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Radiation Biology Branch, National Cancer Institute, Bethesda, MD, 20892
The role of nitric oxide (NO) in toxicology and pathophysiology has received considerable attention since its discovery as an endogenous mediator of different physiological functions. As additional biological aspects of NO have been discovered, speculation has emerged that this diatomic radical plays quintessential roles in various pathophysiological mechanisms. This has lead to considerable effort to understand the underlying mechanisms involving NO during pathophysiological events, to possibly point to therapeutic strategies.
The chemistry is the most important determinant of NO in biological systems. However, deciphering the chemistry of NO in biological systems can be complex with respect to its diverse biological effects and numerous potential chemical reactions. This can leave a large number of possible mechanistic explanations. To reduce these possibilities to a manageable level, we have formulated a scheme called ``the chemical biology of NO.'' This scheme describes the relevant chemical reactions of NO and predicts where they may occur in vivo.
There are two distinct categories in the chemical biology of NO: direct and indirect effects. Direct effects are those chemical reactions where NO reacts directly with its biological target. Conversely, the indirect effects are mediated by reactive nitrogen oxide species (RNOS) which are derived from NO metabolism. The direct effects are very rapid reactions that occur at low NO concentrations (< I uM) and generally involve heme proteins such as guanylate cyclase, cytochrome P450, and hemoglobin. The indirect effects require that NO is first activated by superoxide (O2-) or oxygen to form RNOS which then undergo further reactions with the respective biological target. These RNOS are highly reactive with biological macromolecules such as protein, lipids, and DNA and are thought to be responsible for NO-mediated cell death. These reactions are relevant only when the concentrations of NO are high locally (> 1 uM) for prolonged periods of time, such as in the vicinity of activated leukocytes or other cells sensitive to stimulation by cytokines or pathogenic products.
The indirect effects can be further subdivided into nitrosative and oxidative stress. Traditionally, oxidative stress has referred to conditions where oxygen radical species mediate deleterious effects in vivo. Although enzymes such as cytochrome P450 form powerful oxidants under normal physiological conditions, these chemical reactions are confined to a protein active site. Yet, when oxidants such as O2- and H2O2 reach fluxes that begin to overwhelm the endogenous defense systems, chemical alterations of biological molecules can occur such that the biological system becomes stressed. Recently, it has been shown that RNOS can mediate some of the same kind of chemistry as reactive oxygen species (ROS). This implies that NO may be metabolized to species that mediate oxidative stress.
There are two primary chemical pathways from which oxidative stress is derived: reactive oxygen species or reactive nitrogen oxide species. ROS are formed from O2- and H2O2 through a series of reactions which are referred to as Haber-Wiess chemistry. Specific transition metals, such as iron, react with peroxides/superoxide to form powerful metal-oxo species or hydroxyl radicals. In contrast, oxidative chemistry mediated by RNOS are mediated primarily by two nitrogen oxide species, peroxynitrite (ONOO-) and nitroxyl (NO-). Peroxynitrite originates from the reaction between NO and O2-, while NO- can result from a variety of chemical pathways.
It has long been known that ROS derived from hydrogen peroxide (H2O2), O2-, and alkylhydroperoxide result in powerful oxidants that can mediate cell death. Recently, there has been some discussion as to whether NO augments or abates ROS toxicity. It has been argued that ONOO- formed from the interaction of NO and O2- is a powerful oxidant, and thus must also be a potent cytotoxin . However, comparing the cytotoxicity of ROS and RNOS in fibroblast cells, it shows potency as follows: H2O2, > t-alkylhydroperoxide 
HNO/NO- >> ONOO- (intermediates such as ONOO- formed from the NO/O2- reaction are orders of magnitude less toxic). Moreover, NO is a potent antioxidant and protects cells from ROS mediated cell death. Examination of the effect of NO on ROS levels generated during ischemic reperfusion injury shows that NO dramatically reduces oxidative stress and could provide a potential therapeutic strategy.
Nitroxyl has recently been proposed as an important component of NO metabolism and may be a product of NOS. NO- can oxidize various substrates but does not mediate nitrosative chemistry. Examination of NO- chemistry showed it to be a powerful cytotoxin that mediated double stranded DNA breaks. Further studies showed that interaction between NO- and oxygen is required for cytotoxicity. The chemistry of NO- suggests that the oxidant is not ONOO- but a hydrated species (O(O)NOO3'-).
Nitrosative stress occurs when intermediates are produced from nitrosate thiol, hydroxy and amine groups. Unlike the variety of possible intermediates that could mediate oxidative stress, the primary initiator of nitrosative stress under physiological conditions is N2O3. This species is not as potent an oxidant as those that mediate oxidative stress. Rather, its primary reaction is to donate an equivalent of NO+ to different biological molecules. N2O3 is formed primarily at neutral pH either from the NO/O2 reaction or the NO/O2- reaction when the flux of NO is higher than that of O2-. Due to the rate laws of these and competing reactions, nitrosative stress can only occur in the presence of high localized fluxes of NO. Although at first glance the kinetic rate laws may suggest that nitrosative stress does not occur in vivo there are numerous reports that show nitrosation of amines under inflammatory conditions. Thus nitrosative stress (i.e., N2O3 formation) does exist in vivo.
When mammalian cells were exposed to nitrosative stress by addition of NO donor little toxicity was observed (< 20–40%). Nitrosating intermediates have the greatest affinity for thiols such as glutathione (GSH) suggesting that they are a primary target under nitrosative stress conditions. Cells depleted of GSH were dramatically more susceptible to toxicity from nitrosative stress. In addition, proteins rich in thiols such as metallothionein were also shown to be protective against N2O3. However, if toxic metals such as cadmium are sequestered in metal I othionein, nitrosative stress resulted in release of metal and a subsequent marked enhancement of metal mediated toxicity. It has been reported using Raman spectroscopy that a RSNO adduct is formed upon exposure to NO in an aerobic environment. From these studies, it was suggested that the formation of S-nitrosothiol adducts under conditions of nitrosative stress play a key role in various toxicological mechanisms.
In several studies, the formation of S-nitrosothiol adducts with protein has been suggested as the important step in the inhibition of a variety of enzymes. For example, S-nitrosothiol adduct stimulates ADP ribosylation of glyceraldehyde phosphate dehydrogenase. Additionally, S-nitrosation inhibits DNA alkyl transferase activity both in vitro and in vivo and results in the potentiation of the toxicity of alkylating agents such as chemotherapeutic agents. Proteins containing zinc finger motifs lose their structural integrity upon exposure to NO, resulting in inhibition of enzyme activity.
References
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