Diabetes, Vol 46, Issue 8 1291-1295, Copyright © 1997 by American Diabetes Association

ARTICLES

Mitochondrial DNA in beta-cells is a sensitive target for damage by nitric oxide

GL Wilson, NJ Patton and SP LeDoux
Department of Structural and Cellular Biology, The University of South Alabama, Mobile 36688, USA.

Increasing evidence indicates that nitric oxide (NO) may play a role in immune-mediated injury to beta-cells. One site for the action of this agent is the mitochondrion. Although the exact targets for damage within this organelle have yet to be fully elucidated, a potential location for injury is mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by NO. Both exogenous NO generation (spermine/NO adduct [sper/NO]) and endogenous production of NO (IL-1beta) were studied. To study the effects of exogenously produced NO, neonatal rat islet cells in monolayers were exposed to varying doses of sper/NO for 30 min. Total cellular DNA was isolated and treated with alkali to produce strand breaks at abasic sites resulting from exposure to NO. Damage to mtDNA was evaluated using a quantitative Southern blot technique. The results showed that sper/NO caused dose-dependent damage to mtDNA. Additionally, mtDNA was found to be more sensitive to injury generated by either source than a similarly sized fragment of nuclear DNA. To evaluate the effects of endogenously produced NO, beta-cell cultures were treated with IL-1beta for 18 h. Other cultures were treated with IL-1beta and an inhibitor of the inducible form of nitric oxide synthase, aminoguanidine. DNA was evaluated as described for the sper/NO studies. IL-1beta caused appreciable damage to mtDNA, and this damage was reduced in mtDNA from cultures treated with IL-1beta and aminoguanidine. These studies show that mtDNA is a sensitive target for NO generated both endogenously and exogenously and that this DNA is more vulnerable to NO-induced damage than nuclear DNA.
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