Endogenous nitric oxide induced by interleukin‐1β in rat islets of Langerhans and HIT‐T15 cells causes significant DNA damage as measured by the ‘comet’ assay

Abstract

We have used the comet assay (single cell gel electrophoresis) to measure nitric oxide-induced DNA damage in rat islets of Langerhans and insulin-containing HIT-T15 cells. Damage was induced following treatment with the nitric oxide donor SIN-l, which also releases superoxide, but was not reduced by exogenous superoxide dismutase, suggesting that nitric oxide itself, rather than superoxide or peroxynitrite may be the active species. The DNA damaging effect of nitric oxide was easily detectable at the earliest time point tested (15 min). Damage also resulted following induction of nitric oxide synthase by the cytokine interleukin-I/? in both islets and HIT-T15 cells and was prevented by replacing the substrate, arginine, with nitromonomethyl arginine. Thus intracellular levels of nitric oxide generated by interleukin-l/l-induced nitric oxide synthase were sufficient to cause DNA damage in islet cells and HIT-T15 cells.

FoxO1 and SIRT1 Regulate β-Cell Responses to Nitric Oxide

“…Comet Assay-DNA damage was assessed using the comet assay (single cell gel electrophoresis) as described previously (10,32). Briefly, cells were harvested and embedded in 0.6% low melting agarose on slides precoated with 1.0% agar.…”

“…FoxO1 Is Required for Nitric Oxide-induced GADD45␣ Expression and DNA Repair-Nitric oxide is a known inducer of oxidative DNA damage (10,11,47). The comet assay was used to evaluate both the extent of nitric oxide-mediated DNA damage and the ability of ␤-cells to repair this damage (32).…”

“…The result is a 4-fold reduction in cellular ATP concentration (2, 7) that leads to the inhibition of glucose-induced insulin secretion due to the inability to generate sufficient levels of ATP to close the ATPsensitive K ϩ channels, an event required for ␤-cell depolarization and Ca 2ϩ -dependent exocytosis (8,9). In addition to the inhibition of ␤-cell function, nitric oxide induces DNA strand breaks and oxidative DNA damage (10,11).…”

Sensitivity of human pancreatic islets to peroxynitrite‐induced cell dysfunction and death

“…DNA strand breakage was quantified using the alkaline version of the comet assay (single cell gel electrophoresis) (for recent review on this technique see [17]), performed as previously described for islet tissue [18], with the modifications described below. After the appropriate treatments, groups of 100 islets were rinsed in Hanks buffered saline solution (HBSS) and then gently dispersed into single cells with trypsin.…”

“…Fax: (46) (18) 556401. E-mail: Carol Delaney@medcellbiol.uu.se the susceptibility to pancreatic ~-cell injury, with human islets being less susceptible to several different assaults than islets from rat or mouse [9][10][11].…”