title: In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells.
abstract: OBJECTIVE
  The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials.
  
  
  METHODS
  Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope.
  
  
  RESULTS
  The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation.
  
  
  CONCLUSIONS
  The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the effectiveness of UV-absorbing ophthalmic biomaterials, including contact lenses and intraocular lenses.
authors.affiliation: School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Hyun-Yi
authors.initials: HY
authors.lastname: Youn
authors.forename: David J
authors.initials: DJ
authors.lastname: McCanna
authors.forename: Jacob G
authors.initials: JG
authors.lastname: Sivak
authors.forename: Lyndon W
authors.initials: LW
authors.lastname: Jones
mesh_terms: D017304:Annexin A5 / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D001672:Biocompatible Materials / Q000737:chemistry; D001681:Biological Assay / Q000379:methods; D002138:Calibration; D002478:Cells, Cultured; D003315:Cornea / Q000528:radiation effects*; D006801:Humans; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000528:radiation effects*; D018613:Microscopy, Confocal / Q000379:methods; D010078:Oxazines / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000528:radiation effects*; D012168:Retinal Pigments / Q000378:metabolism; D020112:Rhodamine 123 / Q000494:pharmacology; D014466:Ultraviolet Rays; D014966:Xanthenes / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D001562:Benzimidazoles; D001672:Biocompatible Materials; D010078:Oxazines; D012168:Retinal Pigments; D014966:Xanthenes; C005843:resazurin; D020112:Rhodamine 123; C017807:bisbenzimide ethoxide trihydrochloride