Porphyrin photosensitization and reactive oxygen species scavengers protection in 5-aminolevulinic acid-based photodynamic therapy

MARTINEZ M DEL C.,; TEIJO MJ,; FUKUDA H,; BATLLE A.

Mar del Plata

Congreso; - LX I REUNIÓN ANUAL Sociedad Argentina de Investigación Clínica (SAIC); 2016

SAIC_SAI-SAFE

Photosensitization of endogenously synthesized porphyrins after administration of 5- aminolevulinic acid (ALA), is used in Photodynamic Therapy of tumours and microorganisms infections. Irradiation with appropriate wavelength light triggers photochemical reactions and ROS production are induced, leading to cell death. Several ROS scavengers (ascorbate , trolox, mannitol, GSH, l-tryptophan) were assayed to test their ability to modulate ALA-PDT performed on A549 human lung adenocarcinoma cells, by incubating 3h with 1mM ALA, followed by irradiation with or without 1 h pre-incubation with the modulators. The ratio between cell survival after ALA-PDT in the presence and in the absence of the scavenger agent (protection grade: PG) was determined, and the concentrations showing no cytotoxicity/ photoactivity and providing the highest PG were used. ALA-PDT alone induced a high percentage of apoptotic cell death (98.4±3.5 %). Pre-incubation with modulators at their highest PG concentration significantly reduced apoptotic cells to 48.3±2.7% (ascorbate), 58.8±4.2% (trolox), 78.5±3.1% (GSH), 64.3±1.6% (mannitol), and 74.6±2.3% (l-tryptophan). ROS production in early cell death induction after ALA-PDT was tested by flow cytometry; ROS increased after ALA-PDT (H2-DCFDA + cells: control: 1.1±0.1%; 10 min-PDT: 69.3 ± 5.6%; MVP positive cells: control: 0.65±0.35%; 10 min-PDT: 83.5±1.9%). Ascorbate prevented peroxide formation and singlet oxygen increased, whereas trolox limited peroxides generation, but did not affect significantly singlet oxygen production. These results suggest that undesired photodamage to normal tissue might be attenuated by antioxidant agents administration, showing a strong correlation between porphyrins, ROS production, scavengers and cell death, reinforcing the photodynamic properties of endogenously synthesized porphyrins, which are of high relevance in the photosensitization of porphyric patients.