16. Hydroxyl Radical Formation Mediated by Spin Trapping Agent, DMPO, in Uroporphyrin Photodynamic Reaction in the Presence of Biological Reductants
Keizo Takeshita, Chiho Nishizawa*, Jun-ichi Ueda, Kazuo T. Suzuki* and Toshihiko Ozawa
(* Graduate School of Medical and Pharmaceutical Sciences, Chiba University)
Keywords: hydroxyl radical, singlet oxygen, spin trapping, DMPO, DEPMPO
The photodynamic reaction has been actively used for the treatment of malignant disease (photodynamic therapy, PDT). The oxidation of biomaterials caused by reactive oxygen species (ROS) is believed to be one of the potent mechanisms of PDT. The spin trapping/ESR technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been commonly used to detect oxygen radicals. However, the reaction of DMPO in photosensitization has not been characterized enough. We observed that the presence of reducing agents such as NADPH, glutathione and Trolox (a water-soluble tocopherol derivative) remarkably increased the ESR signal of hydroxyl radical (·OH) adduct of DMPO (DMPO/·OH) in the uroporphyrin photosensitizing reaction with visible light. The inhibition experiments with ROS scavengers indicated that the formation of DMPO/·OH results from singlet oxygen (1O2)-mediated generation of free ·OH. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), neither NADPH, gluthathione nor Trolox increased the ESR signal of ·OH adduct of DEPMPO (DEPMPO/·OH). However, the addition of DMPO to the reaction mixture together with DEPMPO remarkably increased the signal of DEPMPO/·OH regardless of the presence of the reducing agents, accompanying a distinct DMPO/·OH signal only in the presence of the reducing agents. The production of ·OH was also determined with hydroxylation of salicylic acid. The presence of DMPO increased the amount of 2,3-dihydrobenzoic acid (a product of ·OH reaction with salicylic acid). The increase in the presence of the reducing agent was almost the same as that in the absence of the reducing agent. These results indicated that the 1O2-mediated ·OH formation occurs DMPO-dependently to form DMPO/·OH regardless of the presence of the reducing agents, and that the DMPO/·OH declines in the absence of reducing agent.