title: 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 (5mM) 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-sensitive 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 incubation with D-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively). Incubation of HUVECs with aminoguanidine (100 microM) or pyridoxamine (1mM), 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 microg/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.
authors.affiliation: Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. shimoi@smail.u-shizuoka-ken.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Shimoi
authors.forename: A
authors.initials: A
authors.lastname: Okitsu
authors.forename: M H
authors.initials: MH
authors.lastname: Green
authors.forename: J E
authors.initials: JE
authors.lastname: Lowe
authors.forename: T
authors.initials: T
authors.lastname: Ohta
authors.forename: K
authors.initials: K
authors.lastname: Kaji
authors.forename: H
authors.initials: H
authors.lastname: Terato
authors.forename: H
authors.initials: H
authors.lastname: Ide
authors.forename: N
authors.initials: N
authors.lastname: Kinae
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D043382:DNA-Formamidopyrimidine Glycosylase; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005434:Flow Cytometry; D005947:Glucose / Q000633:toxicity*; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011733:Pyridoxamine / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D014471:Umbilical Veins
chemical_list: D006146:Guanidines; D017382:Reactive Oxygen Species; D011733:Pyridoxamine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D005947:Glucose; C004479:pimagedine