title: Difference in superoxide toxicity between 4,7-dicyanobenzofurazan and paraquat.
abstract: The O2-. production by aerobically cultured Escherichia coli in the presence of benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), and 4,7-dicyanobenzofurazan (5) was examined by using the cytochrome c reduction method in order to elucidate the mechanism of cytotoxicity of benzofurazans. Adding compound 5 to E. coli cell suspension caused cytochrome c reduction, which was completely inhibited by superoxide dismutase. The rate of cytochrome c reduction was in the order of 1 = 2 = 3 less than 4 less than 5, which correlates well with that of the reduction potentials of these benzofurazans. Adding glucose to the E. coli cell suspension-compound 5-cytochrome c system accelerated the rate of cytochrome c reduction. The formation of 4,7-dicyanobenzofurazan anion radical in the cell suspension-compound 5-glucose system in the absence of O2 was followed by ESR spectroscopy. The ESR signal of the anion radical disappeared when O2 was added. Compound 5 was shown to have an approximately 10-fold greater increasing effect on the flux of O2-. by E. coli than paraquat (PQ) by the cytochrome c reduction method. The results were confirmed by the electrochemical method with an oxygen electrode. However, compound 5 had a bacteriostatic, but not lethal, effect, while PQ had both effects. The effect of compound 5 and PQ on lethality of E. coli showed a dramatic difference when E. coli was exposed to these two compounds and washed prior to testing the effects of that exposure. This difference probably arose because compound 5 readily leaked from the cells during dilution and plating. Also, the reduced form of compound 5 exits from the cells more readily than the reduced form of PQ and then generates O2-. in the medium by autoxidation. This suggests the importance of the intracellular production of O2-., rather than the extracellular production of O2-., for lethal effect.
authors.affiliation: College of Pharmacy, Nihon University, Chiba, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Takabatake
authors.forename: M
authors.initials: M
authors.lastname: Hasegawa
authors.forename: T
authors.initials: T
authors.lastname: Nagano
authors.forename: M
authors.initials: M
authors.lastname: Hirobe
mesh_terms: D001583:Benzoxazoles / Q000633:toxicity*; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005609:Free Radicals; D005947:Glucose / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010269:Paraquat / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*
chemical_list: D001583:Benzoxazoles; D003574:Cytochrome c Group; D005609:Free Radicals; D013481:Superoxides; C073146:4,7-dicyanobenzofurazan; D005947:Glucose; D010269:Paraquat; D010100:Oxygen