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

In order to investigate the mechanism of the production of oxidative DNA da mage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)- sensitive sites by the comet assay in human umbilical vein endothelial cell s (HUVECs) cultured under various conditions including high glucose. Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 day s 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 L-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sen sitive sites (P < 0.05). Flow cytometric analysis using 6-carboxy-2 ' ,7 ' -dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with L-glucose produced more reactive oxygen species than incuba tion with D-glucose. However, these increases were slight (1.22- and 1.12-f olds, respectively). Incubation of HUVECs with aminoguanidine (100 muM) or pyridoxamine (1 mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive si tes (P < 0.001). However, these inhibitors did not suppress the intracellul ar generation of reactive oxygen species induced by high glucose. These res ults 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 in vestigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the inducti on of FPG-sensitive sites and the intracellular production of reactive oxyg en species in HUVECs. Glyoxal and 3-DG at a concentration of 100 mug/m indu ced FPG-sensitive sites (P < 0.001, P < 0.1, respectively). In contrast, gl yoxal did not generate reactive oxygen species inside HUVECs. The results s hown in this study suggest that glyoxal formed intracellularly or extracell ularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species. (C) 2001 Elsevier Science B.V. All rights reserved.