Protective Effect Of Hyaluronan Against Penetration Enhancers Induced DNA Damage In Corneal Epithelial Cells

Part?Protective effect of hyaluronan against benzalkonium chloride induced DNA damage in corneal epithelial cellsPurposeTo investigate the toxic effects of benzalkonium chloride (BAC), a preservative commonly used in ophthalmic preparations, on DNA damage, reactive oxygen species (ROS), cell survival and cell apoptosis in immortalized human corneal epithelial cells (HCEs), and investigated whether the effect induced by BAC can be inhibited by hyaluronan (HA).MethodsHCEs were exposed to BAC in concentrations ranging from 0.00005% to 0.001% with or without 0.2% HA for 30 min. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. Alkaline comet assay was used to detect DNA single-strand breaks (SSBs).Immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (yH2AX) 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 and cell apoptosis in HCEs was observed after exposure to 0.001% BAC for 30 min (P<0.001). In addition, a significant increase of SSBs, detected by alkaline comet assay, was observed in a dose-dependent manner with BAC exposure in HCEs at concentrations of 0.00005% and higher. Such BAC treatment also exhibited a dose-dependent increase in DSBs, evaluated by number of yH2AX foci. In addition, BAC treatment led to elevated ROS generation (P<0.01). Moreover,0.2% HA significantly decreased BAC-induced cell apoptosis, ROS generation and DNA damage.ConclusionsThe results demonstrated that exposure to BAC in HCEs could induce DNA strand breaks which may be correlated with the cell survival. Moreover, BAC-induced ROS formation may be associated with DNA damage. Furthermore, HA is an effective protective agent that has antioxidant properties and decreased DNA damage induced by BAC. Part?Protective effect of hyaluronan against EDTA induced DNA damage in corneal epithelial cellsPurposeEDTA is a corneal penetration enhancer commonly used in topical ophthalmic formulations. In this study, we have considered the toxic effect of EDTA on DNA damage, reactive oxygen species (ROS), cell survival and cell apoptosis in human corneal epithelial cells (HCEs), and investigated whether the effect induced by EDTA can be inhibited by hyaluronan (HA).MethodsCells were exposed to EDTA in concentrations ranging from 0.00001% to 0.01% for 30 min, or 30 min 0.2% HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of human corneal epithelial cells were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX foci, respectively. ROS production was assessed by the fluorescent probe,2',7'-dichlorodihydrofluorescein diacetate.ResultsEDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in HCEs at concentrations lower than 0.01%(P>0.05). However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in human corneal epithelial cells (P<0.05). In addition, EDTA treatment led to intracellular ROS generation (P< 0.01). Moreover, 30-min preincubation with 0.2% HA significantly decreased EDTA-induced ROS generation and DNA damage. ConclusionsEDTA can induce DNA damage in human corneal epithelial cells, probably through oxidative stress. Furthermore, HA is an effective protective agent that has antioxidant properties and decreased DNA damage induced by EDTA.