Global atmospheric change: potential health effects of acid aerosol and oxidant gas mixtures.

Inhalation toxicology experiments in whole animals have demonstrated a remarkable lack of toxicity of sulfuric acid in the form of respirable aerosols, especially in rats and nonhuman primates. Thus, much of the current experimental emphasis has shifted to the evaluation of the potential health effects of acid aerosols as components of mixtures. Rats have been concurrently exposed to mixtures of ozone or nitrogen dioxide with respirable-sized aerosols of sulfuric acid, ammonium sulfate, or sodium chloride, or to each pollutant individually. Their responses to such exposures have been evaluated by various quantitative biochemical analysis of lung tissue or wash fluids ("lavage fluid") or by quantitative morphological methods ("morphometry"). Such studies have mainly been performed in the acute time frame due to the inherent limitations of the most sensitive assays available and have generally involved exposures for 1 to 9 days, depending on the assays used. Good correlations were found between the most sensitive biochemical indicators of lung damage (protein content of lung lavage fluid or whole lung tissue and lung collagen synthesis rate) and the exposure concentration of oxidant gas present alone or in mixtures with acidic aerosols showing interactive effects. Synergistic interaction between ozone and sulfuric acid aerosol was demonstrated to occur at environmentally relevant concentrations of both pollutants by several of the analytical methods used in this study. Such interactions were demonstrated at concentrations of ozone as low as 0.12 ppm and of sulfuric acid aerosol at concentrations as low as 5 to 20 micrograms/m3.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
One ofthe major global atmospheric changes associated with increased industrialization, and especially with increased combustion of fossil fuels, is the release ofacids and their precursors to the atmosphere. Major concerns in this area involve acids arising from SO, and NO, emissions, including sulfuric acid and bisulfate aerosols. Thus, we might well ask what is known ofthe toxicological impacts ofacid aerosols. In this manuscript I review and discuss specific data bearing on this question based on experiments performed in animals. I consider effects of acid aerosols alone and in combination with ozone and other atmospheric oxidants.

Effects of Sulfuric Acid Aerosols on Lungs of Experimental Animals
Guinea pigs seem to be particularly susceptible to sulfuric acid aerosols compared with other laboratory animals. Guinea pigs respond by showing increased airway resistance after exposure for 1 hr to 100 to 1000 jig/m3 (1). Interestingly, no long-term *Department ofIntemral Medicine, Pulmonary Division, University of California School of Medicine and California Primate Research Center, Davis, CA 95616. effects ofexposure ofguinea pigs to 100 tg/m3 for 52 weeks were reported by Alarie and co-workers (2).
Rats or monkeys are remarkably resistant to observable effects on lung when acutely exposed to respirable aerosols of sulfuric acid (3)(4)(5), even at concentrations of acid at or above 50 to 100 mg/m3. On the other hand, guinea pigs and, to a lesser extent, mice, respond to exposure to lower concentrations of sulfuric acid mist (1,4,5) and presumably represent susceptible species to this agent. Intermittent exposure of mice to 1.4 mg/m3 of sulfuric acid mist in conjunction with carbon particles (1.5 mg/m3; no acid-alone group was exposed) for 20 weeks caused decreased resistance to experimental pulmonary infections (6). No effects of sulfuric acid aerosol exposure at 8 mg/m3 in dogs and at 4 to 14 mg/m3 in sheep were observed for various cardiopulmonary functions examined (7). The relatively high resistance of experimental animals to exposure to sulfuric acid aerosol is assumed to reflect its high absorption by the nasopharynx, thereby reducing the dose reaching the distal lung to very low levels. This is consistent with its high solubility in water. However, sulfuric acid aerosol exposure at relatively low concentrations has been associated with alterations in mucociliary clearance rates for various tracer substances introduced into respiratory airways ofexperimental animals. Fairchild et al. (8) reported that exposure ofguinea pigs for 1 hr to concentrations of acid between 30 and 3000 kg/m3 caused a proximal shift (upwards in the respiratory airways toward nasopharynx and trachea) in the sites of deposition of radioactively labelled streptococcus administered by aerosol. Rabbits exposed for 1 hr to 200 to 1000 tg/m3 of sulfuric acid aerosols show altered rates of mucociliary clearance of radioactive tracers (9,10), as do donkeys (11).
Chronic exposure of monkeys for up to 78 weeks to sulfuric acid aerosol at 1 to 5 mg/m3 resulted in histological changes to the bronchial mucosa and changes in epithelial cells of bronchi and respiratory bronchioles (2,3). These results are consistent with the known sites ofdeposition for particles of about 0.5 to 5 Am, which are preferentially deposited in the deep lung (12).

Effects of Ozone on Lungs of Experimental Animals
Acute exposure to high concentrations of ozone, in excess of about 1 ppm, causes severe pulmonary edema and hemorrhage, which can result in death ofexperimental animals at concentrations in excess of 1.5 ppm for periods of several hours or days. However, with concentrations at or below approximately 1.0 ppm of ozone, effects of ozone related to edema and cellular inflammation are more subtle and are reflected as increased lung weight and as increased lung content of, or lung enzyme activities of, a large variety ofmeasurable parameters and enzymes associated with cellular inflammation and edema (13)(14)(15)(16)(17).
The major changes recorded in experimental animals exposed to moderate concentrations of ozone (below 1 ppm for hours or days) include damage to respiratory tract epithelium, especially loss ofcilia from ciliated cells and cell necrosis. The two major sites of damage appear to be loss of cilia from epithelial cells of the trachea and large bronchi and epithelial cell necrosis in the centriacinar region of the lung. Particularly susceptible cells seem to include the alveolar type I cell and the Clara cells (16). Other changes observed in animals exposed to moderate concentrations of ozone include those typical of an inflammatory cell response, especially in the epithelial layer of small bronchioles in the centriacinar region (16). Chronic exposure to ozone is associated with a continuing bronchiolitis and continuing inflammatory response ofthe centriacinar region (18). Another chronic effect of exposure ofanimals for long periods of time to moderate levels of ozone is pulmonary fibrosis; that is, the accumulation of collagen in the centriacinar region of the lung (19)(20)(21). These results are consistent with the predicted sites ofmaximal deposition ofozone in the respiratory tract based on its known relative solubility in water and established models of ozone dosimetry to the lung (12).
Several laboratories have reported changes in the protein content of lung lavage fluid in animals exposed to ozone. Hu et al. (22) reported increased accumulation of protein in lung lavage fluid from guinea pigs exposed for 72 hr to 0.26, 0.51, or 1 ppm ofozone. No effect was observed at 0.10 ppm. Guth et al. (23) extended these studies by examining concentration x time relationships. The increased protein in lavage fluid seems to originate from serum, suggesting that the changes being measured arise from pulmonary edema. Costa et al. (24) reported that rats exposed to ozone had a higher alveolar permeability to serum albumin than did normal rats. Frank et al. (25) also reported pulmonary edema in rabbits exposed to high quantities ofozone for short periods of time.
Bartlett et al. (26) reported decreased lung tissue elasticity consistent with mild pulmonary fibrosis in young rats exposed to 0.2 ppm ofozone for 30 days. Several groups have reported increased collagen synthesis by lungs ofexperimental animals exposed to ozone (27)(28)(29)(30). The significance ofthese observations with regard to the etiology ofpulmonary fibrosis is controversial. Filipowitz and McCauley (30) have suggested that despite the increase in collagen synthesis rate observed from lungs of rats exposed to ozone, there is no net accumulation of total lung collagen, suggesting increased degradation ofcollagen accompanying (and offsetting) the increase in synthesis.
Effects of Combinations of Ozone and Sulfuric Acid Aerosol on Lungs of Experimental Animals Gardner et al. (31 ) reported that mice exposed sequentially to 0.1 ppm of ozone for 3 hr followed by 900 14g/m3 of sulfuric acid aerosol for 2 hr showed significantly higher mortality when challenged with an aerosol of Streptococcus pyogenes than did mice challenged with either pollutant alone. Osebold et al. (32) reported enhanced antigenicity of protein introduced into the respiratory tract in animals exposed to ozone plus sulfuric acid as compared to mice exposed to either agent alone. They interpreted their findings as indicative of increased epithelial permeability, that is, an enhanced effect of ozone, in animals exposed to the mixture of gas and aerosol.
Last and Cross (33) reported synergism of ozone effects on rat lungs by sulfuric acid aerosol during simultaneous exposure to concentrations ofabout 1 mg/m3 ofthe acid aerosol and 0.4 ppm of ozone for 3 to 14 days. Parameters evaluated included rate of secretion ofmucous glycoproteins by tracheal explants and lung content of water, DNA, RNA, and of various lysosomal enzymes. This same laboratory later showed an enhanced effect of ozone in lungs of rats simultaneously exposed to ozone plus sulfuric acid aerosol as evaluated by elevated lung collagen synthesis rates and morphometric evaluation of lung and inflammatory cell populations at sites of lung lesions (34,35). In all of these studies, sulfuric acid aerosol alone had little or no effect on the parameters being quantified; rather, the acid aerosol significantly increased the effect ofa given concentration ofozone, as if it were increasing the effective dose ofozone delivered to the centracinar region of the lung. Juhos et al. (36) also reported histological changes in lungs of small numbers of rats exposed to 0.9 ppm of ozone plus 2 mg/m3 of sulfuric acid aerosol, consistent with the observations (34,35) described above.
There have also been negative reports of results of exposures to mixtures ofozone and sulfuric acid aerosols. Cavender et al. (5) reported no effects in rats or guinea pigs exposed for up to 7 days to 2 ppm of ozone and/or 10 mg/m3 of sulfuric acid aerosol other than those ascribed to ozone alone. It is, however, noteworthy that control animals lost weight during these exposures, suggestive of illness or other handling problems, and that 2 ppm of ozone for 7 days, which is well in excess ofthe rat LD50 dose, provoked no mortality in this study. Human subjects exposed sequentially to 0.3 ppm of ozone for 2 hr followed by 100 ig/m3 sulfuric acid aerosol for 4 hr showed no effects by pulmonary function testing (37).
A hitherto unexpected synergism between the oxidant air pollutants ozone or nitrogen dioxide and a respirable-sized aerosol ofammonium sulfate [(NH4)2SO41 was observed during controlled exposure of rats to these substances. Response ofrat lungs to these pollutants was quantified by measurement ofapparent collagen synthesis rates in vitro by lung minces from exposed animals. Dose-response curves to either O3 or NO2 were altered in the presence of 5 mg/m3 of (NH4)2S04 aerosol. Morphometric and histologic observations of lungs from rats exposed to high levels of ozone, with and without concurrent exposure to the (NH4)2SO4 particles, confirmed such synergistic effects. In a separate set of experiments, rats were exposed to mixtures of ozone and sulfuric acid aerosol (submicron-sized aerosol). Potentiation ofozone effects on lung collagen synthesis rates was also observed in these experiments (34).
In subsequent studies, cellular populations were examined at lesions in lungs of rats exposed for 3 or 7 days to 0.64 to 0.96 ppm of 03 +5 mg/m3 of ammonium sulfate aerosol (35). These studies gave rise to a hypothesis that the synergistic interaction between ozone and acid aerosols may be mediated by changes in the pH of the lung lining fluid caused by local deposition of acid at or near sites of reaction of 03 or NO2 with molecules within lung cells or lining fluid.
Subchronic Response of Rat Lungs to Ozone-Sulfuric Acid Aerosol Exposure An experiment has been performed to examine the potential relevance of ozone-sulfuric acid interaction beyond the acute time frame (I1-9 days) studied in our earlier cited experiments. We examined the protein content of lungs from rats exposed for 15 or 30 days to 0.2 ppm of ozone + 1 mg/m3 of sulfuric acid aerosol, to ozone or acid aerosol alone, or to filtered air. The results of this experiment are shown in Table 1. There was a significant increase in the protein content of the lungs of rats exposed to ozone after 15 or 30 days of exposure. The relative response was similar at both time points for both ozone alone and for ozone plus acid aerosol. The response to ozone plus acid aerosol was significantly greater than that to ozone alone at 15 days; the difference between the animals exposed to ozone plus .0 (112) 'Data are mean values + SD (n = 6 rats per group) for total lung protein content of rats for the various exposure regimens. Calculations were based on values determined using four replicate 100-yL samples from 4 mL of total homogenate of the right middle plus right lower lobes of rat lung. Each 100-14L sample was precipitated with C13CCOOH and centrifuged, and the pellet was frozen. Protein was then solubilized in 0.5 N NaOH (37°C, 24 hr). NaOH was neutralized with 0.5 mLof 0.5 N HCI. Protein in each sample was determined in duplicate (49). The protein contents for each homogenate were obtained by averaging the four sample protein values (eight determinations). Values in parentheses are percent of controls. acid aerosol and ozone alone was not significant at the 30-day time point, although a trend was apparent. We conclude that total lung protein content may be a suitable measurement of lung alteration (damage and/or repair) during the (sub)chronic time frame to pursue some of the remaining questions about ozoneacid aerosol interaction in future studies. Much more work remains to be done, however, before any conclusions can be drawn regarding the occurrence of such an interaction in the (sub)chronic time frame.

Importance of Aerosol Acidity
Studies ofthe effects ofexposure of rats to various aerosols, alone and in combination with high concentrations (0.96 ppm) ofozone, had shown synergistic interaction between ozone and 5 mg/m3 of ammonium sulfate (pH of stock solution = 5.1 ), with no interaction observed between ozone and sodium chloride or sodium sulfate aerosols (pH of stock solutions = 7.0) (38). These results suggested that the acidity of an aerosol might be the determinant factor as to whether or not it exhibited a synergistic interaction with ozone, and this prompted a series of experiments to more precisely evaluate the role ofaerosol acidity in predicting the response of rats to exposure to mixtures of oxidant gases and aerosols.
We have examined the pH and sulfate concentration of aqueous extracts from filters used to trap sulfuric acid aerosols upon sampling of chamber atmospheres during the course of our experiments, including several of the exposure concentration response experiments discussed above. The results of these measurements are shown in Table 2. The apparent pH of filters used to sample filtered air-only exposure chamber samples as measured using these procedures was 4.8. This pH value is similar to aqueous washes of unused filters [mean + 1 SD was 4.62 + 0.82 (N = 5)]. As the chamber concentration of sulfuric acid aerosol was increased, the pH of the eluate from the filter extract was decreased. When the mean values for analytically determined sulfate mass concentration were plotted against the mean pH values for samples from atmospheres containing 0.1 to 1.0 mg/m3 of sulfuric acid aerosol, a linear relationship was apparent (slope = -0.93, r = 0.98). Interestingly, the pH ofthe samples from chambers containing 40 yg/m3 of sulfuric acid aerosol or less were slightly less acidic than the value calculated from linear extrapolation ofthe data from the higher concentrations of acid aerosol (for example, expected and observed values for 40 Ag/m3 aerosol were 3.74 and 4.27, respectively).
These observations suggest two important conclusions. First, only moderately to strongly acidic aerosols result in a synergistic interaction of lung damage with 03. Based upon the methods for  (39)(40)(41). The correlation coefficient of 0.98 between acid aerosol concentration down to 100 ig/m3 ofacid aerosol and pH of sampling filter eluates in our experiments suggests that any such neutralization ofacid by ammonia in our experiments (down to a sulfuric acid aerosol concentration at or below 0.1 mg/m3) must have been negligible.

Response to NO2
Groups of rats were exposed for 7 days to 10, 5, or 2 ppm of NO2. Rates oflung collagen synthesis as compared with control animals exposed to filtered air were 210, 120, and 99%, respectively. The values at 5 and 10 ppm of NO2 were significantly increased as compared with the controls. Thus, there was a measurable concentration-response relationship by this assay, with an apparent no-effect level at 2 ppm of NO2.
Protein content of lung tissue after 7 days of exposure was significantly increased, to 122 % ofcontrol values, at 10 ppm of NO2. Values observed at 5 and 2 ppm were, respectively, 98 % and 109% ofcontrols; neither value was significantly different from the controls. Thus, an apparent no-effect level, as defined by this assay, was observed at 5 ppm of NO2.

Response to NO2 Plus Sulfuric Acid Aerosol
Additional experiments were also performed, based upon our observed results with ozone-acid aerosol mixtures, to ascertain whether a similar synergistic interaction occurred when NO2 was substituted for ozone. Rats were exposed for 1 or7 days toS ppm of NO2 ± 1 mg/m3 of sulfuric acid aerosol. When rats were exposed to NO2 ± acid aerosol, protein content oflavage fluid was significantly increased, as compared to values in rats exposed to NO2or sulfuric acid alone, to 215 % ofcontrol values afteroneday ofexposure. Lung collagen synthesis rates, measured after7 days ofexposure, were significantly increased in rats exposed to NO2 alone (120% of controls) and not affected by acid aerosol alone (98% ofcontrols); response to NO2plus acid aerosol was 145% ofcontrols, significantly greater than the response to NO2 alone. Rats exposedto2 ppm of'N02 ± 1mg/m3ofsulfuric acid aerosol for 7 days showed the following results as compared to filtered air controls: NO2 alone, 99%; sulfuric acid alone, 98%; NO2 plus acid aerosol, 129 %, a significant increase. We conclude that the no-observable effect level for response of rat lungs to exposure to NO2 plus 1 mg/m3 of sulfuric acid aerosol is below 2 ppm.
Other groups of rats were exposed for 7 days to filtered air, 5 ppm of NO2, 1 mg/rn3 sulfuric acid aerosol, or NO2 plus acid aerosol (Table 3). For all parameters evaluated (Table 3), acid aerosol alone was indistinguishable from filtered air controls, while NO2 alone significantly increased the observed lung inflammatory lesion. The combination of NO2 plus acid aerosol showed significantly higher values than those observed upon exposure to NO2 alone.

N02-NaCI Interaction
There is no observable interaction as evaluated by responses of lungs of rats exposed to ozone (0.96 ppm) and to respirable aerosols of5 mg/m3 of sodium chloride (NaCl) under conditions identical to those that we demonstrated had elicited a synergistic interaction in our experiments with ozone and ammonium sulfate aerosols (38). Rats exposed to 5 ppm of NO2 for 7 days showed a significant increase in lung collagen synthesis rate (120% of control values). Groups of rats exposed to 5 ppm of NO2 and 1 mg/m3 of sulfuric acid aerosol had lung collagen synthesis rates of 145 % ofthe control values, i.e., rats exposed to filtered air, values significantly greater than those observed with the rats exposed to NO2 alone, as reported above (exposure concentration-response experiments). Of interest here, however, is the response ofrats exposed for 7 days to 5 ppm ofNO2 + 1 mg/m3 ofNaCl aerosol.These animals showed a lung collagen synthesis rate that was 165 % ofthe values observed with controls exposed to filtered air. In control experiments we found values of98 and 95% ofcontrol for rats exposed to sulfuric acid aerosols and NaCl aerosols alone, respectively, by this assay.
We also quantified the protein content ofthe lung lavage fluid from rats exposed for 3 days to 5 ppm ofNO2, with and without either 1 mg/m3 of H2SO4 or NaCl aerosol. As compared with filtered air controls, we found values of 175 % ofcontrols in the rats exposed to NO2 alone, 180% of controls in rats exposed to NO2 plus H2SO4 aerosol and 210% of controls in rats exposed to NO2 plus NaCl aerosol. The increase in values for the NO2 plus NaCl group was significant as compared to those observed in rats exposed to NO2 alone (42). As shown in Table 3, the group exposed to NO2 and NaCl aerosol also showed significantly greater morphometric changes than were observed in the rats exposed to NO2 alone.
We interpret these results as suggesting that a reaction product of NO2 and NaCl is responsible for the interaction observed in these experiments because no interaction was observed between NaCl and ozone, which do not react chemically. We hypothesize that the interaction between NO2 and NaCl is due to their reaction to form nitrosyl chloride (NOCI), the mixed anhydride of hydrochloric, nitrous, and nitric acids, which could give rise to strong acids in the centriacinar region of the lung upon hydrolysis (42)(43)(44). The possibility of acid aerosols (or acidogenic aerosols) arising in the atmosphere from sources other than SO2 or direct dissolution of NO2 to give nitric and nitrous acids opens some very interesting vistas toxicologically.

Conclusions
The most important conclusions ofthe studies described here are a) ozone-acid aerosol interaction occurs in rats at concentrations of each agent that approximate to actually encountered ambient levels; b) several sensitive assays may be used to quantify the acute response ofthe lung to oxidants, alone and in combination with respirable acid aerosols; c) there is a reasonably good correlation between the most sensitive biochemical and morphometric indicators oflung response studied; and, d) acidity of an aerosol is apparently a necessary and sufficient condition for it to interact synergistically with an oxidant gas to cause increased lung damage. Table 4 summarizes the results of exposure concentrationresponse studies. The matrix of ozone concentration, sulfuric acid aerosol concentration, and assays performed gives rise to several conclusions. At the highest ozone concentration examined (0.64 ppm), all of the assays tested show significant increases in lungs ofrats exposed to ozone alone. A synergistic interaction between 0.64 ppm of ozone and all three concentrations of sulfuric acid aerosol tested was observed by the criterion of increased lung collagen synthesis rate. Only at the highest concentration of acid aerosol tested, .1 mg/m3, was such a synergistic interaction observed by assay of lung protein content. No significant interaction was observed by assay ofprotein content of lung lavage fluid at 1 mg/m3 of acid aerosol. We interpret these observations as suggesting that the total protein and the whole lung protein content assays cannot discriminate between the lung damage caused by O3 and that by O3 plus acid aerosol exposures at high concentrations of 03.
Total lavagable protein and total lung protein were significantly elevated above control values after rats were exposed to 0.20 ppm of 03. Lung collagen synthesis rates were significantly elevated above control values after exposure ofrats to 0.20 ppm of03 in most ofour experiments. A trend toward higher values "( +) Synergistic interaction observed; (-) interaction not observed; ND, not done; (±), trend observed, but not statistically significant. Values with 0 acid aerosol (i.e., ozone alone) are indicated as (+), (-), or (±) for significant increase, no increase, and trend, respectively, upon exposure to ozone as compared with filtered air controls.
'May be either an additive or synergistic interaction. was observed in the remainder of these experiments. We have previously reported significant increases in lung collagen synthesis rate when rats were exposed to 0.20 ppm of03 for 7 days (45). A significant synergistc interaction was observed between 0.2 ppm ofozone and all ofthe acid aerosol concentrations tested by essentially all ofthe assays performed, consistent with our interpretation that such interaction is most easily evaluated under conditions of minimal lung damage by ozone alone. Rats were exposed to 0. 12 ppm of03, the current peak hourly National Ambient Air Quality Standard for 03, with and without 0.5 mg/m3 of sulfuric acid aerosol. Total lavagableprotein was the only parameter measured that was significantly elevated for the group exposed to 0. 12 ppm of 03 alone. Collagen synthesis rate was significantly elevated in lungs of rats exposed to 0.12 ppm of 03 plus 0.5 mg/m3 of sulfuric acid aerosol. Thus, concentrations of 03 as low as 0. 12 ppm may elicit responses from lungs of exposed rats and may be potentiated by concurrent exposures to acid aerosols.
It is not clear whether the small changes in total lavagable protein and in lung collagen synthesis rate observed in this experiment are necessarily predictive oflong-term damage. In animals exposed to higher concentrations of 03 (0.40 ppm), morphological lesions have been shown to persist in monkey lungs during prolonged exposures and for a 3-month period after termination of exposure. Measurements in rat lungs after exposure to 0.64 ppm of 03 for 1 week showed increases in apparent collagen synthesis rate and in histologically observable collagen consistent with 03-induced fibrosis. The connection (ifany) between the early effects ofexposure to 0. 12 ppm 03 shown here and long-term inflammatory and fibrotic changes has not yet been demonstrated. Based upon results observed after exposure of rats to higher levels of03, it might be prudent to further examine the possibility that these early changes are indeed predictive of long-term effects of ozone on the lung at frequently encountered ambient concentrations.
We can also conclude from these experiments that assays of enzyme activity in cell-free lavage fluid (46,47) are a relatively insensitive method of measurement of ozone-induced lung damage. Acid phosphatase and (3-N-acetylglucosaminidase enzyme activities failed to show significant changes at concentrations of ozone below 0.40 ppm (24 hr); lactate dehydrogenase activity was an even less sensitive assay, with significant increases found only after exposure of rats to 0.64 ppm of ozone for 24 hr. Preliminary experiments suggested that sialic acid content of lavage fluid was also an insensitive (and highly variable) measurement of ozone-induced lung damage, so this assay was not further examined. Increased movement of labeled tracer from blood to a lavagable compartment of the lung was a more sensitive index oflung damage then were any ofthe enzyme activity assays. The most sensitive indicator of ozoneinduced lung response in lavage fluid was an increase in the total protein content (predominantly serum albumin) of the lung lavage fluid, which was significantly increased above control values after 1 or 2 days ofexposure ofrats to 0. 12 ppm ofozone (23,48). Interestingly, the assay of movement of labeled [3H]albumin from blood to lung lavage fluid does not show any synergistic interaction between ozone and acid aerosols under conditions where ozone alone provokes a positive response and where the assay of total lavagable protein content shows synergistic interaction between the mixture of pollutants. It liTble 5 Total lung protein in rts exposed to 0. 2  <0.0001 <0.0001 aerosol 'Data are mean values ± I SD (n = 6 rats per group) for total lung protein content of rats for the various exposure regimens. Data were analyzed for significance by Student's t-test; a value ofp < 0.05 was taken as significant.
would be of interest to see ifthis finding is also true with sequential exposure regimens, but such experiments have not as yet been performed.
To further examine the range of concentrations of ozone and acid aerosol exhibiting synergistic interaction, rats were exposed to 0.20 ppm of 03 in conjunction with 40 14g/m3 ofsulfuric acid aerosol. A synergistic interaction between 0.20 ppm of 03 and 40 ig/m3 of sulfuric acid was demonstrated by assay of tissue protein content after 7 or 9 days ofexposure and by assay oflung collagen synthesis rate (48). We also found interactions between 0.20 ppm of ozone and 20 Ag/m3 of sulfuric acid, with an apparent no-observable effect level by assay of total lung protein content for sulfuric acid aerosol (plus 0.20 ppm ofozone) at or near 5 .g/m3 ( Table 5). The no-observable effect level for ozone (plus 0.5 mg/m3 of acid aerosol) is at or below 0.12 ppm.
The acidic aerosol most likely to occur in polluted urban air under conditions of photochemical smog generation is ammonium sulfate or bisulfate, although ambient concentrations of these aerosols are far lower than those used in most ofthese experiments. We have previously shown that aerosols of neutral salts (sodium chloride or sodium sulfate) do not interact synergistically with O3 (38). Furthermore, we have suggested that the acidity ofan aerosol is the important determinant of whether the aerosol will synergistically interact with 03 and have proposed a mechanism that suggests that the primary determinant for the elicitation of synergy between 03 and acid aerosols is the aerosol acidity [and not the mere presence of the aerosol (35)]. If this mechanism is correct, then weak acids, such as ammonium sulfate, should cause less ofa synergistic response than should strong acids, such as sulfuric acid, in accord with our observations in these studies (compare no-observable effect levels of 1 mg/m3 or above for ammonium sulfate aerosol with about5 g/m3 for sulfuric acid aerosol). Several colleagues helped perform the experiments described in this paper, especially Darren Warren, Dallas Hyde, Joan Gerriets, and Brian Tarkington.