Cell membranes are subject to structural and functional damage when irradiated in the presence of photosensitizing pigments. A prominent form of such damage is lipid peroxidation, which can result in altered fluidity, increased permeability, and other effects. Photosensitized lipid peroxidation in biological systems has not been well studied in terms of reaction pathways, cytotoxic effects, and protective/repair mechanisms. The overall objective of this research is to improve our understanding of membrane lipid peroxidation in each of these areas. A comprehensive and diversified program of work is planned, using model systems initially (liposomes, erythrocyte membranes) and then cultured cells (murine L1210 cells). The sensitizers of interest are non-bound uroporphyrin, and membrane-bound protoporphyrin and hematoporphyrin derivative (HPD). Model Studies: (a) Extensive work is planned involving cholesterol (Ch) as an in situ probe of Type I (free radical) vs Type II (singlet oxygen) pathways of lipid peroxidation. (b) Amplification of photoinitiated lipid peroxidation by ascorbate will be investigated from a system-oriented perspective, e.g. effects of hydrogen peroxide and oxyradicals. (c) Long-range peroxidation (with sensitizer and target lipid in separate membrane compartments) will be studied for the first time. (d) Reductive metabolism (detoxification) of photoperoxides will be examined, using the glutathione reductase/peroxidase system. Cell Studies: (a) The first experiments to exploit Ch as a mechanistic probe in living cells will be carried out using (14C- Ch in HPD-sensitized L1210 cells. (b) The relationship between photoperoxidation and cytolethality will be investigated by (i) comparing lipid peroxide content and survivability, and (ii) determining how depletion of glutathione or glutathione peroxidase affects photosensitivity. The biochemical mechanisms and effects of lipid peroxidation on the integrity and function of biological membranes is of very great fundamental importance. Information that will be derived from this research will have implications in enhancing our basic knowledge of such diverse areas as the mechanisms by which light interacts with photoreceptors, the mechanisms by which leukocytes kill bacteria, and the reasons that cultured cells, when studied under the light microscope for periods of time, may behave differently from cells in the organism.
