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Effect of ozone exposure on prostacyclin synthesis in lung
PROSTAGLANDINS
EFFECT OF OZONE EXPOSURE ON PROSTACYCLIN SYNTHESIS IN LUNG TAKAHIRO KOBAYASHI Division of Basic Medical Sciences, The National Institute for Environmental Studies, Yatabe, Ibaraki 305, Japan ABSTRACT The effect of ozone exposure on prostacyclin (PGI2) synthesis in the rat lung was studied. Male Wistar rats were exposed to 0.2, 0.4, 0.8, 1.2 and 1.8 ppm ozone for 24h. The higher concentration (1.8 ppm) significantly depressed-PG12 synthesizing activity of lung homogenates. Time-courses (1, 3, 5, 7, 14 and 28 days) of the effect of ozone (0.4 and 0.8 ppm) exposure on the PGI2-synthesizing activity of lung homogenates were studied. The PGI2-synthesizing activity of the lung decreased, reaching a maximum at 5 days and then gradually returning to normal by day 14, and remaining normal at day 28, even though the ozone exposure continued. The formation of lipid peroxides due to ozone exposure may cause the depression of PGI2-synthesizing activity of lung. Induction of anti-oxidative enzymes may relate to the recovery of the PGI2-synthesizing activity. INTRODUCTION Photochemical reactions in the atmosphere lead to the formation of oxidants,including ozone which is highly toxic. Ozone appears to react with cellular unsaturated fatty acids to form lipid peroxides [l]. The lung synthesizes predominantly PG12 [2, 31, and lipid peroxides inhibit PG12 synthetase [4]. Nitrogen dioxide (N02) exposure to rats inhibited lung PG12 synthesis [3, 51, probably by the formation of lipid peroxides. NO2 is a strong oxidizing agent like ozone,and can react with unsaturated fatty acids to form lipid peroxides [6]. Thus the present study was designed to investigate 1) the concentration effect of ozone exposure on the PGI2-synthesizing activities of the lung homogenates and 2) the time-course of this effect. MATERIALS AND METHODS Animal Exposure Specific pathogen-free male Jcl:Wistar rats, 13 weeks of age, Exp. 1: The rats were exposed to 0.2, 0.4, were used (6 rats/group). 0.8, 1.2 and 1.8 ppm ozone for 24h. Exp. 2: The rats were exposed to 0.4 or 0.8 ppm ozone for 1, 3, 5, 7, 14 or 28 days. Rats exposed to filtered air for 1, 7 or 28 days were controls for rats exposed to ozone for days 1 and 3; 5, 7 and 14; and 28 respectively. The conditions in the chamber (volume, 1.39 m3) were: temperature, 25kl"C; humidity, 55 *lo%; pressure, -5 mm H 0 relative to atmospheric pressure; total air flow rate, 110-120 m 5/h. The concentrations of ozone
DECEMBER
1983 VOL. 26 NO. 6
1021
PROSTAGLANDINS
were automatically controlled by continuous monitoring with an ozone anaiyzer (Kimoto 806s) using a chemiluminescence method. Assay for the PGI2-synthesizing activity of lung homogenates Immediately after the termination of the exposure, the rats were killed by exsanguination under ether anaesthesia, and lungs were excised. The assay procedure has been described previously [3]. Briefly, a weighed piece of each lung (approx. 600 mg) was homogenized with 2.5 ml cold phosphate buffer (pH 8.2 67 mM) by a polytron homogenizer. The homogenate (1 ml) was incubated with 0.2 1-1 Ci [1-'4C]arachidonic acid (56.4 Ci/mol) and 5 i~-g unlabelled arachidonic acid at 37°C for 15 min. The reaction was terminated by adding 1N HCl to bring the pH of the reaction mixture to 3.0. The reaction mixtures were separated by TLC using the developing solvent system, ethyl acetate / 2,2,4_trimethylpentane / acetic acid / water (11:5:2:10. v/v). The radioactive products were analyzed by thin layer radiochromatography. Protein was determined by the method of Lowry et al [7] with bovine serum albumin as standard. The data are expressed as mean 2 SD and were statistically analyzed using Student's t test with ~~0.05 as the level of significance. RESULTS Fig. 1 shows the relationship between ozone concentration and a ratio of lung wet weight: body weight. 1.2 and 1.8 ppm ozone exposure increased the ratio significantly. Fig. 2 shows the PGI2-synthesizing activity of lung tissue from rats exposed to 0.2, 0.4, 0.8, 1.2 and 1.8 ppm ozone for 24h. In the range of 0.2sO.8 ppm ozone exposure, the PG12 synthesizing activity tended to increase (p= 0.054 at 0.4 ppm). Then the PG12 synthesizing activity decreased. Fig. 3 shows the change in the body weight during 0.4 ppm and 0.8 ppm ozone exposure. The body weight of the rats exposed to 0.8 ppm ozone for 1, 7 or 28 days was less than controls, and decreased dramatically at days 28. With 0.4 ppm ozone, weights were significantly less than controls only at day 1. Fig. 4 shows that the change in the lung wet weight during 0.4 ppm and 0.8 ppm ozone exposure. The lung wet weight increased significantly after 3, 5, 7, 14 or 28 days exposure to 0.8 ppm ozone. With 0.4 ppm ozone, significant increases were observed at days 3 and 28. Fig. 5 shows the relationship between the PGI2-synthesizing activity of homogenized lung and the time of ozone exposure. The PG12 synthesizing activity decreased maximally at about 5 days and then decreased. After 14 days, PG12 synthesizing activity was not significantly different from normal, even though the ozone exposure continued. At 2d days, the level was normal.
1022
DECEMBER
1983 VOL. 26 NO. 6
PROSTAGLANDINS
--
5 A
6
11
IIll
C 0.2 0.4
0.8
1”
1.2
Ozone Concentration
”
1.8
pm
Figure 1 Relationship between 03 concentration and the ratio of lung wet weight: body weight. (Mean+SD, * and ***: ~~0.05 and p
C 0.2 0.4
0.8
1.2
03 Concentration
1.8 ppm
Figure 2 Relationship between 03 concentration and PGI2-synthes izing activity (MeankSD, ** p
DECEMBER
1983 VOL. 26 NO. 6
1023
PROSTAGLANDINS
3oso [
/;,;:; *
250
*** **
9 c.
1.
I I1 1 .I 11. 1 I I 3 5 7 14 Time of 03 exposure
01
,,-
*** 28days
Figure 3 Change in body weight during exposure to 03 (0.8 ppm v and 0.4 ppm 0 ) and filtered air (Ocontrol). Statistical analysis was done at day 1, 7 and 28. (Mean?SD, *, ** and ***: ~~0.05, p< 0.01 and p
9
013 I1
I
I
,I
5
.I
7
1
I,
.
.
.
14 .
Time of 03 exposure
M 28days
Figure 4 Change in lung wet weight during exposure to 03 (0.8 ppm v and 0.4 ppm 0 ) and filtered air (Ocontrol). (Mean+SD, ** and ***: ~~0.01 and p
1024
DECEMBER
1983 VOL. 26 NO. 6
PROSTAGLANDINS
Time of 03 exposure
Fi ure 5
Change in PGI -synthesizing activity of lung by exposure ppmr and 0. Z ppm 0 ) and filtered air (Ocontrol). (Mean* SD, *, ** and ***: p< 0.05, p< 0.01 and p< 0.001 respectively)
kkJT.8
DISCUSSION PG12 synthetase is inhibited by lipid hydroperoxides such as 15-hydroperoxyeicosatetraenoic acid [4]. Since ozone is a strong oxidizing agent, it can readily react with components of lung surface such as unsaturated fatty acids to form peroxygenated intermediates. Roycroft et al. reported a rapid oxidation of arachidonic acid to oxonides, peroxides, and other products by in vitro exposure to ozone [8]. The peroxygenated intermediates may cause the depression of PGI2-synthesizing activity. In the range of 0.2~~0.8 ppm ozone exposure, however, the PGI2synthesizing activity may have been increased (Fig. 2). Lands et al reported that a slight amount of lipid hydroperoxides enhance the cyclooxygenase activity [9]. Several enzymes that supply the reducing equivalents in the form of NADPH are induced during exposure to 0.8 ppm ozone [lo]. The activities of glucose-6-phosphate dehydrogenase (G6PD) and
DECEMBER
1983 VOL. 26 NO. 6
1025
PROSTAGLANDINS
6-phosphogluconate dehydrogenase (GPGD) play important roles of the hexose monophosphate shunt for glucose metabolism, and furnish NADPH. A significant increase in the activities of these enzymes in lung cytosol was observed after exposure to ozone, peaking between 3 and 4 days [lo]. Reduced glutathione is considered to protect against peroxidative damage, by reducing peroxides. Glutathione levels in lung tissue increased and reached a maximum at about 3 days with exposure to 0.8 ppm ozone [lo]. Glutathione peroxidase and superoxide dismutase are also induced by 0.8 ppm ozone exposure [lo]. Therefore increased formation of these enzymes may explain the recovery of the PGI2-synthesizing activity. A continuous formation and a release of PG12 by lung may play a role in preventing platelet aggregation [ll]. Depression of PGI2synthesizing activity of lung by NO2 exposure correlates with a decrease in the number of platelets during NO2 exposure [5]. Therefore further studies on the relationship between the decrease in PG12 synthesis by lung and pathological and physiological changes by ozone exposure may be rewarding. ACKNOWLEDGEMENT I wish to express my gratitude to Dr. K. Kubota and Dr. M. Sagai (The National Institute for Environmental Studies) for encouragement. REFERENCES
1) 2) 3)
4)
5) 6)
7) 8)
1026
Chow, C. K. and A. P. Tappel, An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone-exposed rats, Lipids, 7: 518, 1972. Pace-Asiak, C. R. and B. Rangaraj, Distribution of prostagladin biosynthetic pathways in several rat tissues. Formation of 6keto-prostagladin Flo, Biochim, Biophys. Acta, 486: 579, 1977. Kobayashi, T., I. Morita and S. Murota, Effect of acute nitrogen dioxide exposure on the prostacyclin synthesis in lung, Toxicol. Lett., 9: 373, 1981. Moncada, S. and J. R. Vane, Unstable metabolites of arachidonic acid and their role in homeostasis and thrombosis, Br. Med. Bull., 34: 129, 1978. Kobayashi, T., I. Morita and S. Murota, Effects of nitrogen dioxide exposure of prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats, Prostaglandins,25: 303,1983. Kobayashi, T., The reaction of nitrogen dioxide with lung surface components: The reaction with cis-9-octadecenoic acid, Chemosphere, 12:1317, 1983. Lowry, 0. H., N. H. Rosenbrough, A. L. Farr and K. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193: 265, 1951. Roycroft, H. H., W. B. Gunter and D. B. Menzel, Ozone toxicity: Hormone - like oxidation products from arachidonic acid by ozone catalyzed autooxidation, Toxicol. Lett., 1: 75, 1977.
DECEMBER
1983 VOL. 26 NO. 6
PROSTAGLANDINS
9)
10)
11)
Lands, W. E. M. and M. J. Byrnes, The influence of ambient peroxides on the conversion of 5,8,11,14,17-eicosapentaenoic acid to prostaglandins, Prog. Lipid Res., 20: 287, 1981. Lee, S. D., Eds. Biochemical effects of environmental pollutants, 1974), (Ann Arbor, Michigan: Ann Arbor Science Ptb lishers, Inc., p.74-81. German, R. R., S. Bunting and 0. V. Miller, Modulation of human platelet adenylate cyclase by prostacyclin (PGX), Prostaglandins, 13: 377, 1977. Editor: Alan Bennett
DECEMBER
Received: 9-20-83
1983 VOL. 26 NO. 6
Rcceoted: 11-7-83
1027
EFFECT OF OZONE EXPOSURE ON PROSTACYCLIN SYNTHESIS IN LUNG TAKAHIRO KOBAYASHI Division of Basic Medical Sciences, The National Institute for Environmental Studies, Yatabe, Ibaraki 305, Japan ABSTRACT The effect of ozone exposure on prostacyclin (PGI2) synthesis in the rat lung was studied. Male Wistar rats were exposed to 0.2, 0.4, 0.8, 1.2 and 1.8 ppm ozone for 24h. The higher concentration (1.8 ppm) significantly depressed-PG12 synthesizing activity of lung homogenates. Time-courses (1, 3, 5, 7, 14 and 28 days) of the effect of ozone (0.4 and 0.8 ppm) exposure on the PGI2-synthesizing activity of lung homogenates were studied. The PGI2-synthesizing activity of the lung decreased, reaching a maximum at 5 days and then gradually returning to normal by day 14, and remaining normal at day 28, even though the ozone exposure continued. The formation of lipid peroxides due to ozone exposure may cause the depression of PGI2-synthesizing activity of lung. Induction of anti-oxidative enzymes may relate to the recovery of the PGI2-synthesizing activity. INTRODUCTION Photochemical reactions in the atmosphere lead to the formation of oxidants,including ozone which is highly toxic. Ozone appears to react with cellular unsaturated fatty acids to form lipid peroxides [l]. The lung synthesizes predominantly PG12 [2, 31, and lipid peroxides inhibit PG12 synthetase [4]. Nitrogen dioxide (N02) exposure to rats inhibited lung PG12 synthesis [3, 51, probably by the formation of lipid peroxides. NO2 is a strong oxidizing agent like ozone,and can react with unsaturated fatty acids to form lipid peroxides [6]. Thus the present study was designed to investigate 1) the concentration effect of ozone exposure on the PGI2-synthesizing activities of the lung homogenates and 2) the time-course of this effect. MATERIALS AND METHODS Animal Exposure Specific pathogen-free male Jcl:Wistar rats, 13 weeks of age, Exp. 1: The rats were exposed to 0.2, 0.4, were used (6 rats/group). 0.8, 1.2 and 1.8 ppm ozone for 24h. Exp. 2: The rats were exposed to 0.4 or 0.8 ppm ozone for 1, 3, 5, 7, 14 or 28 days. Rats exposed to filtered air for 1, 7 or 28 days were controls for rats exposed to ozone for days 1 and 3; 5, 7 and 14; and 28 respectively. The conditions in the chamber (volume, 1.39 m3) were: temperature, 25kl"C; humidity, 55 *lo%; pressure, -5 mm H 0 relative to atmospheric pressure; total air flow rate, 110-120 m 5/h. The concentrations of ozone
DECEMBER
1983 VOL. 26 NO. 6
1021
PROSTAGLANDINS
were automatically controlled by continuous monitoring with an ozone anaiyzer (Kimoto 806s) using a chemiluminescence method. Assay for the PGI2-synthesizing activity of lung homogenates Immediately after the termination of the exposure, the rats were killed by exsanguination under ether anaesthesia, and lungs were excised. The assay procedure has been described previously [3]. Briefly, a weighed piece of each lung (approx. 600 mg) was homogenized with 2.5 ml cold phosphate buffer (pH 8.2 67 mM) by a polytron homogenizer. The homogenate (1 ml) was incubated with 0.2 1-1 Ci [1-'4C]arachidonic acid (56.4 Ci/mol) and 5 i~-g unlabelled arachidonic acid at 37°C for 15 min. The reaction was terminated by adding 1N HCl to bring the pH of the reaction mixture to 3.0. The reaction mixtures were separated by TLC using the developing solvent system, ethyl acetate / 2,2,4_trimethylpentane / acetic acid / water (11:5:2:10. v/v). The radioactive products were analyzed by thin layer radiochromatography. Protein was determined by the method of Lowry et al [7] with bovine serum albumin as standard. The data are expressed as mean 2 SD and were statistically analyzed using Student's t test with ~~0.05 as the level of significance. RESULTS Fig. 1 shows the relationship between ozone concentration and a ratio of lung wet weight: body weight. 1.2 and 1.8 ppm ozone exposure increased the ratio significantly. Fig. 2 shows the PGI2-synthesizing activity of lung tissue from rats exposed to 0.2, 0.4, 0.8, 1.2 and 1.8 ppm ozone for 24h. In the range of 0.2sO.8 ppm ozone exposure, the PG12 synthesizing activity tended to increase (p= 0.054 at 0.4 ppm). Then the PG12 synthesizing activity decreased. Fig. 3 shows the change in the body weight during 0.4 ppm and 0.8 ppm ozone exposure. The body weight of the rats exposed to 0.8 ppm ozone for 1, 7 or 28 days was less than controls, and decreased dramatically at days 28. With 0.4 ppm ozone, weights were significantly less than controls only at day 1. Fig. 4 shows that the change in the lung wet weight during 0.4 ppm and 0.8 ppm ozone exposure. The lung wet weight increased significantly after 3, 5, 7, 14 or 28 days exposure to 0.8 ppm ozone. With 0.4 ppm ozone, significant increases were observed at days 3 and 28. Fig. 5 shows the relationship between the PGI2-synthesizing activity of homogenized lung and the time of ozone exposure. The PG12 synthesizing activity decreased maximally at about 5 days and then decreased. After 14 days, PG12 synthesizing activity was not significantly different from normal, even though the ozone exposure continued. At 2d days, the level was normal.
1022
DECEMBER
1983 VOL. 26 NO. 6
PROSTAGLANDINS
--
5 A
6
11
IIll
C 0.2 0.4
0.8
1”
1.2
Ozone Concentration
”
1.8
pm
Figure 1 Relationship between 03 concentration and the ratio of lung wet weight: body weight. (Mean+SD, * and ***: ~~0.05 and p
C 0.2 0.4
0.8
1.2
03 Concentration
1.8 ppm
Figure 2 Relationship between 03 concentration and PGI2-synthes izing activity (MeankSD, ** p
DECEMBER
1983 VOL. 26 NO. 6
1023
PROSTAGLANDINS
3oso [
/;,;:; *
250
*** **
9 c.
1.
I I1 1 .I 11. 1 I I 3 5 7 14 Time of 03 exposure
01
,,-
*** 28days
Figure 3 Change in body weight during exposure to 03 (0.8 ppm v and 0.4 ppm 0 ) and filtered air (Ocontrol). Statistical analysis was done at day 1, 7 and 28. (Mean?SD, *, ** and ***: ~~0.05, p< 0.01 and p
9
013 I1
I
I
,I
5
.I
7
1
I,
.
.
.
14 .
Time of 03 exposure
M 28days
Figure 4 Change in lung wet weight during exposure to 03 (0.8 ppm v and 0.4 ppm 0 ) and filtered air (Ocontrol). (Mean+SD, ** and ***: ~~0.01 and p
1024
DECEMBER
1983 VOL. 26 NO. 6
PROSTAGLANDINS
Time of 03 exposure
Fi ure 5
Change in PGI -synthesizing activity of lung by exposure ppmr and 0. Z ppm 0 ) and filtered air (Ocontrol). (Mean* SD, *, ** and ***: p< 0.05, p< 0.01 and p< 0.001 respectively)
kkJT.8
DISCUSSION PG12 synthetase is inhibited by lipid hydroperoxides such as 15-hydroperoxyeicosatetraenoic acid [4]. Since ozone is a strong oxidizing agent, it can readily react with components of lung surface such as unsaturated fatty acids to form peroxygenated intermediates. Roycroft et al. reported a rapid oxidation of arachidonic acid to oxonides, peroxides, and other products by in vitro exposure to ozone [8]. The peroxygenated intermediates may cause the depression of PGI2-synthesizing activity. In the range of 0.2~~0.8 ppm ozone exposure, however, the PGI2synthesizing activity may have been increased (Fig. 2). Lands et al reported that a slight amount of lipid hydroperoxides enhance the cyclooxygenase activity [9]. Several enzymes that supply the reducing equivalents in the form of NADPH are induced during exposure to 0.8 ppm ozone [lo]. The activities of glucose-6-phosphate dehydrogenase (G6PD) and
DECEMBER
1983 VOL. 26 NO. 6
1025
PROSTAGLANDINS
6-phosphogluconate dehydrogenase (GPGD) play important roles of the hexose monophosphate shunt for glucose metabolism, and furnish NADPH. A significant increase in the activities of these enzymes in lung cytosol was observed after exposure to ozone, peaking between 3 and 4 days [lo]. Reduced glutathione is considered to protect against peroxidative damage, by reducing peroxides. Glutathione levels in lung tissue increased and reached a maximum at about 3 days with exposure to 0.8 ppm ozone [lo]. Glutathione peroxidase and superoxide dismutase are also induced by 0.8 ppm ozone exposure [lo]. Therefore increased formation of these enzymes may explain the recovery of the PGI2-synthesizing activity. A continuous formation and a release of PG12 by lung may play a role in preventing platelet aggregation [ll]. Depression of PGI2synthesizing activity of lung by NO2 exposure correlates with a decrease in the number of platelets during NO2 exposure [5]. Therefore further studies on the relationship between the decrease in PG12 synthesis by lung and pathological and physiological changes by ozone exposure may be rewarding. ACKNOWLEDGEMENT I wish to express my gratitude to Dr. K. Kubota and Dr. M. Sagai (The National Institute for Environmental Studies) for encouragement. REFERENCES
1) 2) 3)
4)
5) 6)
7) 8)
1026
Chow, C. K. and A. P. Tappel, An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone-exposed rats, Lipids, 7: 518, 1972. Pace-Asiak, C. R. and B. Rangaraj, Distribution of prostagladin biosynthetic pathways in several rat tissues. Formation of 6keto-prostagladin Flo, Biochim, Biophys. Acta, 486: 579, 1977. Kobayashi, T., I. Morita and S. Murota, Effect of acute nitrogen dioxide exposure on the prostacyclin synthesis in lung, Toxicol. Lett., 9: 373, 1981. Moncada, S. and J. R. Vane, Unstable metabolites of arachidonic acid and their role in homeostasis and thrombosis, Br. Med. Bull., 34: 129, 1978. Kobayashi, T., I. Morita and S. Murota, Effects of nitrogen dioxide exposure of prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats, Prostaglandins,25: 303,1983. Kobayashi, T., The reaction of nitrogen dioxide with lung surface components: The reaction with cis-9-octadecenoic acid, Chemosphere, 12:1317, 1983. Lowry, 0. H., N. H. Rosenbrough, A. L. Farr and K. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193: 265, 1951. Roycroft, H. H., W. B. Gunter and D. B. Menzel, Ozone toxicity: Hormone - like oxidation products from arachidonic acid by ozone catalyzed autooxidation, Toxicol. Lett., 1: 75, 1977.
DECEMBER
1983 VOL. 26 NO. 6
PROSTAGLANDINS
9)
10)
11)
Lands, W. E. M. and M. J. Byrnes, The influence of ambient peroxides on the conversion of 5,8,11,14,17-eicosapentaenoic acid to prostaglandins, Prog. Lipid Res., 20: 287, 1981. Lee, S. D., Eds. Biochemical effects of environmental pollutants, 1974), (Ann Arbor, Michigan: Ann Arbor Science Ptb lishers, Inc., p.74-81. German, R. R., S. Bunting and 0. V. Miller, Modulation of human platelet adenylate cyclase by prostacyclin (PGX), Prostaglandins, 13: 377, 1977. Editor: Alan Bennett
DECEMBER
Received: 9-20-83
1983 VOL. 26 NO. 6
Rcceoted: 11-7-83
1027