Ultraviolet radiation (UVR) is a major stimulus for reactive oxygen species (ROS) production in the skin. Melanocytes produce the photoprotective pigment melanin, but possess low levels of ROS enzymatic scavengers (antioxidants) and appear to be unusually susceptible to injury by ROS. This paradox will be further examined by studying the regulation of enzymatic antioxidants catalase, glutathione peroxidase and superoxide dismutase and the ROS generating enzyme, xanthine melanocyte damage occurs when the protective antioxidant systems are exhausted. If melanocyte damage occurs repeatedly over a protracted period of time, melanocyte malignant transformation may occur. The long term objective of this project is to understand the effects of UVR-induced ROS on human epidermal melanocyte injury, cytotoxicity and the control of antioxidant enzyme synthesis in these cells. HUman epidermal melanocytes will be exposed to simulated sunlight, UVB or UVA radiation or the inflammatory mediators PGE2, LTB4 or LTC4 or the immune cytokines lL-1, TNF-alpha or TGF-alpha to determine if they cause an increase or decrease in melanocyte enzymatic antioxidants or xanthine oxidase/xanthine dehydrogenase (XO/XD). Antioxidant and XO/XD enzyme activities will be measured by standard biochemical methods. mRNA levels for each antioxidant enzyme and xanthine dehydrogenase will be measured by standard molecular biological techniques before and after the above mentioned treatments. I will then determine if enhanced or diminished antioxidant or XO//XD levels will alter melanocyte resistance to UVR cytotoxicity. Melanocytes will be treated with the tyrosinase-blocking agent, glucosamine (to block any UVR- induced pigmentation), then exposed to UVR or one of the inflammatory mediators or immune cytokines. Twenty-four hours later, the cells will be exposed to a second cytotoxic dose of UVR. Melanocyte survival 24 hours and 7 days after the second UVR exposure will be measured by cell counts and a colorimetric assay of cell survival. The knowledge gained from this work will deepen our understanding of the mechanisms controlling antioxidant systems in melanocytes and ultimately aid us in the development of new treatment strategies for the sequelae of acute UVR exposure of the skin.
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