Kinetics of ultraviolet B irradiation-mediated reactive oxygen species generation in human keratinocytes.

Ultraviolet B (UVB)-mediated oxidative stress in keratinocytes has been accepted as an important factor contributing to skin damage. The present study revealed the kinetics of reactive oxygen species (ROS) production and identified the main specific ROS generated in human keratinocytes exposed to UVB. Keratinocytes were exposed to various doses of UVB, and intracellular ROS kinetics were evaluated by specific oxidant probes, namely, 2',7'-dichlorofluorescein diacetate, dihydroethidium, 10-acetyl-3,7-dihydroxyphenoxazine, and 3'-(p-hydroxyphenyl) fluorescein. Results revealed that UVB-irradiated cells exhibited significantly higher rate of ROS production in the early time period (0-2 h) compared to the nontreated control cells; however, the rate of ROS generation afterward (2-6 h) was similar to that of control cells. Specific ROS, including superoxide anion, hydrogen peroxide, and hydroxyl radical were enhanced in keratinocytes treated with UVB. Results regarding kinetics of specific ROS production revealed that superoxide anion and hydroxyl radical were the main ROS contributing to oxidative stress in the early phase (0-2 h) after UVB treatment in these cells. Further, this study showed the effect of known antioxidant, vitamin C, on ROS kinetics in UVB-exposed keratinocytes.