Equipment and methods
Normal development of the chick embryo
Results
Summary
Author: J. S. SPINDLER , Maria Teresa GARCIA MONGE
Pages: 55 to 60
Creation Date: 1970/01/01
In view of the use and distribution of hallucinogens, especially among young people, a series of studies have been undertaken in recent years as part of the wide and extensive survey of problems arising from such substances. Psilocybine, mescaline and lysergic acid diethylamide (lysergide) (LSD 25) are the best known of these substances, but the last named has aroused particular interest.
The pharmacological properties of LSD 25 [ 1] discovered by Hofmann in the Sandoz Laboratories (Basel) led to research into its effect on the psyche, research which might be relevant to the treatment of certain types of mental diseases (schizophrenia) and useful in analytic therapy [ 2] .
In 1967, Science published the genetic studies carried out by Cohen and his collaborators [ 3] in Buffalo. The addition of LSD 25 to cultures of human leucocytes produces a marked increase in chromosomic deviations and disturbances, such damage being observed in the chromosomes of a patient to whom fifteen doses of 80-200 mcg of the substance were administered over a period of four years. Tests carried out by Irwin and his collaborators [ 4] among a group of volunteers using LSD 25 also showed chromosomic disturbances.
Skakkeback and his collaborators [ 5] have described abnormalities in the meiosis of chromosomes noted in experiments on rats treated with LSD 25.
All this research has produced sufficient evidence to demonstrate the damage to chromosomes caused by LSD 25.
With regard to its teratogenic effect, few cases have been observed so far. H. Zellweger and his collaborators in Iowa have described a deformity of the right leg, an aplastic fibular syndrome, in a girl whose mother took LSD 25 on the twenty-fifth, forty-eighth and ninety-eighth days of pregnancy.
The original of this paper is in Spanish. The work reported was undertaken with the help of the Narcotics Control Department of the Directorate-General of Health, Madrid.
B.A., C.C.N.Y. Research Fellow, School of Medicine, Madrid.
Pharmacologist of the Narcotics Control Department of the Directorate-General of Health - Research Fellow, School of Medicine, Madrid.
Alexander and his collaborators [ 7] experimented by administering a single subcutaneous injection of 5 mcg of LSD 25 per kilogramme of weight to rats during gestation. In the case of a large percentage of the offspring, this resulted in a paralysis of growth, reabsorptions and miscarriages.
Similarly, R. Auerbach and his collaborators [ 8] found that an intraperitoneal injection of the substance into a rat on the seventh day of gestation caused the appearance of a large number of cerebral malformations such as abnormal closure of the medial and posterior regions of the brain and changes in the position of the eyes.
Geber [ 9] administered mescaline, LSD 25 and BOL (the monobromide derivative of LSD 25) to hamsters on the eighth day of gestation, varying the concentration of the drugs and obtaining the following results: exencephalitis, spina bifida, interparietal meningocele, omphalocele, hydrocephalus, myelocele, oedema along the axis of the spine, head and localised cerebral haemorrhages.
According to Warkany and his collaborators [ 10] however, doses of LSD 25 of 1.5 to 300 mcg administered intraperitoneally and orally to rats on the seventh and twelfth day of gestation, produced no abnormalities, from which fact they concluded that LSD is not teratogenic in rats.
The field of research opened by hallucinogens is rapidly widening on account of the appearance of new substances included in this group. One of them, known as DOM and synthesized in the Dow Chemical Laboratories is identical with the hallucinogen known as STP (2.5 dimethoxy - 4 methyl amphetamine).
Chemically it is related to amphetamine and mescaline. Its LD 50 in the rat is 60 mg per kilogramme of weight [ 11] .
Snyder and his collaborators [ 12] have carried out experiments to ascertain the physical and mental effects of DOM on man.
A number of volunteers were given varying doses of DOM hydrochloride dissolved in distilled water.
In the examinations carried out after the substance was taken, the following effects, inter alia, were observed: dilation of the pupils, quickening of the pulse, high oral temperature and high blood pressure. In small doses, DOM produces mild effects of euphoria. In doses of 5 mg or more it produces marked hallucinogenic effects. The authors consider however that DOM is a powerful hallucinogen with a potency which is one thirtieth that of LSD.
We are carrying out a series of studies on the substance DOM (STP) for the purpose of determining its effect on embryonic development.
The present paper relates exclusively to a pilot experiment carried out on chick embryos.
Eggs of the White Leghorn and Ross breeds of hen were used for the experiments.
The DOM (STP) 2,5 dimethoxy-4 methylamphetamine hydrochloride was supplied by the Dow Chemical Company.
Thirty minutes before the administration of the drug to the embryo, two standard solutions are prepared, by diluting 0.5 mg and 5 mg of DOM (STP) in sterile tyrode solution and adjusting the pH to the physiological value of the embryo with 10 per cent sterile solution of bicarbonate of soda. 1/4 cc of this solution contains 0.5 and 0.05 mcg respectively of DOM (STP).
Using as a basis the lethal dose for the chick embryo, the dosage was calculated at less than half of the lethal dose.
The eggs were incubated for thirty hours at 38° C. They were then removed from the incubator in groups of three, and 2 cc of albumen was extracted. Following this, 1/4 cc of the standard solutions was introduced through the opening made in the central part of the longest axis of the egg, and placed beside the blastodisc. The opening was covered with waxed paper.
Four series of experiments were carried out, the same method being used for each one. In each case, 52 embryos at the 5 (-) or 5 stage were used. They were divided into two groups; one received 1/4 cc of the solution, containing 0.5 mcg of DOM (STP) and the other 0.05 mcg of DOM (STP) in the same quantity of solution.
An equal number of controls were set up, to which 1/4 cc of buffered sterile tyrode solution was administered as indicated above.
They were observed daily; at intervals of 24, 48, 72 and 114 hours, in a selected number of eggs, the living embryos were fixed in 10 per cent neutral formol or in Bouin's solution.
The morphological malformations were observed with the naked eye or by means of a binocular magnifier and after the embryos were photographed they were enclosed and cut out for a histological study to be made.
There are critical periods in the development of an embryo, in organs and embryonic tissue, during which they are found to be particularly sensitive to the action of chemical substances and physical agents. For this reason, one of those periods was chosen, the innoculation being carried out during stage 5 (Hamilton and Hamburger) of the development of the chick embryo.
A description is given below of certain fundamental features of the normal development of the chick embryo in each of the stages previously indicated [ 13] , [ 14] .
Stage 5 (30 hours). The primitive streak begins to decrease in length. The notochord grows out of the node.
The neural plate and folds can be seen. The lateral folds of the mesoderm come forward. The front intestinal tube appears.
Stage 13 (53 hours). The prosencephalon forms an angle with the rhombencephalon as a result of flexion. The ectoderm, situated outside the primary vesicle, thickens and becomes the rudimentary lens.
Stage 18 (72 hours). The cerebral hemispheres develop from the telencephalon. The development of the fourth aortic arch is observed.
Because of cephalic flexion, the eyes are behind the otic vesicles and as a result of caudal flexion the tail is at an angle of 90° from the rest of the body.
The outlines of the limbs have become completely distinct.
Stage 28 (144 hours) Three finger bones can be distinguished in the fore limbs and four toes in the hind limbs. The second and third fingers and toes are longer than the others so that the outline of the fore limb, like that of the hind limb, is oval in shape.
The neck has lengthened.
The opening of the exterior anditory canal is very prominent.
The formation of the beak can also be seen.
Each experiment was carried out with 52 white Leghorn and Ross embryos. The DOM (STP) was administered during stage 5 (Hamilton and Hamburger). At about this stage the notochord grows out from the node, and the neural plate and folds can be seen.
A high mortality rate and a large number of abnormalities were noted in the test subjects.
Malformations of the nervous system were so conspicuous that they were taken as the basic theme for this research. Other types of abnormality will be referred to later. A series of embryos which presented the most typical malformations are described below.
Embryo A.15. White Leghorn (see figure 1). Fixed in Bouin's solution at stage 19. The cephalic portion of the nervous system appears exposed on the outside. In it a symmetrical development can be observed of four globular masses forming structures separated amongst themselves by two fissures running horizontally and perpendicularly in the shape of a cross; in the centre of the fissure an opening can be seen. The neural tube has suffered an abnormal fusion, forming a protruding line in the dorsal portion and in the cranic-caudal direction.
The fore limbs can be seen as well as the hind limbs.
In the hind part there is an abnormal torsion to the right.
The loss of normal morphological appearance and the external exposure of the fore, centre and hind brain are characteristic of the anencephalia revealed in this experiment.
Embryo C.27. Ross (see figure 2). Fixed in Bouin's solution at stage 26. The embryo shows incisionary anencephalia dividing the brain from the telencephalon to the rhombencephalon into two parts which project outwards. The abnormal formation of the eye is also to be noted.
The limbs are of normal appearance.
The morphological difference between the embryos in figures 2, 4, 5a and 5b should be noted.
Embryo B.2. White Leghorn (see figure 3). Fixed in Bouin's solution at stage 22.
The folds of the neural tube have failed to fuse, causing the abnormality known as spina bifida.
The open tube is S-shaped, running in a cranio-caudal direction and continuing in its front part in the form of a fissure along the head, thus giving a twisted effect. The development of the head is abnormal as there is no fusion of the cerebrum. The body shows a torsion first to the left then to the right and there is no proportion between the small head and the large body. The outlines of the fore and hind limbs can be seen.
Associated with anencephalia, abnormalities of the eyes have appeared fairly regularly. These exist in varying degrees, from the total absence of the structure of both eyes to partial and abnormal formation of one eye.
Embryo D.43 Ross (see figure 4). Fixed in Bouin's solution at stage 26.
Bilateral anophthalmia and extensive head deformity are observed.
Embryo B.39 Leghorn (see figure 5). Fixed in Bouin's solution at stage 26.
It will be noted that unilateral microphthalmia has occurred in the left eye, which is very small. The pigment is not uniformly distributed but is dense in the upper part of the eye. Figure 5 (a) shows the left side -of the embryo with microphthalmia, and figure 5 (b) shows the right eye which is normal. The difference in size, pigment and development of the lens is to be noted.
Embryos of a large number of the birds used in the experiments are much smaller in size than the embryos of the control group at the same stage. The head is not in proportion with the rest of the body. The outlines of the fore and hind limbs appear to be well shaped. One embryo showed heterotaxy (rotation towards the right side).
Figure 5 (a) and Figure 5 (b)
A high percentage of localized haemorrhages were found mainly in the cephalic, cervical, thoracic and abdominal regions, varying in extent from a small area to a fourth part of the region.
In some cases there was atrophy of the embryo, which remained a shapeless mass with a large superficial heart beating and an extensive area of vascular tissue.
Of the 114 living embryos of the fourth series, 93 showed abnormalities of varying form and degree. The frequency of their occurrence was much higher than in the control group, in which only 0.48 per cent of spontaneous abnormalities were produced.
Anencephalia, which is the predominating abnormality, occurred in 55 of the total number of embryos, followed by microphthalmia with a proportion of 17.3 per cent, i.e. 36 out of the total number of embryos.
The remaining abnormalities occurred in the following proportions: spina bifida 11.52 per cent, microsomia 8.17 per cent and rachischisis 0.48 per cent. A high percentage was also noted of localized haemorrhages which did not occur in the control group (see table II).
The highest mortality rate occurred during the period of long incubation (144 hours) but in the other periods referred to, when incubation was arrested after the introduction of DOM (STP), a relatively small number of embryos died. In the control group, the greatest number of embryos died 48 hours after the introduction of the buffered sterile tyrode solution.
The mortality rate, which increased during the long incubation period, is probably due to the advanced stage of abnormality reached and, in the case of localized haemorrhages, to their extensiveness in different parts of the body.
The total number of dead embryos in the four series was 94 (45.19 per cent). This figure represents three times the number occurring in the control groups (15.38 per cent) (see table I).
|
Number dead |
||||||||
|---|---|---|---|---|---|---|---|---|
|
Experiment |
Breed |
Dose |
Number of embryos used |
Number living |
24 h. |
48 h. |
72 h. |
144 h. |
|
A
|
White Leghorn
|
0.05 mcg | 52 | 30 | 2 |
-
|
2 | 18 |
|
B
|
White Leghorn
|
0.5 mcg | 52 | 28 | 3 | 2 | 2 | 17 |
|
C
|
Ross
|
0.05 mcg | 52 | 30 | 4 | 4 |
-
|
14 |
|
D
|
Ross
|
0.5 mcg | 52 | 26 | 2 | 4 | 4 | 16 |
|
TOTAL
|
208 | 114 | 94 | |||||
|
PERCENTAGE
|
54.80 %
|
45.19 %
|
||||||
|
CONTROLS
|
||||||||
|
a
|
White Leghorn
|
Tyrode 1/4 cc
|
52 | 44 |
-
|
2 | 3 | 3 |
|
b
|
White Leghorn
|
Tyrode 1/4 cc
|
52 | 45 | 1 | 4 | 1 | 1 |
|
c
|
Ross
|
Tyrode 1/4 cc
|
52 | 44 | 2 | 5 |
-
|
1 |
|
d
|
Ross
|
Tyrode 1/4 cc
|
52 | 43 | 4 | 3 |
-
|
2 |
|
TOTAL
|
208 | 176 | 32 | |||||
|
PERCENTAGE
|
84.61%
|
15.38 %
|
||||||
|
No. of embryos |
Number of abnormalities |
|||||||
|---|---|---|---|---|---|---|---|---|
|
Experiment |
Normal |
Abnormal |
Anencephalia |
Spina bifida |
Microphthalmia |
Rachischisis |
Microsomia |
Haemorrhages |
|
A
|
29 | 23 | 13 | 5 | 6 |
-
|
5 | 13 |
|
B
|
27 | 25 | 14 | 6 | 9 | 1 | 5 | 16 |
|
C
|
31 | 21 | 12 | 5 | 9 |
-
|
4 | 15 |
|
D
|
28 | 24 | 16 | 8 | 12 |
-
|
3 | 16 |
|
PERCENTAGE
|
55.28%
|
44.7%
|
26.44%
|
11.52%
|
17.30%
|
0.48%
|
8.17%
|
28.84%
|
|
CONTROL
|
0.48 %
|
|||||||
The two breeds, White Leghorn and Ross, did not show any individual susceptibility to malformations caused by DOM (STP), since the same number of normal and abnormal embryos were observed in the case of each breed.
No special relationship was found between dosage and dead embryos occurring in the two breeds. Nor could any specific relationship be found between dosage and the proportion of abnormalities.
Account must of course be taken not only of the variation in potency among the different hallucinogens but also of the very small doses of DOM (STP) used.
In tables I and II the relationship can be seen between types of abnormality and their percentage of occurrence as well as the frequency of their occurrence both in the test birds and in the control group.
Research has shown that some hallucinogens have teratogenic effects that can influence the normal development and differentiation of the cell, thus causing chromosomic abnormalities [ 3, 4, 5, 6, 7, 8, 9] .
Research is at present being conducted on the hallu- cinogen STP and a series of amphetamine derivatives of similar chemical composition.
A pilot project was set up to determine teratogenic effects of DOM (STP) on the developing chick embryo. DOM (STP) in doses of 0.5 mcg and 0.05 mcg per 1/4 c.c. was introduced into the chick embryo during stage 5 (Hamilton and Hamburger). Incubation was stopped at intervals of 24, 48, 72 and 144 hours and the embryos were fixed in 10 % neutral Formol or Bouin's solutions. These were examined for gross morphological abnormalities. The following results were obtained: the total number of embryos used were 208 of which 115 (55.28%) were normal and 93 (44.70%) displayed varying degrees and types of malformations. The most common anomalies were : anencephalia, microphthalmia, spina bifida, microsomia, rachischisis and localized haemorrhages. These anomalies appeared with a greater frequency than in the control group. These results lead us to believe that DOM (STP) is teratogenic for the developing chick embryo.
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