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J Appl Physiol 91: 1694-1700, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 4, 1694-1700, October 2001

Ozone causes lipid peroxidation but little antioxidant depletion in exercising and nonexercising hamsters

Nancy C. Long1, Jung Suh2, Jason D. Morrow3, Robert H. Schiestl4, G. G. Krishna Murthy1, Joseph D. Brain1, and Balz Frei2

1 Physiology Program and 4 Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115; 2 Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331; and 3 Departments of Pharmacology and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ozone (O3), a major component of urban air pollution, is a strong oxidizing agent that can cause lung injury and inflammation. In the present study, we investigated the effect of inhalation of O3 on levels of F2-isoprostanes in bronchoalveolar lavage fluid (BALF) and on levels of antioxidants in the BALF and plasma of hamsters. Because antioxidants, including urate, ascorbate, GSH, and vitamin E, defend the lungs by reacting with oxidizing agents, we expected to find a decrease in antioxidant levels after O3 exposure. Similarly, we expected an increase in the levels of F2-isoprostanes, which are lipid peroxidation products. Exposure to 1.0 or 3.0 parts/million (ppm) O3 for 6 h resulted in an increase in BALF neutrophil numbers, an indicator of acute inflammation, as well as elevation of BALF F2-isoprostanes. The higher dose of O3 caused an increase in the BALF level of urate and a decrease in the plasma level of ascorbate, but 1.0 ppm O3 had no effect on BALF or plasma antioxidant levels. Exposure to 0.12 ppm O3 had no effect on BALF neutrophils or F2-isoprostanes nor on BALF and plasma antioxidants. We also investigated the effect of O3 exposure of hamsters during exercise on F2-isoprostane and antioxidant levels. We found that exposure to 1.0 ppm O3 during 1 h of exercise on a laddermill increased BALF levels of F2-isoprostanes but had no effect on BALF neutrophils or on BALF and plasma antioxidants. These results indicate that O3 induces inflammation and biomolecule oxidation in the lungs, whereas extracellular antioxidant levels are relatively unchanged.

lung; oxidant injury; prostaglandins; 8-epi-prostaglandin F2alpha ; F2-isoprostanes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CLEAN AIR ACT OF 1970 established limits for six common urban air pollutants: ozone (O3), carbon monoxide, nitrogen oxides, sulfur dioxide, lead, and particles. O3 is not a primary emission. Rather, it is a secondary pollutant produced by the interaction of other pollutants, specifically nitrogen oxides and volatile organic compounds, with sunlight. Consequently, O3 levels are particularly difficult to control. In fact, whereas levels of many pollutants have decreased over the past decade, trophospheric O3 levels have continued to rise. The United States Environmental Protection Agency air quality standard of 0.12 parts/million (ppm) average for 1 h is frequently exceeded in many urban areas in the US and other countries (17). Mexico City, home to more than 20 million people, has O3 levels of between 0.1 and 0.2 ppm year round, and during the autumn and winter, levels >0.3 ppm are common (1).

O3 is one of the most powerful oxidizing agents known (17). Because O3 is so reactive, it may not actually reach or react with the lung epithelium. Instead, it is thought to react with molecules in the respiratory tract lining fluid (RTLF) in airways and alveoli, forming more stable secondary and tertiary products, which, in turn, react with lung tissue (23). O3 could potentially interact with any biomolecule in the RTLF, including surfactant lipids and proteins. However, these molecules are protected by a system of antioxidants that rapidly react with oxidizing agents to form less toxic secondary products, thus minimizing the degree of damage to important biomolecules. Small-molecular-weight antioxidants, such as urate, ascorbate (vitamin C), and GSH, which are present in high concentrations in the RTLF, have high intrinsic reactivities toward O3 and are thought to act as "sacrificial targets," defending the lung against O3-induced injury (16). Alpha-tocopherol (vitamin E) forms a second line of defense by scavenging peroxyl radicals generated by the interaction of O3 with polyunsaturated fatty acids in the RTLF (8).

Usually, these antioxidants are sufficient to protect the lung from the effects of inhaled O3. However, if O3 levels are sufficiently high for a long-enough duration or if antioxidant levels are compromised, these defenses become saturated, allowing oxidation of important biomolecules in the RTLF and oxidative lung injury. Various models of pulmonary exposure to O3 have shown that both proteins and lipids react with this pollutant (3, 14). However, there is some controversy as to which of these biomolecules is most susceptible to O3 damage (14, 20). Nonetheless, the relatively low levels of protein in the RTLF and the abundance of polyunsaturated fatty acids in surfactant mean that lipids are likely targets for attack by O3 (22). Therefore, it is not surprising that lipid peroxidation is thought to be a major mechanism of O3-induced lung injury (8).

We are particularly interested in F2-isoprostane levels as an indicator of lipid peroxidation and as a cause of O3-induced lung injury. Tissue and plasma F2-isoprostanes are formed by free radical-catalyzed peroxidation of arachidonic acid, which occurs independently of the enzyme cyclooxygenase (12). F2-isoprostane levels are a highly specific measure of in vivo lipid peroxidation (21). Interestingly, certain F2-isoprostanes have been shown to exert potent biological activity and, therefore, may contribute to the inflammation observed after O3 exposure (6, 7).

In this study, we examined the effect of O3 exposure on antioxidant depletion and lipid peroxidation in hamsters. We exposed animals to various doses of O3 for 6 h and then measured plasma and bronchoalveolar lavage fluid (BALF) levels of ascorbate, urate, GSH, alpha -tocopherol, and F2-isoprostanes. The 6-h time point was selected because it would allow sufficient time for O3-induced inflammation, which we assessed by counting the number of neutrophils in the BALF, to occur. Thus our analysis of the oxidative consequences of O3 exposure would include not only the immediate effects of O3 exposure, but also the delayed consequences of oxidants released during inflammation.

In a second experiment, we investigated the effect of exercise on these parameters by studying trained hamsters that were running on a laddermill during exposure to either O3 (1.0 ppm) or air for 1 h and comparing them against trained hamsters that were not running during inhalation of O3 or air. Exercise increases the amount of inhaled O3, and the corresponding rise in tidal volume increases the penetration of O3 into the periphery. Therefore, we predicted that the ventilatory changes during exercise would result in a greater degree of inflammation, lipid peroxidation, and antioxidant depletion than in nonexercising hamsters.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

The methods used in this study were evaluated and approved by the Harvard Medical Area Standing Committee on Animals. Male Syrian Golden hamsters (Mesocricetus auratus; Charles Rivers Breeding Laboratory, Wilmington, MA), aged 4-18 mo and weighing between 114 and 190 g, were used in this study. Animals were housed individually and maintained under viral antigen-free conditions in the animal facilities of the Harvard School of Public Health. Purina rat chow and water were provided ad libitum. The animals used in the dose-response study were housed in standard hamster cages. The animals in the exercise study were housed in individual stainless steel cages (25.4 × 15.2 × 12.7 cm) connected by a small doorway to a 35.5-cm (diameter) × 10-cm (width) stainless steel running wheel (Wahmann Manufacturing, Timonium, MD). Each hamster was free to move between the two portions of the cage and to run voluntarily at any time in the self-propelled wheel. Each running wheel was equipped with a mechanical counter mounted on the shaft of the wheel, which recorded total revolutions. The average distance that each hamster ran in 24 h during the training period was 13.29 ± 2.03 km (n = 23). The animals were housed in the running wheel cages for at least 5 wk before being used in the experiment.

O3 Exposure

O3 was generated by passing filtered dry 100% oxygen through a high-voltage (7,000 V) discharge device, producing ultraviolet light, and mixing the resulting gas with a diluting flow of filtered room air in a stainless steel and Plexiglas exposure chamber (45 × 45 × 60 cm in size; ~100 liters). The chamber was designed with a pyramid on top to aid in uniform distribution of the gases. Samples of the chamber atmosphere were continually drawn from the exposure chamber via a sampling port at the level of the hamsters, and the O3 concentration was continually measured throughout the exposure with an O3 chemiluminescent analyzer (model 49, Thermoelectron, Hopkinton, MA). The O3 analyzer was calibrated by reference to an ultraviolet photometer (model 1003, PC S/N 3419, Daisibi, Glendale, CA), which serves as an O3 primary standard as defined by the Environmental Protection Agency. Control hamsters were placed in an identical chamber but were exposed to clean, filtered room air for the same duration. Typical ambient O3 levels in the laboratory were ~10 parts/billion; however, levels may have occasionally reached as high as 50-100 parts/billion.

The running animals ran on a specially designed and constructed small-animal tilted ladder ergometer. The laddermill was enclosed in a 14-liter Lucite box, which allowed us to expose animals to O3 during exercise. The ladder speed was driven by a rheostat-controlled direct-current gear motor, which was located outside the exposure chamber. The laddermill speed could be adjusted to between 1 and 150 m/min. Clean, filtered room air or O3 was continuously drawn through the exposure chamber at a rate of 12 l/min. A small, low-speed circulating fan was used for mixing.

Collection of Blood and BALF

Between 0.5 and 3.5 h after the end of the exposure period, the hamsters were killed by injection of a lethal dose of pentobarbital sodium. Blood was collected into heparinized syringes by cardiac puncture. The trachea was then cannulated with a tubing adapter, and bronchoalveolar lavage was performed by instilling 3 ml of fluid through the tracheostomy tube and then withdrawing the fluid while the chest was massaged. This was repeated 12 times to make a final volume of ~35 ml of BALF. The fluid recovered from the first two washes of BALF was collected in a separate tube from the remaining volume. The lavage fluids and blood were centrifuged (400 g at 4°C for 10 min). The supernatant from the first two washes of BALF and the plasma were removed and stored at -70°C for later assay of antioxidants and F2-isoprostanes.

The pelleted cells from both BALF samples were combined and brought up in saline, and the number and type of cells present in each lavage fluid were determined as follows. A well-mixed sample from each lavage fluid was cytocentrifuged onto microscope slides (Cytospin 2; Shandon Southern Instruments, Sewickley, PA), air dried, and stained with Wright-Giemsa stain (VWB Stat Stain, Brisbane, CA). From these slides, a differential count of 600 cells was performed. The number of cells found in a separate aliquot was counted using a hemocytometer, and, using the differential, the total number of neutrophils in each sample was calculated.

In the study of the effect of various doses of O3, (experiment 1), 100-µl aliquots of blood and the first two washes of BALF were each mixed with 200 µl of 10% perchloric acid, centrifuged (400 g at 4°C for 10 min), and filtered through a 0.2-µm syringe filter (Gelman Sciences, Ann Arbor, MI). The samples were stored at -70°C for later assays for GSH.

Assay of Antioxidants and F2-Isoprostanes

Ascorbate, urate, alpha -tocopherol, and GSH were analyzed by HPLC with electrochemical detection as described (4, 9, 28). F2-isoprostanes were measured after purification and derivatization by capillary gas chromatography/negative ion chemical ionization mass spectrometry as described (13).

Experiment 1: Effect of Various Doses of O3

In this experiment, we studied the effects of three concentrations of O3 (0.12, 1.0, and 3.0 ppm for 6 h) on inflammation, antioxidant status, and lipid peroxidation in hamsters. We assessed the degree of inflammation by measuring the number of neutrophils in the BALF. We also measured levels of the antioxidants ascorbate, urate, and alpha -tocopherol in the BALF and plasma as well as BALF levels of the lipid peroxidation product F2-isoprostane. GSH levels were measured in whole blood and BALF fluid.

Exposure protocol. On the day of the experiments, the hamsters were removed from their home cages and placed in individual cells in a 40 × 40 × 12-cm wire mesh cage that had been divided into 12 compartments. The animals (n = 6 in each group) were placed in the exposure chamber and exposed to either O3 (0.12, 1.0, or 3.0 ppm) or ambient air for 6 h. After the exposure period ended, the animals were removed from the chamber and returned to their home cages.

Data analysis. We used two separate cohorts of animals. The first cohort contained animals that were exposed to 3.0 ppm O3 or air, whereas the second contained animals exposed to 0.12- or 1.0 ppm O3 or air. To control for differences between the air controls in the two cohorts, the data were normalized by dividing each individual data point for each O3-exposed hamster by the mean of its respective air-control cohort. We then performed ANOVAs on the normalized data. When we found significant differences using this test, we performed follow-up t-tests, comparing the O3-exposed animals against the air-exposed controls. We used the Bonferroni correction to correct for multiple comparisons.

Experiment 2: Effect of Exercise on Response to O3

This experiment was performed to assess the effect of exercise by running on a laddermill on the response of hamsters to O3 exposure (1.0 ppm for 1 h). As in the previous experiment, we assessed inflammation by measuring the number of neutrophils in the BALF. Based on the results of the earlier experiment, we decided to measure levels of ascorbate, urate, and alpha -tocopherol in the plasma, and BALF levels of ascorbate, urate, and F2-isoprostanes.

Exposure protocol. The animals were randomly assigned to one of four groups: O3/running (n = 7), O3/nonrunning (n = 6), air/running (n = 5), and air/nonrunning (n = 5). At the start of each experiment, one animal was placed on the laddermill, the laddermill chamber was sealed, and the flow of O3 or air was turned on. The running animals were given ~5 min to accommodate to the laddermill before the start of the exposure. After the laddermill was turned on, most of the hamsters ran comfortably, maintaining a position near the front end of the moving ladder. The hamsters ran for 10 min, then the laddermill was turned off, and they were allowed to rest for 5 min. This cycle was repeated four times, giving the animals a total of 40 min of running time. Each hamster was exercised at near the maximal running speed that it could sustain during each 10-min bout of exercise, ~25 m/min. No aversive treatments, such as electric shock, were used in either the training or experimental phases of this study. The nonrunning hamsters exposed to O3 or room air were placed in small wire mesh cages, which were oriented toward the front end of the laddermill, to maintain exposure positions as close as possible to those of the exercising hamsters.

Data analysis. The BALF samples from this study were analyzed in two separate F2-isoprostane assays. Because there can be some variability between runs of this assay, the results of each test were normalized by dividing the F2-isoprostane value from each hamster by the mean of the air controls run in the same assay. Within each assay, there were no significant differences between the air-exposed groups (running and nonrunning). Therefore, we combined the two groups and used the mean F2-isoprostane level in each assay for normalization. Once the data were normalized, we performed an ANOVA to detect an overall difference among the groups and then follow-up t-tests to compare the individual treatments. We performed a Bonferroni correction to correct for multiple comparisons. We analyzed the number of neutrophils and the levels of antioxidants in the same way, by normalizing each data point against the mean of the air controls.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1: Effect of Various Doses of O3

Table 1 shows the number of neutrophils and the levels of F2-isoprostanes in the BALF of hamsters exposed to air or 0.12 or 1.0 ppm O3 and to air or 3.0 ppm O3. The data are reported separately, because they were collected from separate cohorts of animals. The results of our analyses are shown in Figs. 1 and 2.

                              
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  		Table 1.   Neutrophil and F2-isoprostane levels in BALF of hamsters exposed to air or O3 for 6 h



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  		Fig. 1.   Neutrophils (as percentage of air controls) in bronchoalveolar lavage fluid (BALF) of hamsters exposed to air or 0.12, 1.0, or 3.0 parts/million (ppm) ozone (O3) for 6 h. Animals were killed and samples were collected between 0.5 and 3.5 h after the end of the exposure. Values are means ± SE; n = 6 in each group. Significant difference vs. air: * P < 0.05, *** P < 0.005.



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  		Fig. 2.   Levels of F2-isoprostanes (as percentage of air controls) in the BALF of hamsters exposed to air or 0.12, 1.0, or 3.0 ppm O3 for 6 h. Animals were killed and samples were collected between 0.5 and 3.5 h after the end of the exposure. Values are means ± SE; n = 6 in each group. Significant difference vs. air, *** P < 0.005.

We found a significant effect of exposure to O3 on the number of neutrophils in the BALF (Fig. 1). Exposure to 1.0 ppm resulted in a roughly 2.5-fold increase in neutrophil number (P < 0.05), whereas 3.0 ppm caused a nearly fivefold increase over the air controls (P < 0.005). Animals exposed to 0.12 ppm also had elevated neutrophil counts compared with controls; however, this difference was not statistically significant.

Similarly, we found that the same doses of O3 caused a significant increase in BALF levels of F2-isoprostanes (Fig. 2). Exposure to 1.0 ppm caused F2-isoprostane levels to increase roughly threefold (P < 0.005), and exposure to 3.0 ppm caused an increase of more than 13-fold over the air controls (P < 0.005). The lowest dose of O3 (0.12 ppm) also increased F2-isoprostane levels but not significantly.

The effects of O3 exposure on BALF and plasma antioxidant levels are shown in Tables 2 and 3. In our analysis of the data, we normalized the values as described above for neutrophil count and F2-isoprostane levels. The two lower doses of O3 (0.12 and 1.0 ppm) had no effect on antioxidant levels in either BALF or plasma (Table 2). However, 3.0 ppm O3 caused a significant increase in BALF levels of urate and a significant decrease in plasma levels of ascorbate (Table 3).

                              
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  		Table 2.   BALF and plasma antioxidant levels after exposure to air or 0.12- or 1.0-ppm O3 for 6 h


                              
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  		Table 3.   BALF and plasma antioxidant levels after exposure to air or 3.0-ppm O3 for 6 h

Experiment 2: Effect of Exercise on Response to O3

Table 4 shows the number of neutrophils and levels of F2-isoprostanes and antioxidants in the BALF and levels of antioxidants in the plasma of running and nonrunning hamsters after exposure to air or 1.0 ppm O3. In our analysis of these data, we combined the two air-exposed groups (running and nonrunning), normalized the data by dividing the values collected from each O3-exposed animal by the mean of the air-exposed controls, and then performed an ANOVA. The results are shown in Figs. 3 and 4.

                              
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  		Table 4.   BALF neutrophils, F2-isoprostanes, and antioxidant levels and plasma antioxidant levels of running or nonrunning hamsters exposed to air or 1.0 ppm O3 for 1 h



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  		Fig. 3.   Neutrophils (as percentage of air controls) in the BALF of running or nonrunning hamsters exposed to air or 1.0 ppm O3 for 1 h. Values are means ± SE; n = 10 for air, n = 6 for O3-no run, and n = 7 for O3-run. There were no significant differences among any of the treatment groups.



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  		Fig. 4.   Levels of F2-isoprostanes (as percentage of air controls) in the BALF of running or nonrunning hamsters exposed to air or 1.0 ppm ozone for 1 h. Values are means ± SE; n = 10 for air group, n = 6 for O3-no run, and n = 7 for O3-run. Significant difference vs. O3-run: * P < 0.05, ** P < 0.01.

We found no significant effect of 1 h of exposure to 1.0 ppm O3 on the number of neutrophils in the BALF of running or nonrunning hamsters. However, hamsters that were running during O3 exposure did have significantly higher levels of F2-isoprostanes in their BALF than did either air-exposed animals (P < 0.05) or nonrunning O3-exposed animals (P < 0.01). There were no significant differences among the four groups of animals in the BALF or plasma levels of any of the antioxidants studied.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data in the present study show that, in nonexercising hamsters, 6 h of O3 exposure resulted in a dose-dependent increase in the number of neutrophils in the BALF and in BALF levels of the lipid peroxidation product F2-isoprostanes but have relatively little effect on BALF or plasma antioxidant levels. Exercise during a 1-h exposure to O3 increases BALF levels of F2-isoprostanes but has no effect on BALF neutrophil numbers or on BALF and plasma antioxidant levels. These results are consistent with the ability of O3 to induce inflammation and biomolecule oxidation. Unexpectedly, however, O3 had only a small effect on small-molecule antioxidants, which are thought to be an important defense against oxidant injury in extracellular fluids. This suggests that antioxidant molecules are generously supplied.

Many previous studies have shown that O3 induces pulmonary injury and inflammation (10, 19, 24, 31). Acute exposure to O3 has been shown to increase lung permeability (31) and to trigger proliferation of the airway epithelium (24). In vitro work has shown that O3 exposure causes airway epithelial cells to become activated and to produce a variety of inflammatory mediators and enzymes (10). These changes in the epithelium facilitate the movement of neutrophils into the respiratory tract, which furthers the inflammatory process (19).

The powerful oxidative actions of O3 could induce injury and inflammation in a number of ways. O3 may act directly on the RTLF to oxidize biomolecules. These oxidized biomolecules may decompose to cytotoxic nonradical species, such as aldehydes and ozonides, which could cause further lung damage (8). In addition, oxidative stress may enhance the inflammatory response by promoting the expression of inflammatory mediators. This possibility is supported by the work of Shi and co-workers (25-27), who have shown that oxidative stress regulates expression of macrophage inflammatory proteins-1alpha and -2 and KC by turning on transcription of the genes for these chemokines and by posttranscriptionally stabilizing the resulting mRNA. Other investigators have shown that oxidative stress increases plasma levels of TNF-alpha measured in mice after intravenous injection of lipopolysaccharide (2).

Our results show that O3 exposure increases BALF levels of F2-isoprostanes. This finding is consistent with the work of other researchers showing that O3 exposure results in the production of lipid peroxidation products (reviewed in Ref. 8). O3 exposure of exercising humans has also been shown to result in increased levels of 8-epi-PGF2alpha , a class of F2-isoprostanes, in the airway lavage fluid (5). In addition, other types of oxidative stress, including carbon tetrachloride and diquat poisoning, have been shown to increase production of these compounds in a cyclooxygenase-independent manner in rats (12). Increased F2-isoprostane levels have also been observed in the plasma of smokers (11).

F2-isoprostanes and other lipid peroxidation products may themselves be important contributors to the inflammatory response to O3. The F2-isoprostane 8-epi-PGF2alpha causes vasoconstriction and bronchoconstriction in an isolated perfused rat lung model (6), constriction of guinea pig airways in vitro (7), as well as airflow obstruction and airway plasma exudation in guinea pigs in vivo (18). In all cases, the effects were abolished by treatment with thromboxane receptor antagonists, demonstrating that 8-epi-PGF2alpha acts through the same receptor as the naturally occurring PG, thromboxane A2.

In the exercise study, we saw an increase in BALF levels of F2-isoprostanes but no increase in BALF neutrophil numbers. The most likely explanation for the latter observation is that, because the animals were lavaged immediately after the 1-h exposure, there was not sufficient time for the neutrophils to move into the airways. In rats instilled with endotoxin, a particularly strong inflammatory stimulus, neutrophils did not begin appearing in the airways until 2 h postinstillation and did not reach peak levels until 6 h postinstillation (29). The fact that F2-isoprostane levels rise before neutrophil accumulation suggests that, in combination with exercise, O3 exposure may cause oxidative damage directly, rather than via neutrophil activation. However, it is also possible that neutrophils that were in transit across the airway wall were responsible for F2-isoprostane production.

Because O3 has been shown to cause antioxidant depletion in in vitro models of RTLF (15, 30), we were surprised to find that O3 exposure conditions that caused significant inflammation and lipid peroxidation had relatively little effect on BALF or plasma levels of antioxidants. After 6 h of exposure to the highest dose of O3 (3.0 ppm), we found a decrease in plasma levels of only one antioxidant, ascorbate. At the same dose, we observed an increase in BALF levels of urate. One possible explanation for the latter finding is that cell lysis and release of intracellular urate into the BALF occurred as a result of O3-induced injury. Another possibility is that increased vascular permeability in the lungs, which may have occurred as a result of O3-induced inflammation, allowed more urate to leak from the plasma into the BALF.

It is possible that the doses and time points that we selected were not optimal for assessing the effects of O3 on antioxidants. Because exposure to 1 ppm O3 for 6 h did not alter antioxidant levels, it is not surprising that we also saw no change in these parameters in the exercise study, in which running or nonrunning animals were exposed to O3 for 1 h. Because higher doses of O3 and longer exposure times are not well tolerated by exercising animals, it was not possible to determine whether exposure to higher levels of this pollutant would have caused antioxidant depletion.

The 6-h time point in the dose-response study, which we selected because it allowed enough time for observable inflammation to occur, may have also provided sufficient time for intracellular and tissue stores of antioxidants to be mobilized, keeping plasma and BALF levels of these compounds steady. Furthermore, if antioxidants are able to move from the plasma to the RTLF in inflamed lungs, it is possible that RTLF antioxidants were in fact depleted by O3 exposure and then replenished from the plasma. Further studies using intermediate time points would provide a more complete picture of the dynamics of antioxidant depletion and repletion.

In conclusion, O3 exposure in hamsters results in the formation of the lipid peroxidation products F2-isoprostanes in BALF but causes relatively little change in BALF or plasma levels of small-molecule antioxidants. Exercising hamsters show increased levels of F2-isoprostanes after just 1 h of O3 exposure, more rapidly than other inflammatory indicators become detectable. F2-isoprostanes are not only useful biomarkers of oxidant exposure, but may also mediate some of the inflammatory and pathophysiological changes observed with O3 exposure.


    ACKNOWLEDGEMENTS

We are grateful for the technical assistance of Roslyn Hennessey.


    FOOTNOTES

This work was supported by National Institutes of Health Grants ES-06593, DK-48831, GM-42056, GM-15431, CA-68485, and DK-26657. J. D. Morrow is the recipient of a Burroughs-Wellcome Fund Clinical Scientist Award in Translational Research.

Address for reprint requests and other correspondence: N. C. Long, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (E-mail: nlong{at}hsph.harvard.edu).

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. Section 1734 solely to indicate this fact.

Received 3 August 1999; accepted in final form 25 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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5.   Hazbun, ME, Hamilton R, Holian A, and Eschenbacher WL. Ozone-induced increases in substance P and 8-epi-prostaglandin F2alpha in the airways of human subjects. Am J Respir Crit Care Med 9: 568-572, 1993.

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J APPL PHYSIOL 91(4):1694-1700
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