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

Abstract

UVB-mediated oxidative stress in keratinocytes has been accepted as an important factor contributing to skin damages. The present study revealed the kinetics of ROS production and identified 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, DCFH2-DA, DHE, Amplex Red, and HPF. Results revealed that UVB-irradiated cells exhibited significantly higher rate of ROS production in the early time period (0-2 h) compared to the non-treated 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 main ROS contributing to oxidative stress in the early phase (0-2 h) after UVB treatment in these cells. Furthermore, this study demonstrated the effect of known antioxidants vitamin C, vitamin E, and silymarin on ROS generating kinetics in UVB-exposed keratinocytes. The results indicated that all antioxidants could suppress ROS generation in response to UVB irradiation in a time-dependent manner ascompared to non-antioxidant treated cells. In addition, the linear regression analysis revealed that the rate of ROS generation in early period (0-2 h) correlated with apoptotic cell death of keratinocytes.These findings revealed the novel information regarding ROS generating kinetic in UVB-exposed keratinocytes, which may benefit the development of new strategies in protecting of skin damage.