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Purpose: To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA).Methods: Cells were exposed to EDTA in concentrations ranging from 0.00001% to 0.01% for 60 min, or 30 min high molecular weight 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 HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (H2AX) foci, respectively.Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2, 7-dichlorodihydrofluorescein diacetate.Results: EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%.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 HCEs.In addition,EDTA treatment led to elevated ROS generation.Moreover, 30-min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage.Conclusions: EDTA could induce DNA damage in HCEs, probably through oxidative stress.Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA.