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Development of the chick embryo following PGE1 treatment
Prostaglandins and Medicine 6: 621-626, 1981
DEVELOPMENT OF THE CHICK EMBRYO FOLLOWING PGEl TREATMENT 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 OW3 (reprint request to TVNP). ABSTRACT Chick embryos were treated with prostaglandin El (10, 20, 30, and 50 ug) at 48 and 72 hours incubation. Live embryos were recovered on day 9, staged, and examined for external malformations after weighing. There was no significant increase in embryonic mortality and malformation rates compared to the controls. Embryonic growth was also not affected. INTRODUCTION Although the prostaglandins are widely used for the termination of pregnancy at all stages of gestation, few studies have been undertaken in order to evaluate possible teratogenic side effects (l-2). The chick embryo is considered a highly sensitive system for the screening of drugs for possible teratogenicity. 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 maternal placenta are conveniently avoided (3-4). This report describes the effects of prostaglandin El on the early development of the chick embryo. MATERIAL AND METHODS Fertile White Leghorn Chick eggs (Shamrock Farms, New Brunswick, N.J.) were used in the study. The eggs were incubated in a forced-draft incubator at a temperature of 38'C. PGEl was dissolved in 10% ethyl alcohol and injected into the air sac at dose levels of 10, 20, 30, and 50 ug per egg. All eggs received a single dose of PGEl administered at either 48 or 72 hours of incubation. The total volume of injected material was 0.1 ml per egg. Control eggs were injected with
621
The an equivalent volume of 10% ethyl alcohol only (0.1 ml/egg). total number of eggs used in this study was 188, 94 each at 48 and at 72 hours incubation, respectively. On day 9 the live embryos were removed from the eggs, and these were weighed and examined for the All live embryos were fixed in 10% presence of gross malformations. neutral formalin. Statistical analysis of the data was carried out using the chi-square test with Yates' correction for continuity. RESULTS AND DISCUSSION Embryonic mortality and the incidence of malformations following treatment at 48 hours incubation was not significantly increased at any of the dose levels, compared to the solvent-treated controls (Table 1). The mean embryonic weight following treatment at the different dose levels was less than that of the controls, but the differences were not statistically significant (Table 3). At 72 hours incubation, there was a significant increase in embryonic mortality following treatment with the prostaglandin at doses of 10 and 50 ug, respectively (Table 2). In the solvent-treated control group, all the embryos recovered appeared morphologically normal, in contrast to the control group at 48 hours incubation, where 13 per cent of the surviving embryos were abnormal. The incidence of malformations was higher than in the control groups at both incubation periods, but not significantly different from those treated at 48 hours incubation. In addition, embryonicgrotith was not affected; the weight of the embryos following prostaglandin treatment at all dose levels did not differ significantly from that of the control groups (Table 3). Despite the higher incidence of malformations observed in embryos following prostaglandin El treatment at both periods of incubation, there is little evidence of an embryopathic effect because of the malformation rate in the control group at 48 hours incubation. Ethyl alcohol is known to have a deleterious effect on the development of the chick embryo (5) which probably accounts for the comparable levels of malformations in embryos of both the prostaglandin and control groups. We have shown in previous studies that prostaglandin F2c was not teratogenic in the chick when administered at doses of 10, 40 and 100 ug at 48 and 72 hours incubation (6). On the other hand, prostaglandin E2 induced a high incidence of malformations in embryos treated with 100 ug at 48 hours incubation (7). In both studies, there was no evidence of growth retardation. Prostaglandin El, not unlike PGE2, is rapidly inactivated during its passage through the circulatory system (8). Because more than 90% of the prostaglandin disappears from the circulatory system during one passage, the chick embryo was considered particularly suited for the teratological screening of this prostaglandin.
622
87
13
Percent survival
Abnormal embryos (%)
mations:
hemorrhage reduced body abnormal beak
26
Live embryos on day 9
Types of malfor-
30
Number of eggs treated
20
93
14
15
10
everted viscera reduced body hemorrhage
Solvent-treated controls (10% ethyl alcohol)
exencephaly everted viscera
7
80
12
15
20
Dosage
Effect of PGEl on developing chick embryos at 48 hours incubation
TABLE 1
35
76
13
17
30
everted viscera reduced body
(Ug)
reduced tail everted viscera hemorrhage reduced body
29
82
14
17
50
Types of malformations:
0
Abnormal embryos
everted viscera hemorrhage
33
73(p
93
Percent survival (%)
11
28
Live embryos on day 9
15
30
Number of eggs treated
10
abnormal beak reduced tail & body everted viscera hemorrhage
0
27
short neck reduced body everted viscera
19
75(p
88
16
50
12
17
30
14
tug)
14
16
20
Dosage
chick embryos at 72 hours incubation.
Solvent-treated controls (10% ethyl alcohol)
Effect of PGEl on developing
TABLE 2
TABLE 3 Effect
of
PGE~ on embryonic weight in the chick (day 9)
48 hrs. incubation (Mean ? S.E.)
72 hrs. incubation (Mean + S.E.
Solvent-treated controls (10% ethyl alcohol)
1.34 + 0.10
1.2
+ 0.07
10 &I
1.19
0.01
1.2
+ 0.04
20 )lq
1.12 + 0.04
1.13 t 0.01
30 uq
1.14 f 0.03
1.15 2 0.02
50 uq
1.08 + 0.02
1.13 ? 0.06
+
Compared to prostaqlandins E2 and Fz~, PGEl was found to be most potent in inhibiting implantation when tested in rats and hamsters (9). Teratological studies with PGEl in laboratory rodents have not yet been reported. The problem of rapid metabolism during a single passage through the circulation would prevent the use of laboratory rodents and other species for such studies. In conclusion, the present results indicate that prostaglandin El is not teratoqenic in the chick. Any attempt to discuss our limited findings further, particularly in terms of human teratogens, would only be speculative. 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. REFERENCES 1.
Persaud TVN. Reproductive and teratological studies with prostaglandins. p. 161 in Advances in the Study of Birth Defects. Vol. 2, Teratological Testing (TVN Persaud ed) University Park Press, Baltimore, 1979.
2.
Persaud TVN. Embryonic and fetal development. p. 175 in Prostaglandins. Vol. 2 (PW Ramwell ed) Plenum Press, New York, 1974.
3.
Persaud TVN. Teratogenesis. Gustav Fischer Verlag, Jena, 1979.
625
4.
Gebhardt DOE. The use of the chick embryo in applied teratology. p. 97 in Advances in Teratology. Vol.5 (DHM Woollam ed) Academic Press, New York, 1972.
5.
Sandor S. The influence of aethyl alcohol on the developing embryo. Rev Roum Embryo1 Cytol Ser 5: 167, 1968.
6.
Matthews GBP, Persaud TVN. Non-teratogenicity of prostaglandin F2c in the chick. Res Comm Chem Path01 Pharmacol 13: 141, 1976.
7.
Persaud TVN, Mann RA, Moore KL. Teratological studies with prostaglandin E2 in chick embryos. Prostaglandins 4: 343, 1973.
8.
Ferreira SH, Vane JR. Prostaglandins: and release into the circulation.
9.
Labhsetwar AP. Prostaglandins and studies related to reproduction in laboratory animals. p. 241 in Prostaglandins and Reproduction (SMM Karim ed) University Park Press, Baltimore, 1975.
626
chick
their disappearance from Nature 216: 868, 1967.
DEVELOPMENT OF THE CHICK EMBRYO FOLLOWING PGEl TREATMENT 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 OW3 (reprint request to TVNP). ABSTRACT Chick embryos were treated with prostaglandin El (10, 20, 30, and 50 ug) at 48 and 72 hours incubation. Live embryos were recovered on day 9, staged, and examined for external malformations after weighing. There was no significant increase in embryonic mortality and malformation rates compared to the controls. Embryonic growth was also not affected. INTRODUCTION Although the prostaglandins are widely used for the termination of pregnancy at all stages of gestation, few studies have been undertaken in order to evaluate possible teratogenic side effects (l-2). The chick embryo is considered a highly sensitive system for the screening of drugs for possible teratogenicity. 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 maternal placenta are conveniently avoided (3-4). This report describes the effects of prostaglandin El on the early development of the chick embryo. MATERIAL AND METHODS Fertile White Leghorn Chick eggs (Shamrock Farms, New Brunswick, N.J.) were used in the study. The eggs were incubated in a forced-draft incubator at a temperature of 38'C. PGEl was dissolved in 10% ethyl alcohol and injected into the air sac at dose levels of 10, 20, 30, and 50 ug per egg. All eggs received a single dose of PGEl administered at either 48 or 72 hours of incubation. The total volume of injected material was 0.1 ml per egg. Control eggs were injected with
621
The an equivalent volume of 10% ethyl alcohol only (0.1 ml/egg). total number of eggs used in this study was 188, 94 each at 48 and at 72 hours incubation, respectively. On day 9 the live embryos were removed from the eggs, and these were weighed and examined for the All live embryos were fixed in 10% presence of gross malformations. neutral formalin. Statistical analysis of the data was carried out using the chi-square test with Yates' correction for continuity. RESULTS AND DISCUSSION Embryonic mortality and the incidence of malformations following treatment at 48 hours incubation was not significantly increased at any of the dose levels, compared to the solvent-treated controls (Table 1). The mean embryonic weight following treatment at the different dose levels was less than that of the controls, but the differences were not statistically significant (Table 3). At 72 hours incubation, there was a significant increase in embryonic mortality following treatment with the prostaglandin at doses of 10 and 50 ug, respectively (Table 2). In the solvent-treated control group, all the embryos recovered appeared morphologically normal, in contrast to the control group at 48 hours incubation, where 13 per cent of the surviving embryos were abnormal. The incidence of malformations was higher than in the control groups at both incubation periods, but not significantly different from those treated at 48 hours incubation. In addition, embryonicgrotith was not affected; the weight of the embryos following prostaglandin treatment at all dose levels did not differ significantly from that of the control groups (Table 3). Despite the higher incidence of malformations observed in embryos following prostaglandin El treatment at both periods of incubation, there is little evidence of an embryopathic effect because of the malformation rate in the control group at 48 hours incubation. Ethyl alcohol is known to have a deleterious effect on the development of the chick embryo (5) which probably accounts for the comparable levels of malformations in embryos of both the prostaglandin and control groups. We have shown in previous studies that prostaglandin F2c was not teratogenic in the chick when administered at doses of 10, 40 and 100 ug at 48 and 72 hours incubation (6). On the other hand, prostaglandin E2 induced a high incidence of malformations in embryos treated with 100 ug at 48 hours incubation (7). In both studies, there was no evidence of growth retardation. Prostaglandin El, not unlike PGE2, is rapidly inactivated during its passage through the circulatory system (8). Because more than 90% of the prostaglandin disappears from the circulatory system during one passage, the chick embryo was considered particularly suited for the teratological screening of this prostaglandin.
622
87
13
Percent survival
Abnormal embryos (%)
mations:
hemorrhage reduced body abnormal beak
26
Live embryos on day 9
Types of malfor-
30
Number of eggs treated
20
93
14
15
10
everted viscera reduced body hemorrhage
Solvent-treated controls (10% ethyl alcohol)
exencephaly everted viscera
7
80
12
15
20
Dosage
Effect of PGEl on developing chick embryos at 48 hours incubation
TABLE 1
35
76
13
17
30
everted viscera reduced body
(Ug)
reduced tail everted viscera hemorrhage reduced body
29
82
14
17
50
Types of malformations:
0
Abnormal embryos
everted viscera hemorrhage
33
73(p
93
Percent survival (%)
11
28
Live embryos on day 9
15
30
Number of eggs treated
10
abnormal beak reduced tail & body everted viscera hemorrhage
0
27
short neck reduced body everted viscera
19
75(p
88
16
50
12
17
30
14
tug)
14
16
20
Dosage
chick embryos at 72 hours incubation.
Solvent-treated controls (10% ethyl alcohol)
Effect of PGEl on developing
TABLE 2
TABLE 3 Effect
of
PGE~ on embryonic weight in the chick (day 9)
48 hrs. incubation (Mean ? S.E.)
72 hrs. incubation (Mean + S.E.
Solvent-treated controls (10% ethyl alcohol)
1.34 + 0.10
1.2
+ 0.07
10 &I
1.19
0.01
1.2
+ 0.04
20 )lq
1.12 + 0.04
1.13 t 0.01
30 uq
1.14 f 0.03
1.15 2 0.02
50 uq
1.08 + 0.02
1.13 ? 0.06
+
Compared to prostaqlandins E2 and Fz~, PGEl was found to be most potent in inhibiting implantation when tested in rats and hamsters (9). Teratological studies with PGEl in laboratory rodents have not yet been reported. The problem of rapid metabolism during a single passage through the circulation would prevent the use of laboratory rodents and other species for such studies. In conclusion, the present results indicate that prostaglandin El is not teratoqenic in the chick. Any attempt to discuss our limited findings further, particularly in terms of human teratogens, would only be speculative. 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. REFERENCES 1.
Persaud TVN. Reproductive and teratological studies with prostaglandins. p. 161 in Advances in the Study of Birth Defects. Vol. 2, Teratological Testing (TVN Persaud ed) University Park Press, Baltimore, 1979.
2.
Persaud TVN. Embryonic and fetal development. p. 175 in Prostaglandins. Vol. 2 (PW Ramwell ed) Plenum Press, New York, 1974.
3.
Persaud TVN. Teratogenesis. Gustav Fischer Verlag, Jena, 1979.
625
4.
Gebhardt DOE. The use of the chick embryo in applied teratology. p. 97 in Advances in Teratology. Vol.5 (DHM Woollam ed) Academic Press, New York, 1972.
5.
Sandor S. The influence of aethyl alcohol on the developing embryo. Rev Roum Embryo1 Cytol Ser 5: 167, 1968.
6.
Matthews GBP, Persaud TVN. Non-teratogenicity of prostaglandin F2c in the chick. Res Comm Chem Path01 Pharmacol 13: 141, 1976.
7.
Persaud TVN, Mann RA, Moore KL. Teratological studies with prostaglandin E2 in chick embryos. Prostaglandins 4: 343, 1973.
8.
Ferreira SH, Vane JR. Prostaglandins: and release into the circulation.
9.
Labhsetwar AP. Prostaglandins and studies related to reproduction in laboratory animals. p. 241 in Prostaglandins and Reproduction (SMM Karim ed) University Park Press, Baltimore, 1975.
626
chick
their disappearance from Nature 216: 868, 1967.