Elsevier

Free Radical Biology and Medicine

Volume 67, February 2014, Pages 248-254
Free Radical Biology and Medicine

Original Contribution
Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides

https://doi.org/10.1016/j.freeradbiomed.2013.10.012Get rights and content

Highlights

  • Oxidative/protective effects of NO on prokaryotes under oxidative stress were studied.

  • NO exerts pro-oxidative synergy with H2O2 toward both gram+ and gram bacteria.

  • Nitroxide SOD mimic precludes the H2O2/NO synergy.

  • NO might release redox-active metals from cellular stores fueling the Fenton reaction.

Abstract

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60 min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a “shoulder” sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-μM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.

Graphical abstract

The combination of NO having bacteriostatic effect and H2O2 having moderate bactericidal activity results in a dramatic synergic toxicity toward microbial pathogens.

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Section snippets

Chemicals

Diethylamine NONOate sodium salt (DEA/NO) was purchased from Cayman Chemicals (Ann Arbor, MI, USA); sodium nitroprusside (SNP), 2,2,6,6,-tetramethylpiperidine-N-oxyl (TPO), and 4-hydroxy-2,2,6,6,-tetramethylpiperidine-N-oxyl (TPL) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 3-Carbamoyl-2,2,5,5-pyrrolidine-N-oxyl (3-CP) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NO was purchased from Matheson Gas Products and was purified by passing the gas through a series of

The synergic effect of NO

Addition of NO to E. coli cells subjected to H2O2 induced a synergic cytocidal effect. Fig. 1 displays survival curves observed for exponentially growing E. coli at ca. 2×106 cells ml−1 subjected to H2O2 or NO-donor DEA/NO alone and in combination. NO alone induced a bacteriostatic [53], though not bactericidal, effect. Similarly, H2O2 at ca. 1 mM inhibited the growth rate but hardly caused cell killing. The growth arrest was documented also by respective termination of the increase in OD660 nm.

Discussion

Previous studies demonstrated opposite results and arrived at contradictory conclusions characterizing the effects of NO on microorganisms subjected to oxidative stress. Further elucidation of the NO effect is important because if it enhances the bactericidal effect of H2O2 it represents a therapeutic gain in combating invading pathogens by NO without harming host cells. Conversely, bacterial defense by NO from oxidative stress implicates the bacterial NO synthase in protection of such

Conclusions

This research demonstrates that (i) NO, even at sub-μM/min flux, exerts a dramatic pro-oxidative synergic effect, which greatly potentiates H2O2-induced killing of both gram-negative and gram-positive microbial cells; (ii) NO, in the absence of H2O2, induces a bacteriostatic effect in these cells; (iii) for A. viscosus and S. mutans, and A.a., the synergic effect of NO progressively increases with the duration of exposure, possibly with a concomitant progressive loss of cellular GSH; (iv) the

Acknowledgments

This work was supported by the Queens College Research Enhancement Funds and PSC-CUNY Research Awards. This work was completed using equipment in the Queens College Core Facility for Imaging, Cellular and Molecular Biology.

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