PRODUCTION OF LIPID-DERIVED FREE-RADICALS IN L1210 MURINE LEUKEMIA-CELLS IS AN EARLY OXIDATIVE EVENT IN THE PHOTODYNAMIC-ACTION OF PHOTOFRIN(R)

Photofrin(R) photosensitization initiates a sequence of oxidative even ts that begins with singlet oxygen formation and ultimately leads to c ell death, We hypothesize that membrane lipid-derived free radical for mation is an early event in this process, In the presence of iron and ascorbate, lipid free radicals are generated during cellular photosens itization of L1210 cells as detected by electron paramagnetic resonanc e spin-trapping techniques, Tocopherol levels decline in an inverse ma nner to lipid radical formation, Trypan blue dye exclusion by membrane s also decreases inversely to lipid radical formation but at an initia lly slower rate than alpha-tocopherol depletion. Propidium iodide nucl ear staining as an alternative measure of cell integrity was a later e vent, occurring when alpha-tocopherol levels had fallen by 90%, trypan blue survival had decreased to below 10%, and lipid radical formation was nearing plateau levels. Likewise, the formation of cellular debri s did not occur substantially until alpha-tocopherol was virtually exh austed and radical intensity had nearly reached a maximum, These tempo ral observations suggest the following sequence of events that leads t o Photofrin(R) photosensitization-induced cytotoxicity in the presence of iron and ascorbate: (1) singlet oxygen-derived lipid hydroperoxide formation and subsequent radical production; (2) cellular alpha-tocop herol depletion; (3) trypan blue-detectable membrane leakage; (4) nucl ear exposure to propidium; (5) cell disintegration. These observations are consistent with membrane lipid-derived free radical formation bei ng an early and perhaps seminal event in photosensitization by Photofr in(R), which leads to a concatenated series of events terminating in c ell destruction.