Phototoxicity of Riboflavin and Rose bengal on Cultured Bovine Lens Epithelial Cells in vitro

Cho, Kyung Sun; Joo, Choun Ki; Lee, Jin Hak

Abstract

Light may induce various kindsof oxygen free radicals in the eye and show a toxic effect on lens epithelial cells (LEC)as well as cataract formation. In this study, we examined the oxygen free radical injury of LEC using photosensitizer like riboflavin (RF) or rese bengal (RB) and blocking effect of antagonists on photoxicity of LEC. We cultured bovine lens epithelial cells (BLEC) to 24 well plate at the concentration of 5*10 in each well. BLEC were exposed with a illumination of 1200 lux for 2 hours under th concentration of 10, 25, 50, 75 and 100 uM of RF and 5, 10, 15, and 20 uM of RB, respectively. The pattern of BLEC death was observed with inverted light microscope and the quantitation of BLEC cytotoxicity was calculated using 3-(4, 6-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide(MTT) assay. The blocking effects of catalase, sodium azide, vitamin C, and vitamin E on BLEC phototoxicity were also evaluated. The concentration of RF inducing a prominent phototoxicity with a light of 1200 lux for 2 hours was determined to 50uM and that of RB was determined to 10uM(p<0.05). The quantiative cytotoxicity of BLEC was prominent at 24 hours dark adaptation following 2 hours illumination. In the pattern of phototoxicity of BLEfC, RF induced a cell death showing shrinkage of cell membrane and arborization of process, while RB induced a cell death showing swelling of cytoplasm. In the experiment of the scavengers` effects on phototoxicity of BLEC, catalase (3.75 U/ml) attenuated selectively a phototoxicity of BLEC induced by RF and sodium azide (imM) attenuated selectively a phototoxicity induced by RB. Conclusively, we could find hydrogen peroxide and singlet oxygen were main oxygen free radicals induced by RF and RB, respectively and the patterns of cell death induced by photosensitizer of RF and RB were cellular shrinkage and swelling, repectively.