Hoebeke M, Schuitmaker H J, Jannink L E, Dubbelman T M, Jakobs A, Van de Vorst A
Institute of Physics, University of Liège, Belgium.
Photochem Photobiol. 1997 Oct;66(4):502-8. doi: 10.1111/j.1751-1097.1997.tb03180.x.
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.
以细菌叶绿素a（BCA）作为敏化剂的光动力疗法在体内会诱导红细胞损伤。为了评估活性氧（ROS）的贡献程度并确定可能的反应机制，在作为模型系统的人红细胞和磷酸盐缓冲液中进行了与各种ROS猝灭剂或/和增强剂的竞争实验。在红细胞实验中，将2%的悬浮液与BCA孵育1小时，用磷酸盐缓冲盐水洗涤，重悬，随后用二极管激光以2.65 mW/cm²的光通量率照射。钾泄漏和溶血呈光和BCA剂量依赖性。在照射前向红细胞悬浮液中加入色氨酸（3.3 mM）、叠氮化物（1 mM）或组氨酸（10 mM）可延迟钾泄漏和溶血的发生，表明是II型机制。重水不影响钾泄漏和光溶血。加入甘露醇（13.3 mM）或甘油（300 nM）也会延迟钾泄漏和溶血的发生，表明有自由基参与。在磷酸盐缓冲液实验中，使用与自旋捕获技术相关的电子自旋共振（ESR）表明，BCA能够产生超氧阴离子（O₂⁻）和羟基自由基（OH·），而不产生水合电子。在自旋捕获剂5,5 - 二甲基 - 1 - 吡咯啉 - N - 氧化物（DMPO）存在下，对染料进行可见光或紫外线照射，得到了DMPO - 羟基自由基自旋加合物DMPO - OH的特征ESR谱。加入乙醇或甲酸钠由于各自的CH₃CHOH·和CO₂⁻自由基加合物产生了额外的超精细分裂，表明存在游离的OH·。超氧化物歧化酶（SOD）、过氧化氢酶和去铁胺部分抑制了DMPO - OH的产生，表明铁催化的H₂O₂分解部分参与了所观察到的一部分OH·的形成。叠氮化物和9,10 - 蒽二丙酸（ADPA）对DMPO - OH产生的互补抑制与BCA产生单线态氧（¹O₂）一致，随后¹O₂与DMPO反应，中间复合物衰变形成DMPO - OH和游离的OH·。我们所有的结果似乎表明，在缓冲水溶液中BCA是一种50%/50%的I型/II型敏化剂，并证实了染料诱导的红细胞溶血是由I型/II型混合机制引起的细胞损伤。
