William A. Pryor

Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide.

The roles of superoxide (O2.-), peroxynitrite, and carbon dioxide in the oxidative chemistry of nitric oxide (.NO) are reviewed. The formation of peroxynitrite from .NO and O2.- is controlled by superoxide dismutase (SOD), which can lower the concentration of superoxide ions. The concentration of CO2 in vivo is high (ca. 1 mM), and the rate constant for reaction of CO2 with -OONO is large (pH-independent k = 5.8 x 10(4) M(-l)s(-1)). Consequently, the rate of reaction of peroxynitrite with CO2 is so fast that most commonly used scavengers would need to be present at very high, near toxic levels in order to compete with peroxynitrite for CO2. Therefore, in the presence of physiological levels of bicarbonate, only a limited number of biotargets react directly with peroxynitrite. These include heme-containing proteins such as hemoglobin, peroxidases such as myeloperoxidase, seleno-proteins such as glutathione peroxidase, proteins containing zinc-thiolate centers such as the DNA-binding transcription factors, and the synthetic antioxidant ebselen. The mechanism of the reaction of CO2 with OONO produces metastable nitrating, nitrosating, and oxidizing species as intermediates. An analysis of the lifetimes of the possible intermediates and of the catalysis of peroxynitrite decompositions suggests that the reactive intermediates responsible for reactions with a variety of substrates may be the free radicals .NO2 and CO3.-. Biologically important reactions of these free radicals are, for example, the nitration of tyrosine residues. These nitrations can be pathological, but they also may play a signal transduction role, because nitration of tyrosine can modulate phosphorylation and thus control enzymatic activity. In principle, it might be possible to block the biological effects of peroxynitrite by scavenging the free radicals .NO2 and CO3.-. Because it is difficult to directly scavenge peroxynitrite because of its fast reaction with CO2, scavenging of intermediates from the peroxynitrite/CO2 reaction would provide an additional way of preventing peroxynitrite-mediated cellular effects. The biological effects of peroxynitrite also can be prevented by limiting the formation of peroxynitrite from .NO by lowering the concentration of O2.- using SOD or SOD mimics. Increased formation of peroxynitrite has been linked to Alzheimers disease, rheumatoid arthritis, atherosclerosis, lung injury, amyotrophic lateral sclerosis, and other diseases.

Autoxidation of polyunsaturated fatty acids: II. A suggested mechanism for the formation of TBA-reactive materials from prostaglandin-like endoperoxides

The nature and mechanism of formation of the thiobarbituric acid (TBA)-reactive material produced in the autoxidation of polyunsaturated fatty acids (PUFA) or their esters has been studied. On the basis of chemical studies and

The kinetics of the autoxidation of polyunsaturated fatty acids

The kinetics of the autoxidation of a series of polyunsaturated fatty acids (PUFA) with increasing degrees of unsaturation and the mono-, di-and triglycerides of linoleate have been studied in homogeneous chlorobenzene solution at 37 C under 760 torr of oxygen. The autoxidations were initiated by thermal decomposition of azo initiators and followed by measuring the rate of oxygen uptake. The rate of chain initiation was determined by the induction period method using α-tocopherol as the chainbreaking antioxidant. The measured oxidizabilities of the PUFA are linearly dependent on the number of doubly allylic positions present in the molecule. Thus, the oxidizability of linoleate is 2.03×10−2 M−1/2 sec−1/2, and the value for docosahexaenoate is five times greater, 10.15×10−2 M−1/2 sec−1/2. The rate of autoxidation for all PUFA studied and for the mono- and diglyceride is proportional to the substrate concentration and to the square root of the rate of chain initiation, implying that the autoxidation of these compounds follows the usual kinetic rate law. The autoxidation of the triglyceride is more complex and does not appear to follow the same rate law at all substrate concentrations. This deviation from the usual kinetic rate expression may be due to lipid aggregation at low concentrations of the triglyceride.

The role of free radicals in asbestos-induced diseases

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.

Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter.

The health effects of airborne fine particles are the subject of government regulation and scientific debate. The aerodynamics of airborne particulate matter, the deposition patterns in the human lung, and the available experimental and epidemiological data on health effects lead us to focus on airborne particulate matter with an aerodynamic mean diameter less than 2.5 microm (PM(2.5)) as the fraction of the particles with the largest impact in health. In this article we present a novel hypothesis to explain the continuous production of reactive oxygen species produced by PM(2.5) when it is deposited in the lung. We find PM(2.5) contains abundant persistent free radicals, typically 10(16) to 10(17) unpaired spins/gram, and that these radicals are stable for several months. These radicals are consistent with the stability and electron paramagnetic resonance spectral characteristics of semiquinone radicals. Catalytic redox cycling by semiquinone radicals is well documented in the literature and we had studied in detail its role on the health effects of cigarette smoke particulate matter. We believe that we have for the first time shown that the same, or similar radicals, are not confined to cigarette smoke particulate matter but are also present in PM(2.5). We hypothesize that these semiquinone radicals undergo redox cycling, thereby reducing oxygen and generating reactive oxygen species while consuming tissue-reducing equivalents, such as NAD(P)H and ascorbate. These reactive oxygen species generated by particles cause oxidative stress at sites of deposition and produce deleterious effects observed in the lung.

The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products.

Ozone is so reactive that it can be predicted to be entirely consumed as it passes through the first layer of tissue it contacts at the lung/air interface. This layer includes the lung lining fluid (tracheobronchial surface fluid and alveolar and small airway lining fluid) and, where the lung lining fluid is thin or absent, the membranes of the epithelial cells that line the airways. Therefore, the biochemical changes that follow the inhalation of ozone must be relayed into deeper tissue strat by a cascade of ozonation products. Lipid ozonation products (LOP) are suggested to be the most likely species to act as signal transduction molecules. This is because unsaturated fatty acids are present in the lipids in both the lung lining fluid and in pulmonary cell bilayers, and ozone reacts with unsaturated fatty acids to produce ozone-specific products. Further, lipid ozonation products are finite in number, have structures that are predictable from the Criegee ozonation mechanism, and are small, diffusible, stable (or metastable) molecules. Preliminary data show that individual LOP cause the activation of specific lipases, which trigger the release of endogenous mediators of inflammation.

How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts?

A simple method is suggested for calculating the time it takes ozone to traverse a biological region, such as a bilayer or a cell, and comparing this time to the halflife of ozone within that region. For a bilayer the calculations suggest that most of the ozone reacts within a bilayer, but a fraction may exit unreacted. For the lung lining fluid layer (LLFL), the calculations show that ozone cannot cross this layer without reacting where the LLFL is thicker than about 0.1 microns. However, since the LLFL varies from 20 to 0.1 microns in thickness with patchy areas in the lower airways that are virtually uncovered, some ozone could reach underlying cells, particularly in the lower airways. For cells (such as alveolar type I epithelial cells), the calculations show that ozone reacts within the cell too rapidly to pass through and exit unreacted from the other side. These calculations have implications for ozone toxicity. In vivo, the toxicity of ozone is suggested to result from the effects of a cascade of products that are produced in the reactions of ozone with primary target molecules that lie close to the air/tissue boundary. These products, which have a lower reactivity and longer lifetime than ozone itself, can transmit the effects of ozone beyond the air/tissue interface. The variation in thickness of the LLFL may modulate the species causing damage to the cells below it. In the lower airways, where the LLFL is thin and patchy, more cellular damage may be caused by ozone itself; in the upper airways where the LLFL is thicker, secondary products (such as aldehydes and hydrogen peroxide) may cause most of the damage. In vitro studies must be designed in an attempt to model the lung physiology. For example, if cells in culture are studied, and if the cells are exposed to ozone while under a supporting medium solution that contains ozone-reactive substances, then the cells may be damaged by products that are formed in the reactions of ozone with the cell medium rather than by ozone itself.

Mechanisms of radical formation from reactions of ozone with target molecules in the lung

Ozone is known to cause radicals to be formed in biological systems: for example, it initiates lipid peroxidation and vitamin E protects in vitro model systems, cells, and animals against the effects of ozone. Ozone is not itself a radical, and we have asked: With what molecules does ozone react in the lung and how are radicals produced? Ozone reacts by two quite different mechanisms to produce radicals; one involves an ozone-olefin reaction and the other a reaction with electron donors such as glutathione (GSH). The first mechanism splits an R radical out of an olefin with the structure R-CH = CH2. The R then reacts with dioxygen to become a peroxyl radical (ROO), and both carbon- and oxygen-centered radicals can be detected by the electron spin resonance spin trap method. From the effects of temperature, metal chelators, and water, it is concluded that ozone reacts by the Criegee ozonation pathway to give the classical 1,2,3-trioxolane, which then undergoes O--O bond homolysis to form a diradical. This diradical then either undergoes beta-scission to split out the R radical or forms the more usual carbonyl oxide and a carbonyl compound. (See Figure 3 in the text). The low yield of Criegee ozonide that is generally obtained probably is due in part to the reactions forming radicals from the 1,2,3-trioxolane that compete with production of the Criegee ozonide. The second mechanism for radical production involves the reaction of ozone with electron donors. If the electron donor is, for example, GSH or its ion (GS-), this reaction produces the thiyl radical GS. and 0.3-. The ozone radical anion then reacts with a proton to form the hydroxyl radical and dioxygen: O3.- + H+-->HO. and O2. Using 5,5-dimethyl-1-pyrroline-N-oxide, the spin adduct of the hydroxyl radical is detected. Similar reactions are observed with catechol.

FREE RADICAL BIOLOGY: XENOBIOTICS, CANCER, AND AGING*

It was not very long ago that free radical biology was a rather arcane subject, regarded with disinterest (or even disbelief) by most biologists-a sea of speculation, but few islands of solid fact. Three striking and seminal discoveries have drastically changed this field. The first was the elucidation by McCord and Fridovich of the nature, function, and role of superoxide dismutase (SOD].’ In fact, SOD is now the world’s most studied enzyme; journal articles, conference proceedings, and books on superoxide chemistry and biology are appearing at a very rapid pace, but we probably are still just seeing the tip of the iceberg. The second discovery involves the biosynthesis of peroxidic compounds from arachidonic acid-products of what is called the “arachidonate cascade.”‘ Not more than 10 years ago, it was thought that lipoxygenase activity was limited to plants and that lipid hydroperoxides were not formed in animal cells. In fact, there was even concern that lipid hydroperoxides in plants might be in vitro artifacts and play no role in metabolism. Now it is clear that an array of cyclic and bicyclic peroxides as well as acyclic lipid hydroperoxides not only occur in all animal cells, but play a very critical role in bioregulation and in many vital normal and pathological processes. The third discovery, although perhaps more distant from traditional biochemical interests, also is having a major impact on current research. It is becoming clear that many important environmental toxins exert their effects through radical-mediated reaction^.^.^ In fact, a majority of the compounds that are positive in the Ames test may involve radical-mediated reactions. In this article, I will summarize current thinking on the in vivo sources of radicals. I will cover both exogenous sources-xenobiotics, pollutants, carcinogens, etc.-and endogenous sources-superoxide systems and the arachidonic cascade. And I will close by making some remarks about the effects of antioxidants on carcinogenesis and on the retardation of aging. Thus, I intend to skate from thick ice to thinner and thinner ice: with good timing, I should fall into the refreshingly cool water just at the end of my talk.

Mechanisms of Nitrogen Dioxide Reactions: Initiation of Lipid Peroxidation and the Production of Nitrous Acid

The reactions of nitrogen dioxide with cyclohexene have been studied as a model for the reactions that occur between nitrogen dioxide in smoggy air and unsaturated fatty acids in pulmonary lipids. As predicted from earlier studies at high nitrogen dioxide concentrations, this gas reacts with cyclohexene predominantly by addition to the double bond at nitrogen dioxide concentrations of 1 percent (10,000 parts per million) to 40 percent in nitrogen; in the presence of air or oxygen, this reaction initiates the autoxidation of the alkene. However, at concentrations below 100 parts per million in nitrogen, nitrogen dioxide reacts with cyclohexene almost exclusively by abstraction of allylic hydrogen; this unexpected reaction also initiates the autoxidation of the alkene in the presence of oxygen or air, but it leads to the production of nitrous acid rather than of a product containing a nitro group attached to a carbon atom. The nitrous acid can react with amines to produce nitrosamines. Moreover, the nitrite ion produced by the hydrogen abstraction mechanism would be expected to diffuse throughout the body, unlike nitrated lipids that would be confined to the pulmonary cavity. These findings have been confirmed with methyl oleate, linoleate, and linolenate; some of the kinetic features of the nitrogen dioxide—initiated autoxidation of these unsaturated fatty acids have been studied.