Life Sciences Vol . 20, pp . 1187-1192, 1977 . Priatad is the II .S .A.
SUPEROXIDE DIShüITASE AND PULMONARY OZONE TOXICITY J . S . Douglas, G . Curry and S . A . Geffkin John B . Pierce Foundation Laboratory and The Yale University Lung Research Center 290 Congress Avenue . New Haven, Connecticut 06519 (Raceivéd in final form February 23, 1977) SUMMARY Rats and mice were pre-exposed to law concentrations of ozone and subsequently challenged with higher concentrations of this gas . When the mortalities of pre-exposed and control animals were compared after a lethal challenge, there was a significant difference in ozone toxicity . The levels of superoxide dismutase in lung tissue of control and ozone tolerant rats or mice were not significantly different whether the results were expressed as units of activity per mg protein of lung tissue or as total units of enzyme activity per lung . Mice which . were tolerant to ozone were not tolerant to oxygen . The data suggest that ozone tolerance is unrelated to an induction of superoxide dismutase . It is suggested that the induction of tolerance to oxygen and ozone involve biochemically distinct mechanisms . Recently, the development of tolerance in rats to the toxic effects of oxygen has been correlated with an increased superoxide dismutase content of the lung (1) . These data have suggested that oxygen toxicity may be, in part, associated with the production of the superoxide free radical . In addition, lead to a reduced oxidant toxiadaptive changes of superoxide dismutase We examined, therefore, whether city (2) and tolerance to oxidant stress the development of tolerance to the oxidant gas, ozone, could be correlated with increased quantities of superoxide dismutase in lung tissue .
METHODS Pathogen free male mice (Swiss ICR, Yale Colony, 29-39 g) or male rats (Sprague Dawley, Charles River, Mass ., 200-250 g) were used in this study . Ozone was prepared using an ozone generator (Welsbach, Model T408) coupled to a 100% dry oxygen supply . Concentrations of ozone were prepared by mixing the gas with dry, filtered, oil free air in plexiglass mixing chambers regulated with flow meters . The flow rates of the ozone/air mixtures entering the exposure chamber (16 .5 1/min) were sufficient to produce 12-14 changes of the chamber atmosphere per hour . Atmospheric pressure was maintained within the exposure chamber (1 atmosphere t-3 cm water) . The ozone concentration was sampled from at least two sites within the chamber . Gas concentrations were monitored continually at a constant flow rate via flow meters which were attached to a pump . The ozone/air mixtures were collected in 1% neutral potassium iodide for periods of ten minutes . Ozone concentrations were calculated from the optical density of the sample when compared to a potassium iodide/iodine calibration curve (4) . 1187
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Three types of exposure were performed : 1) animals were exposed to a low concentration of ozone for three hours to induce ozone tolerance ; 2) animals were exposed to a high concentration of ozone for thrée hours and the mortality data were recorded subsequently for 72 hours ; 3) mice were exposed In to 98% oxygen for 100 hours and mortalities were recorded every 8 hours . these exposure regimens, equal numbers of animals were exposed to air in a similar chamber and served as controls . Groups of control and pre-exposed mice or rats were killed by stunning and lung tissue caudal to the trachea was removed . Lung weights and body weights were recorded . The lung tissue from individual rats, or pooled tissue from groups of 5 mice, was weighed and homogenized (Virtis "23") at 4°C in sucrose buffer (4 ml buffer/g lung tissue) . The composition of the buffer was 0 .25M sucrose, 0 .05M Tris HC1, 0 .25M KC1, 0 .005M MgC12, pH 7 .5 . The homogenates were centrifuged at 600 g for 10 minutes to remove tissue debris and then a mitochondria? fraction (10,000 g for 30 min) and an organelle free supernatant (100,000 g for 75 min) were prepared . The mitochondria? and supernatant fractions were suspended in the original volume of sucrose buffer and Immediately bewere stored at -20°C until assayed for superoxide dismutase . fore assay, the samples were thawed and sonicated three times (heat systems cell disrupter, model W185) at a power of 55 watts for 10 seconds . Protein concentration of the sonicates was determined using the Folin Phenol procedure (5) . Superoxide dismutase was assayed spectrophotometrically (Varian model In this 635) at 20°C using a xanthine/xanthine oxidase generating system (6) . procedure, superoxide was generated by the addition of 20 ul of xanthine oxidase (1 .2 units/ml) to 2 .9 ml of 0 .5M phosphate buffer, pH 7 .4, containing EDTA (1 mM), xanthine sodium (0 .3 mM) and cytochrome C (0 .3 mM, horse heart ; all from Sigma Chemical Co ., Mo) . The reduction of cytochrrome C by the generated superoxide was followed for at least three minutes by measuring the increase in absorbante at 550 nm . The procedure was repeated three times, in the absence of tissue fractions . This enabled us to obtain a mean rate for the reduction of cytochrome C . New assay solutions were prepared if the absorbance did not increase at a rate of 0 .04 to 0 .05 units/minute when the xanthine oxidase was added to the cuvette . We then determined the mean rates of reduction of cytochrome C when varying amounts of lung mitochondrial suspension or lung supernatant were present in the 3 ml assay cuvette . From these data, we calculated the % inhibition of the reduction of cytochrome C by the tissue fractions . The % inhibition of the reduction of cytochrome C for each sample assayed was plotted against the protein .concentration in the aliquot . In those tissue aliquots where % inhibition was linearly related to protein concentration, we calculated the units of enzyme activity in the samples . One unit of superoxide dismutase activity was defined as that amount which produced a 50% inhibition of the rate of change of absorbante in the 3 ml assay . RESULTS Mice (4 groups, 10 mice/group) exposed to low concentrations of ozone (5 .4 ppm f 0 .3 for 3 hours) showed labored breathing and lethargy . The concentration of ozone was insufficient to induce mortalities . Two days later, 20 of these mice together with 20 mice not pre-exposed to ozone were challenged with a lethal concentration of the gas (36 .9 ppm t 1 .3 for 3 hours) . While all the mice showed labored breathing and lethargy, animals not pretreated with ozone succumbed to the gas . In contrast, only 30% of the preexposed animals died (p < 0 .001) . These data indicate that the pre-exposure of mice to low ozone concentrations had induced tolerance to this gas . Ozone
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3uperozide Msautasa sad Ozone Tolersace
TABLE 1 Superoxide Dismutase in Lung Cytosol of Control and Ozone Tolerant Mice and Rats A, Mouse n ro zone o erant f ~ oso >ro~~, mg ung T4 l,~ 16 * 1 .4 Superoxide Dismutase 5.4 t 0.5 4,5 t 0 .6 Units/mg protein Superoxide Dismutase 73,8 t 4.8 72 .3 t 11 .7 Units/lung B~ Cytosol Protein, mg/lung 145,3 t 22,3 160,1. t 24,3 Superoxide Dismutase 2.3 t 0.2 2,3 t 0 .2 Units/mg Protein Superoxide Dismutase 304 t 27,4 332 .7 t 34.8 Units/lung CytosT ~ract~ons (TOÔ,I~ g supernatants were prepare ron côntro anâ ozone tolerant animals (see Methods, p, 2) Values are the means t SE of cytosol fractions of. lung tissue from rats (N~10) and mice (N-50) . Values were not significantly different when compared using Student's unpaired 't' test, TABLE 2 Superoxide Dismutase in Lung Mitochondrial Fractions of Control and Ozone Tolerant Mice and Rats A, Mousé on ro zone o erant tochondrial Protein, mg Superoxide Dismutase 4 .2 t 0.4 3,8 t 0 .5 Units/mg protein Superoxide Dismutase 10.8 t 1,0 10.8 t 0,6 Units/lung ß. Rat Mitochondrial Protein, mg/lung 21 .7 t 2 .0 19.6 t 1,1 Superoxide Dismutase 3 .4 t 0.2 3.7 t 0,2 Units/mg protein Superoxide Dismutase 72 .8 t 8.1 70 .3 t 5 .1 Units/lung ~nârie~ frâct~nsj10,00~ gpel~etj wére prepäred from côntrol and ozone tolerant animals (see Methods, p . 2) . Yalues are the means t SE of mitochondrial fractions of lung tissue from rats (N-10) and mice (N'50) . Values were not significantly different when compared using Student's 't' test for unpaired variates . tolerant mice were not tolerant to 98~ oxygen . We recorded the first mortalities in control and ozone tolerant mice after 80 hours of exposure to 98x oxygen . Exposure for 96 hours resulted in a 90x iaortality in both groups and all the mice succumbed after 104 hours of exposure . Thus, ozone tolerant mice were not tolerant to a prolonged exposure to oxygen . We pre-exposed rats (2 groups, 16 animals/grou ) to lav concentrations of ozone (3.7 ppm t 0.3 or 2 .03 ppm t 0 .12 for 3 hours . These low concentra-
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taons were sufficiently toxic to result in mortalities in both groups of animals (44% and 38%, respectively) . The survivors together with unexposed rats were challenged with 16 .6 ppm t 0 .8 of ozone for three hours, two days later . None of the pre-exposed rats succumbed to this toxic concentration of ozone while 8 of 9 unexposed rats died within 72 hours of the challenge . Lung tissue from control and pre-exposed mice (50 animals/group) i .e ., ozone tolerant mice, appeared macroscopically normal two days after the preexposure period . The lung weights of the control and pre-exposed animals, when expressed as a percentage of the body weight, were not significantly different (0 .73% t 0 .03 and 0 .79% t 0 .03, respectively) . Similarly, a comparison of the percent lung weight/body weight ratios of control and preexposed rats (10 animals/group) were not statistically significant (0 .75% t 0 .07 and 0 .89% ± 0 .1, respectively) . We also observed small changes in cytosol, mitochondrial and total protein in the lung fractions but these were not statistically significant (Tables 1, 2) . The superoxide dismutase activity of the cytosol and mitochondrial fractions was assayed spectrophotometrically . Superoxide formation proceeded at a linear velocity and optical density changed at a rate of 0 .04 units optical density/minute . The rate of reduction of cytochrome C was inhibited by the presence of superoxide dismutase . The relationship between protein concentration in the mitochondrial fractions of control ( " ) and ozone tolerant mice (0) was linear to 208 mg protein/3 ml assay . Cytoso7 fractions from control (~) and ozone tolerant mice (o) also inhibited the generation of the superoxide anion in a concentration dependent manner up to 177 mg protein/3 ml assay (Fig . 1) . There was no significant difference in the concentration/ inhibition curves of fractions from control or ozone tolerant mice . In rats, the maximal concentrations of mitochondrial and cytosol proteins which inhibited cytochrome C reduction in a concentration dependent manner were 300 mg and 180 mg protein, respectively . We calculated the specific activity of the enzyme in mitochondrial and cytosol fractions as units of activity/mg protein (see Methods) . We also calculated the total number of units of superoxide dismutase/lung . Neither the specific activity nor the total lung units of superoxide dismutase were significantly different in fractions from ozone tolerant and control mice' or rats (Tables 1 and 2) . DISCUSSION An exposure of animals to high concentrations of ozone results in respiratory distress, pulmonary edema and death . The lethal concentrations of ozone used in this study and the resultant mortalities in rats and mice compare well with previous reports (7, 8) . Pre-exposure of mice and rats to low concentrations of ozone for short periods of time reduces the toxicity of subsequent exposures to this gas (9, 10) . The reduced susceptibility of these pre-exposed animals to toxic levels of ozone is demonstrable for a period of 2 to 6 days after the pre-exposure treatment . The increased tolerance to the gas suggests that major deteriorative processes, which normally result from a toxic exposure, are ameliorated or reduced (8) . Tolerance induction to oxidant gases has been associated with increased concentrations of free radical scavérigers in lung tissue, e .g ., glutathione . More recently, it has been suggested that tolerance to ozone and oxygen may be associated with an induction of superoxide dismutase (1, 2, 3) . There is some evidence to suggest a reciprocity between the induction of tolerance to oxygen, i .e ., reduced oxygen toxicity, and the content of superoxide dismutase in lung tissue (1) . However, the relationship between con-
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Superoxide Diamutase and Oaone Tolerance
FI6 . 1 Relationship between the concentration of superoxide dismutase in lung fractions and its inhibition of cytochrome C .reduction by superoxide anion . Cytochrome C was reduced by a superoxide anion generating system (xanthine/xanthine oxidase) and the reduction followed spectrophotametrically at 550 nm . Varying volumes of mouse lung mitochondrial . or cytosol fractions (expressed as u9 protein/3 ml assay volume, abscissa) were added to the generating system and the % inhibition of the cytochrome C reduction was calculated (ordinate) . The % inhibition (mean t SE of 6 experiments) of cytochrome C reduction by varying concentrations of mitochondrial (0, " ) and cytosol (e, ~) protein from control (1, ~) and ozone tolerant animals (0, e) are shown . centrations of this enzyme in lung tissue and the development of tolerance to ozone is less clear since the time course of these events have not been related . Thus, the increased superoxide dis~tase levels seen after prolonged. exposures ~(8 days) to low concentrations of ozone (2,3) may not be specifically related to the induction of tolerance . We have examined the concentrations of superoxide dismutase in lung tissues from animals in which tolerance was induced by a pre-exposure to low concentrations of ozone but where the exposure period was 3 hours rather than 8 days . The earliest reports of tolerance induction used protocols similar to the procedure we have employed (7,8) . Superoxide dismutase levels in mitochondrial and cytosol fractions of lung tissue from these ozone tolerant mice or rats and appropriate control preparations were not significantly different
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(Tables 1 and 2) . Recently, it has been reported that the concentrations of superoxide dismutase are increased in lung tissues of animals tolerant to nitrogen dioxide . However, those animals which were tolerant to nitrogen dioxide were not tolerant to oxygen and vice versa (11) . Similarly, we were unable to demonstrate a cross tolerance between oxygen and ozone . These observations suggest that tolerance to oxygen may occur by a mechanism biochemically distinct from that which induces tolerance to other oxidant gases . Our data suggest that it is essential to specifically relate changes in the specific activity of an enzyme with the time course of the induction of the tolerance in order to eliminate other but unrelated adaptive changes in enzyme concentrations . ACKNOWLEDGEMENT This work was supported in part by USPHS Grant ~YRR06592 . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
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