title: High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells.
abstract: OBJECTIVE
  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 (γH2AX) 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.
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital, Zhejiang University School of Medicine, #88 Jiefang Road, Hangzhou, Zhejiang, China. yejuan@zju.edu.cn
authors.forename: J
authors.initials: J
authors.lastname: Ye
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: X
authors.initials: X
authors.lastname: Shi
authors.forename: J
authors.initials: J
authors.lastname: Yang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology*; D003315:Cornea / Q000166:cytology / Q000187:drug effects*; D019610:Cytoprotection / Q000502:physiology; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000737:chemistry / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects*; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects
chemical_list: D002614:Chelating Agents; D006820:Hyaluronic Acid; D004492:Edetic Acid