Flow cytometry study of the role of superoxide anion and hydrogen peroxide in cellular photodestruction with 5-aminolevulinic acid-induced protoporphyrin IX.

Flow cytometry was used to investigate the participation of reactive oxygen species, other than singlet oxygen, in the cytotoxic effect of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in vitro in A-431 squamous cell carcinoma (SCC) cells and human skin fibroblasts (HSF). We used propidium iodide to determine cellular cytotoxicity, hydroethidine to measure intracellular superoxide anion (O2-) and dihydrorhodamine 123 to assess intracellular hydrogen peroxide (H2O2) content. Our data support the importance of the incubation time with ALA in the selectivity of PDT with ALA against SCC cells, inducing minimum damage on normal HSF. Photoradiation mortality curves of the response of these cell lines to ALA-induced PpIX photosensitization correlated with the extent of photosensitizer accumulation. Intracellular O2- production correlated with cell death, increasing both in a light dose-dependent fashion in ALA treated cells. This correlation was not observed with H2O2-intracellular production. These results suggest the effectiveness of PDT with ALA in vitro in SCC, the significant participation of O2- in its phototoxic mechanism, and the usefulness of flow cytometry in the study of the cytotoxic effect of ALA-induced PpIX PDT.