Synergistic toxicity of reactive oxygen species

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Hydrogen peroxide is a bio-specific toxin, inert with common organic or inorganic molecules, but in sufficient concentrations killing any type of cells almost on contact, using cellular iron to produce highly-reactive oxidizing species (Fenton's reaction). Chromosomal DNA is the main cellular target of HP poisoning, although the nature of lethal chromosomal damage is still unknown. Even more confusingly, HP turns out to be a surprising choice for a bio- weapon, as the killing concentrations are 1,000-fold higher than the physiological ones, while accumulation of HP in a particular cellular compartment is problematic, because its small size and uncharged nature facilitates diffusion through membranes. Yet, our immune cells somehow use these much lower HP concentrations to efficiently kill invading microbes, by unclear mechanisms. Remarkably, HP toxicity is synergized by other simple molecules, like nitric oxide (NO) or cyanide (CN). We hypothesize that our immune cells use otherwise insufficient HP concentrations to kill bacteria by accumulating potentiator molecules (for example, NO) in the compartments where high [HP] does not accumulate. However, the metabolic mechanisms behind potentiated HP toxicity, thought to elevate the intracellular free iron, are inconsistent with the newest results, while the mechanisms of irreparable chromosomal damage remain mostly unclear. Our recent studies of HP+CN co-toxicity uncovered new aspects of the phenomenon, like iron recruitment from intracellular depots directly to DNA, or catastrophic chromosome fragmentation, which is responsible for the cell killing. We propose to study HP+NO co-toxicity with three specific aims: 1) the nature of this synergistic toxicity and the targets of NO action; 2) the DNA mechanisms behind the catastrophic chromosomal fragmentation; 3) hunger shock as a metabolic potentiator of HP toxicity. The major difference of our approach from the previous work is the emphasis on DNA and chromosome damage, on the significantly expanded genetic scope and on testing specific ways to enhance Fenton. Our long-term goals in this project are to understand the mechanisms behind the catastrophic chromosome fragmentation on the one hand, and those responsible for potentiation of HP toxicity on the other.