USE OF THE COMET ASSAY TO INVESTIGATE POSSIBLE INTERACTIONS OF NITRIC-OXIDE AND REACTIVE OXYGEN SPECIES IN THE INDUCTION OF DNA-DAMAGE AND INHIBITION OF FUNCTION IN AN INSULIN-SECRETING CELL-LINE

We have previously used the comet assay to demonstrate that the nitric oxide donor 3-morpholinosydnonimine (SIN-I) produces DNA damage in ra t islets of Langerhans and in the SV40-transformed insulin-secreting h amster cell line, HIT-T15. Damage is not prevented by the addition of superoxide dismutase (SOD). In the present study, we have compared SIN -1, which generates nitric oxide, superoxide anion and hydrogen peroxi de, with two other nitric oxide donors, S-nitrosoglutathione (GSNO) an d the tetra-iron-sulphur cluster nitrosyl, Roussin's black salt (RES). We have used the comet assay as a highly sensitive method to measure DNA-damaging ability, and also measured inhibition of DNA synthesis an d inhibition of insulin secretion. We have examined the effect of SOD and catalase on each of these endpoints in HIT-T15 cells following a 3 0-min exposure to the compounds (24 h for DNA synthesis), All compound s produced a significant dose-dependent increase in strand-breakage fo rmation and all inhibited DNA synthesis and glucose-stimulated insulin secretion. RES was the most potent. SOD did not reduce the responses observed with any of the compounds. Catalase largely prevented DNA str and breakage, inhibition of DNA synthesis and inhibition of insulin se cretion by SIN-1, but had no effect on responses to GSNO or RES. Addit ion of SOD together with catalase gave no greater protection against S IN-1 than catalase alone, The nitric oxide and superoxide anion produc ed by SIN-1 are thought to combine to form highly reactive peroxynitri te. In addition, H2O2 may be formed in the presence of SIN-1 and may f orm hydroxyl radical in the presence of a transition metal, such as Fe 2+. It appears that in insulin-secreting cells, the effects of SIN-1 a re largely mediated by this latter mechanism. In contrast, GSNO and RE S appear to act by a different mechanism, not overtly involving reacti ve oxygen species. GSNO and H2O2 show no significant interaction in th e induction of DNA strand breaks. Both nitric oxide and H2O2 are effec tive, directly or indirectly, as DNA strand-breaking agents, inhibitor s of DNA synthesis and inhibitors of insulin secretion.