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Ectopia cordis (ectocardia) and gastroschisis induced in rats by maternal administration of the lathyrogen, beta-aminopropionitrile (BAPN)
Ectopia cordis (ectocardia) and gastroschisis in rats by maternal administration of the lathyrogen, beta-aminopropionitrite (BAPN) Mark V. Barrow, M.D., Linda S. Willis Gainesville, Flu.
L
induced
Ph.D.
athyrism includes two disorders.’ The first, neurolathyrism, occurs in man or other animals that ingest the seed of plants of certain lathyrus species, including Lathyrus sativus, L. &era, and L. clymenum. A spastic paraplegia develops and pathological changes have been noted in the spinal cord. At least three neurolathyrogens have been isolated from these plants thus far.’ The other type of lathyrism has been called osteolathyrism. This disorder has not been described in man but has been well described in young or growing rats, and several other species fed L. odoratus (flowering sweetpea) seed as part of the diet. Osteolathyritic effects include kyphoscoliosis of the spine, curvature of the long bones, exostoses of bone at muscle insertion sites, slipped epiphyses and aortic aneurysms (this last abnormality called by some, angiolathyrism, to distinguish this effect from the bone changes). The toxic principle of L. odoratus has been shown to be beta-aminopropionitrile (BAPN) with the formula HZN CHzCHz C=N. This substance interferes with the formation of intermolecular and intramolecular crosslinks in the formation of collagen and e1astin.r Since bone and aortic tissues con-
tain large quantities of one or both of these substances the osteolathyritic effects, including the angiolathyritic ones, have been explained on the basis of abnormalities in these connective tissues. These notions have been substantiated in a variety of ways-for a detailed review of these concepts the reader is referred to our article.’ Several other agents, including aminoacetonitrile, several hydrazines, hydrazides and ureides, have the ability to produce osteolathyrism in young and growing animals. These same agents have also been shown to be teratogenic.rT2 In the chick and turkey embryo osteolathyrogens produce typical bony effects and aortic aneurysms.3-6 Mammals (rats have been the common experimental animal) fed during late gestation also develop kyphoscoliosis and aortic aneurysms, ‘-I3 abnormalities which are similar to the effects seen when the agent is fed to young animals. Earlier treatment, however, produces different effects, these being somewhat unique and interesting. These results have been recently summarized and discussed in experimentation with pregnant rats using BAPN (Table I) .rs2 When given prior to
From the Division of Cardiology, Department of Medicine, University of Florida, Gainesville, Fla. This work was supported by Florida Heart Grant No. 70AG8. Received for publication June 28. 1971. Reprint requests to: Dr. Mark V. Barrow. Div. of Cardiology. Department of Medicine, University of Florida, ville. Ma. 32601.
518
American
Heart
Journal
April, 1972
Vol.
Gaines-
83, No. 4, pp. 518-526
Volume Number
83 4
BAPN
Table I. Summary of teratogenic efects of beta-aminopropionitrile (from Barrow and associates2)
2* day(s)
Ectocardia
Gastroschisis
-
up to 14 14 and 15 16 After 16
+ -
+ -
Table II. Beta-aminopropionitrile
Dose (wlk’g.
1
(BAPN)
Controll 12 13
5,000 5,000
111 5 6
79 43 3.5
13-14 13-14 13-14 13-15 14 14 14 14-15 14-15 14-1.5 14-15 15
3,000 3,500 4,000 2,500 4,000 5,000 5,500 2,500 3,000 3,500 4,000 2,500
8 5 4 12 5 9 7 30 11 12 9 13
42 4.5 31 63 14 54 73 188 87 91 89 4
15 15 15 15
3,500 4,000 4,500 5,000
6 1.5 8 4
16 95 66 36
*Refers
to per cent of viable
Both
Cleft palate
-
-
vi)
(6.6) (81)
(no. )
+
1124 10
in fetal rats
fetuses
(%) (W;)
Total Ectocardia (no.1
Gas&o-
Both (no.1
-
-
-
-
(39)
-
-
I
1
(53)
:
-t
(53) (66)
-i
(6)
rats
Moderate to mere osteolathyritic effects
induced ectocnrdia and/or gastroschisis Viable
519
in pregnant
+ -
Litters (no. ) (no.1
(BAPN)
+ -
Resorptions v days
induced ectocardia
1
1
3 -
-!
m*, (0)
-f 9 1
<6)
_
(13) (4)
(2) (95)
4 y;
(32)
11 4
-
3
11 -
2.5 4
11
--
- 1
:
(19)
(5)
fetuses.
implantation, no significant effects are observed, but when administered during the period of organogensis (8 to 13th day in the rat), a time when most teratogens operate, only decreased fetal weights or increased mortality (seen as resorptions) are noted. Treatment of the pregnant rat on the 14th and 15 th days of gestation produces ectopia cordis (ectocardia) and/or gastroschisis in the fetuses examined six to seven days later at term. Administration of BAPN on the 16th day results in almost all fetuses developing a cleft palate.2 The purpose of this experiment was to evaluate the development of the ectocardia produced by
BAPN given on days 14 and 15. Embryos were examined histologically on days 14, 15, 1.6, 17, and 21 of gestation after either saline or treatment on days 14 and 15 with varying doses of BAPN. Methods
Both Sprague-Dawley and Long-Evans rats were used. In rat breeding rooms with 12 hour light cycles and constant temperature, females weighing 180 to 240 grams were caged overnight with males of the same strain. Nine o’clock the following morning was considered day 1 of gestation either if plugs were noted or if sperm were
Barrow
520
Fig.
1. Term
central
oigan
Am. Heart .I. 1972 April,
and Willis
rat fetuse
ram
is the I&r.
(eniarged
of body
weight
BAPN
via
intubation
on ges
X 2.j
optimal in producing these defects (Table I I). Higher doses predominantly produced resorptions. The optimal dose was then used in 20 other pregnant rats and groups of four animals were killed, the first group on day 14 after the first dose, the second group on day 15 after both doses, and the third, fourth, and fifth groups on days 16, 17, and 21, respectively. Appropriate saline intubated controls were also obtained. On the day of sacrifice, uteri were removed and opened and fetuses were fixed in 10 per cent formalin for 48 hours and then embedded in paraffin. Sgittal sections were made of each embryo and stained with hematoxylin and eosin and examined under the light microscope. Photographs were taken when appropriate. Fig. 2. Live term fetus with ectocardia showing the heart in systole and diastole. The pregnant rat was treated on days 14 and 15 with 2,500 mg. per kilogram of body weight BAPN and killed on day 21. (Enlarged X 2.)
found in the vagina. Pregnant rats were treated via intubation with BAPN fumurate* dissolved in water in several dosage regimes (Table II). Using different dosages, the rate of ectocardia, gastroschisis, or both in near-term fetuses (day 21 of gestation) was obtained for each dose and ultimately a dose of 2500 mg. per kilogram of body weight on days 14 and 15 was found to be *Obtained
from
.4ldrich
Chemical
Co.,
Milwaukee,
Wise.
Results Gross findings. Table II summarizes the results. The optimum dose which gave the highest rate of ectocardia and gastroschisis without an excessively high resorption rate was 2,500 mg. per kilogram of body weight on both days 14 and 15. At this dose BAPN produced a 16 per cent incidence of either ectocardia or gastroschisis or both (Figs. 1 and 2). Twenty-five out of 154 viable fetuses were affected and, of these, eleven had ectocardia only, three had gastroschisis only, and eleven had both. In the fetuses with ectocardia the hearts were beating normally (Fig. 2) and the fetuses were alive and lived for 15 to 20 minutes before ex-
BAPN
induced ectocardia
521
Fig. 3. Control 15, 16, and 17 day embryos. a, Mid-sagittal section of a U-day-old embryo. (Enlarged X11.) b, Mid-sagittal section of a 16-day-old embryo. (Enlarged X9.) c, Mid-sagittal section of a 17-day-old embryo. Note small bowel is still not within the abdomen. (Enlarged X5.) Mesenchymal cell condensation that will form cartilaginous plates form between day 16 and 17. a!, Insert from a showing anterior chest wall 4 to 5 cell layers thick. (Enlarged X600.) e, Insert from b. Anterior chest wall is still thin. (Enlarged X425.) f, Insert from c. Chest wall has become much thicker, mesenchymal plates are condensing to form precartilaginous tissue of the future sternum. (Enlarged X60.)
piring. Higher doses on these two consecutive days resulted in an extremely high resorption rate without a significantly increased rate of ectocardia, suggesting a double threshold, one for ectocardia and/or gastroschisis and the other for embryolethality. Single doses on either day 14 or 15 occasionally produced ectocardia but at a substantially lower rate. Histological studies. Sagittal sections using control fetuses on gestational days 14, 15, 16, and 17 were obtained first. On day
14 the anterior chest wall was a thin membrane some 4 to 6 cell layers thick. This anterior chest wall remained several cell layers thick on day 15 and 16 (Fig. 3, a, b, d, and e) but by day 17 (Fig. 3, c andf) the mesenchymal tissues were forming precartilagenous plates destined to become the sternum several days later. Following these control evaluations, treated fetuses were studied. While the anterior chest wall of most treated fetuses did not appear to be histologically abnor-
522
Barrow
Fig. 4. a, Mid-sagittal of body weight BAPN X9.) b, Insert showing
Am. Heart J. April, 1972
and Willis
section of 16-day-old embryo from a pregnant on days 14 and 15 without ectocardia showing an intact wall. (Enlarged X425.)
ma1 (Fig. 4), about 20 per cent (an incidence comparable to the 16 per cent incidence when term. fetuses were examined) appeared to have forms of ectocardia or gastroschisis in various stages of development (Fig. 5). The thin anterior chest wall appeared to have ruptured on either day 14 or 15 in these fetuses so that it was no longer present on day 16 or 17. At times the wall was missing in front of the heart only, with the heart protruding anteriorly (conceivably by its contracting and relaxing repeatedly) (Fig. 5, b and d) while in others the heart, liver, and gut appeared to be uncovered (Fig. 5, a and c). Examination of day 17 and day 20 fetuses (Figs. 5, d and 6) revealed no epithelial lining covering the heart and also showed a ruptured anterior chest wall with the heart and, at times, other tissues protruding. No abnormalities of the spine, which might push the heart forward, were evident nor were anatomical abnormalities of the heart or aorta evident, using either histological or gross sectioning techniques.15
rat treated with 2,500 mg. per kil logram the intact anterior chest wall. (EI tlarged
Discussion
BAPN has been shown to have significant embryotoxic (increased resorption rate and decreased fetal weight) and teratogenic effects.2 It is not a typical teratogen that produces malformations when given during the period of organogenesis as does radiation,16 trypan blue,l7 or kidney antiserum,r4 but it does produce a distinctive array of malformations when administered later in gestation (Table I). Ectocardia and gastroschisis occurs only when BAPN is given on the 14th and/or 15th day of gestation. Normally on days 14, 15, and 16 of gestation the anterior chest and abdominal walls are a few cell layers thick and only between day 16 and 17 (Fig. 3) and thereafter does the wall become thickened to hundreds of cell layers. Mesenchymal condensations which will later form the sternal cartilages, bone, and muscles of this area also begin to be noticeable histologically between the 16th and 17th day and thereafter. It is conceivable that a rupture of the anterior wall of the chest or abdomen
Volxme Number
83 4
BAPN
induced ectocardia
523
Fig. 5, a and b. Mid-sagittal sections of treated fetuses (2,500 mg. per kilogram of body weight on days 14 and 1.5) showing various stages of ectocardia and gastroschisis formation. a, 15 day fetus. Heart and liver are not covered. (Enlarged X 13.) b, 16 day fetus. Ectocardia is present and the anterior chest wall is missing. The abdominal wall is present over the liver. The small bowel is normally extraabdominal until day 17. (Enlarged X10.)
would tend to occur during the period when the wall was least strong (Fig. 5). We have concluded that ectocardia was produced by the beating heart protruding through the anterior chest wall and rupturing the thin membrane on either day 14 or 15. The degree of rupture and reparative process determined whether only ectocardia (Fig. 5, b and d), gastroschisis, or both (Fig. 5, b and c) resulted. This mechanism of the development of ectocardia and/or gastroschisis is compatible with BAPN’s known biochemical effect of inhibiting the cross-linking of both collagen and elastin, in this instance decreasing the tensile strength of the anterior chest wall. We hypothesize that at times this wall may rupture and, in certain instances, the beating heart may play a significant role in instigating the rupture. Pratt and associates’* showed that BAPN reaches the rat fetus before day 16 of gestation and that it
was rapidly metabolized to cyanoacetic acid (HOOC CH2 C = N) and excreted by the pregnant rat within 24 hours. They also demonstrated that embryonic tissue during this stage contained significant amounts of collagen which was solubilized in the treated animals so that an inhibition of crosslinking had occurred. Waddell and Steffekrg injected r4C labelled BAPN into pregnant mice on day 13 of gestation (with a 20 day gestation, compared to a 21 day gestation in rats) and showed that radioactivity accumulated in the connective tissues of the embryo. Thus, it is clear that BAPN reaches the embryo and that it interferes with collagen cross links in the embryos during a gestational period when the anterior chest wall is quite thin and the embryo is rapidly enlarging. Why BAPN does not result in teratogenie effects prior to day 14 remains speculative at the moment. It is possible that
524
Barrow
Am. Heart J. April, 1972
and Willis
Fig. 5, c and d. c, 16 day fetus with both ectocardia and gastroschisis. ectocardia. The old rupture site of the anterior chest wall is present the heart (Enlarged X 30.)
Fig. 6. 21 day old fetus with ectocardia. of epithelial lining and the way cartilage
(Enlarged X10.) d, 17 day fetus with (arrow). No epithelial membrane covers
a, Sagittal section (Enlarged X3.) b, Enlarged view showing and bone forms above and below the heart. (Enlarged X 15.)
the lack
Volume Number
83 4
BAPN may not reach the embryo prior to the 14th day but it is also conceivable that mature collagen and elastin are not formed prior to this day. The teratogenic effects observed are compatible with either possibility. Future work should clarify whether collagen is present in the embryo prior to the 14th day and whether BAPN reaches the embryo during organogenesis. While ectocardia, gastroschisis, cleft palate, bone, and aorta abnormalities may seem remotely related at first glance, a distinctive relationship may exist along the following lines. All of these tissues contain abundant amounts of collagen and elastin and interference with tissue formation and tensile strength by BAPN could cause the following: (1) an anterior chest wall rupture when given on day 14 and 15, and secondarily, ectocardia and/or gastroschisis, (2) interference with cleft palate closure when given on day 16 only (since the palate fuses on day 16 in the rat), or (3) kyphoscoliosis, curvatures of bones (since bone contains large amounts of collagen), and aortic aneurysms (by decreasing strength of the media) when administered after day 16 or to young animals. Summary
Beta-aminopropionitrile (BAPN) was given to pregnant rats via intubation on days 12 through 15 or using combinations on two consecutive days between 13 and 15. Given on both days 14 and 15 to 30 litters using a dose of 2,500 mg. per kilogram of body weight, BAPN produced 55 per cent resorptions and a 16 per cent incidence (in viable fetuses) of either ectocardia (11 fetuses), gastroschisis (3 fetuses), or both (11 fetuses). Larger doses produced predominantly resorptions. Single doses resulted in occasional animals with ectocardia. Control and treated litters (using 2,500 mg. per kilogram of body weight on days 14 and 15) were then killed on days 14, 15, 16, 17, and 21 and sagittal sections were examined histologically. Control animals showed a thin anterior chest wall membrane until day 17 when the wall thickened and mesenchymal condensations which would form the future sternum appeared. Twenty per cent of the BAPN-treated animals showed a rupture of the anterior chest or abdominal
BAPN
induced ectocardia
52.5
wall at various stages in the development of ectocardia or gastroschisis. An hypothesis to explain the pathogeneis of these malformations is as follows: BAPN, which interferes with collagen and elastin synthesis, interferes with the tensile strength of the anterior chest and abdominal wall. At times this wall may rupture and, in certain instances, the beating heart may play a significant role in instigating the rupture. The rupture, depending on the degree of rupture and the reparative’process, may result in ectocardia, or in both ectocardia and gastroschisis, and rarely in gastroschisis alone.
REFERENCES 1. Barrow, M. V., Simpson, C. F., and Miller, E. I.: Lathvrism: A review. Medicine (Submitted for publication) 2. Barrow, M. V., Steffek, A. J., and King, C. T. G.: Teratogenic and embryotoxic effects of beta-aminopropionitrile (BAPN) in pregnant rats, Teratology. (Submitted for publication) 3. Rosenberg, E. E.: Teratogenic effects of betaamino-propionitrile in the chick embryo, Nature 180:706, 1957. 4. Levine, C. I.: Morphologic and chemical studies on lathyrism in the chick embryo, Fed. Proc. 17:455, 1958. 5. Zackin, S. T., and Goldhaber, P.: The effect of aminoacetonitrile on the chick embryo mandible, J. Dent. Res. 88:741, 1959. 6. Cameron, J. M.: Experimental lathyrism in turkey embryos, Nature 194:210, 1962. 7. Azumis, E. : Experimentally produced scoliosis of vertebrae, J. Osaki City Med. Center 4:19, 1955. nutrition and experimental 8. Baba, T.: Maternal malformations, Acta Pathol. Jap. 5:485, 1955. 9. Pjiorala, K., Seppala, T., and Punsar, S.: Aortic and joint changes in rat foetus treated during pregnancy with lathyritic agents given to the mother, Acta Pathol. Microbial. Stand. 38:273. 1956. 10. Ferm, V. H.: Osteolathyrogenic effects on the developing rat foetus, J. Embryo]. Exp. Morphol. 8:94, 1960. G. L.: Lathyrism in the 11. Herd, K., and Orbison, fetal rat. Arch. Pathol. 81:60. 1966. 12. McCallum, H. M.: Lathyrism in mice, Nature 182:1169, 1958. 13. Zahor, Z., and Czabanova, D.: The frequency and pathogenesis of aortic fibroelastosis in foetal lathyrism in rats, J. Pathol. 84:209, 1962. 14. Barrow, M. V., and Taylor, W. J.: The production of congenital defects in rats using antisera, J. Exp. Zool. 176:41, 1971. 15. Barrow, M. V., and Taylor, W. J.: A rapid method of detecting malformations in rat fetuses, J. Morphol. 127:291, 1969. 16. Hicks, S. P., and D’Amato, C. G.: Effect of
526
17. 18.
Barrow
Am. Heart I. April, 1972
and Willis
ionizing radiations on mammalian development, Adv. Teratology 1:196, 1966. Beck, F., and Lloyd, G. B.: The teratogenic effects of azo dyes, Adv. Teratology 1:131, 1966. Pratt, R. M., Wilk, A. G., Horigan, E. A., and King, C. T. G.: Inhibition of cross-linking in embryonic collagen and &aminopropionitriIe
19.
(BAPN) induced cleft palate in the rat, Teratology 4:239, 1971. Waddell, W. G., and Steffek, A. J.: The distribution of Iathyrogenic agents, p-aminopropionitrilel4C (BAPN’4C) in pregnant A/J mice, Teratology 4:243, 1971.
L
induced
Ph.D.
athyrism includes two disorders.’ The first, neurolathyrism, occurs in man or other animals that ingest the seed of plants of certain lathyrus species, including Lathyrus sativus, L. &era, and L. clymenum. A spastic paraplegia develops and pathological changes have been noted in the spinal cord. At least three neurolathyrogens have been isolated from these plants thus far.’ The other type of lathyrism has been called osteolathyrism. This disorder has not been described in man but has been well described in young or growing rats, and several other species fed L. odoratus (flowering sweetpea) seed as part of the diet. Osteolathyritic effects include kyphoscoliosis of the spine, curvature of the long bones, exostoses of bone at muscle insertion sites, slipped epiphyses and aortic aneurysms (this last abnormality called by some, angiolathyrism, to distinguish this effect from the bone changes). The toxic principle of L. odoratus has been shown to be beta-aminopropionitrile (BAPN) with the formula HZN CHzCHz C=N. This substance interferes with the formation of intermolecular and intramolecular crosslinks in the formation of collagen and e1astin.r Since bone and aortic tissues con-
tain large quantities of one or both of these substances the osteolathyritic effects, including the angiolathyritic ones, have been explained on the basis of abnormalities in these connective tissues. These notions have been substantiated in a variety of ways-for a detailed review of these concepts the reader is referred to our article.’ Several other agents, including aminoacetonitrile, several hydrazines, hydrazides and ureides, have the ability to produce osteolathyrism in young and growing animals. These same agents have also been shown to be teratogenic.rT2 In the chick and turkey embryo osteolathyrogens produce typical bony effects and aortic aneurysms.3-6 Mammals (rats have been the common experimental animal) fed during late gestation also develop kyphoscoliosis and aortic aneurysms, ‘-I3 abnormalities which are similar to the effects seen when the agent is fed to young animals. Earlier treatment, however, produces different effects, these being somewhat unique and interesting. These results have been recently summarized and discussed in experimentation with pregnant rats using BAPN (Table I) .rs2 When given prior to
From the Division of Cardiology, Department of Medicine, University of Florida, Gainesville, Fla. This work was supported by Florida Heart Grant No. 70AG8. Received for publication June 28. 1971. Reprint requests to: Dr. Mark V. Barrow. Div. of Cardiology. Department of Medicine, University of Florida, ville. Ma. 32601.
518
American
Heart
Journal
April, 1972
Vol.
Gaines-
83, No. 4, pp. 518-526
Volume Number
83 4
BAPN
Table I. Summary of teratogenic efects of beta-aminopropionitrile (from Barrow and associates2)
2* day(s)
Ectocardia
Gastroschisis
-
up to 14 14 and 15 16 After 16
+ -
+ -
Table II. Beta-aminopropionitrile
Dose (wlk’g.
1
(BAPN)
Controll 12 13
5,000 5,000
111 5 6
79 43 3.5
13-14 13-14 13-14 13-15 14 14 14 14-15 14-15 14-1.5 14-15 15
3,000 3,500 4,000 2,500 4,000 5,000 5,500 2,500 3,000 3,500 4,000 2,500
8 5 4 12 5 9 7 30 11 12 9 13
42 4.5 31 63 14 54 73 188 87 91 89 4
15 15 15 15
3,500 4,000 4,500 5,000
6 1.5 8 4
16 95 66 36
*Refers
to per cent of viable
Both
Cleft palate
-
-
vi)
(6.6) (81)
(no. )
+
1124 10
in fetal rats
fetuses
(%) (W;)
Total Ectocardia (no.1
Gas&o-
Both (no.1
-
-
-
-
(39)
-
-
I
1
(53)
:
-t
(53) (66)
-i
(6)
rats
Moderate to mere osteolathyritic effects
induced ectocnrdia and/or gastroschisis Viable
519
in pregnant
+ -
Litters (no. ) (no.1
(BAPN)
+ -
Resorptions v days
induced ectocardia
1
1
3 -
-!
m*, (0)
-f 9 1
<6)
_
(13) (4)
(2) (95)
4 y;
(32)
11 4
-
3
11 -
2.5 4
11
--
- 1
:
(19)
(5)
fetuses.
implantation, no significant effects are observed, but when administered during the period of organogensis (8 to 13th day in the rat), a time when most teratogens operate, only decreased fetal weights or increased mortality (seen as resorptions) are noted. Treatment of the pregnant rat on the 14th and 15 th days of gestation produces ectopia cordis (ectocardia) and/or gastroschisis in the fetuses examined six to seven days later at term. Administration of BAPN on the 16th day results in almost all fetuses developing a cleft palate.2 The purpose of this experiment was to evaluate the development of the ectocardia produced by
BAPN given on days 14 and 15. Embryos were examined histologically on days 14, 15, 1.6, 17, and 21 of gestation after either saline or treatment on days 14 and 15 with varying doses of BAPN. Methods
Both Sprague-Dawley and Long-Evans rats were used. In rat breeding rooms with 12 hour light cycles and constant temperature, females weighing 180 to 240 grams were caged overnight with males of the same strain. Nine o’clock the following morning was considered day 1 of gestation either if plugs were noted or if sperm were
Barrow
520
Fig.
1. Term
central
oigan
Am. Heart .I. 1972 April,
and Willis
rat fetuse
ram
is the I&r.
(eniarged
of body
weight
BAPN
via
intubation
on ges
X 2.j
optimal in producing these defects (Table I I). Higher doses predominantly produced resorptions. The optimal dose was then used in 20 other pregnant rats and groups of four animals were killed, the first group on day 14 after the first dose, the second group on day 15 after both doses, and the third, fourth, and fifth groups on days 16, 17, and 21, respectively. Appropriate saline intubated controls were also obtained. On the day of sacrifice, uteri were removed and opened and fetuses were fixed in 10 per cent formalin for 48 hours and then embedded in paraffin. Sgittal sections were made of each embryo and stained with hematoxylin and eosin and examined under the light microscope. Photographs were taken when appropriate. Fig. 2. Live term fetus with ectocardia showing the heart in systole and diastole. The pregnant rat was treated on days 14 and 15 with 2,500 mg. per kilogram of body weight BAPN and killed on day 21. (Enlarged X 2.)
found in the vagina. Pregnant rats were treated via intubation with BAPN fumurate* dissolved in water in several dosage regimes (Table II). Using different dosages, the rate of ectocardia, gastroschisis, or both in near-term fetuses (day 21 of gestation) was obtained for each dose and ultimately a dose of 2500 mg. per kilogram of body weight on days 14 and 15 was found to be *Obtained
from
.4ldrich
Chemical
Co.,
Milwaukee,
Wise.
Results Gross findings. Table II summarizes the results. The optimum dose which gave the highest rate of ectocardia and gastroschisis without an excessively high resorption rate was 2,500 mg. per kilogram of body weight on both days 14 and 15. At this dose BAPN produced a 16 per cent incidence of either ectocardia or gastroschisis or both (Figs. 1 and 2). Twenty-five out of 154 viable fetuses were affected and, of these, eleven had ectocardia only, three had gastroschisis only, and eleven had both. In the fetuses with ectocardia the hearts were beating normally (Fig. 2) and the fetuses were alive and lived for 15 to 20 minutes before ex-
BAPN
induced ectocardia
521
Fig. 3. Control 15, 16, and 17 day embryos. a, Mid-sagittal section of a U-day-old embryo. (Enlarged X11.) b, Mid-sagittal section of a 16-day-old embryo. (Enlarged X9.) c, Mid-sagittal section of a 17-day-old embryo. Note small bowel is still not within the abdomen. (Enlarged X5.) Mesenchymal cell condensation that will form cartilaginous plates form between day 16 and 17. a!, Insert from a showing anterior chest wall 4 to 5 cell layers thick. (Enlarged X600.) e, Insert from b. Anterior chest wall is still thin. (Enlarged X425.) f, Insert from c. Chest wall has become much thicker, mesenchymal plates are condensing to form precartilaginous tissue of the future sternum. (Enlarged X60.)
piring. Higher doses on these two consecutive days resulted in an extremely high resorption rate without a significantly increased rate of ectocardia, suggesting a double threshold, one for ectocardia and/or gastroschisis and the other for embryolethality. Single doses on either day 14 or 15 occasionally produced ectocardia but at a substantially lower rate. Histological studies. Sagittal sections using control fetuses on gestational days 14, 15, 16, and 17 were obtained first. On day
14 the anterior chest wall was a thin membrane some 4 to 6 cell layers thick. This anterior chest wall remained several cell layers thick on day 15 and 16 (Fig. 3, a, b, d, and e) but by day 17 (Fig. 3, c andf) the mesenchymal tissues were forming precartilagenous plates destined to become the sternum several days later. Following these control evaluations, treated fetuses were studied. While the anterior chest wall of most treated fetuses did not appear to be histologically abnor-
522
Barrow
Fig. 4. a, Mid-sagittal of body weight BAPN X9.) b, Insert showing
Am. Heart J. April, 1972
and Willis
section of 16-day-old embryo from a pregnant on days 14 and 15 without ectocardia showing an intact wall. (Enlarged X425.)
ma1 (Fig. 4), about 20 per cent (an incidence comparable to the 16 per cent incidence when term. fetuses were examined) appeared to have forms of ectocardia or gastroschisis in various stages of development (Fig. 5). The thin anterior chest wall appeared to have ruptured on either day 14 or 15 in these fetuses so that it was no longer present on day 16 or 17. At times the wall was missing in front of the heart only, with the heart protruding anteriorly (conceivably by its contracting and relaxing repeatedly) (Fig. 5, b and d) while in others the heart, liver, and gut appeared to be uncovered (Fig. 5, a and c). Examination of day 17 and day 20 fetuses (Figs. 5, d and 6) revealed no epithelial lining covering the heart and also showed a ruptured anterior chest wall with the heart and, at times, other tissues protruding. No abnormalities of the spine, which might push the heart forward, were evident nor were anatomical abnormalities of the heart or aorta evident, using either histological or gross sectioning techniques.15
rat treated with 2,500 mg. per kil logram the intact anterior chest wall. (EI tlarged
Discussion
BAPN has been shown to have significant embryotoxic (increased resorption rate and decreased fetal weight) and teratogenic effects.2 It is not a typical teratogen that produces malformations when given during the period of organogenesis as does radiation,16 trypan blue,l7 or kidney antiserum,r4 but it does produce a distinctive array of malformations when administered later in gestation (Table I). Ectocardia and gastroschisis occurs only when BAPN is given on the 14th and/or 15th day of gestation. Normally on days 14, 15, and 16 of gestation the anterior chest and abdominal walls are a few cell layers thick and only between day 16 and 17 (Fig. 3) and thereafter does the wall become thickened to hundreds of cell layers. Mesenchymal condensations which will later form the sternal cartilages, bone, and muscles of this area also begin to be noticeable histologically between the 16th and 17th day and thereafter. It is conceivable that a rupture of the anterior wall of the chest or abdomen
Volxme Number
83 4
BAPN
induced ectocardia
523
Fig. 5, a and b. Mid-sagittal sections of treated fetuses (2,500 mg. per kilogram of body weight on days 14 and 1.5) showing various stages of ectocardia and gastroschisis formation. a, 15 day fetus. Heart and liver are not covered. (Enlarged X 13.) b, 16 day fetus. Ectocardia is present and the anterior chest wall is missing. The abdominal wall is present over the liver. The small bowel is normally extraabdominal until day 17. (Enlarged X10.)
would tend to occur during the period when the wall was least strong (Fig. 5). We have concluded that ectocardia was produced by the beating heart protruding through the anterior chest wall and rupturing the thin membrane on either day 14 or 15. The degree of rupture and reparative process determined whether only ectocardia (Fig. 5, b and d), gastroschisis, or both (Fig. 5, b and c) resulted. This mechanism of the development of ectocardia and/or gastroschisis is compatible with BAPN’s known biochemical effect of inhibiting the cross-linking of both collagen and elastin, in this instance decreasing the tensile strength of the anterior chest wall. We hypothesize that at times this wall may rupture and, in certain instances, the beating heart may play a significant role in instigating the rupture. Pratt and associates’* showed that BAPN reaches the rat fetus before day 16 of gestation and that it
was rapidly metabolized to cyanoacetic acid (HOOC CH2 C = N) and excreted by the pregnant rat within 24 hours. They also demonstrated that embryonic tissue during this stage contained significant amounts of collagen which was solubilized in the treated animals so that an inhibition of crosslinking had occurred. Waddell and Steffekrg injected r4C labelled BAPN into pregnant mice on day 13 of gestation (with a 20 day gestation, compared to a 21 day gestation in rats) and showed that radioactivity accumulated in the connective tissues of the embryo. Thus, it is clear that BAPN reaches the embryo and that it interferes with collagen cross links in the embryos during a gestational period when the anterior chest wall is quite thin and the embryo is rapidly enlarging. Why BAPN does not result in teratogenie effects prior to day 14 remains speculative at the moment. It is possible that
524
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and Willis
Fig. 5, c and d. c, 16 day fetus with both ectocardia and gastroschisis. ectocardia. The old rupture site of the anterior chest wall is present the heart (Enlarged X 30.)
Fig. 6. 21 day old fetus with ectocardia. of epithelial lining and the way cartilage
(Enlarged X10.) d, 17 day fetus with (arrow). No epithelial membrane covers
a, Sagittal section (Enlarged X3.) b, Enlarged view showing and bone forms above and below the heart. (Enlarged X 15.)
the lack
Volume Number
83 4
BAPN may not reach the embryo prior to the 14th day but it is also conceivable that mature collagen and elastin are not formed prior to this day. The teratogenic effects observed are compatible with either possibility. Future work should clarify whether collagen is present in the embryo prior to the 14th day and whether BAPN reaches the embryo during organogenesis. While ectocardia, gastroschisis, cleft palate, bone, and aorta abnormalities may seem remotely related at first glance, a distinctive relationship may exist along the following lines. All of these tissues contain abundant amounts of collagen and elastin and interference with tissue formation and tensile strength by BAPN could cause the following: (1) an anterior chest wall rupture when given on day 14 and 15, and secondarily, ectocardia and/or gastroschisis, (2) interference with cleft palate closure when given on day 16 only (since the palate fuses on day 16 in the rat), or (3) kyphoscoliosis, curvatures of bones (since bone contains large amounts of collagen), and aortic aneurysms (by decreasing strength of the media) when administered after day 16 or to young animals. Summary
Beta-aminopropionitrile (BAPN) was given to pregnant rats via intubation on days 12 through 15 or using combinations on two consecutive days between 13 and 15. Given on both days 14 and 15 to 30 litters using a dose of 2,500 mg. per kilogram of body weight, BAPN produced 55 per cent resorptions and a 16 per cent incidence (in viable fetuses) of either ectocardia (11 fetuses), gastroschisis (3 fetuses), or both (11 fetuses). Larger doses produced predominantly resorptions. Single doses resulted in occasional animals with ectocardia. Control and treated litters (using 2,500 mg. per kilogram of body weight on days 14 and 15) were then killed on days 14, 15, 16, 17, and 21 and sagittal sections were examined histologically. Control animals showed a thin anterior chest wall membrane until day 17 when the wall thickened and mesenchymal condensations which would form the future sternum appeared. Twenty per cent of the BAPN-treated animals showed a rupture of the anterior chest or abdominal
BAPN
induced ectocardia
52.5
wall at various stages in the development of ectocardia or gastroschisis. An hypothesis to explain the pathogeneis of these malformations is as follows: BAPN, which interferes with collagen and elastin synthesis, interferes with the tensile strength of the anterior chest and abdominal wall. At times this wall may rupture and, in certain instances, the beating heart may play a significant role in instigating the rupture. The rupture, depending on the degree of rupture and the reparative’process, may result in ectocardia, or in both ectocardia and gastroschisis, and rarely in gastroschisis alone.
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ionizing radiations on mammalian development, Adv. Teratology 1:196, 1966. Beck, F., and Lloyd, G. B.: The teratogenic effects of azo dyes, Adv. Teratology 1:131, 1966. Pratt, R. M., Wilk, A. G., Horigan, E. A., and King, C. T. G.: Inhibition of cross-linking in embryonic collagen and &aminopropionitriIe
19.
(BAPN) induced cleft palate in the rat, Teratology 4:239, 1971. Waddell, W. G., and Steffek, A. J.: The distribution of Iathyrogenic agents, p-aminopropionitrilel4C (BAPN’4C) in pregnant A/J mice, Teratology 4:243, 1971.