Investigation of calcitriol and paricalcitol effects on oxidative DNA damage and repair in human umbilical vein endothelial cells

Increasing levels of oxidative stress caused by endogenous and exogenous agents may lead to DNA damage, besides other macromolecules in the living cells. In case of unrepaired DNA damage, a variety of mutations and genomic instability might occur. Vitamin D deficiency is thought to play a role in the development of chronic diseases associated with oxidative damage. Active vitamin D calcitriol, besides its known classic effects, is reported recently to have non-classical effects such as antioxidant, regulation of cell growth and differentiation, etc. However, calcitriol has hypercalcemic effects at supra-physiological doses. Paricalcitol, a synthetic vitamin D analog, has physiological properties similar to calcitriol, but shows less hypercalcemic effects. Calcitriol and paricalcitol exerts most of their actions via binding to vitamin D receptor (VDR). The relation between vitamin D and oxidative DNA damage is noteworthy due to the high prevalence of vitamin deficiency and importance of genomic instability. The aim of this study is: to investigate the dose-dependent effects of calcitriol and paricalcitol on the formation of ROS, levels of oxidatively induced DNA damage and expressions of DNA repair enzyme APE1 and VDR in a model of hydrogen peroxide (H2O2)-generated oxidative stress in human umbilical vein endothelial cells (HUVEC). HUVECs were pretreated for 24 h with physiologic (0.1 nM) and supra-physiologic (10 nM and 100 nM) doses of calcitriol and paricalcitol, before exposure to H2O2 (300 µM) for half an hour. Also, to evaluate the role of VDR, experiments were repeated in the presence of 100 nM ZK159222 antagonist. The generation of ROS was assessed fluorometrically using the probe dichlorofluorescein diacetate (DCFH-DA). Damaged DNA nucleosides (8-OH-dG, R-cdA and S-cdA) were measurement using liquid chromatography-tandem mass spectrometry. mRNA expressions of DNA repair enzymes APE1 and VDR were measured by real-time qPCR. Regarding fluorometric ROS measurement data, we have found that, all doses of calcitriol and paricalcitol significantly reduced ROS formation. Tandem mass spectrometric assay results revealed that, both calcitriol and paricalcitol pretreatments attenuated damaged DNA nucleosides 8-OH-dG, R-cdA and S-cdA in HUVECs, compared to only H2O2 treatment group, especially in supra-physiological doses. Effects of paricalcitol on ROS formation and reduction of damaged nucleoside S-cdA levels, were dose-dependent. These useful effects of calcitriol and paricalcitol were inhibited in the presence of ZK159222. For all doses of calcitriol and paricalcitol no effect on APE1 expression was observed. Physiological dose of calcitriol increased mRNA expression of VDR compared to only H2O2 treatment group, but not other doses. This study demonstrated that, calcitriol and paricalcitol protect from H2O2 generated oxidative stress in human umbilical vein endothelial cells, being paricalcitol more effective. These protective effects of calcitriol and paricalcitol in preventing oxidative DNA damage have been shown to be mediated by VDR. We think that our findings will enlighten further studies investigating oxidative DNA damage-vitamin D relationship and will also create a basis for the therapeutic approaches in the prevention and treatment of oxidative stress related diseases.