title: Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical.
abstract: The generation of oxygen reduction products by Neisseria gonorrhoeae FA1090 upon exposure to streptonigrin (SNG) and paraquat (PQ2+) and their toxicity was examined. N. gonorrhoeae exhibited maximal cyanide-insensitive respiration, which was employed as an indicator of superoxide (O2-) formation, in the presence of 0.064 mM streptonigrin and 90 mM PQ2+, respectively. Using the concentrations of SNG and PQ2+ described above, complete lethality (greater than 10(8) cells/ml) was observed among cells exposed to SNG, whereas PQ2+ reduced viability by only 3 logs. In an attempt to determine the oxygen radical species generated by gonococci when exposed to SNG, dimethyl sulfoxide, Fe3+, KCN, and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect .OH manifested as the methyl adduct (DMPO-CH3). The production of the latter species was not inhibited by catalase, suggesting intracellular .OH generation. When PQ2+ was substituted for SNG, only low levels of DMPO-CH3 were observed, the production of which ceased within 8 min. SNG and PQ2+, added to a O2(-)- generating system in the presence of Fe3+, promoted increased .OH generation. The iron chelator diethyl-enetriaminepentaacetic acid enhanced the generation of spin-trapped .OH and O2- in the presence of PQ2+. The addition of catalase to this system, however, eliminated the DMPO-CH3 signal, showing that the .OH in this system was extracellular. PQ2+-mediated generation of extracellular .OH in the presence of Fe3+-diethylenetriaminepentaacetic acid EDTA did not enhance the killing of gonococci by PQ2+. These data show that the lethality of SNG relative to PQ2+ is due to the inherent ability of SNG to catalyze the formation of critical levels of intracellular .OH, detectable through the use of spin trapping techniques.
authors.affiliation: Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: D J
authors.initials: DJ
authors.lastname: Hassett
authors.forename: B E
authors.initials: BE
authors.lastname: Britigan
authors.forename: T
authors.initials: T
authors.lastname: Svendsen
authors.forename: G M
authors.initials: GM
authors.lastname: Rosen
authors.forename: M S
authors.initials: MS
authors.lastname: Cohen
mesh_terms: D003486:Cyanides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006878:Hydroxides / Q000378:metabolism*; D017665:Hydroxyl Radical; D007700:Kinetics; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000254:growth & development; D010101:Oxygen Consumption / Q000187:drug effects; D010269:Paraquat / Q000494:pharmacology*; D013308:Streptonigrin / Q000494:pharmacology*
chemical_list: D003486:Cyanides; D005609:Free Radicals; D006878:Hydroxides; D013308:Streptonigrin; D017665:Hydroxyl Radical; D010269:Paraquat