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INVITED REVIEW
1Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana; 2Department of Chemistry and Biochemistry, University of California, Los Angeles, California; 3Department of Pharmacology, University of California at Los Angeles School of Medicine, Center for the Health Sciences, Los Angeles, California; 4Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, Birmingham, Alabama; and 5Ethel Percy Andrus Gerontology Center and Division of Molecular and Computational Biology, University of Southern California, Los Angeles, California
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antioxidants; antioxidant defense and repair; nitric oxide; carbon dioxide; hydrogen sulfide; carbon monoxide; ozone; hydrogen peroxide; dissolved gases; oxidants
The mechanism by which oxygen expresses its toxicity is a long-studied problem. It is now known that the superoxide radical ion (O2·–) is the key to understanding this classic problem. Therefore, we also discuss this radical ion, which is present in all aerobic cells. Superoxide can damage certain types of species directly. For example, certain easily oxidized molecules such as ascorbate and some phenols and thiols can be oxidized by superoxide, and this can lead to inactivation of enzymes. Often, superoxide is converted to hydrogen peroxide by reduction or by dismutation, and then hydrogen peroxide is converted to the hydroxyl radical (HO·). This conversion most often involves metal ions, generally iron. The hydroxyl radical is one of the most reactive species known in chemistry, able to abstract an unactivated hydrogen atom at room temperature, and HO· reacts with most of the species it collides with at a rate constant very near the diffusion limit.
Superoxide can lead to the production of hydrogen peroxide, which is found in human fluids and exhaled breath (128, 133, 222, 232) but can be toxic. Hydrogen peroxide and superoxide may be separately regulated and, although each can cause pathological damage, both also function in normal cell regulation and signaling (48, 109, 196, 217, 218, 225). The interaction of superoxide-producing systems with iron is important in understanding superoxide chemistry, and we spend some time on this system.
We also discuss ·NO, peroxynitrous acid, and its conjugate base, peroxynitrite. Nitric oxide, a stable free radical, is a hormone-like species that contains no carbon atoms. (This remarkable discovery was recognized in 1998 with the award of a Nobel Prize to Robert Furchgott, Louis Ignarro, and Ferid Murad.) Peroxynitrite (–O-O-N
We also discuss nitrogen dioxide, long studied in smog, but also formed in vivo when nitric oxide is present. It has recently been emphasized (210) that the combination of nitrogen dioxide (·NO2) and superoxide forms peroxynitrate (O2N-O-O–), the conjugate base of peroxynitric acid (O2N-OO-H).
There are two other forms of reactive oxygen: singlet oxygen, an excited form of oxygen in which all the electrons are paired, and ozone, O3, a nonradical that is a potent oxidant in photochemical smog. Ozone is known to greatly accelerate the air oxidation of biomolecules (50, 170, 178, 179, 182).
We also discuss carbon monoxide and hydrogen sulfide, which have fundamental cell signaling properties and effect oxidative stress. There are other gases that either respond to, or directly affect, the level of radicals, oxidants, and oxidative stress. Included in the list are ethylene (13, 25, 183) and a number of others. Perhaps this account will encourage others to take up the biochemistry of these other gases.
Our purpose here is to group together a discussion of these gases and to focus attention on the surprising fact that nature chose gases to perform vital biological functions involving oxidative stress.
Free radical biology has often been controversial. The word "radical" itself may be part of the problem, evoking visions of unkempt men stirring up revolution in coffeehouses. Vitamin E, nature's favorite antioxidant (19, 101), also has had a controversial history, since the original biodetection method, rat fetus resorption, led to the early association of vitamin E with human sexual function, despite the fact that vitamin E deficiency does not cause fetus resorption in mice, humans, or most other species.
1) Oxygen: Triplet And Singlet
The electronic ground state of dioxygen is a triplet (3O2). Since it has two unpaired electrons, both with the same spin, it also is a diradical. Pairing these electrons to give one with "up" spin and one "down" gives a singlet state (1O2). This takes energy, and the singlet is 1 eV (23 kcal/mol or 94 kJ/mol) higher in energy than the ground state triplet.
The triplet state of oxygen (its ground state), 3O2, acts as a diradical, a one-electron oxidant (albeit a fairly poor one), whereas singlet 1O2 acts as a more potent, often two-electron, oxidant that adds to 
bonds, undergoes ene reactions, and can insert into CH bonds. The nearly unique triplet ground state of oxygen occurs because the two highest energy electrons reside in identical * molecular orbitals. In such a case, the triplet state is lower in energy because electrons with the same spin cannot occupy the same region of space (the Pauli exclusion principle). Electrons with opposite spin, as in the singlet state, can occupy the same region of space, but because the electrons are closer together on average, repulsion between these electrons is greater, and so the singlet state is higher in energy than the triplet.
Oxygen atoms are present in a variety of minerals, and oxygen is the most abundant element in the Earth's crust (93). Triplet oxygen supports metabolism through respiration. In mammalian systems, oxygen binds to hemoglobin, a marvelously tuned carrier of oxygen; hemoglobin moves oxygen through the organism and uses cooperative binding to maximize uptake and release to tissues that require O2. When released, O2 is involved in a series of complex redox cycles, ultimately being reduced to water and giving ATP.
Singlet oxygen is not produced by simple thermal processes from 3O2, but requires intervention of high energy molecules. Singlet oxygen can be produced by energy transfer from the excited triplet states of aromatic dyes, like Rose Bengal, and also by fullerenes. These reactions occur with little or no activation enthalpy; Diels-Alder reactions, ene reactions, and 2+2 cyclo-additions with electron-rich alkenes (D = electron donor) are common singlet oxygen reactions, as shown in the three reactions below (reactions 1–3) (62).
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| (1) |
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| (2) |
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| (3) |
Note these products are peroxides, and like typical peroxides these can generate radicals that can undergo subsequent reactions.
Singlet oxygen is produced in several physiological processes, but whether it has any natural function or physiological relevance has been a matter of controversy. Neutrophils produce hypochlorous acid, HOCl, and hydrogen peroxide, H2O2, and these can react to generate singlet oxygen (reaction 4).
 | (4) |
Singlet oxygen has a rather long lifetime in the gas phase but decomposes more rapidly in solution by a variety of nonradiative processes. Nevertheless, at one time, it was suggested that superoxide dismutase (SOD) was actually singlet oxygen decontaminase (161), a claim that has since faded away (204). SOD has nothing to do with singlet O2!
Singlet oxygen does have a role in medical interventions in biology, as the active species formed in photodynamic therapy. Sensitizers such as porphyrins in tumor cells are irradiated and 7sensitize the formation of singlet oxygen that ultimately kills tumor cells. A disease, porphyria, is related to a deposition of porphyrins that can cause deleterious formation of singlet oxygen upon exposure to sunlight on skin. The legend of vampires may have developed from porphyria-stricken people, who had to avoid sunlight to avoid photosensitized singlet oxygen generation.
Recently it was proposed by Wentworth et al. (242, 243) that singlet oxygen from neutrophils can oxidize water through antibody catalysis. These workers proposed that antibodies might not only have a long-reorganized pathogen recognition function but antibodies might also have once had a killing function through the generation of hydrogen peroxide (reaction 5) (242, 243).
 | (5) |
This reaction, as shown above, is endergonic by 28.1 kcal/mol, according to thermodynamic values in the 2005 NIST Chemistry WebBook database (2) or high-level quantum mechanical calculations (K. N. Houk and P. R. McCarren, unpublished observations). Because of unfavorable energetics, a research team at Scripps (242) has proposed that two or three singlet oxygens are required for each two hydrogen peroxide molecules produced (reactions 6 and 7). The energetics of these processes are:
 | (6) |
 | (7) |
Wentworth et al. (242) have performed calculations that show how water molecules oriented in an antibody could catalyze H2O2 formation. Interestingly, these reactions involve both hydroperoxyl and hydrotrioxyl radicals as intermediates. However, no mechanism has been proposed to explain how the conversion of two singlet oxygens to two triplet oxygens can be coupled to the reaction of singlet oxygen with water to form hydrogen peroxide. More recent publications (244, 245) have suggested that trioxygen species, including ozone, are produced by antibody catalysis. These reports are described below in the fourth section, titled Ozone.
2) Oxygen Toxicity and Superoxide
It has been known for many years that oxygen can be toxic, but the detailed mechanisms of oxygen toxicity are still under investigation. An enormously influential, watershed advance was made in 1969, when Joe McCord and Irwin Fridovich (139) reported the isolation of an enzyme from bovine red blood cells. They named the enzyme superoxide dismutase, SOD, and showed that it catalyzed the dismutation of superoxide (reaction 8) to form oxygen and hydrogen peroxide (139).
 | (8) |
A search for "superoxide" in the biological literature for 1968 and prior years gives 520 "hits," with 56 publications in the year 1968 alone, the year of the publication of McCord and Fridovich. (The name perhydroxide, HOO·, used in the early years, does gather some hits for 1968 and prior years; SciFinder Scholar gives 9 hits for this term for during this period.) At the beginning of 2006, there were 113,692 hits on the word "superoxide"! The study of this species is now important in the fields of chemistry, biochemistry, biology, and medicine.
The very existence of SOD implied a revolutionary concept. The notion that superoxide plays a role in biology and medicine presupposes that oxygen, the prototypical oxidant in nature, can form superoxide, a species that usually reacts as a reductant. In the mid-20th century when SOD was discovered, radicals were regarded as too reactive and unselective to play any role in biology. (Radicals were thought to have a role that was limited to the chemistry that occurs in the stratosphere and in smog in the biosphere.) Radicals were believed to be too uncontrollable to occur in any reactions involving an enzyme; radicals in cells were viewed as a "bull in a china shop."
Even the notion of organic "free" radicals had been controversial. The 19th century literature on the "impossibility" of radicals existing in nature is amazing to read in the light of current knowledge (167, 171). The existence of radicals was finally made real for organic chemists by the work of Gomberg on triphenylmethyl radicals in the years 1900–1910 and for physical chemists by the work of Paneth on small aliphatic radicals in 1929 (166). In the 20th century, early proponents of free radicals in biology argued that all redox reactions must occur one electron at a time (because coincidence of electron movement would be unexpected), and therefore all redox reactions must produce free radicals. For example see Michaelis (141, 168) and Szent-Györgyi and colleagues(67, 219–221). In the 1950s, Westheimer et al. (246) published a series of papers that implied that the oxidation of ethanol to acetaldehyde as catalyzed by the enzyme alcohol dehydrogenase occurred by a direct, stereospecific transfer of hydrogen (55) and therefore involved the hydride ion moving with two electrons at one time without free radical formation. This discovery, as well as the mistaken belief that all radicals are extremely reactive and very short lived, led to the acceptance of the incorrect idea that radicals1 could not exist in vivo (169, 171).
This analysis overlooked the difference between the fleeting transfer of electrons one-at-a-time and actually observing and/or isolating free radicals, which would require a lifetime of more than just a few vibrations. Now that fast flash and pulse methods are available to identify very short-lived intermediates, the difference between the theoretical occurrence of one-electron intermediates and the actual observation and/or isolation of radical intermediates is understood.
Another problem with the early analysis is the assumption that all radicals are "a bull in a china shop." In fact, radicals vary in reactivity (and therefore lifetimes) by some 10 orders of magnitude! Some radicals, such as the hydroxyl radical, react near the diffusion limit with the first molecule they bump into, whereas others, such as semiquinones, are stable for days, weeks, or months (172). So the "bull" may be reactive but also has his contemplative moments.
Hydrogen peroxide, which is an oxidant but not a radical, is produced in the SOD-catalyzed dismutation of superoxide (reaction 8). For this reason, catalase, which catalyzes the dismutation of hydrogen peroxide to oxygen and water (reaction 9), often is codistributed in tissue along with SOD.
 | (9) |
The discovery of SOD did two things that had enormous impact. First, the ready availability of SOD allowed simple tests to be devised to prove the presence of the superoxide radical in biological systems and thus provided a powerful tool to resolve the long-standing debate on whether radicals could exist in cells. And second, as described in a review by Imlay (100), the role for superoxide in biology allowed the demonstration of the molecular mechanisms for oxygen toxicity and the involvement of superoxide.
The discovery of SOD was unquestionably an event of such enormous consequence and influence that it should have resulted in a Nobel Prize. Why has this Prize not been awarded? Initially the role of SOD was greeted with skepticism by some. Various arguments were advanced to support the idea that SOD must have another function, including the fact that superoxide dismutates spontaneously and does not "require" a catalyst (53). This argument is spurious; hydrogen peroxide is relatively unreactive as an oxidant, but there has never been any doubt that catalase exists to catalyze the dismutation of hydrogen peroxide to water and oxygen. The arguments by McCord and Fridovich (139), that SOD does not have the function assigned to it, have been demolished with a flood of publications on SOD, covering every aspect of its chemistry and biology. The tone of the debate on the subject of the role of SOD can be judged from a 1981 discussion of a paper presented by Fee (53).
In these early years when the role of SOD in biology was being debated, the role of nitric oxide and peroxynitrite in biology was not yet known. Now that the oxidizing power of peroxynitrite is known, it can be appreciated that SOD removes superoxide from systems containing nitric oxide and thus prevents the rapid reaction of nitric oxide and superoxide to form the potent oxidant peroxynitrite (discussed in more detail below) (180).
2.1) Oxidative stress. Sies stated, "A disturbance in the pro-oxidant/antioxidant systems in favor of the former may be denoted as an oxidative stress" (202). Many of the gases (and derived species) we review are biological oxidants that can initiate reactions that ultimately cause oxidative stress. Indeed, oxidative stress is a mechanism of toxicity (in many cases the major mechanism) for most of the gases we discuss. Oxidative stress can result from increased exposure to oxidizing gases or from a lessened protection against oxidants; both problems may occur simultaneously. Oxidative stress can cause damage to proteins, lipids, carbohydrates, and nucleic acids (21, 22, 43, 61, 83, 202). Thus cellular enzymes and structural proteins, membranes, simple and complex sugars, and DNA and RNA are all susceptible to oxidative damage (21, 22, 43, 61, 83, 202). Surviving an oxidizing environment is actually one of the greatest challenges faced by living organisms. The concept that we gradually surrender to a form of organic "rust" led to the free radical theory of aging (86, 159).
2.2) Antioxidant defense and repair systems. The first-level cellular response to oxidative stress is to use antioxidant defense and repair systems to minimize the damage that occurs and to remove or repair whatever cellular components were damaged (21, 22, 43, 61, 80, 83, 159, 202, 231, 237). Although living organisms may eventually succumb to oxidant-induced aging, oxidatively damaged cellular constituents are maintained at very low levels for most of an organism's life span. Damage accumulation is kept low by multiple interacting systems of antioxidant compounds, antioxidant enzymes, damage-removal enzymes, and repair enzymes.
Antioxidant compounds include the vitamins C and E, ubiquinone, uric acid, glutathione, and others. Antioxidant compounds are sacrificially oxidized to protect more important cellular components (21, 22, 43, 83, 202). Antioxidant enzymes, such as superoxide dismutases, glutathione peroxidases, and quinone reductases, act catalytically to convert oxidants to less reactive species (43, 61). The essential cytoplasmic and nuclear proteolytic enzyme, proteasome, recognizes and selectively degrades oxidized proteins (43, 80, 159). Oxidized membrane lipids are recognized and selectively removed by lipases, particularly phospholipase A2 (43, 159, 231). Oxidized DNA is subject to removal or excision repair by a wide series of DNA repair enzymes including endonucleases, glycosylases, polymerases, and ligases (43, 159, 237).
Oxygen concentration is tightly controlled in multicellular organisms, directly limiting oxidation of biopolymers by oxygen-centered oxidants. In humans, oxygen concentration falls from atmospheric levels (20%) in the lungs to 2–5% in the tissues. The very low Michaelis constant, Km, of mitochondrial cytochrome oxidase for oxygen (23) assures that most oxygen in cells is bound and consumed by the cytochrome oxidase complex. Thus one component of oxidative stress defense is to keep cellular oxygen tension to a minimum. This fact is often overlooked by scientists performing cell culture experiments at atmospheric oxygen levels.
As described above, aerobic organisms have evolved a multitude of ways to protect themselves from the deleterious aspects of an aerobic existence. It has also become increasingly clear that cells have the capacity to adapt to the presence of oxidative stress via the activation of signaling pathways regulated by oxygen and oxygen-derived species. Moreover, it appears that oxygen-derived species are part of normal cell signaling processes. In the following discussion, examples of these types of effects are presented.
2.3) Mitogenic effects of low oxidant concentrations. Exposure of dividing mammalian cells in culture to very low concentrations of many oxidants actually stimulates cell growth and division. This fascinating mitogenic effect is seen, for example, with exposure of fibroblasts to hydrogen peroxide at 3–15 µM, i.e., 0.1–0.5 µmol/107 cells (17, 18, 23, 247). Presumably such low oxidant concentrations do not cause a true oxidative stress. In all probability, low oxidant levels are signals for mitosis, although the mechanism is not fully understood. Interestingly, this growth-stimulatory effect of very low oxidant concentrations is also seen with bacteria (26, 46) and yeast cells (42).
2.4) Temporary growth arrest as a defense against mild-to-moderate oxidative stress. Although very low oxidant exposure is mitogenic, mild-to-moderate oxidative stress (e.g., 120–150 µM hydrogen peroxide, or 2–5 µmol/107 cells) typically causes a temporary growth arrest in mammalian fibroblasts (247). This temporary growth arrest lasts for some 2–4 h and appears to be caused by expression of the gadd45, gadd153 (12, 57, 58), and adapt15 genes (37, 38). Many investigators have treated all growth-arrest responses as toxic outcomes of oxidant exposure, but it is now clear that temporary growth arrest is actually a defense mechanism. During oxidant-induced temporary growth arrest, the expression of many housekeeping genes is halted, while expression of a select group of shock or stress genes is induced. Similarly, proliferating cells exposed to mild or moderate oxidative stress shut off expression of all but the most essential shock/stress genes, supercoil their DNA to protect it against oxidation, and conserve resources for future use, during temporary growth arrest.
In this review the term "mild-to-moderate oxidative stress" is defined by cellular responses, not by hydrogen peroxide concentrations. Mild stress involves little or no cell death (either by apoptosis or necrosis), and no long-term inhibition of cell division, DNA synthesis, or protein synthesis. In fact, cells exposed to mild-to-moderate oxidative stress exhibit adaptive responses, with increased expression of protective and repair genes.
2.5) Transient adaptation with mild-to-moderate oxidative stress.
Very important early studies on transient adaptation to oxidative stress were performed by Spitz et al. (206) and by Laval (129). After 4–6 h of temporary growth arrest, many cells exposed to moderate oxidative stress (e.g., 120–150 µM hydrogen peroxide, or 2–5 µmol/107 cells) undergo further changes that can be characterized as transient adaptation to oxidative stress. In mammalian fibroblasts (32, 33, 35–38, 129, 130, 206, 240, 247) maximal adaptation is seen 
18 h after initial exposure to hydrogen peroxide, i.e., some 12–14 h after they exit from temporary growth arrest. In bacteria such as Escherichia coli and Salmonella, maximal adaptation is seen 20–30 min after oxidant exposure (26, 33, 46), whereas yeast cells require some 45 min (33, 42).
The adaptation referred to in the title of this section means increased resistance to mild-to-moderate oxidative stress, as measured by cell proliferation capacity. Furthermore, the adaptation is transient, lasting some 18 h in mammalian fibroblasts, 90 min in yeast, and only 60 min in E. coli. It is especially important to avoid selecting for preexisting resistant cells in the population, by repeatedly checking that transiently adapted cells can actually de-adapt. In both prokaryotes and eukaryotes, transient adaptation to oxidative stress depends upon transcription and translation. A large number of genes undergo altered expression during the adaptive response. Some genes are upregulated, some are downregulated, and some are modulated early in the adaptation, while the expression of others is affected at later times. Inhibiting either transcription or translation during the adaptive response greatly limits the development of increased H2O2 resistance. If both transcription and translation are inhibited, then little or no adaptation will occur. Therefore, the transient adaptive response to oxidative stress depends largely on altered gene expression but partly on increased translation of preexisting mRNAs. It further appears that message stabilization (for some mRNAs), increased message degradation (for other mRNAs), and altered precursor processing, are all involved in altered translational responses (32, 33, 35–38, 42, 43, 130, 240, 247). Studies in E. coli and Salmonella have shown that two particular regulons are responsible for many of the bacterial adaptive responses to oxidative stress: the oxyR regulon (214) and the soxRS regulon (79). In mammalian cells no "master regulation molecules" have been found, but at least 40 gene products are involved in the adaptive response. Several of the mammalian adaptive genes are involved in antioxidant defenses, and others are damage removal or repair enzymes. Many classic shock or stress genes are involved very early in adaptive responses. As indicated in section 2.4 (Temporary growth arrest as a defense against mild-to-moderate oxidative stress) above, gadd153, gadd45, and adapt15 play important roles in inducing temporary growth arrest, which is a very important early portion of the adaptive response to oxidative stress (12, 37, 38, 57, 58, 247). The transcription factor, adapt66 (a mafG homologue) is probably responsible for inducing the expression of several other adaptive genes (36). A number of other "adapt" genes have recently been discovered, but their functions are not yet clear. One of these genes is the calcium-dependent adapt33 (240), and another is adapt73, which appears to also be homologous to a cardiogenic shock gene called PigHep3 (32). Adapt78 has also been called DSCR1 and RCAN1 (35, 130) and, in addition to its induction during oxidative stress adaptation, now appears to be involved in Down syndrome and Alzheimer disease (49, 87).
Numerous other genes have been shown to be inducible in mammalian cell lines following exposure to moderate oxidative stress that will cause transient adaptation. These include the protooncogenes c-fos and c-myc (30), c-jun, egr, and the platelet-derived growth factor-inducible "early" gene JE (147, 150, 199). Similar oncogene induction has also been reported following exposure to tert-butylhydroperoxide (147, 150). The induction of heme oxygenase by many oxidants may have a strong protective effect, as proposed by Keyse and Tyrrell (115). Other gene products that have been reported to be induced by relatively mild oxidative stress in dividing mammalian cell cultures include the following: the CL100 phosphatase (114); interleukin-8 (45); catalase, glutathione peroxidase, and mitochondrial mangano-superoxide dismutase (201); natural killer-enhancing factor-B (118); mitogen-activated protein kinase (81); and 
-glutamyltranspeptidase (252). Relatively low levels of nitric oxide have also been shown to induce expression of c-jun (102), c-fos (102, 144), and zif/268 (144). The list of oxidant stress-inducible genes is much longer than the space limitations of this review article will allow.
Investigators also have chronically exposed cell lines to various levels of oxidative stress over several generations and have selected for preexisting or mutant phenotypes that confer oxidative stress resistance, such as high catalase activity (207). Stable oxidative stress resistance may tell us a great deal about the importance of individual genes to overall cellular survival, and the value of such cell lines should not be underestimated. It should be clear, however, that transient adaptive responses in gene expression and stable stress resistance are quite different.
2.6) Permanent growth arrest at higher levels of oxidative stress. If dividing mammalian cells are exposed to higher oxidant levels than those that cause temporary growth arrest and transient adaptation, then they can be forced into a permanently growth-arrested state. Thus, cells exposed to H2O2 concentrations of 250–400 µM, or 9–14 µmol/107 cells, will never divide again (247).
Countless cytotoxicity studies have measured "cell death" by loss of proliferative capacity. These include studies with oxidizing agents, alkylating agents, heavy metals, and various forms of radiation. The common assumption of such investigations is that loss of divisional competence equals cell death. It is true that many cells exposed to sufficient stress will both stop dividing and die (see next two sections, sections 2.7 and 2.8). It is clearly incorrect to conclude, however, that all permanently growth-arrested cells will die as a direct consequence of toxicant exposure; cells may survive an oxidative stress yet be permanently growth-arrested (24, 247).
Studies of both cell populations and individual cells have revealed that cultured mammalian cells (normal doubling time of 24–26 h) can survive for many weeks, or even months, following exposure to higher oxidative stress levels without dividing again (247). In the past, studies estimating percent cell viability by growth curves or colony formation measurements alone have completely missed this permanent growth-arrest response. Arrested cells still exclude trypan blue, maintain membrane ionic gradients, utilize oxygen, and make ATP; in other words, these cells are alive but do not divide. Interestingly, such permanently growth-arrested cells may make good cellular models for certain aging processes (24). Whether the cessation of proliferation induced by oxidative stress (or other stressful exposures) in some way mimics the loss of divisional competence typical of terminally differentiated cells remains to be seen.
2.7) Cell suicide or apoptosis at high levels of oxidation stress. A fraction of cells exposed to high levels of oxidative stress (e.g., H2O2 concentrations of 0.5–1.0 mM, or 15–30 µmol/107 cells) will enter the apoptotic pathway. The mechanism of oxidative stress-induced apoptosis appears to involve loss of mitochondrial transmembrane potential (252), release of cytochrome c to the cytoplasm (187), loss of bcl-2 (106), downregulation and degradation of mitochondrial encoded mRNA, rRNA, and DNA (31, 34, 39), and diminished transcription of the mitochondrial genome (126). Current thinking about toxicant-induced apoptosis suggests that, in multicellular organisms, the repair of severely damaged cells represents a major drain on available resources for the "host" organism. To avoid this difficulty, individual cells within organisms (or organs or tissues) will "sacrifice" themselves for the common good of the many. Apoptotic cells are characterized by "blebbing", nuclear condensation, and DNA laddering (6). Such cells are engulfed by phagocytes, which prevent an immune reaction and recycle usable nutrients (6, 31, 34, 39, 51, 106, 126, 164, 187, 252). Certain toxicants, such as staurosporine, can induce widespread apoptosis in fibroblast cell cultures, with greater than 80% cell suicide (34, 126). Even higher levels of apoptosis (98% or more) are routinely observed upon withdrawal of IL-2 from in vitro cultures of T lymphocytes (34, 126). In contrast, the highest levels of apoptosis induced in fibroblasts by H2O2 exposure never exceed 30–40% (34). The cause of such disparity is not at all clear, but it may suggest either slightly divergent pathways to apoptosis or different efficiencies of repair processes for various toxicants. The apoptotic pathway may be very important in several age-related diseases such as Parkinson disease, Alzheimer disease, and sarcopenia. Importantly, many mitochondrial changes, including loss of membrane potential (252) and downregulation and degradation of mitochondrial polynucleotides (31, 34, 39), are common to apoptosis directly induced by oxidants and to apoptosis induced by staurosporine or withdrawal of IL-2. Furthermore, overexpression of the p53 gene has been seen to result in induction of multiple "redox-related" gene products and initiation of apoptosis (164). These observations support a strong involvement of oxidative stress mechanisms in general apoptotic pathways.
2.8) Cell disintegration or necrosis at very high levels of oxidative stress. At concentrations of hydrogen peroxide of 5.0–10.0 mM or 150–300 µmol/107 cells, most cells simply disintegrate or become necrotic. Membrane integrity breaks down at such high oxidant stress levels, and all is then lost (100). Studies that purport to examine cellular responses to extremely severe oxidative stress are really not looking at the responses of cells but rather at the release of components those cells originally contained. At a sufficiently high level of oxidative stress, all mammalian cell cultures will turn into a necrotic "mess" (247). Oxidation-induced necrosis may play a significant role in ischemia-reperfusion injuries such as heart attacks, strokes, ischemic bowel disease, and macular degeneration. Unfortunately, necrotic cells cause inflammatory responses in surrounding tissues. Such secondary inflammation (also an oxidant stress) may be particularly important in many autoimmune diseases such as rheumatoid arthritis and lupus.
In most cases so far, we have discussed the hallmarks and biology of oxidative stress as it relates to the presence of oxygen and the generation of oxygen-derived species. In the following sections, we discuss in more detail the chemical nature of these oxidants and the possible mechanisms of their generation.
3) The Role of Iron Ions in Producing the Hydroxyl Radical
Study of the reduction of hydrogen peroxide by ferrous ions dates to the work of Fenton, Haber, Weiss, and Willstatter at the end of the 19th century (124, 125, 236). Early concerns were the nature of the iron-hydrogen peroxide interaction and the intermediates produced. It was thought that an outer-sphere electron transfer was involved (236), as shown in reaction 10.
 | (10) |
After a century of study, the mechanism of the reaction is still debated. Current thinking is that the net Fenton reaction is a simplification of a more complex process. The simplest way to envision the reaction is a single electron donation from Fe2+ to hydrogen peroxide (an outer sphere mechanism), as shown in reaction 10. However, this reaction is thermodynamically unfavorable, and it is thought an inner sphere mechanism is more likely (76).
The reaction in vivo is a chain reaction in which the ferric ion produced in reaction 10 is reduced back to ferrous, as in reaction 11.
 | (11) |
The nature of the electron donor in reaction 11 has been the subject of a considerable amount of study. At first it was thought that the electron donor was superoxide, providing another rationalization for the existence of SOD (60, 139). However, the reduction of ferric ions by superoxide was shown to be far too slow (236). The reducing agent in reaction 11 now is agreed to be the reducing agents that are ubiquitous in biosystems, including thiols and other more complex sulfur compounds and ascorbate and similar one-electron transfer agents.
Now that the chemistry of nitric oxide has caught up with the advances in superoxide chemistry, we recognize the vital role of SOD in preventing the combination of nitric oxide and superoxide to form peroxynitrite, as in reaction 12. Thus, the need to protect biological systems against superoxide buildup is the result of both superoxide toxicity itself and the conversion of superoxide to peroxynitrite, a potent oxidant toward certain biological species.
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\mathrm|<|O|>|_|<|2|>|^|<||<|\cdot|>||<|-|>||>||<|+|>|\mathrm|<|NO|>|^|<||<|\cdot|>||>||<|\rightarrow|>|^|<||<|-|>||>|\mathrm|<|O|>|-\mathrm|<|O|>|-\mathrm|<|N|>|
| (12) |
4) Ozone
Ozone, O3, is a gas in the stratosphere that protects organisms on Earth from the most harmful wavelengths of solar radiation ("good" ozone). Ozone also is a significant pollutant near the Earth's surface ("bad" ozone), where it is produced in the series of smog reactions and can oxidize bioorganic compounds and initiate the autoxidation of unsaturated fatty acids, leading to membrane destruction (228). Wentworth et al. (242–245) recently reported the startling claim that antibodies produce ozone in vivo. This group first reported this for the catalytic conversion of singlet oxygen to ozone and then in atherosclerotic plaques. Wentworth et al. (242–245) reported what they considered to be definitive evidence for ozone production through the identification of oxidation products that they propose are biomarkers for the presence of ozone.
Significant reservations about these conclusions have been expressed (203, 204). The suggestion that O3 was involved in atherosclerosis arose from the discovery that 3
-hydroxy-5-oxo-5,6-secocholestanol-6-al and 3,5-dihydroxy-5-B-norcholestane-6-carboxaldehyde are present in atherosclerotic plaques. The first compound, cholesterol secoaldehyde, exists in equilibrium with its intramolecular aldol condensation product (see Fig. 1). Wentworth et al. (244) identified these species as their 2,4-dinitrophenyl hydrazones and considered them proof for the participation of ozone in the systems they studied.
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In aqueous systems, such as occur in liposomes and biomembranes, the ozonation of double bonds gives poor yields of Criegee ozonides and instead favors formation of carbonyl products, produced by the hydrolysis of the ozone-derived products by water (211). This suggests that if ozone were to react with cholesterol in vivo, then cholesterol secoaldehyde (C-seco) and its aldol condensation product would be major products, and the Criegee ozonide (C-oz) would be only a minor product, since most of the cholesterol resides in the lipid bilayer but water traps most of the intermediate carbonyl-oxide before the fragments can recombine to form the Criegee ozonide (211). (See Fig. 2.) Thus, although the direct hydrolysis of C-oz, as well its reduction, does yield C-seco and its aldol condensation product, it is also possible that the reaction of the cholesterol ozonide with 2,4-dinitrophenylhydrazine gives the same hydrazone products as does the hydrazine's reaction with C-seco and its aldol condensation product.
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Another concern in the report of Wentworth et al. (244) is that the hydrazone derivatives were identified by mass spectrometry by means of their [M-H]– negative ion at m/z 597. However, as remarked by Smith (204), the hydrazone derivative of 3,5-dihydroxy-5
-cholestan-6-one, an oxidation product of cholesterol that is formed even in the absence of ozone, will yield the same [M-H]– negative ion.
Yet another concern with the claim that cells produce ozone is that the formation of ozone requires substantial energy. The heat of formation of ozone in the gas phase is 34.1 kcal/mol; the gas phase reaction of three singlet oxygen molecules to form two ozone molecules (by an unknown mechanism) is exothermic by 1 kcal.
A new study reports that H2O3 is produced from "oxone," which is a mixture of ozone plus hydrogen peroxide used in water purification (151). These authors report that the reaction of ozone with hydrogen peroxide is of biological interest and that this reaction, wherein H2O3 is a reaction intermediate, is a source of singlet oxygen (151). Nieva and Wentworth (149) have proposed that H2O2, H2O3, 1O2, and O3, all of which are highly reactive oxidants, can be endogenously produced by antibody catalysis of water oxidation. In advancing this hypothesis, these authors fail to compare, for example, the rate constants for the reactions of O3 with H2O2 (k = 0.065 M–1·s–1) (1) with the rate constants for the reactions of O3 with small molecular weight antioxidants, such as glutathione (k = 7 x 108 M–1·s–1) and ascorbate (6 x 107 M–1·s–1) (68, 175). Taking into consideration that all these reaction rate laws are first order in O3 and first order in these substrates and that these small molecular weight antioxidants are considerably more abundant than H2O2 in biological systems, one can conclude the reaction of O3 with H2O2 is at least 10 billion times slower than the combined rate of O3 with these antioxidants and that it will hardly occur in vivo. Thus, these claims are thought-provoking, but they remain highly controversial.
Lipid ozonation products (LOP) should be considered in the light of the observation of cholesterol oxidation (41, 59, 103, 104, 173, 181). LOP include ozone-derived lipid peroxides that can undergo homolysis and generate oxy-radicals that can oxidize unsaturated compounds. They oxidize polyunsaturated lipids (PUFA) faster than lipids such as cholesterol, and unsaturated lipids probably are more plentiful than is cholesterol. Some LOP can initiate PUFA oxidation. It is possible that any oxidative stress (including ozone exposure) will result in cholesterol oxidation. So the oxidation/ozonation of cholesterol may well be the result of a complex, multistep process.
5) Nitric Oxide
The discussions above have, for the most part, focused on oxygen and oxygen-derived species. However, there is another relevant radical, nitric oxide, ·NO. Nitric oxide is the first gaseous species unequivocally identified as an endogenously generated cell signaling/effector agent (65, 99, 160). This was a significant finding since it established a new paradigm in cell signaling whereby an endogenously generated, small, ordinarily gaseous molecule, which is toxic at moderate concentrations, is used as a cell-signal species. This discovery encouraged the suggestion that other small molecules (many of which were also known primarily for their toxicity) could also be biosynthesized and possess important physiological functions (see below). The first established physiological role for ·NO was as a vasorelaxant. The ability of ·NO to elicit vasorelaxation is due to its ability to increase intracellular levels of cGMP in smooth muscle tissue. Another important aspect of ·NO, also due to increased intracellular cGMP levels, is its ability to inhibit cell (platelet) adhesion and aggregation. However, it appears that the effects and function of ·NO go beyond its ability to regulate vascular tone and/or cell adhesion, as other aspects of ·NO physiology and pathophysiology have been postulated (for a review, see Ref. 98). For example, ·NO is made in the brain, as a part of immune response, and in mitochondria, and the role of ·NO in these systems probably is not related to its specific effects on the vascular system or cell adhesion. Unlike the established role of ·NO in vascular physiology, the function of ·NO in these other systems is not well-understood. Below is a brief review of ·NO biology and chemistry with focus on the role of ·NO in biological free radical chemistry.
5.1) Nitric oxide biosynthesis and physiology. Nitric oxide (·NO) is biosynthesized by a family of enzymes referred to as the nitric oxide synthases (NOS) (for a review, see Ref. 216). There are three primary isoforms of NOS that originate from separate genes and differ in their subcellular localization and mode of regulation. They are typically designated as endothelial (eNOS), neuronal (nNOS), and immunological/inducible NOS (iNOS), although these designations do not strictly reflect their tissue expression or utility. eNOS and nNOS are constitutively expressed; nevertheless, their levels can change in response to a variety of physiological events (e.g., hormonal influences). As the name indicates, iNOS is highly induced in a variety of cells, often as a result of immune stimulation (e.g., lipopolysaccharide, cytokines). Both eNOS and nNOS are regulated primarily by Ca2+ via the actions of calmodulin. In contrast, iNOS is not regulated by Ca2+.
Regarding the vascular effects of ·NO, it has been known for almost 140 years that some nitrogen oxide-containing species relieve the symptoms associated with coronary artery disease. For example, in 1867 it was reported that amyl nitrite could be used to alleviate the chest pain associated with angina (16). Although not known in these early studies, it is now established that the ability of "nitrovasodilators" such as amyl nitrite and nitroglycerin to relax coronary arteries is due to their biotransformation to ·NO (for example, see Ref. 54). The mechanism by which ·NO elicits vasorelaxation is known to occur via activation of the enzyme soluble guanylate cyclase (sGC) (for a review, see Ref. 89). sGC is a heterodimeric heme-containing soluble protein, which exists in the cytosolic fraction of virtually all mammalian cells and acts as a principal intracellular target for ·NO. (Homologous enzymes exist in lower species such as Drosophila and Caenorhabditis elegans.) Activation of sGC by ·NO results in an increased conversion of GTP to the second messenger, cGMP, which governs many aspects of cellular function via interaction with cGMP-dependent protein kinases, cyclic nucleotide gated ion channels, or cyclic nucleotide phosphodiesterases (89, 195). As a consequence, sGC has become accepted as the primary ·NO receptor, and the ·NO-sGC-cGMP signal transduction pathway has been established as an important signal transduction system. Because of its ubiquitous nature, aberrations in sGC-mediated signaling may be fundamental to the etiology of a wide variety of pathological disorders; this is exemplified in the cardiovascular system by disease states such as sepsis and atherosclerosis (90, 91). Nitric oxide-mediated activation of sGC occurs by ·NO ligation to the regulatory heme site on the protein. The heme of sGC does not directly participate in the catalytic chemistry of the enzyme and appears to serve a purely regulatory function. Nitric oxide is a unique ligand for heme proteins in that the ferrousnitrosyl adduct prefers to exist as a 5-coordinate, square pyramidal complex resulting in the release of an axial histidine ligand in sGC, which, presumably, is critical for enzyme activation (226, 227).
5.2) Physiological and pathophysiological chemistry of ·NO.
As mentioned above, ·NO is a unique ligand for heme proteins, and, indeed, the distinctive coordination chemistry of ·NO allows it to be an especially potent activator of sGC. Nitric oxide possesses other unique and important chemical properties that are also critical to aspects of its biology. Nitric oxide has one unpaired electron located in an anti-bonding orbital and therefore is a radical. Nitric oxide is almost exclusively a monomeric radical species at room temperature and pressure, so its reactions with other radicals (such as O2·– or alkyl radicals) are extremely facile. The inherent radical nature of ·NO and its reactions with other free radicals present in biological systems are important to some of its possible biological actions. For example, it has been proposed that ·NO can be toxic through reaction with superoxide (O2·–) to generate peroxynitrite (O
. Further comments on the toxicity of O
The reaction of ·NO with O2 generates species such as nitrogen dioxide (NO2·) and dinitrogen trioxide (N2O3) that may have biological significance. Dinitrogen trioxide is a potent nitrosating agent that can alter protein function via nitrosation of critical nucleophilic residues (reactions 13–17).
 | (13) |
 | (14) |
 | (15) |
 | (16) |
 | (17) |
Note that reaction 17 can lead to protein nitrosation and altered protein function.
The NO2· formed in reaction 15 is also a free radical species that, unlike ·NO, is a fairly potent oxidant [E° for the NO2·/NO2– couple = 1.04 V, vs. normal hydrogen electrode (NHE)]. There are a variety of potential reaction pathways by which NO2· can cause oxidation to biological molecules: hydrogen atom abstraction, addition to unsaturated bonds and electron transfer reactions (for review of NO2· oxidation chemistry, see Ref. 96). The kinetics for the ·NO + O2 reactions (first order in O2 and second order in ·NO to form NO2·) requires fairly high concentrations of ·NO for this reaction to be physiologically relevant. However, it has been postulated that the lipophilicity of ·NO and O2 allows their concentration to be high enough for this reaction to occur in cell membranes (132).
The chemistry of ·NO and derived species with thiols appears to be an important aspect of ·NO biology (212). Modification of biological molecules by ·NO may occur via reaction with a thiol function; for example, nitrosothiols (RSNO) can be formed by reaction with ·NO or, more likely, ·NO-derived species. RSNO formation can be accomplished by reaction 17 (with a nucleophilic thiol). Also, RSNO formation has been postulated to occur via direct reaction of ·NO with a thiol, a reaction first proposed by Pryor and coworkers (174) followed by reaction of the thiol-·NO intermediate with O2 (reactions 18 and 19) (77).
 | (18) |
 | (19) |
This route to RSNO formation has not, however, been established to be physiologically relevant. Finally, RSNO formation can occur via metal-mediated processes whereby the metal binds ·NO and acts as an electron acceptor when reacted with a thiol (see, for example, Refs. 56, 82, 235, 241) (reactions 20 and 21). This chemistry can be accomplished by, for example, ferric heme proteins, ultimately resulting in the generation of a ferrous nitrosyl adduct (reaction 22, Mn = Fe3+-heme, Mn–1 = Fe2+-heme) in a process referred to as "reductive nitrosylation."
 | (20) |
 | (21) |
 | (22) |
Thus, there are a variety of potential physiologically relevant mechanisms that lead to the generation of S-nitrosothiols, and there is little doubt that they are formed in vivo. Once formed, RSNO compounds can react further with a variety of biological species. For example, RSNO can react with another thiol, R'SH, to transfer the equivalent of a nitrosonium ion, NO+ (10, 11) (reaction 23).
 | (23) |
RSNO can also react with thiols to give, instead, the corresponding disulfide and HNO (reaction 24) (249).
 | (24) |
The factors governing the site of attack on the RSNO functional group have not been established.
Modification of protein or peptide thiols by ·NO and ·NO-derived species is of possible biological significance to metal metabolism/metal homeostasis, since the binding of metals to proteins often involves thiol ligation. Thus, nitrogen oxide-mediated thiol modification can potentially disrupt metal-protein interactions, leading to possible metal release and subsequent metal-derived free radical chemistry. Thiols ligated to metals may be specific targets for modification by ·NO and would indicate an ability of ·NO to disrupt/regulate metal metabolism even in the presence of high levels of non-metal-bound thiols such as glutathione (see, for example, Ref. 200).
5.3) Nitric oxide as an oxidant and antioxidant.
As indicated above, ·NO can react with O2 and O2·– to generate potentially deleterious oxidants such as O
Free radical chain processes occur in membranes because the membrane PUFA are susceptible to radical initiation processes and undergo the well-known PUFA radical chain autoxidaton (166, 170). Lipid alkoxyl (LO·) and peroxyl (LOO·) radicals are important intermediates in these lipid autoxidation processes. Nitric oxide can behave as an antioxidant or as a prooxidant in lipid autoxidations, depending on the experimental conditions (88, 152, 153). The antioxidant action of ·NO occurs by chain-breaking termination reactions of ·NO with LO· and LOO· radicals, as in reactions 25 and 26.
 | (25) |
 | (26) |
Thus, depending on conditions, ·NO can act as an oxidant or antioxidant. The reaction of nitric oxide with LOO· results in the formation of an alkyl peroxynitrite, LOONO, which can homolyze to generate a geminate radical pair, ·NO2 and an alkoxyl radical, LO·. Both of these radicals can initiate further radical reactions (for example, see Refs. 75 and 255). About 86% of these radical pairs from LOONO rapidly recombine to give unreactive alkyl nitrates, LONO2 (75), indicating that ·NO can be an effective antioxidant. However, the remaining 14% of the radical pairs formed in the homolysis of LOONO become free ·NO2 and LO· radicals (see reactions 27 and 28).
 | (27) |
 | (28) |
Thus, 14% of the ·NO and LOO· radicals that react to form LOONO get effectively converted into ·NO2 and LO·, a much more reactive pair. For instance, in abstracting a hydrogen atom from a doubly allylic position, the rate constants for LOO· and LO· are 31 M–1·s–1 and 1 x 107 M–1·s–1 (4), respectively, and although ·NO cannot abstract a hydrogen atom from a doubly allylic position in a PUFA, the rate constant for the reaction of ·NO2 with linoleic acid is 2 x 105 M–1·s–1 (165). In summary, through the sum of reactions 26–28, 86% of the LOO· and ·NO radicals go on to form the stable product LONO2, and 14% go on to form the more reactive radicals LO· and NO2·, revealing the Janus-like behavior of ·NO.
The rate constant for the homolysis of LOONO with L = CH3 can be estimated using group increment rules, using a procedure similar to the procedure applied by Richeson et al. (189) to estimate the corresponding rate constant for HOONO. Thus, using a basis set containing 
fH° and S° for HOONO (–1 ± 1 kcal/mol and 68 ± 1 cal/K·mol, respectively, from Ref. 189), and H2O2, CH3OH, CH3OOH, CH3OOCH3, and CH3ONO (from Ref. 2), fH° (CH3OONO) can be computed by replacing H by CH3, H by CH3O, or H by NO in various ways, affording a mean value of fH° (CH3OONO) = 1 ± 1 kcal/mol. Similarly, S° (CH3OONO) can be computed from the entropy gain for H 
CH3, H CH3O, or H NO as a mean value of S° (CH3OONO) = 78 ± 1 cal/K·mol. Combining these values with CH3O· and ·NO2, one can compute thermodynamic values for the equilibrium shown in equation 29 as H29° = 11 ± 1 kcal/mol, S29° = 36 ± 1 cal/K·mol, and G29° = 0 ± 1 kcal/mol.
 | (29) |
The gas phase high-pressure limit, independent of temperature, for k–29 (with L = CH3) has been determined to be 1.2 x 1010 M–1·s–1. Accordingly, the preexponential A29 at 298 K can be computed from A29/s–1 = A–29(0.224T)–1exp(S29°/R) = (1.2 x 1010)(1.5 x 10–2)(7.37 x 107) = 1.3 x 1016. Since H29°
= H29° = 11 ± 1 kcal/mol, the rate constant for homolysis of CH3OONO in the gas phase is computed as k29 = (1.3 x 1016)exp(–11/RT) = 1.5 x 108 s–1. If one assumes a retarding solvent cage effect (142) of a factor of about 102 and small solvent effects for the homolysis process, then one would expect an overall solution rate constant of decomposition for CH3OONO on the order of 106 s–1, contrasting sharply with the rate of decomposition for HOONO of about 1 s–1. These calculations are in good agreement with a recent report by Goldstein et al. (75), who estimated this rate constant at about 5 x 105 s–1. Recent calculations by Zhao et al. (255) predict a H for homolysis of HOONO of 18 kcal/mol beginning from the cis conformer. The corresponding activation energy for homolysis of CH3OONO is 12 kcal/mol.
In contrast to LONO, LNO2, and LONO2, which are relatively stable, LOONO is not stable, and the rapid homolysis of LOONO into LO· and ·NO2 will be reflected in the oxidative effects of ·NO. Another important aspect of lipid-nitrogen oxide interaction is the finding that lipid nitration, likely via ·NO2, generates a nitrolipid (LNO2), which has been reported to have biological signaling properties that are cGMP-dependent and -independent (29). Recently, nitrolipids had been found in red blood cell membranes in surprisingly high levels and appear to represent a novel class of lipid-derived signaling mediators. The mechanism by which these nitrolipids are formed in vivo is not known with certainty. However, ·NO2 had been found to yield vinylnitro- as well as allylnitro-lipids under a variety of conditions (66) (Fig. 3).
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-substituted nitro-fatty acid, where X could be, for example, -OH, -OOH, -ONO, -NO2, -ONO2, etc. This -substituted nitro-fatty acid could then undergo elimination of HX and yield a vinylnitro-fatty acid. Nitrogen dioxide (or a more reactive radical) also can react with unsaturated fatty acids by abstracting an H atom from an allylic position yielding an allylic radical which can then react with ·NO2 to yield an allylnitro-fatty acid. Finally, vinyl- and allylnitro-fatty acids may isomerize and equilibrate, as has been reported for several homologous compounds (223). Of course, lipid radical intermediates can also lead to biologically active isoprostanes (191). 5.4) Oxidation and nitration in nitric oxide-producing systems. Nitric oxide can play a role in cell physiology, for example, by acting as a cell signaling agent. Signaling by ·NO is usually accompanied by some degree of oxidative and nitrative stress in which the superoxide radical and dissolved gases (e.g., oxygen and carbon dioxide) can play modulatory roles. Moreover, as we have shown, peroxynitrite, peroxynitrate, and their esters, which are known to play a role as reactive intermediates in smog, can also occur downstream from the formation of ·NO in vivo. The delicate interplay of these reactions is briefly reviewed below.
5.5) Peroxynitrite, CO2, and selectivity among free radicals. The discovery in 1995–1996 (47, 134, 230) that CO2 reacts rapidly with peroxynitrite and alters its reactivity was vital in understanding its reactions. In the absence of CO2, peroxynitrite slowly decomposes to form the free radicals HO· and ·NO2 and is a powerful but slow oxidant and a poor nitrating reagent (208). In humans, fluids and tissues contain 1–2 mM CO2, and the reaction of peroxynitrite with CO2 is 50 to 100 times faster than its rate of decomposition to form HO· and ·NO2. Furthermore, other biological molecules, such as heme proteins, also react with peroxynitrite at considerable rates. Thus, in vivo, peroxynitrite does not decompose to form HO· and ·NO2, because this reaction is too slow.
The modulation of peroxynitrite reactivity by CO2 described above is shown in reactions 30 and 31. In the absence of CO2, peroxynitrite slowly decomposes to give the HO· and ·NO2 radicals, but in its presence peroxynitrite generates the CO3·– and ·NO2 radicals in a fast reaction.
 | (30) |
 | (31) |
As discussed above, some but not all free radicals are short-lived and reactive. Certain free radicals, such as HO·, react with virtually all biomolecules as fast as they collide and consequently live only microseconds. For this reason, biological damage by the HO· radical is random, inefficient, and widespread, and generally does not affect critical cellular targets. In contrast, other radicals, such as CO3·– and ·NO2, display various degrees of selectivity for biological molecules (7, 208, 210). Furthermore, when CO3·– and ·NO2 react in concert, as when these radicals are produced together from the reaction of peroxynitrite with CO2, these two radicals constitute an efficient nitrating system that is pivotal to biological nitration of protein tyrosine residues. More than 50 human diseases show elevated 3-nitrotyrosine levels (78, 84).
The different reactivities of the free radicals HO·, CO3·–, and ·NO2 are shown in Table 1, where second-order rate constants for the reactions of these free radicals with selected biological target molecules are given. These rate constants were taken either from published work and databases of rate constants (72) or, when not available, were estimated from the reactivities of related compounds (69, 117). Thus, the HO· radical reacts at or near the rate limit for diffusion with most biomolecules, whereas the CO3·– and ·NO2 radicals react more slowly and consequently have longer diffusion distances. Furthermore, unlike the HO· radical, the CO3·– and ·NO2 radicals the have a wide range of reactivities.
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The modulation of peroxynitrite reactivity caused by carbon dioxide is dramatic. It leads to the less reactive CO3·– and ·NO2 radicals, which have longer diffusion distances than the HO· radical. Beckman et al. (14) proposed in 1990 that peroxynitrite will be formed in biological systems in which both ·NO and superoxide are produced. Yet, the mechanism for the reaction of peroxynitrite with CO2 was not understood until 1995–1996, and the impact that this reaction has in the oxidative biochemistry of peroxynitrite was not immediately recognized. As a result, the role of CO2 was ignored for several years, and the reactions of peroxynitrite in biological and model systems were not investigated in solutions that contained
