Production of lipid-derived free radicals in L1210 murine leukemia cells is an early oxidative event in the photodynamic action of Photofrin.

Photofrin photosensitization initiates a sequence of oxidative events that begins with singlet oxygen formation and ultimately leads to cell death. We hypothesize that membrane lipid-derived free radical formation is an early event in this process. In the presence of iron and ascorbate, lipid free radicals are generated during cellular photosensitization of L1210 cells as detected by electron paramagnetic resonance spin-trapping techniques. Tocopherol levels decline in an inverse manner to lipid radical formation. Trypan blue dye exclusion by membranes also decreases inversely to lipid radical formation but at an initially slower rate than alpha-tocopherol depletion. Propidium iodide nuclear staining as an alternative measure of cell integrity was a later event, 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 debris did not occur substantially until alpha-tocopherol was virtually exhausted and radical intensity had nearly reached a maximum. These temporal observations suggest the following sequence of events that leads to Photofrin photosensitization-induced cytotoxicity in the presence of iron and ascorbate: (1) singlet oxygen-derived lipid hydroperoxide formation and subsequent radical production; (2) cellular alpha-tocopherol depletion; (3) trypan blue-detectable membrane leakage; (4) nuclear exposure to propidium; (5) cell disintegration. These observations are consistent with membrane lipid-derived free radical formation being an early and perhaps seminal event in photosensitization by Photofrin, which leads to a concatenated series of events terminating in cell destruction.