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Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen during the Photohemolysis of Human Erythrocytes with Bacteriochlorin a
Author(s) -
Hoebeke Maryse,
Schuitmaker Hans J.,
Jannink Lies E.,
Dubbelman Tom M. A. R.,
Jakobs Andreas,
Vorst Albert
Publication year - 1997
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1997.tb03180.x
Subject(s) - chemistry , radical , spin trapping , photochemistry , singlet oxygen , hydroxyl radical , electron paramagnetic resonance , hydrogen peroxide , potassium phosphate , hemolysis , rose bengal , oxygen , nuclear chemistry , chromatography , nuclear magnetic resonance , biochemistry , organic chemistry , physics , immunology , biology
— Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate‐buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm 2 . Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 m M ), azide (1 m M ) or histidine (10 m M ) to the erythrocyte suspension before illumination delayed the onset of K‐leakage and hemolysis suggesting a type II mechanism. The D 2 O did not affect K‐leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K‐leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin‐trapping techniques that BCA is able to generate 0 2 ∼* and OH* radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5‐dimethyl‐1‐pyrroline‐iV‐oxide (DMPO) gave an ESR spectrum characteristic of the DMPO‐hydroxyl radical spin adduct DMPO‐OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH 3 CHOH * and CO 2 ‐1 radical adducts, indicating the presence of free OH * . Production of DMPO‐OH was partly inhibited by superoxide dismutase (SOD), catalase and desferoxamine, suggesting that the iron‐catalyzed decomposition of H 2 O 2 was partly involved in the formation of one part of the observed OH * . The complementary inhibition of DMPO‐OH production by azide and 9,10‐anthracenedipropionic acid (ADPA) was consistent with 1 O 2 production by BCA followed by reaction of 1 O 2 with DMPO and decay of the intermediate complex to form DMPO‐OH and free OH * . All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye‐induced hemolysis of erythrocytes was well caused by a mixed type 1/type 2 mechanism.