Protective Effect Of Hyaluronan And Hydroxypropyl Methylcellulose Against Preservative Induced DNA Damage In Ocular Surface Epithelial Cells And The DNA Damage Response Mechanism

Part I Thimerosal and sodium perbarate induce DNA damage in Chang conjunctival epithelial cellsPurposeTo investigate the toxic effects of thimerosal (Thi) and sodium perbarate, two preservative commonly used in ophthalmic preparations, on DNA damage, reactive oxygen species (ROS) and cell survival in immortalized human Chang conjunctival cells, and compare the genotoxicity between two preservative.MethodsCells were exposed to Thi and sodium perbarate in concentrations ranging from0.00005%to0.001%for30min. The cell viability was measured by the MTT test. Alkaline comet assay was used to detect DNA single-strand breaks (SSBs). Immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci indicated DNA double-strand breaks (DSBs). Reactive oxygen species production was assessed by the fluorescent probe,2’,7’-dichlorodihydrofluorescein diacetate.ResultsA significant change of the relative cell survival rate in cells was observed after exposure to0.001%Thi for30min (P<0.001). In addition, a significant increase of SSBs, detected by alkaline comet assay, was observed in a dose-dependent manner with Thi exposure in cells at concentrations of0.00005%and higher. Such Thi treatment also exhibited a dose-dependent increase in DSBs, evaluated by number of H2AX foci. In addition, intracellular ROS induced by Thi was dose-dependent (P<0.01), except, at0.001%induced less ROS than at0.0005%. Sodium perbarate didn’t induce the decrease of cell survival in every concentration, but could induce DSBs, SSBs and ROS generation in0.001%concentration (P<0.001)ConclusionsThe results demonstrated that exposure to Thi in Chang conjunctival epithelium cells could induce DNA strand breaks which may be correlated with the cell survival. Moreover, Thi-induced ROS formation may be associated with DNA damage. Sodium perbarate didn’t induce the decrease of cell survival in every concentration while DNA damage and ROS generation was detected in high concentration, suggesting new type oxidative preservative was less toxicity than old type preservative. Part II Protective effect of hyaluronan and hydroxypropyl methylcellulose against preservative induced DNA damage in Chang conjunctival epithelial cellsPurposeTo investigate genotoxicity of the preservative thimerosal (Thi), and the cytoprotective and antioxidant effects of hyaluronic acid (HA) and hydroxypropyl methylcellulose (HPMC) on Chang conjunctival cells.MethodCells were divided into three groups. One group was exposed to Thi at various concentrations (0.00001%～0.001%) for30min; the other two groups were pretreated with0.3%HA or0.3%HPMC for30min before the Thi exposure. After cell viability evaluated, alkaline comet assay and the phosphorylated form of histone variant H2AX (yH2AX) foci detection were used to detect DNA damage. Reactive oxygen species (ROS) production was assessed by the fluorescent probe,2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA).ResultsA significant change of cell viability was observed after exposure to0.001%Thi for30min. DNA single-and double-strand breaks was significantly increased in a dose-dependent manner with Thi exposure. In addition, Thi treatment led to elevated ROS generation (P<0.05). However, cells pretreated with0.3%HA or0.3%HPMC showed significantly increased cell survival, and decreased DNA damage and ROS production compared to cells that with Thi alone.0.3%HA was found to be even more protective than0.3%HPMC.ConclusionThi can induce DNA damage in human conjunctival epithelial cells, probably related to oxidative stress.0.3%HA and0.3%HPMC are protective agents that have antioxidant properties and can decrease DNA damage induced by Thi.0.3%HA may be more protective of the ocular surface than0.3%HPMC. Part Ⅲ Acute exposure of thimerosal induce antiproliferative properties, apoptosis and autophagy activation in Chang conjunctival epithelial cellsPurposePreviously we have shown that acute exposure to thimerosal (Thi) could induce oxidative stress and DNA damage in a human conjunctival cell line. However, the long-term effect of Thi on Chang conjunctival cells is not clear. Therefore, the aim of this study was to further investigate the fate of the cells after acute exposure to Thi.MethodCells were first exposed to various concentrations of Thi (0.00001%-0.001%) for30min, and then cells were assessed after a24-h recovery period. Morphologic changes were observed under a light microscope and cell viability was evaluated. Cell apoptosis, cell cycle distribution and mitochondrial membrane potential (MMP)(rhodamine123assay) were detected by flow cytometry analysis. Poly (ADP-ribose) polymerase (PARP), Activation of caspase-3and microtubule-associated protein light chain3(LC-3) was examined by western blot analysis.ResultsDNA strand breaks were significantly increased in a dose-dependent manner with30min exposure to Thi, although no significant cell death was detected. However, after24-h recovery, the ratio of apoptotic cells was significantly increased in0.0005%and0.001%Thi treated groups (p<0.001compared to the control group). Apoptosis was confirmed by the cleavage of PARP and caspase-3activation. In addition, G2/M cell cycle arrest and decrease of MMP were recorded. Finally, the LC-3results indicated the occurrence of autophagy in Thi-treated cells. ConclusionAcute exposure to Thi can induce DNA damage, and eventually can lead to cell death, probably through the caspase-dependent apoptosis pathway, while autophagy might also be involved.