Oxygen toxicity

REVIEW

Oxygen Toxicity

LEE FRANK, M.D., Ph.D. DONALD MASSARO, M.D. Miami. Florida

From the Pulmonary Toxicology Laboratory, the V.A. Hospital, Miami, Florida; and the Pulmonary Division, Oak Asthma Research and Treatment Facility, University of Miami School of Medicine, Miami, Florida. This study was supported in part by NIH Grants HL07283, HL20366 and V.A. Research Funds. Requests for reprints should be addressed to Dr. Lee Frank, Pulmonary Division R-120, University of Mi-

ami. School of Medicine, P.O. Box 016960, Miami, Florida 33101. Manuscript accepted January 24,198O.

The “free radical theory of oxygen toxicity” attributes the damaging effects of hyperoxia to highly-reactive metabolic products of oxygen (Or) that can inactivate enzymes in the cell, damage DNA and destroy lipid membranes. To protect the organism from these cytotoxic O2 metabolites, an array of cooperative antioxidant defense systems have evolved. These include the enzyme superoxide dismutase (SOD), catalase and glutathione peroxidase, plus other endogenous antioxidants such as ascorbate and vitamin E. The increased levels of 02-free radicals produced during hyperoxia may overwhelm these normal antioxidant defense systems. The ability to respond rapidly to hyperoxic challenge with increased antioxidant activity has been shown repeatedly to be associated with relative tolerance to Oainduced injury and lethality. Treatment with exogenous SOD or vitamin E has had some clinical success in combating Os-free radical-mediated tissue injury. Treatment with small doses of bacterial endotoxin has provided almost complete protection to rats against pulmonary O2 toxicity; this agent seems to act by facilitating rapid increases in endogenous antioxidant enzyme levels. An increased understanding of the molecular mechanisms of 02 toxicity and of the endogenous defenses that organisms have evolved against 02-free radical injury may lead to a more rational basis for the clinical use of O2 and to the development of therapeutic measures effective in preventing or ameliorating O2 toxicity. These measures may involve (1) tests aimed at estimating a person’s tolerance to hyperoxia at any given time, [z) means to effectively deliver exogenous antioxidants and (3) methods to augment endogenous antioxidant defenses. It has been nearly a decade since Haugaard [l], and Clark and Lambertson [z] published their excellent reviews of oxygen toxicity. In this review we shall stress some of the newer experimental information on the mechanism(s) of 02 toxicity and proposed means of protection against its detrimental actions. We shall emphasize what we believe is the major development of the past 10 years: the free radical theory of 0s toxicity. Finally, we shall discuss future approaches to therapy that may evolve from further understanding of the innate defenses of the organism against 02-free radical injury.

THE OXYGENIC THEORY OF EVOLUTION With his first tentative cry, the newborn assumes complete dependence upon his own respiratory function and opens his lungs to the alien environment of 21 per cent 02 for the first time. If ontogeny does recapitulate phylogeny, then birth represents a leap of millions of years:

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the span of time when primordial creatures emerged from the sea to breathe the relative hyperoxia of the ambient air. A fascinating evolutionary history has preceded the newborn and prepared him for adaptation to the 02 environment. Although O2 has been present in the earth’s atmosphere an estimated 5 billion years, its concentration is believed to have been infinitesimal until about 2 l/2 billion years ago when photosynthetic organisms first appeared [3,4]. Joining the hypotheses about the progressive increase in the 02 content of our atmosphere with the classic Darwinian hypothesis of evolution leads to the interesting concept that the progressive evolution of life forms on earth was directed by and paralleled the evolution of our 02-containing atmosphere [4]. It has been proposed that the gradual increase of atmospheric O2 concentration not only permitted the further evolution of animal forms dependent on aerobic metabolism, but also may have actually acted as a driving force for the evolution of extant life forms: favored for survival were those forms which were best adapted for protection against the increasing oxidant stress of a changing O2 atmosphere [4]. Haugaard [l] has refined this oxygenic theory suggesting that the evolution of organisms dependent on aerobic metabolism required that biochemical defenses be evolved to protect them from the potential for excess oxidation of cellular constituents by OZ. Fridovich [5] has discussed how the evolution of efficient cellular electron transfer molecules (cytochrome oxidases) has reduced the potential for O2 toxicity during normal cellular 02 consumption. The development of effective antioxidant defenses is, therefore, one more homeostatic requirement to enable adapting organisms to buffer the internal milieu from potentially toxic metabolic products [5,6]. The oxygenic theory of evolution has interesting implications for fetal development. It suggests the need for early development to proceed in the special hypoxic uterine environment until the fetus has evolved mechanisms sufficient to defend its tissues from the higher 02 concentrations that it must adapt to at birth. The lungs must be prepared to provide oxygenation to the other tissues of the body while resisting 02 injury to their own cellular structure. Recent work showing that the activities of antioxidant enzymes in the lungs [7,8] and in the blood [8-lo] increase in late gestation tend to support these ideas. The physiologic stimulus for the prenatal increase in antioxidant defensive capacity is not known, teleologic explanations aside. However, we can speculate that the marked increase in the metabolic activity of the lung that occurs during late gestation [11,12] and the increasing total 02 consumption of the growing fetus [13,14] could provide sufficient increases in Ox-free radical production to stimulate increases in antioxidant enzyme activity during the late stages of gestation, preparatory to birth. The oxygenic theory of evolution focuses our attention on the development and importance of antioxidant 118

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defense mechanisms. It also provides a foundation for exploring how organisms might adapt in more extreme 02 concentrations than those of their current evolutionary niche. PATHOLOGY: OXYGEN-INDUCED DAMAGE TO TISSUES The lung is the organ most severely damaged by exposure to hyperoxia at 1 atmosphere (atm) pressure. The pathologic changes in the lung associated with hyperoxic exposure have been systematically worked out in various animal models [15-181. In brief, these are often separable into an acute or exudative phase of 02 toxicity and a subacute or chronic proliferative phase of O2 toxicity. The acute phase is associated with perivascular, peribronchiolar, interstitial and alveolar edema, and alveolar hemorrhage, with variably extensive necrosis of the pulmonary endothelium and type I epithelial cells. The proliferative phase is characterized by progressive resorption of exudates and thickening of the alveolar septums due to hyperplasia of interstitial cells and the relatively 02-resistant type II alveolar lining cells. A variable degree of collagen and elastin deposition in the interstitum of the alveolar walls contributes to the thickened gas exchange areas of the lung. Animals that survive prolonged O2 exposure may show near complete resolution of these changes in the lung [18], but, more often, permanent alterations of lung structure and function due to diffuse scarring and/or emphysematous changes are the outcome [15,17]. Detailed examination of the alterations in the lungs of human subjects treated with prolonged high levels of fractional concentration of oxygen is inspired gas (FiOa) suggests that a very similar sequence of pathologic changes occursin man [1,2,15,19-221. The detrimental effects of hyperoxic exposure on other organ systems have not received much experimental attention since the review of Clark and Lambertson [2] appeared. A more recent review by Balentine [l5] does provide some additional information, and a very thorough review of 02 toxicity and the developing retina (retrolental fibroplasia or RLF) has also appeared P3l. HYPERBARIC HYPEROXIA The pulmonary effects of hyperbaric 02 exposure in experimental animals have been detailed in several recent reviews [1,2,15,24,25]. These effects are essentially an accelerated onset and course of the Oz-induced changes described herein for hyperoxic exposure at
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these studies in human volunteers stress the marked individual variability of response to hyperbaric exposure. The unique toxic problem associated with exposure to O2 at elevated pressure compared to normobaric hyperoxia, and the factor which currently limits the duration of its use in man, is its effect on the central nervous system. In animal studies, grand mal convulsions are a reproducible effect when high 02 pressures are tested: and the seizure-free period progressively decreases as the level of hyperbaria used increases [1,2,15.25]. In studies in man, grand ma1 seizure activity has been induced with exposure conditions ranging from 3 atm for 35 to 95 minutes to 7 atm. for only 5 minutes [1,2,24,27]. Clark 1241and others [25] have studied this problem closely in order to determine reasonable safety limits in man to avoid central nervous system toxicity. These guidelines indicate that exposure to 12.8 atm Oz appears safe for a duration not to exceed 2 hours. Many basic questions remain about the mechanism(s) responsible for the toxic central nervous system effects of hyperbaric OZ. questions which extensive animal studies have still left unresolved: What is the metabolic basis for the changes in the central nervous system; what is the role of altered neurotransmitter turnover [2,25,28]; the role of changes in the central nervous system in the production of pulmonary toxicity [1,15,29]; and, are there permanent lesions in the central nervous system due to increased O2 tensions? [1.15,30]. These unresolved issues assume an increased importance with the renewal of interest in the application of hyperbaric medicine in the past decade or so. In addition to the successful use of hyperbaric treatment in underwater and altitude decompression sickness [25.31], other uses of hyperbaric O2 treatment are being clinically tested. The use of 02 therapy as a treatment for gas gangrene due to Clostridial infection dates back to 50 years [32]. Recently, there is a great deal of clinical interest in the use of hyperbaric 02 as adjunctive treatment for other anaerobic infections and for indolent infections such as chronic osteomyelitis. The successful elimination of refractory foci of osteomyelitis. resistant to standard surgical and antibiotic management, has been reported from a number of centers [33,34]. The efficacy of this adjunctive treatment is believed to depend on several effects of 02, including stimulation of osteoblastic/ osteoclastic activity, vascular proliferation and correction of hypoxia at the infected site, and perhaps an additional direct bacteriostatic or tidal action of increased O2 tension. Hyperbaric 02 treatment for severe carbon monoxide poisoning has also been proved to be of clinical value [35]. In human volunteers it has been shown that the half-time for elimination of carbon monoxide from the blood can be reduced from 68 to 19 minutes with exposure to 02 at 2.5 atm [36]. Clinical trials of hyperbaric O2 treatment for other pathologic conditions have not yet produced the same definitive results seen in the

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research laboratory. However, some clinical successes have been reported with the application of hyperbaric 02 therapy: in combination with radiation for head and neck tumors [35,37]; to increase O2 delivery to marginally-vascularized skin-pedicle grafts [35,38]; in the management of burn patients [35,39]; in the treatment of indolent decubitus ulcers [35]; and combined with hypothermia to prolong the viability period of organs for transplantation [35]. As experience with these applications of hyperbaric medicine increases, optima1 parameters for treatment and safer guidelines for treatment appear to be evolving simultaneously. MECHANISM OF OXYGEN CELLULAR DEFENSES

TOXICITY

AND

“A better understanding of the fundamental mechanism involved inclines us to marvel at the continuous and powerful defenses against oxygen, rather than to be surprised at its potential destructive action.” Rebeca Gem&man, 3964 [6]. The mechanism of O2 toxicity at the molecular level is now generally attributed to 02-free radical reactions with cellular components. This so-called “free radical theory of 02 toxicity” attributes the damaging effects of hyperoxia to highly reactive metabolites of molecular Oz. These 02-free radicals should not be regarded as biologic curiosities for they arc products of normal cellular oxidation-reduction processes [1,40]; under conditions of hyperoxia. their production increases markedly [41,42]. The inherent nature of the O2 molecule makes it susceptible to univalent reduction reactions in the cell to form superoxide anion (02-), a highly reactive cytotoxic free radical [43-461. In turn, other reactive products of 02 metabolism including hydrogen peroxide (H202), the hydroxyl radial (OH-) and singlet oxygen (‘02) can be formed from subsequent intracellular reactions of 02-. These short-lived species are capable of various toxic activities including inactivation [oxidation) of sulfhydryl enzymes, interactions with DNA and lipid peroxidation of cellular membranes [1.43,44] (See Figure 1). A large amount of evidence has appeared recently about the endogenous defense systems evolved by organisms to protect their biologic integrity from free radical destruction. These intrinsic biochemical defensive systems include the antioxidant enzymes SUperoxide dismutasc (SOD), catalase [CAT] and glutathione peroxidase (GSH pcroxidasc or GP); the lipid membrane constituent, vitamin E; and other intracellular antioxidant compounds such as ascorbate, and sulfhydryl-containing components like glutathione. cysteine and cysteamine. Although the existence of H202-detoxifying agents in biologic systems (catalases and peroxidases) has been known for mom than a century, the ubiquity and importance of the protective enzyme, SOD, has been established for less than a decade. McCord and Fridovich

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(‘$0, + 0;

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*OH-+

OH.

+ ‘02)

SULFHYDRYL ENZYME INACTIVATION LI

INTERACTION WITH DNA

\

POLYUNSATURATEDFATTY ACIDS cL,p,oPtROX,OL,,OW) CELL ORGANELLE MEMBRANES

I

Non-Toxic

HYDROXY

F.A./~OXlLllZED

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/

\

NADPHJ

Figure 1. Oxygen free radicals and antioxidant defense systems. Illustrated are the highly reactive O2 metabolites: Oz-, superoxide anion; OH., hydroxylradical; H202, hydrogen peroxide; and 102, singlet oxygen. Also shown are the antioxidant enzyme defense systems: SOD, superoxide dismutase; catalase; and, glutathione (GSH) peroxidase, glutathione (GSH) reductase, and glucose-6-phosphate dehydrogenase (GG-PD). These enzymes function to protect the cell from the types of damaging biochemical interactions with 02 radicals listed: critical sulfhydryl oxidations; DNA scism, alteration: membrane lipid peroxidations.The reaction between

H202 and 02- to form other toxic O2 metabolites is the socalled Haber-Weiss reaction (in parentheses). This reaction requires trace amounts of metals such as iron.

[45] isolated SOD from erythrocytes and showed that it functioned to detoxify the superoxide radical (02-l by the reaction: ZOs- + ZH+--SOD+H20s + OZ. With a KM of 2 X log M-l set-I, SOD is an extremely efficient Os- radical scavenger [4l]. Fridovich and co-workers [46] demonstrated the presence of SOD in a large variety of aerobic bacteria and its absence,in anaerobic organisms for which 02 exposure is rapidly toxic [46]. They also demonstrated the inducibility of SOD enzyme activity in prokaryotes by exposure to hyperoxia and showed that microorganisms with induced high levels of antioxidant enzyme were able to resist subsequent exposure to normally lethal extremes of hyperbaric 02 [47]. The involvement of antioxidant enzymes in the development of tolerance to hyperoxia was extended to eukaryotes by Crapo and Tierney [48]. They showed an increase in SOD activity in the lung during exposure of rats to a sublethal level of 0s (85 per cent) and prolonged survival in preexposed rats transferred to a normally lethal 100 per cent O2 environment. Re-entry to room air resulted in a gradual return to normal SOD levels in the lung and a parallel loss of tolerance to hyperoxia [48]. By demonstrating that the lungs of 02-tolerant newborn animals respond to hyperoxic exposure with rapid and significant increases in lung SOD activity, Frank et al. [49,50] and Yam et al. [5l] have provided a possible biochemical explanation for the well-known tolerance of newborn animals to hyperoxic challenge. By then

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treating rat pups with diethyldithiocarbamate (DDC], an SOD inhibitor, it was shown that the newborn animal’s relative tolerance to hyperoxia could be severely compromised by blocking the normal rise in pulmonary SOD activity during 0s exposure [52]. A variety of in vitro studies have confirmed the biologic importance of SOD in the prevention of Oz-free radical cytotoxicity [44,53-55). Glutathione peroxidase (GSH peroxidase or GP) is able to convert toxic lipid peroxides (formed through free radical attack on unsaturated lipid in the cell) into nontoxic products [56-581 [Figure 1). GP utilizes the reducing equivalents (electrons) from reduced glutathione (GSH) for these reactions. In turn, the enzyme glutathione reductase (GSH reductase) transfers the reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to regenerate intracellular supplies of reduced GSH. NADPH is then regenerated through the action of the pentose phosphate shunt enzyme, glucose-6-phosphate dehydrogenase (G-6-PD) or the action of other enzymes such as malic enzyme or 6-phosphogluconate dehydrogenase. In addition to this detoxifying action, GP is apparently the primary enzyme for H202 removal in red blood cells [59]. McCay et al. [60] and Fong et al. [58] have proposed that lipid peroxidation in biologic membranes is not only checked by GP activity on lipid peroxides but also that lipid peroxidation may actually be prevented by the direct scavenging of the highly reactive hydroxyl radical (OH.) by this enzyme. The role of the GP system in protection from Os-free radical toxicity is also supported by a large number of studies examining the correlation between 0s tolerance/susceptibility and lung enzyme activity [57,61-631. Tolerance is associated with a high level of enzyme activity and especially with the ability to rapidly increase enzyme activity during hyperoxic challenge. Susceptibility to hyperoxia is associated with low levels of GP activity or with an inability to increase the level during exposure to toxic concentrations of 0s. The importance of catalase [CAT) in the antioxidant armamentarium is less clearly established than are the roles of SOD and GP. Many aerobic bacteria contain negligible CAT activity yet tolerate 0~ exposure satisfactorily, and substantial concentrations of CAT are present in some obligate anaerobic microorganisms for which O2 exposure is lethal [47]. Theoretically, rapid removal of Hz02 should be a very important factor in limiting cellular Os-free radical injury. The rapid removal of Hz02 would serve to diminish the production of the highly reactive OH- radical via the Haber-Weiss reaction (0~~ + HsOs+OH+ OH- + OZ] (Figure l] [40]. Under hyperoxic conditions, with excess HzOz formation, CAT may assume a more important antioxidant role than it has under normoxic conditions, when GP is able to handle the catalysis of low concentrations of HzOz more efficiently. Evidence does exist for the importance of CAT during hyperoxic exposure; for example, significantly increased CAT activity in the lungs

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of Oz-exposed neonatal animals was associated with prolonged survival in hyperoxia [50,51]. In addition, increased CAT activity was measured in the lungs of rats tolerant to 02 or nitrogen dioxide (NOs] in the recent cross-tolerance studies of Crapo et al. [64]. A variety of in vitro studies also suggest the importance of CAT activity in the over-all antioxidant defensive capacity of the cell [53,55,65,66]. Increases in G-6-PD activity in responses to oxidant exposure would have a dual importance. Greater enzyme activity could provide for increased amounts of NADPH both for use by the GP antioxidant system (Figure 1) and also for the biosynthesis of nucleotides and fatty acids critical to the repair of oxidant-induced cell damage [57,62,67]. In many of the studies mentioned herein increased G-6-PD activity has been demonstrated in the lungs of hyperoxic-stressed animals [51,61,63,64,67]. This increased activity, however, does not appear to be directly correlated to protection from oxidant toxicity. Instead, the change in G-6-PD activity seems to be a general biochemical response to cell injury caused by a variety of toxic agents in addition to oxidants [63]. Both Oz-tolerant neonatal animals and OZ-susceptible adult animaIs had significant increases in G6-PD lung enzyme activity during exposures to 295 per cent 0s [5l]. Studies of 02 toxicity in primates also showed that increased levels of G-6-PD (in the absence of increased SOD, GP and CAT) failed to protect the animals from the development of severe lung injury [681. Vitamin E is thought to be the main lipid-phase antioxidant protective mechanism of the cell. It acts by donating hydrogen to peroxides that form in lipid membranes; this Iimits their reaction with neighboring polyunsaturated fatty acids, thereby confining lipidperoxidation chain reactions and resultant membrane damage [69,70]. Mustafa and Tierney [63] have provided a compilation of the important studies confirming the influence of vitamin E in the prevention of oxidant injury. Over-all, the evidence for aggravation of O2 damage in experimental animals that are made vitamin E deficient is much more convincing than is the evidence for protection afforded to vitamin E-replete organisms that are given supplemental vitamin E prior to or during oxidant exposure. Yam and Roberts [71], and other investigators [63,72,73]. failed to show any protective effects of supplemental vitamin E against Oz-induced lung damage or lethahty in adult rats and mice. Nonetheless, vitamin E has been shown to be a very effective antitoxidant against free radical-mediated damage in the following test systems: amelioration of hyperoxic injury to the eye in the premature kitten model of retrolental fibropIasia [74,75]: protection from oxidant damage, lipid peroxidation, hemolysis and hemoglobin oxidation to methemoglobin in red blood cells exposed to oxidant stress in vitro [69.70,76]. Clinical evidence for vitamin E as an effective antioxidant is subsequently discussed herein.

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The antioxidant role of water-soluble vitamin C (ascorbate) is not clear-cut. Early studies indicated a protective effect from ozone and hyperbaric O2 exposure, but prolongation of survival in the treated animals was not very striking [l,2]. Although there is some evidence that ascorbate can react directly with 0~~ to produce HzOz, its quantitative biologic importance is unknown [77,78]. Indeed, several investigators have discussed how ascorbate may have an ambiguous role; considering evidence for ascorbate as a biologic protectant against 0~ free radical damage or as potential promotor of lipid peroxidation reactions [63,79]. Theoretically, the presence of excess thiol compounds (such as cysteine and cysteamine], which can donate electrons from their sulfhydryl groups, can reduce Oz-free radical injury to more important cellular components by competitively preventing sulfhydryl-enzyme inactivation (oxidation). In one very recent study significant protection from acute hyperoxic lung injury has been shown in rats treated with continuous infusions of cysteamine [80]. PHARMACOLOGIC APPROACHES TO PROTECTION FROM O2 TOXICITY Although clinically effective agents to help circumvent O2 toxicity are still unavailable, some promising new studies in animals and preliminary clinical reports of efficacy merit attention. Most of these have involved efforts to augment the normal antioxidant protective systems by exogenous means. Vitamin E appears to be the most promising prophylactic agent against 02 toxicity, especially its use in premature infants who are normally deficient in vitamin E. Administration of vitamin E shortly after birth has been reported to ameliorate and reduce the incidence of retrolental fibroplasia in O&reated premature infants [81,82] and to reduce the destruction of red blood cells that characterizes hemolytic anemia in the premature infant and which is accelerated by 02 treatment [83,84]. Vitamin E treatment has also been recently reported to reduce the incidence of bronchopulmonary dysplasia (BPD) in premature infants who require prolonged high OZ treatment immediately after birth [85]. These preliminary results of a protective effect from pulmonary O2 toxicity (BPD) will need confirmation in larger series which utilize different methods of ventilation because the method used in this study resulted in an unusually high incidence of bronchopulmonary dysplasia in the control group of infants (46 per cent) and because of criticism of the roentgenologic criteria used to make the diagnosis of bronchopulmonary dysplasia [86]. Other means of increasing the antioxidant defense capacity of humans subjects have been limited to trials in very desperate situations in which the efficacy is difficult to evaluate due to the delayed initiation of treatment. An example is the administration of SOD for attempted interruption of severe pulmonary damage following paraquat ingestion [87,88]. Paraquat is a commonly used herbicide whose lung toxicity is be-

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a problem

1

I

Figure 2. Vulnerability of pulmonary endothelium to oxygen toxicity. Plasma membrane of endothelial cells are subjected to two-pronged attack by oxygen free radicals (02-): (1) increased intracellular production of 0~~ due to increased O2 tension, and (2) extracellular 0~~ released by circulating blood cells. At right, platelets have attached to Os- damaged site on endothelial plasma membrane, aggregated, and released additional toxic 02- radicals. Type I (and type II) epithelial cells are removed from intimate contact with bloodborne 02- radicals. This proposed schema may help to explain why pulmonary endothelial cells are the earliest cells of the lung damaged by hyperoxic exposure.

lieved to be mediated by 02-free radicals [89-911. Animal studies designed to test the protective effect of exogenous SOD versus 02-free radical toxicity have provided conflicting results [89,92-951. Since the large protein molecule (MW 32,000) SOD enters cells poorly [95,96], the protective effect of exogenous SOD may be exerted mainly against extracellular Os- radicals. Nonetheless, this might result in an important measure of protection for the functions of the endothelial cell plasma membrane. This notion is supported by studies which demonstrate that exogenous SOD can scavenge free radicals from the extracellular compartment before cytotoxicity occurs [96-991, and that SOD treatment protects the serotonin (5-HT) uptake function of lung endothelial cells during hyperoxic exposure [loo]. Steinberg et al. [loll provided additional evidence of the efficacy of exogenous SOD using the isolated, perfused lung preparation. Toxicity and loss of pulmonary endothelial cell plasma membrane function that occurs when an Os--generating system is present in the perfusate was blocked by the addition of SOD to the perfusate [loll. Direct injury to endothelial cells by 0~~ free radical release from complement-activated white blood cells has also been demonstrated very recently by Sacks et al. [102]. A commercial preparation of SOD (Orgotein, Diagnositc Data Inc., Palo Alto, California), has had successful preliminary clinical trials as an anti-inflammatory agent, functioning to detoxify 02released by phagocytic cells at sites of inflammation [98,99,103]. These new studies may have some direct bearing on 122

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that has been perplexing investigators of 0s toxicity for many years, namely, why the pulmonary endothelium is more vulnerable to hyperoxic lung damage than the alveolar epithelium which is also directly exposed to the high 0s tensions of alveolar gas. To explain this, researchers have theorized t.hat some blood or blood cell-endothelial cell interaction may occur which multiplies the toxic attack on the endothelium [104,105]. It may well be then that the pulmonary endothelium is made especially vulnerable to 0s toxicity because of a two-pronged attack by increased 0~~ radicals: those generated within the endothelial cell itself due to increased 0s tensions, and those radicals released from circulating blood cells as they pass directly in apposition to the capillary endothelium in the lung [106]. Since platelets also are known to release 0~~ radicals during clumping and metamorphosis [107,108], the attachment of platelets to Os-damaged endothelial cells may result in an additional exacerbation of 02radical attack. This process could play a role in the accelerated destruction of the endothelium that occurs in pulmonary 02 toxicity, initiating the cascade of pulmonary edema and hemorrhage responsible for the death of the hyperoxic-exposed animal. If these events do indeed take place in vivo during exposure to high concentrations of 02, then infusion therapy to maintain a high blood level of SOD could become an important means of warding off extracellular Os- radical attack on the endothelial barrier of the lung (see Figure 21. A comparable protective effect against retrolental fibroplasia might be anticipated by infusions designed to elevate plasma SOD levels, since 02--induced lipid peroxidation in developing retinal vascular membranes has been proposed as a possible mechanism initiating the vaso-obliterative phase of retrolental fibroplasia [23,109]. Clark and Lambertson [2], and Haugaard [l] review some other pharmacologic agents reported in the older literature to have some protective effect [but also toxicity of their own) against hyperoxia. The phenomenon of “tolerance” to 100 per cent 0s exposure induced in rats by a pre-exposure to sublethal concentrations of 0s and the “tolerance” achieved in animals after pretreatment with the lung toxins alpha-naphthylthiourea (ANTU) and oleic acid will be omitted from discussion, since very recent summaries of this work are available [63,73]. Also, because substantial lung damage still occurs with these treatment regimens, the possible clinical applicability of such treatment is unpromising. In contrast to these experimental manipulations is the marked protective effect observed in adult rats treated during 02 exposure with small dosages of bacterial endotoxin [110-1121. The 72-hour survival rate of rats in 295 per cent 0s has been increased from 30 per cent to 97 per cent with daily injections of endotoxin in dosages equivalent to only about 1/5Oth to l/lOOth of the median lethal dose (LDsO)for the species. Survival of rats during more prolonged seven-day exposures to hyperoxia was improved from less than 15 per cent in the untreated

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animals to 95 per cent [112]. More recently, it was shown that a single dose of endotoxin administered even up to 24 to 36 hours after the start of the hyperoxic exposure period resulted in significantly increased survival of the treated animals and in reductions in the acute exudative changes seen in the lungs of the untreated 02-exposed animals [113]. It is of interest that whereas untreated adult rats in 100 per cent 02 fail to show changes in pulmonary antioxidant enzyme activity, the simultaneously-exposed endotoxin-treated animals consistently show significantly increased pulmonary SOD, CAT, and GP activity during 02 exposure [110.112.113]. FVMJRE

PROSPECTS

Knowledge about the mechanism of action of a toxic process usually precedes the development of effective measures or agents to counter the process. Our understanding of the free radical theory of 02 toxicity as it has evolved over the past decade or so has not only provided us with a rational perspective on the mechanism of O2 toxicity at the molecular level, but also a direction for future therapeutic measures designed to circumvent O2 toxicity. Interference with the initiation of O2 toxic changes may come about with the use of agents that function either intracellularly or extracellularly to reinforce the endogenous antioxidant protective systems. The studies cited herein in which exogenous SOD preparations showed experimental efficacy suggest that SOD infusions could offer some degree of protection from 02-free radical attack caused by 0~~ release from formed blood elements, especially if less rapidly cleared enzyme preparations with longer plasma half-lives become available [89,93,94.98,100]. Furthermore, the development of chemically modified preparations of SOD, with increased solubility in lipid membranes (liposomal encapsulation?) might provide a means to augment the intracellular concentrations of this important antioxidant defense against 02 cytotoxicity. Further investigation into the mechanism of action of experimental protective agents like bacteria1 endotoxin, which appear to facilitate increases in endogenous antioxidant enzyme activity, could provide another means of achieving this same end [llO-1131. Alternately, it has been hypothesized that cells with increased sources of exogenous polyunsaturated fatty acids (PUFA) or sulfhydryl compounds may establish intracellular pools of these expendable agents to trap 02-free radicals and thereby deflect their attack from important structural components of the cell [1.2,114-116). Preliminary studies with PUFA dietary supplementation and with cys-

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teamine infusion therapy have suggested the feasibility of protection from O2 lung damage in rats by these means [80,115]. The future use of synthetic chemical antioxidant agents, including specific OH- radical scavengers with proved experimental protective effects, will probably depend on the chemists’ ability to produce nontoxic preparations of such agents which retain efficacy. Based on the encouraging preliminary trials in premature infants [81-851, vitamin E treatment to provide additional resources to interrupt the propagation of destructive peroxidation reactions caused by 02 radicals deserves further clinical evaluation in larger groups of patients. One of the most puzzling aspects of O2 toxicity is the unexplained individual variation in susceptibility/ resistance to the development of tissue damage and physiologic alterations in animals (or human subjects] exposed for prolonged periods to hyperoxic conditions [1,2,20,21,24,26,117,118]. A few studies have suggested a genetic basis for the significant differences in 02 susceptibility/resistance observed within various strains of the same animal species [119-1211. An important consequence of the marked individual variation in susceptibility to O2 toxicity is invalidation of attempts to define minimal toxic threshold doses for hyperoxic treatment in man. Relatively safe levels of O2 exposure in one patient population may be associated with severe toxic complications in other patients treated in a similar manner. If it should become possible to pretest for individual differences in resistance to hypcroxia (either innate or acquired during the course of an illness), this information could provide an important rationale for therapeutic decisions in complicated clinical situations. Finally, it has recently been shown, somewhat surprisingly, that animals pre-exposed to levels of FiOz commonly used to treat patients, i.e., 40 per cent O2 or 60 per cent OZ. do much more poorly when placed in 100 per cent O2 than do animals exposed directly to 100 per cent O2 [122,123]. These findings should not, in any manner, alter currently accepted guidelines for the administration of 02. However, they should, and have, stimulated research that may eventually provide information which will require that we re-examine the current assumptions held about the clinical administration of OZ. ACKNOWLEDGMENT We thank Katherine assistance.

V. Frank for her skilled editorial

REFERENCES 1. 2. 3.

Haugaard N: Cellular mechanisms of oxygen toxicity. Physiol Rev 1988; 48: 311. Clark jM, Lambertson Cl: Pulmonary oxygen toxicity: a review. Pharmacol Rev 1971; 23: 37. Berkncr LV. Marshall LC: The history of oxygenic concentration in the earth’s atmosphere. Faraday Discuss Chem Sot 1984: 37: 112. July 1969

Shanklin DR: A general theory of oxygen toxicity in man. Perspcct Rio1 Mecl 1969; 13: 80. 5. Fridovich I: The biology of oxygen radicals. Science 1978; 201: 875. 6. Cerchman R: Biological effects of oxygen. In: Dickens F. Neil E, eds. Oxygen in the animal organism. New York: McMillan & Co, 1984; 475.

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7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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of rat lung lipid during development and following agedependent lipid peroxidation. Lipids 1977: 12: 596. Winter PM. Smith G: The to;cicitv of oxvnen. AnesthesioloavII 1972; 37:.210. Caldwell PRB. Lee Jr WL, Schildkraut HS. et al.: Changes in lung volume. diffusing ca city, and blood gases in men breathing oxygen. J Appl Pr ysiol1966: 21: 1477. Wood JD: Development of a strain of rats with greater than normal susceptibility to oxygen poisoning. Can J Physiol Pharmacol1966: 44: 259. Hill GB. Osterhout S, O’Fallon WM: Variation in response to hvuerbaric oxvgen among inbred strains of mice. Proc Soc~Exp Biol Med1966: 125 687. Shanklin DR: On the pulmonary toxicity of oxygen. II. The relationship of oxygen content to the effect of oxygen on the lung. Lab Invest 1969: 21: 439. Hayatdavoudi G, Crapo JD. Fescue HA, et al.: Evidence of the toxicity of 60% oxygen on rat lungs. Am Rev Respir Dis 1978; 117: 346. Frank L, Massaro D: Accelerated 02 toxicity in 100% 02 after pre-exposure to lower FIOz: protection with endotoxin treatment (abstract). Clin Res 1980: 28: 425A. _I