Oxygen toxicity in control and H2O2-resistant Chinese hamster fibroblast cell lines

Following exposure to 95% oxygen, clonogenic cell survival was assayed and qualitative morphologic changes were observed in a Chinese hamster fibroblast cell line (HA-1). The time in 95% O2 necessary to clonogenically inactivate 90% of the cells was inversely related to the cell density of the cultures at the beginning of hyperoxic exposure (from 1 to 6 X 10(4) cells/cm2). The O2-induced loss in clonogenicity and evidence of morphologic injury were shown to be significantly delayed (17-22 h) in an H2O2-resistant variant of the parental HA-1 cell line. After the delay in onset of clonogenic cell killing or morphologic injury, the process of injury proceeded in a similar fashion in both cell lines. The H2O2-resistant cell line demonstrated significantly greater catalase activity (20-fold), CuZn superoxide dismutase activity (2-fold), and Se-dependent glutathione peroxidase activity (1.5-fold). The greater activities of CuZn superoxide dismutase and catalase were accompanied by similarly greater quantities of immunoreactive protein as determined by immunoblotting. These data demonstrate that the cells adapted and/or selected for growth in a highly peroxidative environment also became refractory to O2-induced toxicity, which may be related to increased expression of antioxidant enzymes. However, the magnitude of this cross-resistance to O2 toxicity was less than the magnitude of the cellular resistance to the toxicity of exogenous H2O2, suggesting that in this system the toxicity of 95% oxygen is not identical to H2O2-mediated cytotoxicity.     

Biosynthesis of oxygen-detoxifying enzymes in Bdellovibrio stolpii.

Axenically grown Bdellovibrio stolpii (i.e., grown independently of the host) was examined for superoxide dismutase, catalase, and peroxidase activities. Kinetics of enzyme synthesis were determined for aerobically grown cultures and for cultures exposed to 100% oxygen. Enzymatic activities varied with the age of the culture. Normally grown cultures exhibited maximum activity during the first 10 h of growth and again as the stationary phase was approached, beginning at about 48 h. Polyacrylamide gel electropherograms of cell-free extracts revealed that B. stolpii contained one major band (1) and two minor bands (II, III) of superoxide dismutase activity. Each of these enzymes was inactivated by H2O2, indicating that they were iron-containing enzymes. Manganese-containing superoxide dismutase was not detected in B. stolpii. Increased oxygenation did not appreciably stimulate enzyme synthesis, for only superoxide dismutase was induced, reaching maximum activity at 10 h and then rapidly falling to normal levels. Superoxide dismutase appears to be the main enzymatic defense against oxygen toxicity in B. stolpii. Induction of superoxide dismutase with 100% oxygen was manifested as an increase in the intensities of the two minor bands of activity, suggesting that isozyme I is constitutive, whereas isozymes II and III are inducible. The induction of isozymes II and III by 100% oxygen was prevented by an inhibitor of protein biosynthesis, chloramphenicol.     

Manganese and Defenses against Oxygen Toxicity in Lactobacillus plantarum

Lactobacillus plantarum is aerotolerant during log-phase growth on glucose, but is an obligate aerobe on polyols. Respiration was cyanide resistant and under certain conditions was associated with the accumulation of millimolar concentrations of H(2)O(2). On glucose, optimal growth was observed in the absence of O(2). Extracts of L. plantarum did not catalyze the reduction of paraquat by reduced nicotinamide adenine dinucleotide, but plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was readily reduced. Such extracts produced O(2) (-) in the presence of NADH plus plumbagin. Plumbagin caused a 10-fold increase in the rate of respiration of intact cells in the presence of glucose and also imposed a loss of viability which was dependent upon both glucose and O(2). Although extracts of L. plantarum were devoid of true superoxide dismutase activity, this organism was comparable to superoxide dismutase-containing species in its resistance toward hyperbaric O(2) and toward the oxygen-dependent lethality of plumbagin. L. plantarum required Mn-rich media and actively accumulated Mn(II). Soluble extracts were found to contain approximately 9 mug of Mn per mg of protein and 75 to 90% of this Mn was dialyzable. Such extracts exhibited a dialyzable and ethylenediaminetetraacetic acid-inhibitable ability to scavenge O(2) (-). This O(2) (-)-scavenging activity was due to the dialyzable Mn(II) present in these extracts and could be mimicked by MnCl(2). Cells grown in Mn-rich media were enriched in dialyzable Mn and were more resistant toward oxygen toxicity and toward the oxygen-dependent plumbagin toxicity than were cells grown in Mn-deficient media. L. plantarum exhibited no nutritional requirement for iron and little or no iron was present in these cells, even when they were grown in iron-rich media. L. plantarum thus appears to use millimolar levels of Mn(II) to scavenge O(2) (-), much as most other organisms use micromolar levels of superoxide dismutases.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Superoxide Dismutase and Oxygen Toxicity in a Eukaryote

Saccharomyces cerevisiae var. ellipsoideus contained 6.5 times more superoxide dismutase and 2.3 times more catalase when grown under 100% O(2) than when grown anaerobically. Growth under oxygen caused equal increases in both the cyanide-sensitive and the cyanide-insensitive superoxide dismutases of this organism. Experience with other eukaryotes has shown that cyanide sensitivity is a property of the cupro-zinc superoxide dismutase of the cytosol, whereas cyanide insensitivity is a property of the corresponding mangani-enzyme found in mitochondria. Cu(2+), which has been shown to increase the radioresistance of yeast, also caused an increase of both of the superoxide dismutases of S. cerevisiae. Yeast which had been grown under 1 atm of O(2) were more resistant toward the lethal effects of 20 atm of O(2) than were yeast which had been grown in the absence of O(2). Escherichia coli K-12 his(-) responded to growth under 1 atm of O(2) by increasing its content of catalase and of peroxidase, but not of superoxide dismutase. This contrasts with E. coli B, which was previously shown to respond to O(2) by a striking increase in superoxide dismutase. E. coli K-12 his(-) did not gain resistance toward 20 atm of O(2) because of having been grown under 1 atm of O(2). Once again, this contrasts with the behavior of E. coli B. These data indicate that, in both prokaryotes and in eukaryotes, superoxide dismutase is an important component of the defenses against oxygen toxicity.     

Oxygen Toxicity and the Superoxide Dismutase

Oxygen caused an increase in the amount of superoxide dismutase in Escherichia coli B but not in Bacillus subtilis. E. coli B cells, induced by growth under 100% O(2), were much more resistant to the lethal effects of 20 atm of O(2) than were cells which contained the low uninduced level of this enzyme. In contrast, B. subtilis, which could not respond to O(2) by increasing its content of superoxide dismutase, remained equally sensitive to hyperbaric O(2) whether grown under 100% O(2) or areobically. The catalase in these organisms exhibited a reciprocal response to oxygen. Thus, the catalase of E. coli B was not induced by O(2), whereas that of B. subtilis was so induced. These results are consistent with the view that superoxide dismutase is an important component of the defenses of these organisms against the toxicity of oxygen, whereas their catalases are of secondary importance in this respect. The ability of streptonigrin to generate O(2) (-), by a cycle of reduction followed by spontaneous reoxidation, has been verified in vitro. It is further observed that E. coli B which contain the high induced level of superoxide dismutase were more resistant to the lethality of this antibiotic, in the presence of oxygen, than were E. coli B which contained the low uninduced level of this enzyme. This difference between induced and uninduced cells was eliminated by the removal of O(2). These results are consistent with the proposal that the enhanced lethality of streptonigrin under aerobic conditions may relate to its in vivo generation of O(2) (-) by a cycle of reduction and spontaneous reoxidation. In toto, these observations lend support to the hypothesis that O(2) (-) is an important agent of oxygen toxicity and that superoxide dismutase functions to blunt the threat posed by this reactive radical.     

Induction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone

Oxyradicals have been implicated in ozone (O3) toxicity and in other oxidant stress. In this study, we investigated the effects of O3 on the biosynthesis of the antioxidant enzymes catalase and superoxide dismutase in Escherichia coli to determine their role in the defense against ozone toxicity. Inhibition of growth and loss of viability were observed in cultures exposed to ozone. Results also showed an increase in the activities of catalase and superoxide dismutase in cultures exposed to ozone, which was shown to be due to true induction rather than activation of preexisting apoproteins. Cessation of O3 exposure resulted in 30 min of continual high rate of catalase biosynthesis followed by a gradual decrease in the level of the enzyme approaching that of control cultures. This decrease was attributed to a concomitant cessation of de novo enzyme synthesis and dilution of preexisting enzyme by cellular growth. Ozonation of cell-free extracts showed that superoxide dismutase and catalase are subject to oxidative inactivation by ozone. In vivo induction of these enzymes may represent an adaptive response evolved to protect cells against ozone toxicity.     

Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h(-1)). Accumulation of H(2)O(2) in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H(2)O(2) accumulated only temporarily during the lag phase. H(2)O(2) is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min(-1) mg of protein(-1)), which reduced oxygen to water, whereby superoxide was formed as a by-product. H(2)O(2) originated from the dismutation of superoxide by superoxide dismutase. Although H(2)O(2) was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H(2)O(2)-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H(2)O(2) and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H(2)O(2).     

Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect.

Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.     

Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli.

Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.     

Oxygen toxicity in Streptococcus sanguis. The relative importance of superoxide and hydroxyl radicals

Streptococcus sanguis, whose growth appears to be independent of the availability of iron, makes no hemes, contains neither catalase nor peroxidase, and can accumulate millimolar concentration levels of H2O2 during aerobic growth. It possesses a single manganese-containing superoxide dismutase whose concentration can be varied over a 50-100-fold range by manipulating the availability of oxygen during growth. Cell extracts contain a soluble NADH-plumbagin diaphorase which mediates O2- production in vitro and presumably also in vivo. Plumbagin increased oxygen consumption by S. sanguis and imposed an oxygen-dependent toxicity. Cells grown aerobically and containing elevated levels of superoxide dismutase were resistant to this toxicity. Dimethyl sulfoxide, which was shown to permeate S. sanguis freely, was used as an indicating scavenger of OH. An in vitro enzymic source of O2- plus H2O2 generated formaldehyde from dimethyl sulfoxide, an indication of OH. production. Either superoxide dismutase or catalase inhibited this OH. production and iron salts augmented it. Intact, aerobic cells of S. sanguis also gave evidence of OH. production, in the presence of plumbagin, but all of it appeared to be generated outside the cells. In addition, 0.5 M dimethyl sulfoxide did not diminish the oxygen-dependent toxicity of plumbagin. We conclude that, in S. sanguis, O2- can exert a toxic effect independent of the production of OH..     

Induction of Superoxide Dismutase by Molecular Oxygen

Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O(2) had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O(2) caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O(2) rather than to pressure, since 20 atm of N(2) was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N(2)-grown cells of S. faecalis were exposed to 20 atm of O(2) at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O(2) during growth. S. faecalis, which had been grown under 100% O(2) and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O(2) than were cells which had been grown under N(2). E. coli grown under N(2) contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O(2). The E. coli which had been grown under N(2) was as resistant to the deleterious effects of 50 atm of O(2) as was S. faecalis which had been grown under 1 atm of O(2). These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.     

Effects of growth conditions on superoxide dismutase and catalase activities inSaccharomyces cerevisiae var.ellipsoideus

The superoxide dismutase (SOD) activity inSaccharomyces cerevisiae var.ellipsoideus increased when the cells were exposed to hyperbaric oxygen tension. Ethanol-grown cells, having a more oxidative metabolism, showed higher SOD activities than did glucosegrown cells. In a glucose-limited chemostat the SOD activity increased with the specific oxygen consumption rate. The increase in SOD activity may be explained by a higher intracellular flow of superoxide radicals at higher respiration rates. The catalase activity decreased with increasing growth rates in a glucose-limited chemostat, and the activity was lower in glucosethan in ethanol-grown cells.     

Superoxide dismutase and O2 lethality in Bacteroides fragilis.

Exposure of midlog Bacteroides fragils (VPI 2393) to 2% O2-98% N2 caused a three- to fivefold increase in superoxide dismutase specific activity within the cells. The increase in specific activity was completed within 90 min after exposure to oxygen and was dependent upon protein synthesis. Cells containing the higher superoxide dismutase level were more resistant to the effects of 5 atm of oxygen tension than were cells containing the lower level of superoxide dismutase but were equally resistant to 5 atm of nitrogen tension. Similar results were observed upon comparing viability experiments with B. fragilis and B. vulgatus. Superoxide dismutase activity in sonic extracts of B. fragilis was rapidly inactivated by exposure to 5 mM H2O2 and was inhibited by 1 mM NaN3 but not 5 mM NaCN. The inhibition pattern is identical to the pattern demonstrated for the purified iron-containing enzyme from Escherichia coli B and suggests that the superoxide dismutase in B. fragilis is an iron enzyme.     

Glucose suppresses superoxide generation in metabolically responsive pancreatic beta cells

High rates of glucose metabolism and mitochondrial electron transport have been associated with increased mitochondrial production of reactive oxygen species (ROS). This mechanism was also proposed as a possible cause for dysfunction and death of pancreatic beta cells exposed to high glucose levels. We examined whether high rates of glucose metabolism increase ROS production in purified rat beta cells. Glucose up to 20 mm did not stimulate H(2)O(2) or superoxide production, whereas it dose-dependently increased cellular NAD(P)H and FADH(2) levels with an EC(50) around 8 mm. On the contrary, glucose concentration-dependently suppressed H(2)O(2) and superoxide formation, with a major effect between 0 and 5 mm, parallel to an increase in cellular NAD(P)H levels. This suppressive effect was more marked in beta cells with higher NAD(P)H responsiveness to glucose; it was not observed in glucagon-containing alpha cells, which lacked a glucose-induced increase in NAD(P)H. Suppression was also induced by the mitochondrial substrates leucine and succinate. Experiments with electron transport chain inhibitors indicate a role of respiratory complex I in ROS production at low mitochondrial activity and low NADH levels. Superoxide production at low glucose is potentially cytotoxic, because scavenging by the superoxide dismutase mimetic agent manganese(III)tetrakis(4-benzoic acid)porphyrin was found to reduce the rate of beta cell apoptosis. Analysis of islets cultured at 20 mm glucose confirmed that this condition does not induce ROS production in beta cells as a result of their increased rates of glucose metabolism. Our study indicates the need of beta cells for basal nutrients maintaining mitochondrial NADH production at levels that suppress ROS accumulation from an inadequate respiratory complex I activity and thus inhibit a potential apoptotic pathway.     

Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B.

Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c. The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase. Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol. This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline. Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. The addition of superoxide dismutase and catalase to the incubation medium partially protected E. coli B against the light-dependent lethality. Preinduction of intracellular superoxide dismutase and catalase substantially protected E. coli B against the phototoxicity of tetracyclines. Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality. Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity. The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical. These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E. coli B.     

Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide

The effects of carbon tetrachloride (CCl4) and paraquat on the growth of Escherichia coli were investigated. Paraquat at 10 mM caused some inhibition of growth of E. coli in trypticase-soy-yeast extract medium. CCl4 enhanced growth inhibition by paraquat in a concentration-dependent manner. In the absence of paraquat, CCl4 had no effect on growth rate or on surviving cell numbers at stationary phase. CCl4 did not prevent the induction of manganese-superoxide dismutase by paraquat. Under anaerobic conditions, CCl4 and paraquat exhibited no effect on E. coli. In the presence of Mn(II) and paraquat, intracellular superoxide dismutase was markedly induced and protected E. coli against the toxicity of CCl4 and paraquat. The reactive free radical CCl3OO-, which can be formed from the reaction of O2- with CCl4, may cause cell damage. The growth-inhibiting effects of polyhalides in the presence of paraquat followed the order CBrCl3 greater than CCl4 greater than CHCl3 greater than CH2Cl2, which is in accord with that of the reaction rates of these compounds with O2- and with their hepatotoxicities. These results suggest that O2- plays a role in the hepatotoxicity of polyhalides.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.