The effect of ozone exposure on the release of eicosanoids in guinea-pig BAL fluid in relation to cellular damage and inflammation

The observed effects after ozone exposure strongly depend on ozone concentration and exposure time. We hypothesized that depending on the O3 exposure protocol, mainly either an oxidant damage or an inflammation will determine the O3 toxicity. We compared two different ozone exposure protocols: an acute exposure (3 ppm 2 h) for studying the oxidant damage and an exposure (1 ppm 12 h) where an inflammatory component is also probably involved. We measured LDH activity and protein and albumin exudation as markers for cellular damage. After the acute exposure an increase in LDH activity was measured and after exposure to 1 ppm ozone for 12 h the exudation of protein and albumin was also enhanced. The histological examinations showed a neutrophilic inflammatory response only after exposure to 1 ppm ozone for 12 h. The acute exposure protocol resulted in an increased release of PGE2, PGD2, PGF2α and 6-ketoPGF1α whereas exposure to 1 ppm ozone for 12 h led to an additional release of LTB4. No effects were measured on the release of TxB2 and LTC4/D4/E4. These changed amounts of eicosanoids will probably contribute to the ozone-induced lung function changes.


Introduction
Ozone is an important constituent of photochemical smog and because of its high oxidation potential is able to produce alterations in various functional, biochemical and morphological properties in the airways of humans and experimental animals. 1 Cellular membranes are sensitive to oxidative stress because they contain important targets like polyunsaturated fatty acids, sulfhydryl groups and amino acids of proteins which can easily be oxidized. Damage to cellular membranes may result in the release of lactate dehydrogenase (LDH) and an accumulation of proteins and in ammatory cells in broncho-alveolar lavage (BAL) uid which are generally seen as biomarkers for lung injury. Also after exposure to ozone these phenomena are observed in animal as well as in human studies. [2][3][4][5][6][7] Prostaglandins and leukotrienes have been shown to be importantly involved in the in ammatory response in the airways 8 -12 and also some studies have been performed to investigate the role of these lipid mediators in the ozone-induced lung function changes. 4,13 -16 However, due to relatively large interspecies differences [17][18][19][20] concerning receptor distribution and the release of eicosanoids the obtained results are rather contradictory. Also the exposure protocols used differ widely among the studies performed to investigate the effects of ozone exposure. The main purpose of the studies carried out in our laboratories is to unravel the complexity underlying the ozoneinduced changes in guinea-pig lung function. The current study deals with the release of eicosanoids in guinea-pig BAL uid in relation to the in ammatory response. The results presented here come from two different ozone exposure protocols; an acute exposure to 3 ppm ozone for 2 h where probably only an oxidative effect is responsible for the observed damage and an exposure to a lower ozone concentration (1 ppm) over a longer period of time (12 h) where the effects are expected to be mainly determined by an in ammatory response. Our objective is to study and compare the release of eicosanoids in both the ozone exposure protocols i.e. oxidant and in ammation mediated toxicity.

Animals
Male Dunkin-Hartley guinea pigs, weighing 300 ±350 g, obtained from Harlan CPB (Zeist, The Netherlands) were kept in a light-and temperature-controlled room (21 18 C, humidity 50 5%). The animals were fed a standard diet (Hope Farms, Woerden, The Netherlands) and were allowed to tap water ad libitum. The animals were adapted to the laboratory housing conditions for at least 1 week before starting the exposure.

Animal exposure
Guinea pigs were placed separately in rectangular stainless steel inhalation chambers 21 with a volume of 0.21 m 3 . Two different exposure conditions were used: exposure to 3 ppm (6 mg/ m 3 ) ozone for 2 h and exposure to 1 ppm (2 mg/ m 3 ) ozone for 12 h (guinea pigs were killed immediately after exposure). The ozone was generated by passing O 2 (pressure 1 atm.) through a pen-ray UV-light generator type 3 SC-9 with a SCT-4 power-supply (Ultra-Violet Products, San Gabriel, CA, USA) in which oxygen is partially converted to ozone. The ozone was diluted with ltered clean air as it was drawn into the exposure chamber with an air ow of 6.0 m 3 / h. The exposure chambers were conditioned at a temperature of 21 18 C and a relative humidity of 50 5%. The ozone concentration within each chamber was constantly monitored using a UV-Photometric analyzer model 9810 (Monitor Labs, San Diego, CA, USA). Calibration was performed before the exposure period using a UV-photometric calibrator (Thermo Environmental Instruments, Franklin, MA, USA). To maintain the ozone concentration at the desired value, a manual control was performed (Mass Flow Controller Type AFC-260, ASM, Bilthoven, The Netherlands).
Control animals were exposed under identical conditions to clean ltered air.

Broncho-alveolar lavage (BAL)
After exposure to ozone lungs were removed, weighed and perfused using saline to remove excessive blood. Lungs were lavaged using 40 ml prewarmed (378 C) 0.9% saline per kg body weight. Three repetitive lavages with the same aliquot were performed by steady instillation and withdrawal through a tracheal cannula. The obtained BAL uid was centrifuged for 10 min at 400 3 g at 48 C and the cell-free lavage uid supernatant was used for further analysis.

Analysis of LDH, protein and albumin
Cell-free BAL uid was analysed for total protein, albumin 22 and lactate dehydrogenase (LDH). Total protein was measured using the bicinchoninic acid (BCA) protein assay reagent according to the manufacturer's instructions (Pierce, Rockford, IL, USA). LDH activity was assayed at 378 C in 50 mM Tris-buffer, pH 7.4, with 5 mM EDTA, 0.15 mM NADH and 1.22 mM pyruvate by measuring the decrease in absorbance at 340 nm.

Analysis of cyclooxygenase and lipoxygenase products from guineapig BAL¯uid
The release of cyclooxygenase products was quanti ed using radioimmunoassays (RIA) for PGE 2 , PGF 2a , PGD 2 , 6-ketoPGF 1a and TxB 2 (antibodies from PerSeptive Diagnostics (USA), tritiated compounds from Amersham (UK), and standards from Sigma Chemicals (Belgium)) according to the manufacturer's instructions. Lipoxygenase products (LTB 4 and LTC 4 / D 4 / E 4 ) were measured using an enzyme immunoassay (EIA) system (Amersham, UK), according to the manufacturer's instructions. Samples were assayed in duplicate and standard curves were run with each assay of unknown samples. The production of cyclooxygenase and lipoxygenase products was expressed as picograms per millilitre (pg/ ml) BAL uid.

Histological examination
After exposure to ozone the lungs were removed and in ated with a 2% solution of glutaraldehyde in 0.1 M phosphate buffer for xation. After embedding in paraf n, 5 mm lung sections were stained with haematoxilin and eosin and examined by light microscopy.

Data analysis
Each experiment was performed in duplicate and results were statistically evaluated using Student's t-test. P , 0·05 was considered signicant.

LDH, protein and albumin measurements
Both exposure protocols (i.e. 3 ppm ozone for 2 h and 1 ppm ozone for 12 h) increased the amount of lactate dehydrogenase as measured in the guinea-pig BAL uid ( Table 1). The amount of total protein, or more speci cally albumin, measured in the BAL uid was not changed after exposure to 3 ppm ozone for 2 h but a signi cant increase was measured after exposure to 1 ppm ozone for 12 h.

Histological examination
In Fig. 2 the histological changes in guinea-pig lung tissue after ozone exposure are shown. In the control situation ( Fig. 2A) a bronchiole, covered with cuboidal epithelium, is shown in cross-section together with expanded alveoli separated from each other by thin septa. Exposure to 3 ppm ozone for 2 h (Fig. 2B) results in a desquamation of the bronchiolar epithelium and thus a naked basement membrane. The bronchiolar lumen is lled with these desquamated epithelial cells. The alveolar lumina are empty and the alveolar septa are thin. A bronchiolitis consisting of polymorphonuclear cells (PMNs) and mononuclear cells is accompanied with a centriacinar in ammation when the animals are exposed to 1 ppm ozone for 12 h. The bronchiolar epithelial layer is intact (Fig. 2C).

Discussion
It is shown in this and other studies that exposure to ozone is attended by an in ammatory response. [3][4][5][6][7] In this study we compared two different ozone exposure protocols (acute exposure to 3 ppm ozone for 2 h and an exposure to a lower concentration for a longer period of time (1 ppm ozone for 12 h) with respect to the in ammatory response coupled to the release of eicosanoids in the guinea-pig BAL uid.
It is shown that the increase in the lactate dehydrogenase (LDH) activity can be seen as an early marker for ozone toxicity. LDH activity is the only biochemical marker we tested that already is affected after the acute exposure to ozone (3 ppm, 2 h). Measuring LDH activity represents an early indication for cellular damage; a slight change in membrane uidity already causes a leakage of LDH from the alveolar cells in the lower airways. This rapid change in LDH release was also observed in an in vitro system using different types of cultured respiratory epithelial cells. 23 Exposure to relatively low ozone concentrations (0.5 ppm ozone for 3 h) already caused membrane injury of the epithelial cell types leading to increased lactate dehydrogenase release. Also in human studies early changes in LDH-release were measured in BAL-uid 24,25 after ozone exposure.
The other two markers for cellular damage, albumin and protein, indicate an increased permeability leading to exudation of the constituents from serum into the airways. This exudation was measurable only after exposure to 1 ppm ozone for 12 h. The increase in serum constituents in BAL uid may be caused by the ozone-induced oxidation of unsaturated fatty acids in lipids and susceptible amino acids in proteins, which results in an alteration of the biological membrane properties. In addition to the increased membrane permeability an in ux of in ammatory cells from the blood stream into the alveolar spaces was shown in our histological studies. After exposure to 3 ppm ozone for 2 h only a desquamation of the bronchiolar epithelial layer was observed whereas after exposure to 1 ppm ozone for 12 h an in ltration of neutrophilic granulocytes could be seen. However, the bronchiolar epithelium remained intact. Cell differentiation performed in guinea-pig broncho-alveolar lavage (BAL) uid after exposure to these ozone exposure protocols shows comparable results (data not shown). Exposure to 1 ppm ozone for 12 h resulted in an increased amount of neutrophilic granulocytes (represented as percentage of the total amount of cells) compared with the control situation as well as compared with the situation where the animals were exposed to 3 ppm ozone for 2 h. These current results are supported by a number of both human 13,25 as well as animal studies. A variety of animal species has been studied in order to examine the ozone-induced injury at tissue level. Ozone-induced tissue neutrophilia was rst demonstrated by Castleman et al. 26 in the bronchiolar wall of Rhesus monkeys after a 4 h exposure to 0.8 ppm ozone. Also in mongrel dogs a neutrophilic in ammation was observed in the tracheal and bronchial mucosa 27 already 1 h after exposure to 2.1 ppm ozone for 2 h which was accompanied by an ozone-induced hyperresponsiveness. In a study where guinea pigs were exposed to 3 ppm ozone during 2 h (identical to our acute exposure protocol) the time course of histological changes was examined. 28 In agreement with our ndings no in ammatory effect was observed immediately after exposure but at 6 h after exposure an increase in neutrophilic granulocytes was measurable peaking at 2 days post-exposure. Also after an exposure to 2 ppm ozone for 4 h a rapid accumulation of polymorphonuclear leukocytes (PMNs) in guinea-pig lung interstitial and airway spaces was observed. 5 The in am-

Mediators of In¯ammation´Vol 6´1997
359 matory response declined to control values within 24 h in lung interstitium whereas the increased amount of in ammatory cells measured in BAL uid remained elevated for 3 days. Very recently a study of Sun and Fan Chung 29 also showed an increased number of neutrophils after single as well as after repeated exposure (exposure on 4 successive days) to 3 ppm ozone for 3 h. The neutrophilic granulocyte is a potential source of a wide variety of mediators, including potent lipid mediators like prostaglandins, thromboxanes, leukotriene B 4 and PAF which might contribute to altered airway responses and/or exacerbation of the in ammatory response. 11 In the current study it was shown that after exposure to 3 ppm ozone for 2 h a signicant increase in the release of PGF 2a , PGE 2 , PGD 2 and 6-ketoPGF 1a (the stable endproduct of PGI 2 ) in guinea-pig BAL uid was observed. After exposure to a lower concentration over a longer period of time (1 ppm ozone for 12 h) an additional increased release of LTB 4 was measured. No effects were observed concerning the release of TxB 2 (stable end product of TxA 2 ) and LTC 4 / D 4 / E 4 .
Although no neutrophilic in ammatory response was observed after exposure to 3 ppm ozone for 2 h, an increased release of some of the lipid mediators was perceived. This observation suggests that the increase in prostaglandins is not coming from the in ux of in ammatory cells, but from cells that are present in the airways under normal conditions, possibly the alveolar macrophages or airway epithelial cells. 30 It has been shown that LTB 4 is a predominant neutrophil chemoattractant 31,32 which is present in alveolar macrophages and alveolar epithelial cells 9,30 and is thought to be responsible for initiating the in ammatory response after ozone exposure. 33 The 5-lipoxygenase pathway in neutrophils selectively generates LTB 4 upon stimulation with a variety of stimuli. 30,34 This could explain the observation that after exposure to 1 ppm ozone for 12 h, when an in ammatory response is present, a three-fold increase in the release of LTB 4 was observed whereas after exposure to 3 ppm ozone for 2 h no effects were measurable.
In our study it was shown that ozone exposure did not affect the release of LTC 4 / D 4 / E 4 . Comparable results were found in a series of experiments where humans were exposed to 0.10 ppm ozone or 0.08 ppm ozone for 6.6 h with moderate exercise (40 l/ min) and where BAL was performed 18 h after exposure. 25 No effect was observed in the release of LTB 4 although a marked in ammatory response was present. The authors suggest that the time course between ozone exposure and BAL measurements (18 h) may be responsible for this observation, since neutrophils are already attracted to the lung as early as 3 h following ozone exposure. 13 It is possible that LTB 4 is present in BAL uid shortly after ozone exposure. Although no clear indication can be found in the literature it may be expected that comparable with the prostaglandin release 17 -20 in the case of leukotriene release a species difference may also account for these observed differences between guinea pig and human studies. LTB 4 itself is not able to contract or relax the airways but it seems able to induce airway hyperresponsiveness by the release of TxB 2 . 31 After LTB 4 inhalation an in ux of neutrophils into the airways was observed but also a striking increase in TxB 2 in lavage uid in dogs. This increased airway responsiveness was prevented by pretreatment with the thromboxane synthase inhibitor OKY-046 whereas the inhibitor did not change the amount of in ammatory cells after LTB 4 -inhalation. 31 Surprisingly, our study did not show any increase in the release of TxB 2 although an increased release of LTB 4 was present after exposure to 1 ppm ozone for 12 h. The amounts of TxB 2 measured in our experiments are, compared with the other eicosanoids, rather high. This might suggest that the neutrophils are not the major source of TxB 2 in our experimental set-up and that basal release of TxB 2 from other cell types in the airways 35 exceeds the release from neutrophils. On the other hand, the release of TxB 2 from thrombocytes may also account for the observed effects since these cells produce very large amounts of TxB 2 36,37 and this might overwhelm the ozoneinduced changes in the TxB 2 release.
In summary, we have shown in this study that the in ammatory response after exposure to ozone strongly depends on ozone concentration and exposure time. LDH seems to be the most sensitive marker for ozone-induced tissue damage whereas the exudation of albumin and protein is only measurable after exposure over a longer period of time. This exudation is accompanied by a neutrophilic in ammatory response and a subsequent increase in the LTB 4 release in guinea-pig BAL uid. The other mediators remained unchanged (TxB 2 and LTC 4 / D 4 / E 4 ) or were already increased after exposure to 3 ppm ozone for 2 h in the absence of an in ammatory response (PGE 2 , PGD 2 , PGF 2a and 6-keto-PGF 1a ). The precise role of these eicosanoids in the ozone-induced changes in airway reactivity in our experimental set-up and exposure protocol requires further investigation.