb-treated cells displaying DNA fragmentation; PTIO(300 μM), an NO scavenger, had effects similar to L-NAME. The intracellular superoxide dismutase mimetic, MnTMPyP, completely blocked DNA fragmentation caused by cytokine+Pb treatment. These results show that cytokine+Pb combinations cause oxidative DNA damage by a mechanism which primarily involves peroxynitrite generation through the simultaneous induction of NOS and generation of superoxide. Pb sensitizes the cells to the actions of ONOO^- , probably because it stimulates DNA synthesis in hepatocytes.