Nitric Oxide Chemistry Contributes To Cystic Fibrosis Research
Because of Lymar’s expertise in inorganic nitrogen chemistry, Hassett’s team sought his assistance in their observation of the bactericidal action of sodium nitrite on the mucoid forms of Pseudomonas aeruginosa.
“I felt that they were up to something potentially very important,” Lymar said. “It has quickly become clear that the species toxic to these bacteria is not the nitrite itself, but one of the downstream products of its decomposition in a slightly acidic environment. This chemistry is complex, including a peculiar mix of very rapid and slow reactions and a number of products and intermediates, so we had to resort to computer simulations to understand what is happening on the relatively long time scales typical of bactericidal assays.”
The project was quite a change of pace for Lymar, who is used to dealing with reactions occurring in microseconds. In this case, computations revealed that the reactions take hours and days to unfold. His modeling clearly pointed to nitric oxide as the most probable toxic species and also helped to design experiments that could test this and other model predictions.
“We knew from experience, that this kind of experiment is very susceptible to artifacts,” Lymar said. “One needs to work cleanly, quantitatively, with the right mixtures of gases -- and completely eliminate contact with air. When all that was done, the model predictions checked out beautifully.”
"For the mucoid bacteria, nitric oxide can be a messenger of death."
-- Sergei Lymar
Both simulations and experiments showed highly desirable properties of acidified sodium nitrite as a time-release capsule drug-delivery system. In a matter of hours, it elevated the nitric oxide content of cell cultures and then maintained these levels over days, constantly exposing bacteria to this active ingredient and killing them. Similar rates of killing were observed in a series of experiments designed to produce the same nitric oxide (NO) levels either by various combinations of nitrite concentration and acidity or by applying nitric oxide-argon gas mixtures in the absence of nitrite. Another remarkable feature of the acidified nitrite chemistry is a built-in “feedback mechanism” where the system will increase the rate of nitric oxide production in response to an attempt by bacteria to get rid of NO.
“Although we are convinced that the bactericidal action of acidic nitrite is due to nitric oxide that it generates, we are less certain about the molecular mechanism of its toxicity,” Lymar said.
One important clue might be in the dramatic decline of the bacterial population upon exposure to NO. One day after treatment, the organism population decreased by roughly 90 percent; the next day, 90 percent of what remained was eliminated.
“This means that treatment wouldn’t need to be long-term,” Lymar said. “But what it also tells us is that nitric oxide might act as a trigger of bacterial death. An organism that has survived to any given time appears “to have no idea” that it has spent a day or two in a toxic environment until something hits it. It is difficult to reconcile such behavior with slowly accumulating damage to a microbe; it rather suggests that only a small number of reactive events, or perhaps even a single event, trigger the bacterial death.”
“This pattern is consistent with a well recognized role of nitric oxide as a regulator in biology. It can trigger biochemical processes, but doesn’t necessarily participate directly in them. For the mucoid bacteria, nitric oxide can be a messenger of death.”
The research was funded by the National Institutes of Health and the Cystic Fibrosis Foundation and Lymar’s work on nitrogen oxides is supported by the U.S. Department of Energy.