Mitochondrial DNA in beta-cells is a sensitive target for damage by nitric oxide.
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.
Wilson
G L
GL
Department of Structural and Cellular Biology, The University of South Alabama, Mobile 36688, USA.
Patton
N J
NJ
LeDoux
S P
SP
D000818:Animals; D000831:Animals, Newborn; D001345:Autoradiography; D015139:Blotting, Southern; D002478:Cells, Cultured; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000032:analysis / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000633:toxicity; D006146:Guanidines / Q000633:toxicity; D007375:Interleukin-1 / Q000633:toxicity; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009153:Mutagens / Q000633:toxicity*; D009573:Nitrites / Q000378:metabolism; D009589:Nitrogen Oxides; D051381:Rats; D017207:Rats, Sprague-Dawley; D013096:Spermine / Q000031:analogs & derivatives* / Q000633:toxicity
D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D006146:Guanidines; D007375:Interleukin-1; D009153:Mutagens; D009573:Nitrites; D009589:Nitrogen Oxides; C091861:spermine nitric oxide complex; D013096:Spermine; C004479:pimagedine