Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen during the Photohemolysis of Human Erythrocytes with Bacteriochlorin a

Abstract

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/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D2O 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 O2-. 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-N-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 CH3CHOH. and CO2-. radical adducts, indicating the presence of free OH.. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferrioxamine, suggesting that the iron-catalyzed decomposition of H2O2 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 1O2 production by BCA followed by reaction of 1O2 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 cell caused by a mixed type 1/type 2 mechanism.

Trapping Effect of Eugenol on Hydroxyl Radicals Induced by L-DOPA in Vitro

“…3F and G, a set of lines due to DMPO-CH 3 CHOH (a N ϭ16.01 G, a H ϭ23.07 G) appeared in the ESR spectra and the ESR signal of DMPO-CH 3 CHOH increased and that of DMPO-OH decreased with increase in the concentration of ethanol. 24) As DMPO-OH could be generated from the reaction of DMPO and hydroxyl radical, we studied the generation mechanism of hydroxyl radical using several conditions. As shown in Fig.…”

Imidazoacridinone Derivatives as Efficient Sensitizers in Photoantimicrobial Chemotherapy

“…Alternatively, in the type II pathway, the energy is transferred directly to O 2 , thus producing a singlet oxygen, a molecule against which there is no detoxifying enzyme known. For porphyrin-and phenothiazinebased PSs, it is generally accepted that they act mainly via singlet oxygen formation; however, O 2 ⅐ Ϫ (superoxide anion) and ⅐ OH (hydroxyl radical) are also implicated (27,28). Bacteriochlorin a (BCA), a structural analog of protoporphyrin IX, was shown to be a 50%/50% type I/type II photosensitizer (28).…”

“…For porphyrin-and phenothiazinebased PSs, it is generally accepted that they act mainly via singlet oxygen formation; however, O 2 ⅐ Ϫ (superoxide anion) and ⅐ OH (hydroxyl radical) are also implicated (27,28). Bacteriochlorin a (BCA), a structural analog of protoporphyrin IX, was shown to be a 50%/50% type I/type II photosensitizer (28). Our experiments concerning survival of C. albicans after PDI in the presence of type I (glycerol) and type II (sodium azide) or mixed (type I/type II) quenchers pointed out that C1330-based PDI acted via both mechanism types (Fig.…”

Progress with heterocyclic photosensitizers for the photodynamic therapy (PDT) of tumours

“…39 Scheme 5 m-THPBC 22 [39,47], which has λ max 735 nm (ε 91,000) in methanol; the oxobacteriochlorin 30, which has λ max 786 nm (ε 64,000) in dichloromethane [48]; and bacteriochlorin a (bacteriochlorin e 6 ) 31 which has λ max 760 nm (ε 32,000) in phosphate buffered saline [49].…”