Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical.
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.
Hassett
D J
DJ
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.
Britigan
B E
BE
Svendsen
T
T
Rosen
G M
GM
Cohen
M S
MS
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*
D003486:Cyanides; D005609:Free Radicals; D006878:Hydroxides; D013308:Streptonigrin; D017665:Hydroxyl Radical; D010269:Paraquat