Repair of Cytokine-induced DNA Damage in Cultured Rat Islets of Langerhans

Abstract

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1β (IL-1β), interferon γ (IFN γ) and tumour necrosis factor α (TNF α) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-β-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18 h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.

• Comet assay
• DNA repair
• Nitric oxide
• Hydroxyurea
• 1-β-d-arabinosyl cytosine
• NG-monomethyl-l-arginine
• 8-OHG, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine)
• AraC, 1-beta-d-arabinofuranosyl cytosine
• Fpg, formamidopyrimidine glycosylase
• HU, hydroxyurea
• IFNγ, interferon γ
• IL-1β, interleukin-1β
• MEM, minimum essential medium with Earle's salts
• NMMA, NG-monomethyl-l-arginine monoacetate
• TNFα, tumour necrosis factor α