Subthreshold UV light-induced peroxide formation in cultured corneal epithelial cells

Purpose: To demonstrate intracellular peroxide formation in cultured corneal epithelial cells by subthreshold ultraviolet (UV) light, and the protective effects of lactoferrin as an antioxidant. Methods: Intracellular oxidative insults and cell viability of rabbit corneal epithelial cells (RCEC) were assessed by dual-color digital microfluorography using carboxydichlorofluorescin (CDCFH) diacetate bis acetoxymethyl ester, a hydroperoxide-sensitive fluoroprobe, and propidium iodide (PI), respectively. The magnitude of UV-induced oxidative insults was calibrated by concentrations of exogenously applied H₂O₂ which evoke compatible levels of CDCFH oxidation. Inhibition of peroxide formation by lactoferrin, a potent iron-chelating protein present abundantly in tear fluids, was evaluated. Results: Exposure of RCEC to low dose UV-B (2.0 mJ/cm²) caused intracellular oxidative changes which were equivalent to those elicited by 250 μM hydrogen peroxide. The UV-induced changes were dose dependent, non-necrotic, and were partially inhibited by lactoferrin (1 mg/ml) but not by iron-saturated lactoferrin. Pretreatment with deferoxamine (2 mM) or catalase (100 u/ml) also attenuated the UV-induced oxidative stress. Conclusions: UV light comparable to solar irradiation levels caused significant intracellular peroxide formation in corneal epithelial cells. The UV-induced oxidative stress was suppressed by lactoferrin, indicating that lactoferrin in tears may have a physiological role in protecting the corneal epithelium from solar UV irradiation.