MECHANISMS OF CHEMICALLY INDUCED PHOTOSENSITIVITY

工作总结：当光线与皮肤和眼睛中的内源性或外源性化学物质相互作用时，就会产生光敏作用。这一过程可能会产生不良的临床后果，如光毒性(夸大的晒伤)、光过敏、光致癌性和/或光致突变；或者它可能具有有益的效果，如肿瘤光动力疗法(PDT)和煤焦油、蒽啉或补骨脂素(PUVA)治疗银屑病。本研究的目的是阐明光敏剂发挥其毒性或治疗作用的光化学机制。氟喹诺酮类(FLQ)是一类相对较新的抗菌药，用于治疗革兰氏阴性细菌感染。当在人体上使用时，氟喹诺酮往往会引起光毒性。最近的研究表明，洛美沙星(LOME)和氟罗沙星(FLER)在注射这些药物并接受UV-A(315-400)nm照射的HR/J小鼠中会导致鳞癌。我们研究了FLQ的光化学性质，以确定为什么这些药物作为一类经常具有光毒性，以及为什么LOME和FLER具有光致癌作用。FQ抗菌剂的单线态氧和超氧化物产量与其光毒潜力无关。然而，对于LOME和FLER，pBR322 DNA的光切割效率至少是单氟化类似物的10倍。根据彗星试验，LOME和FLER还对HaCaT细胞(一种不朽的人类角质形成细胞系)造成了广泛的DNA光损伤。然而，克林沙星和Bay y3118这两种高度光毒的flq在同一检测中甚至更具活性。FLQs的光致突变性可能是由于8-卤素原子作为卤化物的光消除而产生了高活性的卡宾。黄花是一种草本植物，广泛用于洗眼液和护肤水等多种医疗用途，目前正在接受国家毒理学计划的测试。我们已经证实，黄花丹中的主要生物碱黄连素对HaCaT角质形成细胞具有光毒性。用1微克分子黄连素溶液孵育的角质形成细胞经UVA照射10min后，细胞存活率下降50%。通过彗星试验测定，黄连素/长波紫外线也会损伤角质形成细胞DNA。黄连素在水溶液中不光敏单线态氧，但在二氯甲烷中产生单线态氧，量子产率为0.34。这表明，光敏化需要一个疏水环境。我们在苯中检测到黄连素光解产生的氧中心自由基。在黄连素的长波吸收带内，观察到水/醇低温玻璃中的甲醇自由基。在这类酒精玻璃中，我们还检测到了77K时黄连素三联体的EPR信号，而水玻璃中既没有检测到三联体，也没有检测到自由基。我们的数据表明，尽管黄连素在水中是一种微弱的单线态氧光敏剂，但在疏水环境中能够产生自由基。因此，对角质形成细胞的杀伤可能涉及一种光动力学机制，该机制工作在疏水的细胞室(DNA，膜)中，这些细胞室被黄连素光生产生的活性氧物种原位氧化。通过与北卡罗来纳州立大学植物病理系的科学家合作，我们发现，维生素B6对单线态氧产生光毒素的抗性机制与维生素C和E相当。这一发现表明，维生素B6在体内可能起到一种迄今未知的抗氧化剂作用--光敏化、光毒性、光过敏、光致癌

Summary 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