Mitomycin C potentiates ultraviolet-related cytotoxicity in corneal fibroblasts

Purpose: To study the morphologic changes and cytotoxicity in corneal fibroblasts after mitomycin C (MMC) treatment, ultraviolet B (UVB) irradiation, or in combination. Methods: Primary porcine corneal fibroblasts, passages 3-5, were treated with MMC (0.1 or 0.2 mg/mL, ie, 0.01% or 0.02%, for 5 minutes), UVB irradiation (2, 5, or 10 mJ/cm(2)), or in combination. Control cells were treated with culture medium as a sham procedure. Alterations in cell morphology were documented by phase-contrast microscopy; cellular apoptosis was evaluated by Annexin V/propidium iodide staining and analyzed by flow cytometry; cytotoxicity was evaluated by lactate dehydrogenase assay; and cell growth was studied by genomic DNA quantification with the PicoGreen assay. Results: UV irradiation induced significant dose-dependent corneal fibroblast rounding and detachment and cytotoxicity. MMC at 0.1 or 0.2 mg/mL induced considerable cell elongation and retarded cell proliferation at similar rates. MMC treatment alone did not cause significant apoptosis or cytotoxicity. However, MMC treatment before UV irradiation potentiated UV-related cytotoxicity proportional to the UV radiation dose. Conclusions: UV irradiation causes dose-dependent cytotoxicity in porcine corneal fibroblasts. MMC pretreatment potentiates UV-related cytotoxicity.