1 Nelson Institute of Environmental Medicine, New York University Medical Center, New York, New York 10016; and 2 Pulmonary Toxicology Branch, Experimental Toxicology Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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Previous studies have shown that rats late in pregnancy and throughout lactation are more susceptible to ozone (O3)-induced pulmonary inflammation than are prepregnant (virgin) or postlactating rats. The major aim of the present study was to determine whether these differences in response intensity could be accounted for by the O3 dose to the lower region of the lung. The relative O3 dose to the lower lung of groups of pregnant, lactating, and virgin female rats was estimated by measuring the incorporation of the 18O isotope into low-speed (cells) and high-speed (surfactant) pellets of bronchoalveolar lavage fluid immediately after acute exposure to 0.5-1.1 parts/million 18O3. The polymorphonuclear leukocyte (PMN) and protein inflammatory responses were established 20 h after acute exposure of identical physiological groups to 0.5-1.1 parts/million 16O3 (common isotope). A single regression of PMN inflammation data against surfactant 18O concentration for all physiological groups gave a linear relationship, indicating direct proportionality of PMN inflammation with this estimate of relative dose to the lower lung regardless of physiological status. This implies that the chemical species that react with surfactant molecules, i.e., O3 or its metabolites, are the same as or proportional to those chemical species responsible for initiating PMN inflammation. Additional experiments showed that lung tissue ascorbic acid concentration was significantly lower in pregnant and lactating rats than in virgin female rats. Although a causative relationship cannot be assumed, the deficit in tissue ascorbic acid concentration in pregnant and lactating rats compared with virgin female rats is consistent with their greater responsiveness and higher relative surfactant O3 dose.
ozone; ascorbic acid; antioxidant; surfactant; polymorphonuclear leukocyte
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INTRODUCTION |
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SEVERAL STUDIES FROM OUR LABORATORY (5, 6, 29) have demonstrated that rats late in pregnancy and throughout lactation are more susceptible to acute ozone (O3)-induced pulmonary inflammation and damage than virgin female rats, rats early in pregnancy, or postlactating rats. Data from one of these studies (29) suggested that a significant portion of the difference in pulmonary response between lactating and postlactating animals stems from the greater inhaled O3 dose in lactating rats due to their metabolically driven higher ventilation. In addition to the difference in total inhaled O3 dose, a significant difference in regional dose to the lower lung of lactating and postlactating rats must also occur because of the considerably larger tidal volume in lactating rats that drives O3 deeper into the alveolar region (15). It seems probable that O3 delivered deeply into the alveolar region may have a disproportionately greater effect on the induction of inflammatory changes. The earlier study by Weideman et al. (29) did not address this concept because accurate assessment of the regional dose from the available data was not possible. In the study presented here, the relative O3 dose received by the lower lungs of pregnant, lactating, and prepregnant (virgin female) rats is estimated, and the possibility that the relative dose to this region can account for documented differences in O3-induced inflammatory responses among these physiological states is explored.
Biological responses in the lung after O3 exposure are not directly dependent on the concentration of O3 in inhaled air but rather on the rate and duration of delivery of O3 or O3 metabolites to critical reactive sites in the respiratory tract tissues (i.e., O3 dose to the tissue). Among groups of dissimilar animals (e.g., different physiological states) exposed to the same O3 concentration, this critical parameter of dose may vary due to inherent differences in, for example, lung chemistry or ventilation. The lower lung was targeted for dose estimates because it is believed that O3-induced lung inflammation is initiated primarily by reactions in the lower air spaces rather than in the upper airways. The amount of O3 or active O3 metabolite that reacts with specified components of the lower lung was measured by the methods of Hatch and colleagues (7, 8) and Santrock et al. (23) and was assumed to be proportional to the O3 dose to the lower lung. The rationale for this method is that 18O derived from inhaled 18O3 acts as a tracer for O3 or O3 metabolites that react with cellular or acellular components of interest (i.e., 18O3-derived reaction products). For example, Hatch et al. (8) considered 18O incorporated into the bronchoalveolar lavage (BAL) fluid (BALF) cell pellet and the surfactant pellet to be estimates of the O3 dose to the alveolar region of the lung because this is primarily where these targets are located. Experiments from the study presented here show that differences in O3-induced pulmonary inflammation among pregnant, lactating, and virgin female rats were consistent with differences in O3 surfactant dose measured as 18O incorporated into the surfactant pellet collected by BAL (i.e., 18O3-derived reaction products incorporated into the surfactant pellet). Additional experiments comparing the antioxidant status of the lungs of pregnant, lactating, and virgin female rats were performed because the responses to O3 are believed to be modulated by antioxidant factors in the lung (3). These experiments revealed that measurements of surfactant O3 dose and inflammatory response among these physiological groups were inversely correlated with pulmonary ascorbic acid (AA) concentration.
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METHODS |
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Experimental animals. The rats used in this study were Sprague-Dawley supplied by Charles River Laboratories. Pregnant and lactating rats were mated by the breeder at 8-9 wk of age. The rats were received ~1 wk before exposure and were housed singly in solid-bottom cages in a room maintained at 20-23°C and 45-55% relative humidity with a 6 AM to 6 PM light cycle. Purina rodent chow and water were provided ad libitum. On the day of exposure to O3 or air, the pregnant rats were at gestation day 17 (day of vaginal plug called day 1) and lactating rats were 13 days postpartum. At exposure, virgin female rats were between 10 and 14 wk, pregnant rats ~12 wk, and lactating rats 13-14 wk of age.
BAL and nasal lavage. BAL was performed as previously described (5). Briefly, the rats were exsanguinated via the abdominal aorta after an overdose of intraperitoneal pentobarbital sodium. The trachea was cannulated with a blunted 15-gauge needle, and the chest cavity was opened. The lungs were inflated to total capacity with calcium- and magnesium-free phosphate-buffered saline (PBS; pH 7.2) that flowed passively from a reservoir maintained at a height of 25 cm of buffer. The PBS remained in the lungs for 30 s after total inflation (i.e., 30-s dwell time) and was then briskly withdrawn manually with a syringe attached to the tracheal cannula via a three-way adapter. This BALF (BALF 1) was stored on ice, and the procedure was repeated five times. The BALF was centrifuged at 4°C at 450 g (low-speed centrifugation), and the supernatant and cells were processed and saved for later analytic procedures.
After BAL, the nasal passages of some rats were lavaged retrogradely via a blunted, 1.8-cm-long, 15-gauge stainless steel needle that was inserted into the trachea just distal to the larynx, advanced anteriorly through the nasopharyngeal orifice, and secured with suture thread. The jaw of the rat was removed so that pressure could be applied on both sides of the catheter to prevent leakage into the oral cavity during lavage. A syringe containing 1.4 ml of PBS was attached to the needle hub, the contents were slowly forced through the nasal passages, and the nasal lavage (NL) fluid (NLF) was collected as it dripped from the nares.
Animal exposures. The rats were restrained in tube holders and acutely exposed to either 0.5, 0.8, or 1.1 parts/million (ppm) O3 or charcoal-filtered air for 1-4 h beginning at approximately 9 AM in a stainless steel "nose-only" apparatus (CH Technologies, Westwood, NJ). O3 was generated by passing a mixture of 1% O2 (16O or 18O, 99% purity) in argon through an electric arc generator at a precisely controlled flow rate in the range of 30-100 ml/min. The efficiency of conversion of O2 to O3 in this system was ~4%. The O3 output was mixed with a stream of charcoal-filtered air (~15 l/min), and the O3 concentration generated was maintained within ±2.0% of the target by controlling the flow of O3 from the arc generator. The O3 concentration was monitored continuously with a Dasibi 1003-PC O3 analyzer (Dasibi Environmental, Glendale, CA), which was calibrated quarterly with a Monitor Laboratories (San Diego, CA) calibrator. The background O3 concentration in the charcoal-filtered air was 0.00-0.02 ppm.
Separate groups of animals of each status were exposed to identical concentrations of either 18O3 (tracer isotope) or 16O3 (common isotope) and killed at different times after exposure to accurately assess both dose and inflammatory responses. This was necessary because the optimum time for the collection of material for measurement of 18O3-derived reaction products (dose) is immediately after exposure because of the relatively short half-life of these reaction products (~6 h; Ref. 23), whereas the optimum time for detection of inflammatory changes is generally the day after exposure. Exposure to 18O3 for estimation of dose was 3 h in duration, and exposure to 16O3 for measurement of inflammatory changes was 4 h in duration. Because 18O3-derived reaction products were used as a measure of relative dose (as opposed to absolute dose), these estimates of relative dose after 3 h of exposure were valid for rats exposed to O3 for 4 h. All three physiological groups were exposed to 0.5 and 0.8 ppm in both 18O3 and 16O3 exposure regimens; however, only the virgin female group was exposed to 1.1 ppm. Virgin female rats were exposed to the higher concentration of O3 to produce surfactant doses and inflammatory responses that were approximately comparable to those of the pregnant and lactating groups exposed to 0.8 ppm O3.
Preparation of samples for estimation of
dose. Animals acutely exposed to
18O3
were killed immediately (5-10 min) after exposure, and the cell
pellets from the low-speed centrifugation of BALFs
1-6 were combined
in a 1.5-ml microfuge tube and stored at 70°C. The
supernatants from the low-speed centrifugation of BALFs
1-3 were combined and centrifuged at 27,000 g (high-speed centrifugation) for 30 min. The resulting supernatant was discarded, and the pelleted material that contained most of the extracellular surface-active lipid-protein complex (22) was stored at
70°C. Although this procedure
does not give a quantitative recovery of surfactant, it is a frequently employed and accepted method for its collection. Therefore, this high-speed pellet is hereafter referred to as surfactant or surfactant pellet. The surfactant and cell pellet samples were lyophilized, transferred into tared 5 × 3.5-mm-diameter silver capsules, and weighed on a microbalance. The samples were secured by crimping the
capsules and were then stored at
70°C. All samples were
analyzed within 4 mo for 18O
concentration as described in
18O
analysis.
Preparation of samples for measurement of inflammatory
response. Groups of animals acutely exposed to
16O3
(common isotope) were killed 20 ± 1 h after exposure. Aliquots of
BALF 1 supernatant from the low-speed
centrifugation were frozen at 70°C for later protein
analysis (25). The cell pellets from all six lavages
(BALFs
1-6) were
pooled and enumerated, and viability was determined by trypan blue
exclusion at a magnification of ×400. The viability of all groups
was >90%, and there were no differences among groups. Slides of
cells from the BALF pellets were prepared with a cytospin centrifuge,
fixed with methanol, and stained with a Hemacolor stain set (EM
Diagnostic Systems, Gibbstown, NJ) for differentiation of 500 inflammatory cells/rat at a magnification of ×1,000.
Preparation of samples for antioxidant
analysis. Other groups of animals were acutely exposed
to air or 0.8 ppm
16O3
and killed immediately after exposure for BAL and NL. Aliquots of
BALF 1 and NLF supernatants from the
low-speed centrifugations were stabilized in 2.5% perchloric acid and
stored at 70°C. Both stabilized supernatants were later
analyzed for AA and uric acid (UA).
Lung tissue from additional groups of naive rats was prepared for
subsequent determination of several antioxidant species. Approximately
0.2 and 0.3 g wet weight of lung tissue perfused via the pulmonary
artery and devoid of major bronchi were homogenized with a Kinematica
tissue homogenizer (model PT 10-35, Brinkmann Instruments, Westbury,
NY) in 3.0 ml of 3.0% perchloric acid and 3.0 ml of 1.15% KCl-50 mM
Tris buffer, pH 7.6, respectively. The homogenates were centrifuged at
20,000 g for 30 min, and the
supernatants were stored frozen at 70°C pending analysis for
AA, UA, and total glutathione (perchloric acid supernatant) and for
glutathione peroxidase, glutathione reductase, superoxide dismutase,
glucose-6-phosphate dehydrogenase (G6PDH), and catalase (Tris buffer
supernatant). A third piece of perfused lung tissue (~0.5 g wet wt)
was stored at
70°C for subsequent vitamin E analysis.
Antioxidant analyses. Aliquots used for enzymatic activity determinations were clarified after being thawed by centrifugation at 12,000 g for 20 min. Glutathione peroxidase activity was determined from the consumption of NADPH in the presence of tert-butyl hydroperoxide and glutathione reductase activity from the reduction of dithio(bis)nitrobenzoic acid (9). G6PDH activity was determined by the method of Lohr and Waller (13), superoxide dismutase activity by inhibition of the reduction of pyrogallol (14), and catalase by the method of Wheeler et al. (30). All enzymatic analyses were performed with a COBAS FARA autoanalyzer. Perchloric acid supernatants of tissue homogenates and BALFs were clarified by centrifugation at 20,000 g for 20 min and assayed for AA and UA by HPLC with amperometric detection (11). The perchloric acid tissue homogenate supernatant was also analyzed for total glutathione (oxidized plus reduced) with a cycling assay with the COBAS FARA autoanalyzer (1). Tissue vitamin E concentration was determined by HPLC analysis of the concentrated heptane extract of a 80% ethanol tissue homogenate (27).
18O analysis. Determination of the 18O concentration in lyophilized surfactant and cell pellet samples was accomplished by the analytic method described by Hatch et al. (8). In brief, this method consists of mass-spectrophotometric determination of the ratio of 18O to 16O in the sample after conversion of O2 to CO2. The raw data thus determined (18O-to-16O ratios) were corrected for background tissue 18O that results from the presence of ~0.2% of atmospheric O2 in the 18O isomeric form. This background ratio of 18O to 16O is identical in all tissues and can be determined by analysis of plasma from air-breathing rats. The background 18O-to-16O ratio established from rat plasma was subtracted from each surfactant and cell pellet sample 18O-to-16O ratio obtained from rats exposed to 18O3. The resulting corrected ratios represented the fractional enrichment of 18O due to 18O3 exposure. The corrected sample ratios were then converted to "excess micrograms of 18O per gram of dry weight" (8).
Statistics. Protein and polymorphonuclear leukocyte (PMN) responses (see Figs. 1 and 2) and BALF AA as well as NLF AA concentrations (see Table 2) in virgin female, pregnant, and lactating rats exposed to O3 or air were analyzed by two-factor analysis of variance (ANOVA). After detection of significant differences for protein and PMN responses, comparisons among physiological groups exposed to the same O3 concentration were made by single-factor analysis of variance and Tukey-Kramer post hoc test. For BALF AA data, Dunnett's two-tailed post hoc test was used to compare the pregnant and lactating groups with the virgin group, and t-tests were used to compare O3 versus air exposure within each physiological group. The activity of each pulmonary antioxidant factor (see Table 1) was compared among the three physiological groups with a one-way ANOVA. If a significant effect was observed, Dunnett's two-tailed post hoc test was used to compare the pregnant and lactating groups with the virgin group. The slopes of linear regressions (see Figs. 1, 2, 6, and 7) were compared by analysis of covariance (31). ![]() |
RESULTS |
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Comparison of pulmonary responses of virgin female, pregnant, and
lactating rats to acute O3 exposure.
Data on inflammation and damage from rats exposed to 0.5, 0.8, or 1.1 (virgin female rats only) ppm
O3 for 4 h are shown in Figs.
1 (BALF PMN) and
2 (BALF protein). Both responses were
greater in pregnant and lactating rats than in virgin female rats, and the absolute differences increased as the
O3 concentration increased. Two-factor ANOVA showed that there were significant differences with
respect to physiological group, O3
concentration, and the interaction of these two factors for both BALF
protein and PMN responses. Subsequent single-factor ANOVA followed by
post hoc testing indicated significant differences among the
physiological groups at 0.5 and 0.8 ppm
O3 concentrations as shown in
Figs. 1 and 2. This pattern of response is similar to those previously observed after 6-h exposures (5, 29).
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Assessment of relative O3 dose in the
lower lungs of virgin female, pregnant, and lactating rats.
The relative dose of O3
(O3 itself or reactive metabolites
of O3) delivered to reactive
sites in the lower lung was estimated by measuring the concentration of
18O in lavageable surfactant and
inflammatory cells immediately after exposure to
18O3.
To explore the validity of this estimation, the linearity of
incorporation of 18O was
investigated in virgin female rats exposed to 0.8 ppm
18O3
for periods of 1-4 h (Fig. 3). Note
that incorporation of 18O was
reasonably linear in both surfactant and lavaged cells, although the
rate was considerably greater in the surfactant.
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Relationship between relative O3 dose to
the lower lung and pulmonary responses in virgin female, pregnant, and
lactating rats.
To establish the relationship between PMN inflammation and relative
O3 dose in the lower lung, mean
values of BALF PMNs for each physiological group at each concentration
(Fig. 1) were regressed against their respective mean
O3 doses estimated as
surfactant-incorporated 18O (also
termed "relative surfactant O3
dose"). The result, illustrated in Fig.
6, indicates very good correlation between
PMN inflammation and relative surfactant
O3 dose regardless of the
physiological status. Analysis of the PMN values from individual
animals (not mean values) showed that the slopes of the linear
regressions of virgin female, pregnant, and lactating rats were not
significantly different from the common regression slope derived from
all data when the relative dose was used as the regressor
(P > 0.05;
F = 2.94; Fig. 6), whereas the slopes
were significantly different when concentration was used as the
regressor (P < 0.05;
F = 8.90; Fig. 1). Data of BALF
protein were analyzed similarly and are illustrated in Fig.
7. Although the
F ratio improved from 44.51 to 11.26 when relative dose (Fig. 7) was used as the regressor instead of
concentration (Fig. 2), individual regression slopes of the
physiological groups were still significantly different from the common
regression slope (P < 0.05),
probably due mainly to the exaggerated BALF protein response in the
lactating group after 0.8 ppm O3.
It is thought that the protein response in this group was exaggerated
because of localized regions of breakdown of the blood-alveolar barrier
resulting in pockets of air spaces flooded with plasma proteins (see
DISCUSSION).
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DISCUSSION |
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This study confirms that greater air space PMN and protein levels are induced in pregnant and lactating rats than in virgin female rats by acute exposure to the same concentrations of O3. In addition, the present study extends the investigation of responses among these physiological states to include the relationship between pulmonary inflammation and an estimate of relative O3 dose in the lower lung. This estimate consists of surfactant-incorporated 18O reaction products derived from inhaled 18O3. The concentration of 18O in surfactant is thought to be proportional to the O3 molecules that penetrate into the air spaces of the lower lung and escape scavenging by antioxidant molecules in the ELF. Although the chemical identity of the 18O3-derived reaction products is not identified in this analysis, it is probable that incorporated 18O reflects chemically and biologically active O3 metabolites. The validity of surfactant 18O3-derived reaction products as an estimate of O3 dose to the lower lung is supported by the linear incorporation of 18O with respect to time of exposure to 18O3 (Fig. 3). In addition, the greater rate of incorporation of 18O into the surfactant of lactating rats compared with the surfactant of virgin female rats (Fig. 4) is consistent with the higher tidal volumes in lactating rats (28, 29).
Data from three previous studies (8, 10, 24) have shown that there is a direct correlation between 18O3-derived reaction products in the BAL high-speed pellet (surfactant) and indicators of toxicity and inflammation. This positive correlation is confirmed by the results of this study that, in addition, show that the relationship between surfactant dose and PMN inflammation is linear among three physiologically diverse groups of rats exposed to several O3 concentrations (Fig. 6). Thus the data illustrated in Fig. 6 demonstrate that the surfactant O3 dose (18O3-derived reaction products in surfactant) is a reliable measure of the effectiveness of O3 in causing pulmonary PMN inflammation regardless of physiological status. This relationship implies that the O3 and/or metabolites of O3 that react with surfactant are either the same as or proportional to the chemical species responsible for initiating inflammation. It can also be inferred from Fig. 6 that the difference in PMN response to acute O3 exposure among these physiological states (Fig. 1) can probably be explained by factors that determine the surfactant O3 dose in the lower lung, such as ventilation and the concentration of key antioxidants, not by differences in the inherent sensitivity of pulmonary tissues to O3 or its proximal toxicants.
Although not as sensitive to O3 exposure as BALF PMNs, the BALF protein response to acute O3 exposure is a reasonably good linear function of surfactant O3 dose (Fig. 7) provided that the exaggerated response of lactating rats after exposure to 0.8 ppm is excluded. The basis for this exclusion is the suspected localized breakdown of the blood-air barrier in the high-concentration lactating group. Although the majority of airway protein detected in all other groups probably consisted of plasma proteins that transuded a relatively intact epithelial-capillary barrier, in the 0.8 ppm O3-exposed lactating group much of the airway protein is believed to have leaked from ruptured blood vessels through a badly disrupted alveolar epithelium. This is suggested by the relatively large number of red blood cells (RBCs) in the BALF of this group (mean ~20 × 106) compared with the few RBCs in the BALF of all other groups (mean ~1.4 × 106). Such leakage of blood would greatly increase the BALF protein concentration but would not significantly affect the percentage of BALF PMNs because of the low concentration of PMNs in blood relative to other types of leukocytes.
In theory, lavageable inflammatory cells (largely macrophages) present in the RTLF of the normal lung should function similarly to surfactant molecules as reactive sites for O3 and its metabolites. In actuality, however, the concentration of 18O3-derived reaction products in the BALF pellet of inflammatory cells was not as good an indicator of O3 dose as was surfactant 18O (compare Figs. 4 and 5). There are two probable reasons for the unreliability of cell pellet 18O3-derived reaction products as an estimate of dose. First, the rate of incorporation of 18O into the cell pellet is slow relative to its incorporation into surfactant (see Fig. 3). Thus cell pellet-associated 18O is not responsive to relatively small increments in O3 dose. Second, exposure to high O3 concentrations causes some sloughing of epithelial cells, producing cellular fragments or debris of unknown 18O concentration that contaminate the inflammatory cells in the BALF cell pellet.
Other data from this study showed that lung tissue and BALF AA concentrations are lower in pregnant and lactating rats than in virgin female rats (Tables 1 and 2). This observed difference, especially the apparent difference in AA concentration in the ELF, could be an important factor modulating the rate of O3 penetration to surfactant molecules and other reactive sites in the lower lung. The RTLF (ELF plus fluid lining the upper airways) is the initial compartment that inhaled O3 encounters. It is believed that O3 is essentially completely removed from the air phase by reactive absorption in this fluid layer (18, 19). Although the specific reactions of O3 within the RTLF are not known, it is thought that O3 can initiate a cascade of potentially toxic reactions that includes reactions with proteins and sites of unsaturation in fatty acids (19-21). Alternatively, O3 may be scavenged by certain antioxidant molecules, thus limiting entry into the more toxic pathways (21). We hypothesize that an important factor in modulating both the incorporation of 18O into surfactant molecules and the induction of inflammation is the rate of removal of O3 by antioxidants in the RTLF. In rats, the most prominent antioxidant in the RTLF is AA (3). It has been demonstrated in model systems that AA is depleted by O3 and also that AA can effectively remove O3 from an airstream (2, 12, 26). Results from the experiments reported here (Table 2), as well as those from a previously reported study (5), show that AA is depleted in BALF 1 by acute O3 exposure and thus are consistent with the model experiments mentioned above. Furthermore, the results of the present study are consistent with the concept that the BALF 1 AA concentration reflects the concentration of AA in the ELF and the underlying tissues. The reaction of inhaled O3 with AA in the ELF upsets the equilibrium between the ELF and the underlying cells, ultimately depleting AA in the epithelial tissues and compromising their capacity for protection against oxidant damage. Because pregnant and lactating rats have lower concentrations of AA in their ELF and tissues than virgin female rats (Tables 1 and 2), presumably a larger amount of inhaled O3 will enter into toxic reactive pathways in the former physiological states, leading to both greater inflammation and greater incorporation into surfactant molecules. Data from the present study are consistent with, but do not confirm, this scenario because there may be other variables (e.g., ventilation) among pregnant, lactating, and virgin female rats that can affect O3-induced inflammation and incorporation of 18O3-derived reaction products into surfactant.
NL data (Table 2) suggest that AA in the lining fluid of the nasal passages reacts with inhaled O3 and thus may function to protect the lower lung from O3 exposure. A study by others (4) showed that the nasal tissues (tissues anterior to the posterior pharynx) of tidal-breathing humans remove an average of 40% of inhaled O3. Therefore, it follows that factors affecting the efficiency of removal of O3 by the nasal passages have biological significance. One such factor may be the presence of antioxidants in the nasal tissues and lining fluid. The data from this study suggest that AA in the nasal passages of the rat reacts with and removes inhaled O3, much as UA is believed to do in humans (16, 17). The NL data from this study also indicate that, unlike the airways and tissues of the lung, there is no difference in AA concentration in the nasal passage RTLF among naive pregnant, lactating, and virgin female rats. Therefore, the observed differences in inflammation and surfactant O3 dose among these physiological states are unlikely to be caused by differential removal of O3 in the nasal passages by AA.
There are data from other laboratories that suggest the importance of ELF AA concentration in protection against the effects of acute O3 exposure. For example, in a study investigating the effect of dietary restriction on acute O3 toxicity in rats, Kari et al. (10) reported relationships similar to those observed here. Their data showed an inverse relationship between BALF AA concentration and both pulmonary inflammation and incorporation of 18O3-derived reaction products into lung surfactant and cells. In their study, BALF glutathione was also elevated in diet-restricted animals and, therefore, like AA might have influenced the mitigation of O3 effects in these rats.
In conclusion, this study has demonstrated that quantitatively disparate inflammatory responses among prepregnant (virgin), pregnant, and lactating rats acutely exposed to O3 can be explained by proportional O3 doses in the lower lung as estimated by 18O3-derived reaction products incorporated into the surfactant collected by BAL. AA concentration in the BALF and lung tissue correlated inversely with inflammation and surfactant O3 dose in these groups of rats and may, in part, mediate the observed differences.
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ACKNOWLEDGEMENTS |
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We thank Ralph Slade, Kay Crissman, and Judy Richards for antioxidant factor analyses; Linda P. Harris for oxygen-18 analysis; and Allen Bowers for assisting with ozone-18 exposures.
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FOOTNOTES |
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This research was supported by National Institute of Environmental Health Sciences (NIEHS) Grant ES-05939 and is part of a center program supported by NIEHS Grant ES-00260.
This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency (USEPA), and was approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the USEPA nor does mention of trade names of commercial products constitute endorsement or recommendation for use.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. F. Gunnison, New York Univ. Medical Center, Nelson Institute of Environmental Medicine, 57 Old Forge Rd., Tuxedo, NY 10987.
Received 15 July 1998; accepted in final form 9 November 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, M. E.
Determination of glutathione and glutathione disulfide in biological samples.
Methods Enzymol.
113:
48-55,
1985.
2.
Cross, C. E.,
P. A. Montchnik,
B. A. Bruener,
D. A. Jones,
H. Kaur,
B. N. Ames,
and
B. Halliwell.
Oxidative damage to plasma constituents by ozone.
FEBS Lett.
298:
269-272,
1992[Medline].
3.
Cross, C. E., A. van der Vliet, C. A. O'Neill, S. Louie, and B. Halliwell. Oxidants, antioxidants, and
respiratory tract lining fluids. Environ. Health
Perspect. 102, Suppl.
10: 185-191, 1994.
4.
Gerrity, T. R.,
R. A. Weaver,
J. Berntsen,
D. E. House,
and
J. J. O'Neal.
Extrathoracic and intrathoracic removal of O3 in tidal-breathing humans.
J. Appl. Physiol.
65:
393-400,
1988
5.
Gunnison, A. F.,
G. E. Hatch,
K. Crissman,
and
A. Bowers.
Comparative sensitivity of lactating and virgin female rats to ozone-induced pulmonary inflammation.
Inhalation Toxicol.
8:
607-623,
1996.
6.
Gunnison, A. F.,
P. A. Weideman,
and
M. Sobo.
Enhanced inflammatory response to acute ozone exposure in rats during pregnancy and lactation.
Fundam. Appl. Toxicol.
19:
607-612,
1992[Medline].
7.
Hatch, G. E.,
H. Koren,
and
M. Aissa.
A method for comparison of animal and human alveolar dose and toxic effect of inhaled ozone.
Health Phys.
57:
37-40,
1989[Medline].
8.
Hatch, G. E.,
R. Slade,
L. P. Harris,
W. F. McDonnell,
R. B. Devlin,
H. S. Koren,
D. L. Costa,
and
J. McKee.
Ozone dose and effect in humans and rats. A comparison using oxygen-18 labeling and bronchoalveolar lavage.
Am. J. Respir. Crit. Care Med.
150:
676-683,
1994[Abstract].
9.
Jaskot, R. H.,
E. G. Charlet,
E. C. Grose,
M. A. Grady,
and
J. H. Roycroft.
An automated analysis of glutathione peroxidase, S-transferase, and reductase activity in animal tissue.
J. Anal. Toxicol.
7:
86-88,
1983[Medline].
10.
Kari, F.,
G. Hatch,
R. Slade,
K. Crissman,
P. P. Simeonova,
and
M. Luster.
Dietary restriction mitigates ozone-induced lung inflammation in rats: a role for endogenous antioxidants.
Am. J. Respir. Cell Mol. Biol.
17:
740-747,
1997
11.
Kutnink, M. A.,
W. A. Hawkes,
E. E. Schaus,
and
S. T. Omaye.
An internal standard method for unattended high-performance liquid chromatography analysis of ascorbic acid in blood components.
Anal. Biochem.
166:
424-430,
1987[Medline].
12.
Langford, S. D.,
A. Bidani,
and
E. M. Postlethwait.
Ozone-reactive absorption by pulmonary epithelial lining fluid constituents.
Toxicol. Appl. Pharmacol.
132:
122-130,
1995[Medline].
13.
Lohr, G. W.,
and
H. D. Waller.
Glucose-6-phosphate dehydrogenase.
In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. New York: Academic, 1974, vol. 2, p. 636-643.
14.
Minami, M.,
and
H. A. Yoshikawa.
A simplified assay method of superoxide dismutase activity for clinical use.
Clin. Chim. Acta
92:
337-343,
1979[Medline].
15.
Overton, J. H.,
R. C. Graham,
and
F. C. Miller.
A model of the regional uptake of gaseous pollutants in the lung. II. The sensitivity of ozone uptake in laboratory animal lungs to anatomical and ventilatory parameters.
Toxicol. Appl. Pharmacol.
88:
418-432,
1987[Medline].
16.
Peden, D. B.,
R. Hohman,
M. E. Brown,
R. T. Mason,
C. Berkebile,
H. M. Fales,
and
M. A. Kaliner.
Uric acid is a major antioxidant in human nasal airway secretions.
Proc. Natl. Acad. Sci. USA
87:
7638-7642,
1990[Abstract].
17.
Peden, D. B.,
M. Swiersz,
K. Ohkubo,
B. Hahn,
B. Emery,
and
M. A. Kaliner.
Nasal secretion of the ozone scavenger uric acid.
Am. Rev. Respir. Dis.
148:
455-461,
1993[Medline].
18.
Postlethwait, E. M.,
S. D. Langford,
and
A. Bidani.
Determinants of inhaled ozone absorption in isolated rat lungs.
Toxicol. Appl. Pharmacol.
125:
77-89,
1994[Medline].
19.
Pryor, W. A.
How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts?
Free Radic. Biol. Med.
12:
83-88,
1992[Medline].
20.
Pryor, W. A.
Mechanisms of radical formation from reactions of ozone with target molecules in the lung.
Free Radic. Biol. Med.
17:
451-465,
1994[Medline].
21.
Pryor, W. A.,
G. L. Squadrito,
and
M. Friedman.
A new mechanism for the toxicity of ozone.
Toxicol. Lett.
82-83:
287-293,
1995.
22.
Sanders, R. L.
The composition of pulmonary surfactant.
In: Lung Development: Biological and Clinical Perspectives, edited by P. M. Farrell. Orlando, FL: Academic, 1982, vol. 1, p. 193-210.
23.
Santrock, J.,
G. E. Hatch,
R. Slade,
and
J. M. Hayes.
Incorporation and disappearance of oxygen-18 in lung from mice exposed to 1 ppm 18O3.
Toxicol. Appl. Pharmacol.
98:
75-80,
1989[Medline].
24.
Slade, R.,
W. P. Watkinson,
and
G. E. Hatch.
Mouse strain differences in ozone dosimetry and body temperature changes.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L73-L77,
1997
25.
Smith, P. K.,
R. I. Krohn,
G. T. Hermanson,
A. K. Mallia,
F. H. Gartner,
M. D. Provenzano,
E. K. Fujimoto,
N. M. Goeke,
B. J. Olson,
and
D. C. Klenk.
Measurement of protein using bicinchoninic acid.
Anal. Biochem.
150:
76-85,
1985[Medline].
26.
Van der Vliet, A.,
C. A. O'Neill,
J. P. Eiserich,
and
C. E. Cross.
Oxidative damage to extracellular fluids by ozone and possible protective effect of thiols.
Arch. Biochem. Biophys.
321:
43-50,
1995[Medline].
27.
Vandewoude, M.,
M. Claeys,
and
I. DeLeeuw.
Determination of alpha-tocopherol in human plasma by high-performance liquid chromatography with electrochemical detection.
J. Chromatogr.
311:
176-182,
1984[Medline].
28.
Weideman, P. A.
Comparison of the Pulmonary Response of Lactating and Postlactating Rats to Acute Ozone Exposure and Evaluation of the Influence of Inhaled Dose (PhD thesis). New York: New York University Medical Center, 1995.
29.
Weideman, P. A.,
L. C. Chen,
and
A. F. Gunnison.
Enhanced pulmonary inflammatory response to ozone during lactation in rats: evaluation of the influence of inhaled dose.
Inhalation Toxicol.
8:
495-519,
1996.
30.
Wheeler, C. R.,
J. A. Salzman,
N. M. Elsayed,
S. T. Omaye,
and
D. W. Korte, Jr.
Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity.
Anal. Biochem.
184:
193-199,
1990[Medline].
31.
Zar, J. H.
Comparing simple linear regression equations.
In: Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall, 1974, chapt. 17, p. 228-235.
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