High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

Abstract

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 (gH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2 0 ,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.

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis

“…Apart from regulating cytokine release, hyaluronan itself can protect cells from apoptosis by scavenging reactive oxygen species. This has been shown in cultured corneal epithelial cells and keratinocytes treated with either UV radiation or toxic substances (75,76). Furthermore, Wang and co-workers (77) showed that high levels of hyaluronan and Has2 expression were associated with resistance to UVB radiation and serum starvation in mouse fibroblasts.…”

Effects of antiproteolytic agents on corneal epithelial viability and matrix metalloproteinase‐2 and metalloproteinase‐9 activity in alkali‐burned corneas of rats

“…The benefits of EDTA in inhibiting MMPs have been previously reported . However, this substance commonly used as a preservative and corneal penetrating enhancer in ophthalmic solutions induces oxidative DNA damage in the corneal cells at high concentrations . The high concentration used herein (1%) may have caused the significant larger ulcerated area observed at the 6‐h time point following corneal injury, in association with a higher area under the curve in the EDTA treatment group.…”

“…Our experiment failed to show benefits of any tested agent in decreasing the levels of MMP‐2 and MMP‐9 after corneal alkali injury in rats. Despite the possible toxic corneal effects of EDTA and N‐acetylcysteine have been previously discussed herein, they may have acted only in acute phases of the corneal healing, once the corneal epithelium surface tended to appear restored at the end of the study.…”

The Protective Role of Hyaluronic Acid in Cr(VI)-Induced Oxidative Damage in Corneal Epithelial Cells

“…There have been literatures reported that a preincubation of HMW-HA could protect corneal epithelial cells against oxidative damage induced by ultraviolet B [24, 25], benzalkonium chloride [22, 40], EDTA [41], sodium lauryl sulfate [26], and so on. In the present study, we confirmed the protective role of 0.2% 1000 kDa HA against genotoxicity of Cr(VI) on HCE cells.…”