Nitric Oxide in the Cytotoxicity of Metronidazole Against Trichomonas Vaginalis
Oluoch, Antony Onyango
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https://hdl.handle.net/2142/87109
Description
Title
Nitric Oxide in the Cytotoxicity of Metronidazole Against Trichomonas Vaginalis
Author(s)
Oluoch, Antony Onyango
Issue Date
2001
Doctoral Committee Chair(s)
Clarkson, Robert B.
Department of Study
Veterinary Clinical Medicine
Discipline
Veterinary Clinical Medicine
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
Using an amperometric technique (The ISO-NO meter), it was established that in aqueous solutions the rate law for auto-oxidation of NO is second order with respect to the concentration of NO, with a third order rate constant. Using a combination of electron paramagnetic resonance (EPR) techniques and fusinite, it was shown that in the ascorbate/nitrite NO generating system, the rate limiting steps are dependent on the reductant (ascorbate) and oxidant (nitrite) concentrations, and on the pH of the reaction mixture. It was established that Fe-MGD (a complex of N-methyl-D-glucamine dithiocarbarmate (MGD) and reduced iron (Fe2+)) was an efficient trap for NO in EPR. Using Fe-MGD, the reduction of metronidazole in various systems was examined. When metronidazole was incubated for one hour at 37°C with (1) sodium dithionite and (2) the enzymatic mixture consisting of ferredoxin oxidoreductase, ferredoxin, and NADPH, NO was only detected by EPR following the addition of ascorbic acid. On the other hand, the nitro anion radical was consistently produced at concentrations of >40 muM when 50 mM metronidazole was incubated with 109 Trichomonas vaginalis for one hour at 37°C regardless of whether ascorbic acid was added to the system or not. Further, in all three reduction systems, nitrite ions were separately detected using the Griess reagent. In the Lactate dehydrogenase assay, there was a positive correlation between the concentration of metronidazole, NO and the toxicity to Trichomonas vaginalis. On electron microscopy (EM), metronidazole, as well as NO, caused variable cytopathic changes to the trichomonads. On the other hand, nitrite alone at concentrations tested (20 mM) was not toxic to Trichomonas vaginalis in both the LDH assay and EM.
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