Porphyrin photosensitization of the skin is a major health problem for patients suffering from certain types of human porphyria and for individuals treated with hematoporphyrin derivative (HPD) and light (photoradiation therapy) for cancer. Porphyrins are resonating structures (tetrapyrroles) which can absorb radiant energy in the visible spectrum (400-700 nm) and when present in increased amounts in living cells can cause phototoxic damage. It is generally believed that porphyrin photosensitization is a photodynamic process which occurs as a consequence of the generation of reactive species of oxygen; however, little is known regarding the role of these agents in porphyrin photosensitization of the skin. The subcellular sites of cutaneous phototoxic injury evoked by porphyrins have also not been rigorously defined. The hypothesis to be tested in this proposal is that lipid-rich membranous structures of the cell such as plasma membranes, mitochondria, lysosomes and endoplasmic reticulum are potential targets for the injurious response evoked by porphyrin photosensitization in the skin; that one or another species of active oxygen mediates this response; and that peroxidation of lipid-rich subcellular membranes is the fundamental mechanism of phototoxic injury. It is planned to conduct a systematic series of studies in cultured Balb/C mouse keratinocytes and fibroblasts and in the dermis and epidermis of the same strain of animals in an effort to assess the relative phototoxic effect of porphyrin photosensitization on these subcellular fractions. Enriched preparations of plasma membranes, lysosomes and mitochondria will be obtained using a metrizamide gradient technique and endoplasmic reticulum will be prepared by differential centrifugation. Marker enzymes will be assayed to verify the fractionation procedure and to determine the effects of porphyrin photosensitization on particular subcellular membranes. Lipid peroxide malondialdehyde will be measured as a marker for membrane lipid peroxidation. The long range goal of these studies is to determine whether cutaneous phototoxicity evoked by porphyrins of different solubility characteristics produces damage to specific subcellular organelles and to define the species of reactive oxygen that mediate(s) this cutaneous inflammatory response. Knowledge obtained in these studies may lead to the development of new strategies to reduce or eliminate porphyrin photosensitization, resulting in more effective therapeutic approaches to the management of cutaneous porphyria and to an improved therapeutic index for photoradiation therapy of human cancer.
