of Work: Photosensitization can result when light interacts with endogenous or exogenous chemical agents in the skin and eyes. This process can produce undesirable clinical consequences, such as phototoxicity (exaggerated sunburn), photoallergy, photocarcinogenicity and/or photomutagenicity; or it can have beneficial effects as in tumor photodynamic therapy (PDT) and coal tar, anthralin or psoralen (PUVA) therapy for psoriasis. The objective of this research project is to elucidate the photochemical mechanisms whereby photosensitizers exert their toxic or therapeutic effects. Fluoroquinolones (FLQ) are a relatively new class of antibacterials that are useful in the treatment of gram-negative bacterial infections. When used in humans FLQs often cause phototoxicity. Recent studies by others have shown that lomefloxacin (LOME) and fleroxacin (FLER) cause squamous cell carcinomas in HR/J mice injected with these drugs and irradiated with UV-A (315-400) nm. We have studied the photochemical properties of the FLQs to determine why these drugs as a class are often phototoxic and why LOME and FLER are photocarcinogenic. Singlet oxygen and superoxide yields for the FQ antimicrobials do not correlate with their phototoxic potentials. However, photocleavage of pBR322 DNA is at least 10-fold more efficient for LOME and FLER than for monofluorinated analogs. LOME and FLER also caused extensive DNA photo-damage in HaCaT cells (an immortal line of human keratinocytes) as measured by the Comet assay. However, clinafloxacin and BAY y3118, two highly phototoxic FLQs were even more active in the same assay. The photo-mutagenicity of the FLQs is probably due to the generation of a highly reactive carbene as a result of the photo-elimination of the 8-halo atom as halide. Goldenseal is a herb widely used for numerous medical applications including eyewash and skin lotion, which is currently undergoing testing by the National Toxicology Program. We have established that berberine, the main alkaloid in Goldenseal, is phototoxic to HaCaT keratinocyte skin cells. Cell viability decreased by 50% after 10 min UVA irradiation of the keratinocytes incubated with 1micromolar berberine solution. Berberine/UVA also damages keratinocyte DNA as measured by the Comet assay. Berberine does not photosensitize singlet oxygen in aqueous solutions, but singlet oxygen is produced with a quantum yield of 0.34 in dichloromethane. This suggests that a hydrophobic environment is required for photosensitization. We have detected oxygen-centered radicals photogenerated by berberine in benzene. Methanol radicals were observed in water/alcohol low temperature glasses irradiated in the berberine long-wavelength absorption band. In such alcoholic glasses, we have also detected an EPR signal from the berberine triplet at 77K, in contrast to aqueous glasses where neither triplet nor radicals were detectable. Our data show that, although a weak singlet oxygen photosensitizer in water, berberine is able to produce radical species in a hydrophobic environment. Thus the killing of keratinocytes can involve a photodynamic mechanism operating in hydrophobic cell compartments (DNA, membranes) that are oxidized in situ by reactive oxygen species photo- produced by berberine. As a result of a collaboration with scientists in the Plant Pathology Department at North Carolina State University on the mechanism of Cercosopera nicotianae resistance to singlet oxygen generating phototoxins, we have discovered that vitamin B6 quenches singlet oxygen at a rate comparable to vitamins C and E. This finding suggests that vitamin B6 may play a hitherto unknown role in vivo as an antioxidant - photosensitization, phototoxicity, photoallergy, photocarcinogenicity
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