Oxygen-derived Species: Their Relation to Human Disease and Environmental Stress Barry Halliwell'2 and Carroll E. Cross1 1Pulmonary-Critical Care Medicine, University of California-Davis Medical Center, Sacramento, California; 2Pharmacology Group, University of London Kings College, London, England Free radicals and other (ROS) are constantly formed in the human body, often for useful metabolic purposes. defenses protect against them, but these defenses are not completely adequate, and systems that repair damage by ROS are also necessary. Mild oxidative stress often induces antioxidant defense enzymes, but severe stress can cause oxidative damage to lipids, proteins, and DNA within cells, leading to such events as DNA strand breakage and disruption of calcium ion metabolism. Oxidative stress can result from exposure to toxic agents, and by the process of tissue injury itself. Ozone, oxides of nitrogen, and cigarette smoke can cause oxidative damage; but the molecular targets that they damage may not be the same. - Environ Health Perspect 1 02(Suppl 1 0):5-12 (1994) Key words: free , oxygen radical, superoxide, hydroxyl, , oxidative stress, transition metals, ozone, nitrogen dioxide, cigarette smoke

Introduction technique of electron spin resonance is mental air pollutant to appear in large There is considerable current interest in the often used to measure free radicals; it quantities on the planet. role of free radicals, oxygen radicals, and records the energy changes that occur as However, even present-day aerobes suf- oxidative stress as mediators of tissue injury unpaired electrons align in response to a fer oxidative damage if they are exposed to in human disease and of the effects of air magnetic field. A superscript dot ( ) is used 02 at concentrations greater than 21% (2). pollutants, such as ozone (03), nitrogen to denote free radical species. Oxygen toxicity has been demonstrated in dioxide (NO2), and tobacco smoke. This plants, animals and microorganisms. For article explains the meaning of these terms Oxygen-A Free Radical and example, exposure of adult humans to pure and summarizes current knowledge of the Environmental Toxin 02 at 1 atm pressure for as little as 6 hr roles they play in these various situations. When living organisms first appeared on causes chest soreness, cough, and sore the earth, they did so under an atmosphere throat in some subjects; and longer periods What Is a Radical? containing very little 02, i.e., they were of exposure lead to lung damage. The inci- In the structure of atoms and molecules, essentially anaerobes. Anaerobic microor- dence of ocular damage in babies known as electrons usually associate in pairs, each ganisms still survive to this day, but their retrolental fibroplasia ("formation of pair moving within a defined region of growth is inhibited and they can often be fibrous tissue behind the lens") increased space (an atomic or molecular orbital). killed by exposure to 21% 02, the current abruptly in the early 1940s among babies One electron in each pair has a spin quan- atmospheric level. As the 02 content of born prematurely and led to many cases of tum number of +1/2, the other -1/2. A the atmosphere rose (due to the evolution blindness. Not until 1954 was it realized free radical is any species capable of inde- of organisms with photosynthetic water- that retrolental fibroplasia is associated with pendent existence (hence the term "free") splitting capacity) many primitive organ- the use of high 02 concentrations in incu- that contains one or more unpaired elec- isms probably died. Present-day anaerobes bators for premature babies. More careful trons, an unpaired electron being one that are presumably the descendants of those control of 02 concentrations (continuous is alone in an orbital (1). The simplest free primitive organisms that followed the evo- transcutaneous 02 monitoring, with sup- radical is a hydrogen atom, with one pro- lutionary path of "adapting" to rising plementary 02 given only where necessary) ton and a single electron. Table 1 gives atmospheric 02 levels by restricting them- and administration of x-tocopherol have some examples of other free radicals. Note selves to environments that 02 did not decreased its incidence, but the problem has that the gases nitric oxide (NO0) and nitro- penetrate. However, other organisms not disappeared, since many premature gen dioxide (NO;) are free radicals, began the evolutionary process of evolving infants need continuous high 02 to survive whereas ozone (03) is not-no unpaired antioxidant defense systems to protect at all (3). electrons are present. The spectroscopic against 02 toxicity. In retrospect, this was The damaging effects of elevated 02 on a fruitful path to follow since organisms aerobes vary considerably with the organism that tolerated the presence of 02 could studied, age, physiologic state, and diet; and This paper was presented at the Conference on also evolve to use it for metabolic transfor- different tissues are affected in different Oxygen Radicals and Lung Injury held 30 August-2 mations (involving enzymes such as oxi- ways. Thus, cold-blooded animals such as September 1993 in Morgantown, West Virginia. The authors are grateful to National Institutes of dases and oxygenases) and for efficient turtles and crocodiles are relatively resistant Health (grants HL47628, RR-00169, and ES-00628) energy production (by using electron to 02 at low environmental temperatures, and to MAFF (UK) for research support. transport chains with as the terminal but become more sensitive at higher tem- Address correspondence to Barry Halliwell, 02 Pharmacology Group, Kings College, Manresa Road, electron acceptor, such as the mitochon- peratures. Neonatal rats resist lung damage London SW3 6LX, England. Telephone 44 71 333 drial oxidative phosphorylation system). in an atmosphere of 100% 02 far more 4860. Fax 44 71 333 4949. Hence, 02 was probably the first environ- effectively than do adult rats (2).

Environmental Health Perspectives 5 HALLIWELL AND CROSS

Table 1. Examples of free radicals. although the precise amounts generated Name Formula Comments and the steady-state concentrations Hydrogen atom H The simplest free radical known. achieved are still uncertain. Generation of Trichloromethyl CCI5 A carbon-centered radical (i.e., the unpaired electron resides on these species occurs by two types of carbon). CClI is formed during metabolism of CCI4 in the liver processes described below. and contributes to the toxic effects of this solvent (7). "Accidental" Generation. This Superoxide O° An oxygen-centered radical. Hydroxyl OH An oxygen-centered radical. The most highly reactive oxygen radi- encompasses such mechanisms as "leakage" cal known. of electrons onto 02 from mitochondrial Thiyl RS- General name for a group of radicals with an unpaired electron electron transport chains, microsomal residing on sulfur. cytochromes P450 and their electron Peroxyl, alkoxyl RO1, RO- Oxygen-centered radicals formed during the breakdown of organic donating enzymes, and other systems peroxides. (1,5,20). It also includes so-called autoxi- Oxides of nitrogen NO,, NO2 Both are free radicals. NO is formed in vivo from the amino acid in which compounds such L-arginine (8). NOi is made when NO reacts with 02 and is dation reactions found in polluted air and smoke from burning organic materials, as catecholamines, ascorbic acid, and e.g., cigarette smoke. reduced flavins are alleged to react directly with °2 to form O-- (5). In fact, such autoxidations are usually catalyzed by tran- sition metal ions (1). The earliest suggestion made to explain All organisms suffer some exposure to Deliberate Synthesis. The classic 02 toxicity was that 02 is a direct inhibitor OH-, because it is generated in vivo by example of deliberate metabolic generation of enzymes, thereby interfering with metab- homolytic fission of 0-H bonds in water, of ROS for useful purposes is the produc- olism. However, very few targets of direct driven by our continuous exposure to back- tion of O2, HOCI, and H202 by activated damage by 02 have been identified in aer- ground ionizing radiation (6). Hydroxyl phagocytes (21). Hydrogen peroxide is obes. In 1954, Gerschman et al. (4) pro- radical is so reactive with all biological mol- additionally generated in vivo by several posed that the damaging effects of 02 could ecules that it is impossible to evolve a oxidase enzymes, such as glycolate oxidase, be attributed to the formation of oxygen specific scavenger of it-almost everything xanthine oxidase, and D-amino acid oxi- radicals. This hypothesis was popularized in living organisms reacts with OH with dase (18,22). Evidence is accumulating and converted into the superoxide theory of second-order rate constants of 109 to 1010 that 0 - is also produced by several cell 02 toxicity following the discovery of super- M - 1sec l (essentially, if OH- contacts the types other than phagocytes, including oxide dismutase (SOD) enzymes by compound, reaction occurs). Damage lymphocytes (23), fibroblasts (24,25), and McCord and Fridovich (5). In its simplest caused by OH-, once this radical has been vascular endothelial cells (26-28). Such form, this theory states that 02 toxicity is formed, is probably unavoidable and is Op might be involved in intercellular sig- due to excess formation of superoxide radi- dealt with by repair processes (Table 2). nalling and could serve important biologic cal (O2-)' the one electron reduction prod- It is now well established (1,5,18-20) functions, although more information is uct of 02, and that the SOD enzymes are that 0p and H202 are produced in aerobes, needed. important antioxidant defenses because they remove p--. Ironically, with all the Table 2. of oxidative fuss made about oxygen radicals, it must be Repair damage. realized that the diatomic oxygen molecule Representative is itself a free radical, containing two Substrate of damage Repair system recent references unpaired electrons (1). Fortunately, the electronic arrangement in 02 renders this DNA molecule unreactive its free radical All components of DNA can be A wide range of enzymes exist that recog- (9,10) despite attacked by OH', whereas singlet 02 nize abnormalities in DNA and remove nature (1). attacks guanine preferentially. H202 them by excision, resynthesis, and rejoin- Reactive Oxygen Species and °2- do not attack DNA (9). ing of DNA strand (10). in Vivo Proteins Reactive oxygen species (ROS) is a collec- Many ROS can oxidize -SH groups. Oxidized methionine residues may be (12,13) tive term used to include not OH attacks many amino acid residues repaired by methionine sulfoxide reduc- by biologists ( 11). Proteins often bind transition tase. Other damaged proteins may be rec- only oxygen radicals (O-p and hydroxyl metal ions, making them a target of ognized and preferentially destroyed by radical, OH) but also some derivatives of attack by site-specific OH' generation cellular proteases. 02 that do not contain unpaired electrons, (1,11). such as hydrogen peroxide (H202), singlet 02(1Ag), and hypochlorous acid (HOCI)*. Lipids Reactive is of course a relative term: Op is Some ROS (not including °2- or H202) Chain-breaking (especially (15-17) can initiate lipid peroxidation ( 14). ct-toxopherol) remove chain-propagating more reactive than 02, but neither Op nor peroxyl radicals. Phospholipid hydroperox- H202 in aqueous solution is anywhere near ide peroxidase can remove as reactive as OH(1). peroxides from membranes, as can some phospholipases. Normal membrane turnover can release damaged lipids. *HOCI could equally well be regarded as a "reactive chlorine species."

6 Environmental Health Perspectives OXYGEN RADICALS, DISEASE, AND THE ENVIRONMENT

Generation of O-, HOCI, and H202 then O2-, by removing NO- (Equation 3), Fe(II) + H202 -* OH- by phagocytes is known to play an impor- can act as a vasoconstrictor, and this might + OH + Fe(III) tant part in the killing of several bacterial have deleterious effects in some clinical sit- [6] and fungal strains (21). Some other meta- uations (36,37). Although there has been repeated con- bolic roles for H202 have been proposed Considerable debate continues in the troversy in the literature as to whether OH- (29-33). For example, H202 is used by literature as to whether or not the interac- is formed in such reactions at physiologic the enzyme thyroid peroxidase to help tion of O- and NO (Equation 3) is dam- pH, the evidence is now overwhelming make thyroid hormones (30). H202 or aging to cells (38). Peroxynitrite might be (49-51). Copper can also catalyze OH- products derived from it can displace the directly toxic to cells (38,39). It might also formation from H202 (52). inhibitory subunit from the cytoplasmic decompose to form a range of toxic prod- Iron and copper (and other transition gene transcription factor NF-KB. The ucts, including some OH- (38,40). metal ions) in chemical forms that can active factor migrates to the nucleus and decompose H202 to OH- are in very short activates genes by binding to specific DNA ONOO- + H+ - OH-, NOQ, NO+ [4] supply in vivo: organisms are very careful sequences in enhancer and promoter ele- to ensure that as much iron and copper as ments. Thus, H202 can induce expression Note that Equation 4 also produces the possible are kept safely bound to transport of genes controlled by NF-KB. This is of toxic free radical gas nitrogen dioxide. or storage proteins. Indeed, the "sequestra- particular interest because NF-KB can However, the physiologic significance of tion" of metal ions into forms that will not induce the expression of genes of the these reactions is still uncertain, since some catalyze free radical reactions is an impor- provirus HIV-1, the major cause of experiments suggest that NO' may protect tant antioxidant defense mechanism acquired immunodeficiency syndrome against oxidative damage even when O-p is (1,29,53). Sequestration of iron and cop- (33). H202, a nonradical, resembles water being generated (41,42). per ions deters the growth of many bacter- in its molecular structure and is very dif- Superoxide has also been shown to be ial strains in human blood plasma (54): it fusible within and between cells. capable of inactivating several bacterial also ensures that plasma will not convert Much O-p generated in vivo probably enzymes, such as Escherichia coli dihy- O2- and H202 into OH' (29,53). undergoes a dismutation reaction to give droxyacid dehydratase, aconitase, and 6- Prevention of OH' formation may allow H202, as represented by the overall equa- phosphogluconate dehydratase (5,20,43). small quantities of O-p and H202 released tion It appears to attack iron-sulfur clusters at into the extracellular environment (e.g., the enzyme active sites. Whether such reac- from endothelial cells, lymphocytes, and 20i- + 2H+ 3-H202 + 02 [1] tions of Op- occur in mammals is uncer- phagocytes) to perform useful metabolic tain, although in isolated submitochondrial roles, such as intercellular signaling, rather particles, O-p has been claimed to inacti- than causing damage (29). Toxicity of Superoxide and vate the NADH dehydrogenase complex of In any case, any transition metal ions Hydrogen Peroxide the mitochondrial electron transport chain that do become available to catalyze free Experimental data show clearly that (44). The protonated form of O2- radical reactions in vivo will not exist in the removal of O-p and H202 by antioxidant hydroperoxyl radical (HO2), is much more "free" state for very long. Thus, if iron ions defense systems is essential for healthy aer- reactive than O°- in vitro. For example, are liberated, they must bind to a biological obic life (1,5,18,34). Why is this? In HOp- can initiate peroxidation of polyun- molecule or else eventually precipitate out organic media, 0° can be very reactive but saturated fatty acids and decompose lipid of solution as ferric hydroxides, oxyhydrox- in aqueous media it is not, mainly acting as hydroperoxides, which O2- cannot ides, and phosphates. If metal ions bound a moderate reducing agent, e.g., the reduc- (45,46). However, there is no direct evi- to a biological molecule react with O-p and tion of cytochrome c dence that HOp- exerts damaging effects in H202 (Equations 5,6), OH' will be vivo. The pKa of HOp- is about 4.8, so formed upon the molecule. This OH- cyt c (Fe)3++Op-+ cyt c (Fe2+) + 2 [2] only a small fraction of O- is protonated mediated damage is said to be "site at physiologic pH values. specific" (55). Binding of metal ions to a However, O-p, can react with some tar- H202 at low micromolar levels also target means that any OH' generated will gets. In particular, O-p reacts fast with appears poorly reactive (1). However, tend to react with that target rather than nitric oxide in a radical addition reaction higher levels of H202 can attack several with any added scavenger, and the OH- (35). cellular energy-producing systems, e.g., by will be very difficult to intercept by OH' inactivating the glycolytic enzyme glycer- scavengers. 2-p+ NO -* ONOO0 aldehyde-3-phosphate dehydrogenase It follows that a major determinant of peroxynitrite [3] (47). the nature of the damage done by excess It is usually thought that most or all of generation of ROS in vivo is the availabil- the toxicity of O- and H202 involves their ity and location of metal ion catalysts of NO- is known to be produced in vivo conversion into OH' (1,48). Several mech- OH- radical formation (1,29). If, for by vascular endothelial cells, by some cells anisms have been proposed to explain this. example, "catalytic" iron or copper ions are in the brain, and by phagocytes (8). NO The most recent is the interaction of O- bound to DNA in one cell type and to performs useful physiologic functions, such and NO' (Equations 3,4). An earlier pro- membrane lipids in another, then excessive as regulation of vascular smooth muscle posal (5,48) was the superoxide-driven formation of H202 and O°- will, in the tone (hence controlling blood pressure) Fenton reaction first case, damage the DNA and in the action Since other could initiate and neurotransmitter (8). O- + -* + lipid peroxidation. NO- acts upon smooth muscle cells in Fe(III) Fe(II) 02 [5] Evidence for OH' formation in the nucleus blood vessel walls to produce relaxation, of cells treated with H202 has been

Volume 102, Supplement 10, December 1994 7 HALLIWELL AND CROSS

obtained, presumably involving metal ions However, antioxidant defenses exist as a that there is continuous low-level oxidative bound upon, or very close to DNA balanced and coordinated system. Thus, damage in the human body. This creates a (56,57). although SOD is important, an excess of need for repair systems that can deal with E. coli mutants lacking SOD activity are SOD in relation to peroxide-metabolizing oxidatively damaged molecules (Table 2). hypersensitive to damage by H202 (34), enzymes can be deleterious (60-63). This However, if a greater imbalance occurs in and extra SOD can often protect cells has been shown by transfecting cells with favor of the ROS, oxidative stress is said to against damage by H202, provided that it human cDNAs encoding SOD (60). result (19). Most aerobes can tolerate mild can enter the cell (58). These data are con- Transgenic mice overexpressing human oxidative stress: indeed they often respond sistent with a role of O2- in facilitating Cu,Zn-SOD are resistant to elevated 02 to it by inducing synthesis of extra antioxi- damage by H202, and Equations 5 and 6 and to certain toxic agents (62,63) but dant defenses. For example, if rats are grad- provide an explanation. However, many they show certain neuromuscular abnor- ually acclimatized to elevated 02, they can scientists are reluctant to believe that O°- malities resembling those found in patients tolerate pure 02 for much longer than serves only as a reducing agent for metal with Down's syndrome (62). The gene naive rats, apparently due to increased syn- ions since, in general, mammalian tissues encoding Cu,Zn-SOD is located on chro- thesis of antioxidant defenses in the lung are fairly reducing environments. The argu- mosome 21 in humans, and Down's syn- (64,65). Other examples are the complex ments have been rehearsed in detail drome is usually caused by trisomy of this adaptive response of E. coli treated with (1,49,59) but the point is not yet settled. gene, raising tissue Cu,Zn-SOD levels by low concentrations of H202 (66) and the about 50%. The limited data available at activation of NF-KB in oxidatively stressed Antioxidant Defenses present are consistent with the view that mammalian cells (33). Living organisms have evolved antioxidant the excess of Cu,Zn-SOD may contribute However, severe oxidative stress can defenses to remove excess O- and H2O to at least some of the abnormalities in cause cell damage and death. In mam- Superoxide dismutases (SODs) remove O- patients with Down's syndrome (62). malian cells, oxidative stress appears to by greatly accelerating its conversion to In addition to antioxidant defense cause increases in the levels of free Ca2+ H202 (Equation 1). Human cells have a enzymes, some low-molecular-mass free (67) and free iron (68) within cells, e.g., SOD enzyme containing manganese at its radical scavengers exist. Reduced glu- by damaging proteins that normally keep active site (Mn-SOD) in the mitochondria. tathione can scavenge various free radicals these metal ions safely bound. Iron ion A SOD with copper and zinc at the active directly, as well as being a substrate for release can lead to OH- generation, which site (Cu,Zn-SOD) is also present, but GSHPX enzymes. a-Tocopherol is the has been shown to occur within the largely in the cytosol (5). Catalases in the most important free radical scavenger nucleus of H202-treated cells (56). An peroxisomes convert H202 into water and within membranes. Attack of reactive radi- excessive rise in intracellular free Ca2+ can 02 and help dispose of H202 generated by cals, such as OH-, upon membranes can also activate endonucleases and cause DNA peroxisomal oxidase enzymes (18). damage them by setting off a free radical fragmentation (67). However, the most important H202- chain reaction called lipid peroxidation. a- Hence, oxidative stress results in dam- removing enzymes in human cells are glu- Tocopherol (a-TH) inhibits this by scav- age to DNA, proteins, lipids, and carbohy- tathione peroxidases (GSHPX), which enging peroxyl radicals (Table 1), drates (67,68). The relative importance of require selenium (as an active site selenocys- intermediates in the chain reaction. damage to these different molecular targets teine residue) for their action. GSHPX in mediating cell injury or death depends enzymes remove H202 by using it to oxidize a-TH + LOO- -* aT. + LOOH [7] upon what degree of oxidative stress reduced glutathione (GSH) to oxidized glu- occurs, by what mechanism it is imposed, tathione (GSSG). Glutathione reductase, a However, the tocopherol thereby for how long, and the nature of the system flavoprotein enzyme, regenerates GSH from becomes a radical, aT-. This illustrates a stressed. For example, lipid peroxidation GSSG, with NADPH as a source of reduc- fundamental principle of free radical chem- appears to be an important consequence of ing power (18). Another important antioxi- istry: when radicals react with nonradicals, oxidative stress in human atherosclerotic dant defense already referred to is the new radicals are generated. Only when two lesions (69). Several halogenated hydrocar- sequestration of transition metal ions into radicals meet and join their unpaired elec- bons (such as CC14 and bromobenzene) forms that will not catalyze free radical reac- trons are the radicals lost (termination reac- appear to exert some, or all, of their toxic tions (1,29,53). This is particularly impor- tions). An example is the reaction of O- effects by stimulating lipid peroxidation in tant in the extracellular environment, where with NO (Equation 3). vivo (7). However, for most other toxic levels of SOD, GSH, GSHPX, and catalase Overall, antioxidant defenses seem to agents causing oxidative stress, lipid peroxi- are often very low (53). be approximately in balance with genera- dation is not the major mechanism of pri- Antioxidant defense enzymes are essen- tion of oxygen-derived species in vivo. mary cell injury: damage to proteins and tial for healthy aerobic life. For example, There appears to be no great reserve of DNA is usually more important (1,47,68). SOD-negative mutants of E. coli will not antioxidant defenses in mammals, perhaps For example, it has often been assumed grow aerobically unless given a rich growth because, as pointed out previously, some that lipids are a major target of damage by medium, due to impaired biosynthesis of oxygen-derived species perform useful inhaled ozone, but proteins may be equally certain amino acids. Even when so supple- metabolic roles. or more important (see below). mented, SOD E. coli cells grow slowly, suf- fer membrane damage, are abnormally Oxidative Stress: A Definition Causes of Oxidative Stress: sensitive to damage by H202 (perhaps Generation of ROS and the activity of Toxic Agents because of Equations 5 and 6) and show a antioxidant defenses appear more or less Oxidative stress can be imposed in several high mutation rate (34). balanced in vivo. In fact, the balance may ways. Thus, severe malnutrition can deprive be slightly tipped in favor of the ROS so humans of the minerals (e.g., Cu, Mn, Zn,

8 Environmental Health Perspectives OXYGEN RADICALS, DISEASE, AND THE ENVIRONMENT

Table 3. Why cigarette smoke can impose oxidative stress. age. This seems to happen in the inflamed (1) Smoke contains many free radicals, especially peroxyl radicals, that might attack biological molecules and joints of patients with rheumatoid arthritis deplete antioxidants, such as and x-tocopherol. (72) and in the gut of patients with (2) Smoke contains oxides of nitrogen, including the unpleasant nitrogen dioxide (N10-). inflammatory bowel diseases (73). Tissue (3) The tar phase of smoke contains hydroquinones. These are lipid-soluble and can -cycle to form °2- and injury can release metal ions from their stor- H20; They can enter cells and may even reach the nucleus to cause oxidative DNA damage. Some hydro- age sites within cells, leading to OH- genera- quinones may release iron from the iron-storage protein ferritin in lung cells and respiratory tract lining fluids. tion (72,74). Thus, the main question (4) Smoking may irritate lung macrophages, activating them to make 0-.a about ROS in human disease is not "can we (5) Smokers' lungs contain more neutrophils than the lungs of nonsmokers, and smoke might activate these cells demonstrate oxidative stress?" but rather to make 02-.a "does the oxidative stress that occurs make a (6) Smokers often eat poorly and drink more alcohol than nonsmokers and may have a low intake of nutrient significant contribution to disease activity?" antioxidants. The answer to the latter question appears to be "yes" in at least some cases, including "The effects of cigarette smoke on phagocytes are dose-related. Low levels may stimulate them, but high levels atherosclerosis, rheumatoid arthritis, and may poison them and so depress their activity. inflammatory bowel disease (1,69,172-74). However, it may well be "no" in many oth- ers. Elucidating the precise role played by Se) and vitamins (e.g., riboflavin-needed of prematurity) in premature babies (3). free radicals has not been easy because they for the FAD cofactor of glutathione reduc- However, there are many papers in the bio- are difficult to measure, but the develop- tase, and cx-tocopherol-needed for antiox- medical literature suggesting a role for ment of modern assay techniques is helping idant defense) (1). More usually, however, oxidative stress in other human diseases to solve this problem (72). the stress is due to production of excess [over 100 at the last count (1)]. ROS. Tissue damage by disease, trauma, poi- Causes of Oxidative Stress: Several drugs and toxins impose oxida- sons, and other causes usually lead to forma- Environmental Air Pollutants tive stress during their metabolism. tion of increased amounts of putative The role offree radicals in the toxicity of °2 Carbon tetrachloride is one example "injury mediators," such as prostaglandins, and of cigarette smoke (Table 3) has already (Table 1). Another is paraquat, a herbicide leukotrienes, interleukins, interferons, and been discussed, but oxidative damage is fre- that causes lung damage in humans. Its tumor necrosis factors (TNFs). All of these quently suggested to be involved in the dele- metabolism within the lung leads to pro- have at various times been suggested to play terious effects of 03 and NO2 (75,76). 03 duction of large amounts of 0;- and important roles in different human diseases. is not a free radical, but it can oxidize many H202 (1). Gas-phase cigarette smoke also Currently, for example, there is much inter- biological molecules directly and, in addi- imposes some oxidative stress. Some of the est in the roles played by TNFa, NO-, and tion, it reacts slowly with water at alkaline reasons for this are summarized in Table 3 interleukins in adult respiratory distress syn- pH to give OH- (77). It has also been sug- (70,71). drome and septic shock (8,72). ROS can be gested to produce singlet 02 when it reacts placed in the same category, i.e., tissue dam- with biological molecules (78). Causes of Oxidative Stress: age will usually lead to increased ROS for- The first biological fluids that come Disease and Tissue Injury mation and oxidative stress. Figure 1 into contact with inhaled 03 are the respi- Does oxidative damage play a role in summarizes some of the reasons for this. ratory tract lining fluids (RTLFs), that pre- human disease? Many of the biologic con- Indeed, in most human diseases, oxidative sumably serve to absorb and detoxify some sequences of excess radiation exposure may stress is a secondary phenomenon, a conse- of the inhaled 03 so as to lower the amount be due to OH--dependent damage to pro- quence of the tissue injury. That does not that enters the more vulnerable peripheral teins, DNA, and lipids (6). Oxidative mean it is not important (1,72). For exam- gas exchange regions of the lung. Some damage (resulting from exposure to ele- ple, excess production of O°- 202, and information is available about the antioxi- vated 02 in incubators) may account for other species by phagocytes at sites of dants of these fluids (79,80) but the prob- damage to the retina ofthe eye (retinopathy chronic inflammation can cause severe dam- lems of sampling them (by the techniques of respiratory tract lavage) have hindered elucidation of their precise comparative * Increase in radical-generating enzymes (e.g. antioxidant capabilities, since lavage itself xanthine oxidase) and/or their substrates (e.g. hypoxanthine) produces considerable and variable dilution of RTLFs and some of their constituents - Activation of phagocytes may be oxidized during the procedures. In Heat - Activation of phospholipases, addition, the antioxidants present depend Trauma cyclooxygenases, and lipoxygenases Ultrasound upon which part of the respiratory tract is Infection * Tissue I *- Dilution and destruction of antioxidants being sampled (79), i.e., nasal passages, air- Radiation Damage Elevated 02 - Release of "free"metal ions ways, bronchioles, alveoli. Often a mixed Toxins from sequestered sites fluid is obtained. Exercise to excess lschemia * Release of heme proteins (hemoglobin, By contrast, the antioxidant defenses of myoglobin) human plasma have been well characterized -. Disruption of electron transport chains and (53). To approach an understanding ofhow increased electron leakage to form 02' 03 might interact with a complex biological fluid, the reactions of 03 with freshly pre- Figure 1. Some of the mechanisms by which tissue damage can cause oxidative stress. pared human plasma have been studied.

Volume 102, Supplement 10, December 1994 9 HALLIWELL AND CROSS

Table 4. A comparison of the effects of cigarette smoke (CS), 03, and NO2 on human plasma. L-H + LOO --> L + LOOH [10] Event CS 03 N02 Initiation of peroxidation by NOQ Oxidation of ascorbate + + + + + + + + + + + + (Equation 8) presumably sets up the auto- Breakdown of uric acid + + + + + + + + + Loss of plasma -SH groups + + + + + + + + + catalytic chain reaction of lipid peroxida- Formation of protein carbonyls + + + + + + 0 tion (Equations 9,10) resulting in the Depletion of a-tocopherol + + 0 + + accumulation of lipid peroxides (LOOH). Lipid peroxidation + 0 + + Uric acid and ascorbate were found to be important antioxidants protecting plasma against oxidative damage by NO;, but the Indeed, oxidant injury to the lung causes Since uric acid is a major constituent of lipid-soluble antioxidants ct-tocopherol increased influx of plasma constituents, so upper airway lining fluid in humans (79), it and ubiquinol probably also play an that the lung lining fluids become more like could act as a "scrubber," decreasing the important protective role in limiting lipid plasma in composition. Uric acid and ascor- concentration of 03 in inhaled air so as to peroxidation (86). bic acid were found to be the major plasma protect the more vulnerable alveolar regions Hence, as summarized in Table 4, 03 scavengers of 03 (81). Although it is often ofthe lung (81). and NO2 damage different molecular tar- assumed that lipids are a major target of By contrast with the effect of 03, NO0 gets, which could be one reason why they attack by 03, no evidence of substantial did not generate protein carbonyls in might sometimes exert synergistic damag- lipid damage by 03 was obtained (81), in plasma, although nitration of aromatic ing effects in vivo. For comparison, Table 4 keeping with other studies in the literature amino acids took place and protein -SH also includes the effects of cigarette smoke (82,83). Instead, oxidative protein damage groups were lost, presumably by direct upon plasma. Ascorbic acid is rapidly oxi- was observed in 03-exposed plasma, as -SH reaction with NO (86). Again unlike 03, dized and damage to both proteins (car- group loss and protein carbonyl formation NO2 induced lipid peroxidation in plasma, bonyl formation, loss of -SH groups) and (84). However, it will be necessary to study presumably by the reactions lipids (lipid peroxide formation) occurs. human lung lining fluids to substantiate this Ascorbic acid protects the plasma against conclusion, especially as Cueto et al. (85) L-H + NO - HN02 + L [8] lipid damage by ozone, but does not pro- found end products of lipid damage in lung tect against protein damage (87,88). lining fluids from rats after 03 exposure. L- + 02-4 LOO [9]

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