Oxidative DNA damage induced by high glucose and its suppression in human umbilical vein endothelial cells

Abstract

In order to investigate the mechanism of the production of oxidative DNA damage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)-sensitive sites by the comet assay in human umbilical vein endothelial cells (HUVECs) cultured under various conditions including high glucose. Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 days in high glucose (45 mM) compared with normal glucose (5 mM) medium (P<0.001). FPG-sensitive sites increased in a time-dependent manner under high glucose treatment (3 days: P<0.05, 5 days: P<0.001), whereas Image-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sensitive sites (P<0.05). Flow cytometric analysis using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with Image-glucose produced more reactive oxygen species than incubation with Image-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively). Incubation of HUVECs with aminoguanidine (100 &micro;M) or pyridoxamine (1 mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive sites (P<0.001). However, these inhibitors did not suppress the intracellular generation of reactive oxygen species induced by high glucose. These results indicate that FPG-sensitive sites induced by high glucose are not due to intracellular reactive oxygen species. In order to clarify what caused the induction of FPG-sensitive sites, we investigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the induction of FPG-sensitive sites and the intracellular production of reactive oxygen species in HUVECs. Glyoxal and 3-DG at a concentration of 100 &micro;g/ml induced FPG-sensitive sites (P<0.001, P<0.01, respectively). In contrast, glyoxal did not generate reactive oxygen species inside HUVECs. The results shown in this study suggest that glyoxal formed intracellularly or extracellularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species.