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Embryopathic effects of thromboxane B2 in the chick
Prostaglandins and Medicine 6: 627-631, 1981
EMBRYOPATHIC EFFECTS OF THROMBOXANE B2 IN THE CHICK S.H. Gilanil and T.V.N. Persaud2. 'Department of Anatomy, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, N.J. 07103, U.S.A. 'Department of Anatomy, University of Manitoba, 730 William Avenue, Winnipeg, Canada, R3E 0W3 (reprint request to TVNP). ABSTRACT Several prostaglandins of the E and F series are now known to be involved in reproduction and developmental events. However, there is as yet no such evidence for any of the other arachidonate metabolites. Thromboxane B2 is the stable metabolic end product of the biologically active but unstable intermediate thromboxane A. Administered to developing chick embryos at 48 and 72 hours incubation, thromboxane B2 caused growth retardation and induced a high incidence of anomalies, in particular,everted viscera. INTRODUCTION Thromboxane A2 is the unstable intermediate formed by the cyclooxygenase enzyme during the conversion of arachidonic acid and cyclic endoperoxides to thromboxane B2. It is biologically active, causing platelet aggregation and the constriction of arteries, in contrast to the stable metabolite thromboxane B2 which is considered to be devoid of biological activities. Thromboxane A2 is synthesized in a wide variety of tissues (l), including the uterus and placenta of the rat and human. In addition, thromboxane B2 was found in the microsomal fraction of rat decidual tissue (2). The significance of these substances in reproductive physiology is not known. In order to determine whether thromboxane may influence embyronic development, early chick embryos were treated with thromboxane B2. Similar studies with thromboxane A2 were not feasible because of the very short biological half-life (32 seconds) of this compound. MATERIALS AND METHODS Fertile white leghorn chick eggs (Shamrock Farms, New Brunswick, N.J.) were used in these studies. Eggs were incubated in a forced-draft incubator at a temperature of 38'C. Thromboxane B2 was dissolved in
627
physiological saline and injected into the air-sac at dose levels of 10, 30, and 50 pg per egg. All eggs received a single dose of thromboxane B2 administered either at 48 or 72 hours of incubation. The total volume of injected material was 0.1 ml per egg. Control eggs were injected with an equivalent volume of physiological saline only (0.1 ml/ In all, 169 eggs were used in this investigation. On day 9, the egg). live embryos were removed from the eggs. These were staged, weighed and subsequently examined for the presence of gross malformations. The embryos were fixed in 10% neutral formalin. 84 eggs were treated at 48 hours and 85 at 72 hours, respectively. Statistical analysis of the data was carried out using the chi-square test with Yates' correction for continuity. RESULTS AND DISCUSSION Following treatment of the chick embryos with thromboxane B2 at 48 hours incubation an increasing level of mortality was observed, but the difference from the control group was significant only at the highest dose level. The incidence of abnormal embryos was dose related and significantly increased at all dose levels. Everted viscera and a reduced body form were observed at the lowest dose level; at the 30 and 50 ug dose levels, the only malformation was everted viscera (Table 1). At 72 hours incubation, all of the control embryos survived and were normal. In contrast, the survival rate of treated embryos varied with the dose levels and in all groups was significantly different from that of the controls. The incidence of malformations increased with the dose of the substance. The most frequent anomaly was everted viscera which was induced at all dose levels (Table 2). At both periods of incubation, thromboxane B2 treatment caused an overall growth retardation. The mean weight of embryos exposed to thromboxane B2 was significantly less than that of the corresponding control groups (Table 3). The chick embryo is a highly sensitive system for the screening of drugs for possible embryopathic effects. The technique is inexpensive, simple to perform, and exposes the rapidly developing embryo to direct contact with the test substance. Furthermore, the influences of the mother and placenta are conveniently avoided (3-4). In particular, the use of this experimental model was ideally suited for studying the effects of thromboxane B2 on embryonic development because of the limited availability of the substance. The results of the present investigation revealed an adverse effect on the development of the chick embryo which must be attributed to the substance itself. How these substances might be related to normal embryogenetic processes cannot be determined from the results that are available.
628
95
Percent survival
Types of malformations:
5
18
Live embryos on day 9
Abnormal embryos ($1
19
Number of-eggs treated
Solvent-treated controls (physiological saline)
everted viscera
everted viscera reduced body
everted viscera
IO
31 21
16
20
50
8O(p
19
22
Dosage (wg) 30
86
96
22
23
10
Effect of thromboxane B2 on developing chick embryos at 48 hours incubation.
TABLE 1
embryos
C%)
Types of malformations:
Abnormal 0
100
20
Live embryos on day 9
Percent survival
20
Number of eggs treated
twisted neck reduced body hemorrhage microphthalmia everted viscera twisted limbs abnormal beak
18
82(p
18
22
10
39
7O(p
16
23
(us) 30
hemorrhage reduced body microphthalmia everted viscera
Dosage
chick embryos at 72 hours incubation.
Solvent-treated controls (physiological saline)
Effect of thromboxane B2 on developing
TABLE 2
everted viscera short & twisted neck exencephaly reduced body
45
65(p
13
20
50
TABLE 3 Effect of thromboxane B2 on embryonic weight in the chick (day 9).
48 hrs. incubation (Mean f S.E.)
72 hrs. incubation (Mean 2 S.E.)
Solvent-treated controls (physiological-saline)
1.31 * 0.07
1.36 f 0.022
10 ug
1.19 t 0.02
1.20 + 0.0047
30 !q
1.17 f 0.05
1.19 f 0.0439
50 ug
1.15 2 0.04
1.17 f 0.0186
ACKNOWLEDGEMENTS We are extremely grateful to Mr. Joseph J. Giovinazzo for his excellent technical assistance. This investigation was partly supported by an institutional grant from the College of Medicine and Dentistry of New Jersey, New Jersey Medical School to SHG. RFFEXENCES 1.
Moncada S and Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2 and prostacyclin. Pharmacol Rev 30: 293, 1979.
2.
Williams KI and Downing I. Prostaglandin and thromboxane production by rat decidual microsomes. Prostaglandins 14: 813, 1977.
3.
Persaud TVN.
4.
Gebhardt DOE. The use of the chick embryo in applied teratology. p. 97 in Advances in Teratology. Vol. 5 (DRM Woollam ed) Academic Press, New York, 1972.
Teratogenesis. Gustav Fischer Verlag, Jena, 1979.
631
EMBRYOPATHIC EFFECTS OF THROMBOXANE B2 IN THE CHICK S.H. Gilanil and T.V.N. Persaud2. 'Department of Anatomy, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, N.J. 07103, U.S.A. 'Department of Anatomy, University of Manitoba, 730 William Avenue, Winnipeg, Canada, R3E 0W3 (reprint request to TVNP). ABSTRACT Several prostaglandins of the E and F series are now known to be involved in reproduction and developmental events. However, there is as yet no such evidence for any of the other arachidonate metabolites. Thromboxane B2 is the stable metabolic end product of the biologically active but unstable intermediate thromboxane A. Administered to developing chick embryos at 48 and 72 hours incubation, thromboxane B2 caused growth retardation and induced a high incidence of anomalies, in particular,everted viscera. INTRODUCTION Thromboxane A2 is the unstable intermediate formed by the cyclooxygenase enzyme during the conversion of arachidonic acid and cyclic endoperoxides to thromboxane B2. It is biologically active, causing platelet aggregation and the constriction of arteries, in contrast to the stable metabolite thromboxane B2 which is considered to be devoid of biological activities. Thromboxane A2 is synthesized in a wide variety of tissues (l), including the uterus and placenta of the rat and human. In addition, thromboxane B2 was found in the microsomal fraction of rat decidual tissue (2). The significance of these substances in reproductive physiology is not known. In order to determine whether thromboxane may influence embyronic development, early chick embryos were treated with thromboxane B2. Similar studies with thromboxane A2 were not feasible because of the very short biological half-life (32 seconds) of this compound. MATERIALS AND METHODS Fertile white leghorn chick eggs (Shamrock Farms, New Brunswick, N.J.) were used in these studies. Eggs were incubated in a forced-draft incubator at a temperature of 38'C. Thromboxane B2 was dissolved in
627
physiological saline and injected into the air-sac at dose levels of 10, 30, and 50 pg per egg. All eggs received a single dose of thromboxane B2 administered either at 48 or 72 hours of incubation. The total volume of injected material was 0.1 ml per egg. Control eggs were injected with an equivalent volume of physiological saline only (0.1 ml/ In all, 169 eggs were used in this investigation. On day 9, the egg). live embryos were removed from the eggs. These were staged, weighed and subsequently examined for the presence of gross malformations. The embryos were fixed in 10% neutral formalin. 84 eggs were treated at 48 hours and 85 at 72 hours, respectively. Statistical analysis of the data was carried out using the chi-square test with Yates' correction for continuity. RESULTS AND DISCUSSION Following treatment of the chick embryos with thromboxane B2 at 48 hours incubation an increasing level of mortality was observed, but the difference from the control group was significant only at the highest dose level. The incidence of abnormal embryos was dose related and significantly increased at all dose levels. Everted viscera and a reduced body form were observed at the lowest dose level; at the 30 and 50 ug dose levels, the only malformation was everted viscera (Table 1). At 72 hours incubation, all of the control embryos survived and were normal. In contrast, the survival rate of treated embryos varied with the dose levels and in all groups was significantly different from that of the controls. The incidence of malformations increased with the dose of the substance. The most frequent anomaly was everted viscera which was induced at all dose levels (Table 2). At both periods of incubation, thromboxane B2 treatment caused an overall growth retardation. The mean weight of embryos exposed to thromboxane B2 was significantly less than that of the corresponding control groups (Table 3). The chick embryo is a highly sensitive system for the screening of drugs for possible embryopathic effects. The technique is inexpensive, simple to perform, and exposes the rapidly developing embryo to direct contact with the test substance. Furthermore, the influences of the mother and placenta are conveniently avoided (3-4). In particular, the use of this experimental model was ideally suited for studying the effects of thromboxane B2 on embryonic development because of the limited availability of the substance. The results of the present investigation revealed an adverse effect on the development of the chick embryo which must be attributed to the substance itself. How these substances might be related to normal embryogenetic processes cannot be determined from the results that are available.
628
95
Percent survival
Types of malformations:
5
18
Live embryos on day 9
Abnormal embryos ($1
19
Number of-eggs treated
Solvent-treated controls (physiological saline)
everted viscera
everted viscera reduced body
everted viscera
IO
31 21
16
20
50
8O(p
19
22
Dosage (wg) 30
86
96
22
23
10
Effect of thromboxane B2 on developing chick embryos at 48 hours incubation.
TABLE 1
embryos
C%)
Types of malformations:
Abnormal 0
100
20
Live embryos on day 9
Percent survival
20
Number of eggs treated
twisted neck reduced body hemorrhage microphthalmia everted viscera twisted limbs abnormal beak
18
82(p
18
22
10
39
7O(p
16
23
(us) 30
hemorrhage reduced body microphthalmia everted viscera
Dosage
chick embryos at 72 hours incubation.
Solvent-treated controls (physiological saline)
Effect of thromboxane B2 on developing
TABLE 2
everted viscera short & twisted neck exencephaly reduced body
45
65(p
13
20
50
TABLE 3 Effect of thromboxane B2 on embryonic weight in the chick (day 9).
48 hrs. incubation (Mean f S.E.)
72 hrs. incubation (Mean 2 S.E.)
Solvent-treated controls (physiological-saline)
1.31 * 0.07
1.36 f 0.022
10 ug
1.19 t 0.02
1.20 + 0.0047
30 !q
1.17 f 0.05
1.19 f 0.0439
50 ug
1.15 2 0.04
1.17 f 0.0186
ACKNOWLEDGEMENTS We are extremely grateful to Mr. Joseph J. Giovinazzo for his excellent technical assistance. This investigation was partly supported by an institutional grant from the College of Medicine and Dentistry of New Jersey, New Jersey Medical School to SHG. RFFEXENCES 1.
Moncada S and Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2 and prostacyclin. Pharmacol Rev 30: 293, 1979.
2.
Williams KI and Downing I. Prostaglandin and thromboxane production by rat decidual microsomes. Prostaglandins 14: 813, 1977.
3.
Persaud TVN.
4.
Gebhardt DOE. The use of the chick embryo in applied teratology. p. 97 in Advances in Teratology. Vol. 5 (DRM Woollam ed) Academic Press, New York, 1972.
Teratogenesis. Gustav Fischer Verlag, Jena, 1979.
631