ABSTRACT
Axolotl embryos at various stages of development were placed in different concentrations of chlorpromazine for varying periods of time. The embryos were then reared up to stage 46 and examined with a binocular microscope.
The deleterious effect of CPZ on the embryos was found to be greater the higher the concentration and the longer the time of treatment.
The following degrees in the potency of the effect were noted: (a) almost immediate stoppage of development followed by death and cytolysis; (b) development up to stage 37–38, followed by cessation of development and degeneration; (c) development to the final stage (46) accompanied by microcephaly and pronounced defects in the eyes, gills, pigmentation, and axial organs, as judged by the external appearance of the larvae; (d) development to the final stage (46) of normal larvae with a tendency to a reduced amount of melanin in the melanophores of certain parts of the body (particularly the top of the head); (e) the development of larvae which appeared to be completely normal.
In the discussion an attempt was made to coordinate the arrest of development at stages 37–38 with Løvtrup and Werdinius’s respiration curve of developing axolotl embryos. It was found that these critical stages correspond to the second period of constant oxygen consumption prior to a rapid rise in respiration. The assumption was formulated that as a result of the effect of CPZ the embryos are unable to increase their respiratory rate in subsequent stages, with resultant cessation of development followed by degeneration.
A comparison stressing both similarities and differences between the effects of CPZ and other teratogenic substances was made.
INTRODUCTION
Chlorpromazine (CPZ) is a potent tranquillizer and is employed as such as an adjuvant in the treatment of psychiatric disorders. Because of its anti-emetic action it is used to alleviate the nausea and vomiting associated with a variety of disease processes. The potentiating effects of CPZ on the hypnotics, sedatives, analgetics, and anaesthetics have led to its use in combination with these agents (Grollman, 1960). CPZ is known to cause, occasionally, toxic reactions as hepatitis with jaundice, hypoplastic anaemia, agranulocytosis, ataxia, and dermatitis (Bernhard et al., 1955; Cares et al., 1957; Hall et al., 1956). Malformations of the offspring resulting from the use of CPZ during human pregnancy have, however, not been reported.
Numerous experiments have shown the growth-retarding effect of CPZ at various developmental stages in mammals, chickens, and other organisms (Decourt, 1953; Chambon, 1955, 1956; Maki, 1958), yet the absence of malformations is generally stressed. Only Roux in 1959 reported a higher incidence of malformation in the offspring of CPZ-treated pregnant rats.
In the present work an attempt has been made to elucidate the possible effects produced by CPZ on developing amphibian embryos, since these lend themselves to continuous observation.
METHODS
A single batch of Axolotl (Ambystoma mexicanum) eggs was used for each of the four experiments. Abnormal and undeveloped eggs were discarded. The remaining normally developed eggs were first washed for a few seconds in 70 per cent, alcohol, and then several times in sterile tap-water.
In the first three experiments the sensitivity of initial stages 10–11, 15–16, 29–30 (according to Harrison) to treatment by varying concentrations of CPZ for various periods of time was examined. For each experiment all the eggs of the same batch which had reached the required initial stage were collected and introduced in groups of 30–35 eggs into dishes, each of which contained 60–70 ml. of one of the CPZ concentrations, prepared afresh from Specia’s CPZ chlorhydrate powder. The following concentrations were used: 352×10−6 M (0·125 mg./ml.), 113×10−6 M (0·04 mg./ml.), 65×10−6 M (0·023 mg./ml.), 34 × 10−6 M (0·012 mg./ml.), and 3·5 × 10−6 M (0·00125 mg./ml.). No decapsulation of the eggs was performed in order to reduce the danger of infection as the CPZ solutions contained no antibiotics. At fixed intervals (1 hr., 2 hrs., 5 hrs., 24 hrs., and 96 hrs., respectively) 5–6 eggs were transferred from each concentration into a dish containing 60–70 ml. of sterile tap-water for at least 15 minutes. They were then transferred into a second dish of water containing 0·1 per cent, sulphadiazine and 0-020 per cent, streptomycin, in which they were allowed to develop. For each experiment controls were reared in tap-water containing the same antibiotics.
The eggs were examined every second day for their rate of development and typical morphological features. The stages were determined according to Harrison’s normal table for A. punctatum, with due regard to variations in the species used. The larvae were fixed in Lenhossek’s solution at stage 46. Before fixation, the largest and smallest specimen of each dish was photographed.
In a fourth experiment, likewise carried out on a single large batch of eggs, six different initial stages: (stages 1, 2·3, 7, 10·11, 15·16, and 29·30) were selected and merely introduced into CPZ concentration of 3·5 × 10−6 M.
RESULTS
The results of the first three series of experiments are summed up in three tables, each showing the development of eggs of one initial stage kept at different CPZ concentrations for varying immersion periods. The salient feature noted in all three tables is that the eggs show a more rapid decline in viability as the CPZ concentration rises.
CPZ concentration of 352 × 10−6 M
When kept in this concentration for 24 hours, almost complete absence of development and consequent rapid disintegration of the eggs of all initial stages occurred. The eggs of initial stage 10–11 reached stage 13 with a very crumpled animal side (Text-fig. 1a) before disintegrating. Eggs of initial stage 15–16 reached stage 17 and subsequently disintegrated, whereas eggs of initial stage 29–30 failed to develop at all before the onset of disintegration.
(a) Egg of initial stage 10–11 after 24 hours’immersion in concentration of 352 × 10−6 M of CPZ. Note crumpled animal side preceding disintegration at stage 13. (b) Egg of same initial stage after 48 hours’ immersion in concentration of 113 × 10−6 M of CPZ. Reached stage 15 with abnormal neural plate. Later disintegration set in.
(a) Egg of initial stage 10–11 after 24 hours’immersion in concentration of 352 × 10−6 M of CPZ. Note crumpled animal side preceding disintegration at stage 13. (b) Egg of same initial stage after 48 hours’ immersion in concentration of 113 × 10−6 M of CPZ. Reached stage 15 with abnormal neural plate. Later disintegration set in.
Eggs of all three stages placed in a concentration of 352 × 10−6 M for a period of 5 hours continued to develop during the first few days. Eggs of initial stage 10–11 reached stage 36 after 6 days (similar to the controls) before development ceased, and degeneration followed by disintegration occurred. In eggs of initial stage 15–16 an immediate retardation in development was noted, followed by death. Fixation of the last remaining embryo had to be carried out at stage 35. In eggs of initial stage 29–30, retardation in development was likewise noted immediately. Disintegration set in at stage 32–33.
The eggs of all three initial stages kept in CPZ concentration of 352 × 10−6 M for 2 hours showed a trend similar to that of eggs kept in the same concentration for 5 hours. Eggs of initial stage 10–11 developed normally at first, and then showed a marked lack of uniformity. Some embryos were considerably retarded and highly defective, whereas others corresponded to the controls. During the subsequent days disintegration of the epidermis and gills started. Most of the embryos did not develop further than stage 37–38. This was later followed by death. In eggs of initial stage 15–16 development was likewise normal during the first 4 days, after which embryos of various developmental stages could be recognized, which later died as a result of infection. Eggs of initial stage 29–30 developed normally for about 3 days, after which the typical signs of degeneration appeared at stage 37 (Plate 1, fig. A) and fixation had to be carried out.
The eggs of all three initial stages left in the same concentration for only 1 hour also failed to reach stage 46, and were blocked in stage 37–38.
On summing up the reaction of the eggs of the three initial stages to CPZ concentration of 352 × 10−6 M, it may be concluded that eggs introduced into the solution at a later initial stage (29–30) did not develop to a more advanced stage than those introduced earlier. Sometimes it appeared as though eggs of later initial stages were more sensitive to CPZ, and terminated their lives earlier than eggs immersed at an early initial stage.
The developmental stage of the embryos, as it appears in the tables, was determined mainly according to the shape of the gills and the fin. Classification was easy when development proceeded normally. However, in all those embryos which failed to develop normally and which were arrested at an early stage, degeneration usually set in, affecting predominantly the gills and fin, so that their stage could not be determined with certainty (as indicated by a question mark in the tables). The degeneration became more pronounced the longer the embryo was kept in CPZ and the later the initial stage. A picture of a larva immersed in CPZ concentration of 352 ×10−6 M for 2 hours at initial stage 29–30 (Plate 1, fig. A) shows the typical peeling of the epidermis as well as the characteristic deformation of the tail-fin which had at first commenced developing normally. As a result of repeated cell peelings no fin remained, the tail being represented by its stick-like axis. The general effect of this concentration of CPZ solution on differentiation, expresses itself in a tendency to microcephaly of the larvae, noticeable even after immersion of the eggs for 1 hour and becoming more pronounced after 2 hours’ immersion. At the same time, a marked retardation in melanophore development takes place. Whereas in the controls the melanophores have reached the form of branched cells organized in wide dorsoventral strips, they appear as dark dots devoid of the typical pattern in the experimental animals. In most larvae affected by CPZ treatment, the blastopore persists in the form of a wide opening from which the yolk plug still protrudes even at stage 37. These larvae furthermore exhibit a typical pronounced curvature of the body axis.
CPZ concentration of 113 × 10−6 M
While at a concentration of 352 × 10−6 M the eggs of all three initial stages failed to reach the ‘final stage’ (46) even after only 1 hour’s immersion, apparently normal larvae were obtained at a concentration of 113 × 10−6 M after 1 hour’s treatment. On longer treatment, differences between the three initial stages were noted.
From eggs of initial stage 10–11, small and defective larvae of stage 45+ on fixation were already obtained after 2 hours’ immersion in a concentration of 113 × 10−6 M (controls and 1-hour treated embryos reached stage 46). The main defects were curvatures of the body axis, small heads, degenerated eyes and gills, and defects in pigmentation consisting of a decrease in both the number of melanophores and the amount of melanin. Eggs of initial stage 10–11 left in this concentration for 5 hours survived until stage 37-38, while those kept in the same concentration for 24 hours only reached stage 22 before disintegration started. Eggs subjected to longer treatment (48 and 72 hours respectively) reached stage 14–15 within 2 days as abnormally shaped and crumpled open neurulae (Text-fig. 1b). Eggs of initial stage 15–16 reached the final stage in a macroscopically apparently normal state after 5 hours’ immersion in CPZ concentration of 113 × 10−6 M, whereas 24 hours’ treatment in the same concentration caused cessation of development at stage 37-38. When immersion was further prolonged, the development of eggs of this initial stage was blocked at still earlier stages according to the duration of treatment (see Table 2). Eggs of initial stage 29–30 developed normally after 1 and 2 hours treatment in a concentration of 113×10−6 M, while after 5 hours their development was blocked at stage 37–38. Only one larva, which was in an exceptionally good condition, was kept until the controls reached the end of the experiment. This larva had a severely curved axis, very small and abnormal eyes, and pronounced defects in pigmentation (Plate 1, fig. C). After immersion for longer periods the development of the eggs was arrested earlier in all three initial stages.
Initial stage 10–11
The numbers represent the stage (according’to Harrison) reached by the larvae on the day indicated at the top of each column.

Initial stage 15–16
The numbers represent the stage (according to Harrison) reached by the larvae on the day indicated at the top of each column.

CPZ concentration of 65×10−6 M
A more or less normal development was obtained for eggs of initial stage 10–11 after 1, 2, and 5 hours’ immersion, respectively, while after 24 hours’ immersion development was arrested at stage 37–38. Eggs of initial stage 15–16 developed normally after 1, 2, and 5 hours’ immersion in this concentration. While some of the eggs immersed in the same concentration for 24 hours were arrested, others survived until the end of the experiment, but again showed a curvature of the body axis and defects in eyes, gills, and pigmentation. In eggs of initial stage 29–30 a similar situation was encountered. After treatment in CPZ concentration of 65 × 10−6 M for 1–5 hours the larvae were normal, but of those which underwent 24 hours’ immersion only one larva survived until the end of the experiment, showing the characteristic defects (Plate 1, fig. D).
CPZ concentration of 34×10−6 M
The eggs of all initial stages developed normally even after 24 hours’ immersion. After 48 hours’ treatment, eggs of initial stage 10–11 were arrested at stage 37–38 in a state ranging from the practically normal to the microcephalie. Larvae developed from eggs of initial stage 15–16 were badly affected after 48 hours’ immersion and disintegrated as a result of infection. All larvae developed from eggs of initial stage 29-30 kept in a concentration of 34 × 10−6 M for 48 and 72 hours,were defective, thus occupying an intermediate position between normal and arrested development. In Plate 1, fig. E a defective larva is shown which developed from an egg treated for 48 hours. It shows the typical curvature, almost complete absence of melanin, degeneration of the fin, and reduction of the eyes. The larva in Plate 1, fig. F developed from an egg treated for 72 hours. Similar curvature is seen, the head is noticeably short, and the eyes are much reduced, but the melanin is unaffected.
CPZ concentration of 3·5 × 10−6 M
Defects first appeared in the group of initial stage 10–11 after 96 hours’ treatment. Eggs of initial stages 15-16 and 29-30 developed into macroscopically normal larvae even after 96 hours’ treatment. In this concentration some eggs of each initial stage were left permanently in CPZ solution. None of them was able to reach stage 46. However, the permanently immersed eggs of initial stage 29–30 succeeded in reaching stage 44–44+ in a defective and poor condition, and subsequently died.
A comparative experiment at a concentration of 3·5 × 10−6 M
The fourth experiment was carried out in a different way. Each of the three previous experiments had been limited to eggs of a certain initial stage, submitted to several CPZ concentrations. It was therefore difficult to draw up an accurate comparison between the reactions of the various initial stages to CPZ, as each of them had been collected from a different batch of eggs which might have had a slightly different degree of sensitivity to CPZ. It was therefore decided to perform a complementary experiment on a number of initial stages selected from one batch, and treated by the same CPZ concentration. In view of the relatively small number of eggs thus available, CPZ concentration of 3·5 × 10−6 M alone was chosen as the one permitting a relatively favourable development. In view of the data obtained from previous experiments it was decided to make only three transfers of eggs from the CPZ solution, namely after 24,48, and 96 hours. Some of the eggs were kept in CPZ solution permanently. The solution in each dish was replaced daily by a new CPZ solution prepared afresh from powder. The sterile tap-water (plus antibiotics), in which the control and experimental animals were kept after transfer, was likewise changed daily.
In this experiment all the larvae which developed from eggs treated up to and including 96 hours reached the final stage (46). Among these, the larvae which developed from initial stages 1 and 2–3 were normal, except that some of the 96-hours treated larvae showed a defect in the pigmentation of the head region. The photographs in Plate 2 show the gradual disappearance of melanin from the dorsal region of the head. In larva B (initial stage 2–3, 96 hours’ treatment) the melanophores have a centre devoid of melanin, as compared with the control larva A, while in larva C (initial stage 1, 96 hours’ treatment) there is a conspicuous ‘bald spot’ on the dorsal surface of the head, due mainly to a reduction in the amount of melanin, but possibly also to the decrease in the number of melanophores in this region.
A strong reduction in pigment appeared also in the first three experimental series, where the phenomenon was more marked at the higher concentrations, as seen in three of the defective larvae of initial stage 29–30 (Plate 1, C, D, E). In these cases the damage to pigmentation was mostly acute and noticeable also in other parts of the body, as in animal C, where a strong reduction in the number of melanophores all over the body occurred. An intermediate stage between this and the normal condition was represented by a larva of initial stage 29–30 after 48 hours’ treatment in CPZ concentration of 34 × 10−6 M (Plate 2, fig. E). Here the lateral melanophores were very pale owing to the paucity of pigment. In this case the pallor of animal E as compared to the control D was not due to the presence of a lower number of melanophores, but to the scanty amount of melanin in the melanophores.
The development of the larvae of the 4 later initial stages treated for up to 96 hours in CPZ concentration of 3·5 × 10−6 M was generally normal, but the results were not as uniform as in the two earlier initial stages. Even after 24 hours’ treatment, random cases of larvae with defective pigmentation or relatively smaller eyes appeared. The daily follow-up of all experimental groups indicated that the development of larvae of initial stages 1, 2–3, and 29–30 was uniform within each group subjected to a certain treatment period, whereas the development of larvae of the initial stages 7, 10–11, and 15–16 was non-uniform. In the latter larvae different stages of development were reached as soon as 4 days after the beginning of the experiment.
The behaviour of the embryos immersed permanently in a concentration of 3·5 × 10−6 M was identical in each of the first five initial stages. The initial development was normal and equivalent to that of the controls, and in some cases the treated embryos even seemed to overtake the controls in their rate of development during the first few days. Normal development continued until the larvae reached approximately stage 38. At this stage degeneration set in without any direct relation to the duration of immersion in CPZ (which varied according to the initial stage, being shorter as the initial stage becomes more advanced). The phenomena characterizing this degeneration were uniform for all initial stages. The larvae started losing their mobility which had previously corresponded to their stage of development. Extensive peeling of the epidermis commenced until it disintegrated completely. As a result fistulae were formed in the abdominal region and the yolk extruded from the intestines. The peeling process affected particularly the gills and fin, which gradually disappeared. At stages later than 37–38, rapid differentiation of the head region normally occurs, but in the permanently immersed larvae it was soon noted that the head failed to develop normally, exhibiting a marked tendency to microcephaly and a gradual transition to an expanded anterior body region. The eyes were as a rule rudimentary. At this stage disturbances in the hitherto normal blood circulation occurred; haemorrhages were noted in various regions and it seemed as though all blood-cells gradually disappeared from circulation until finally the heart stopped beating. The larvae which showed signs of degeneration were transferred to a fresh CPZ solution, but when it was found that there was no chance of survival it was decided to transfer them to ordinary water to see whether they might then survive. The larvae of initial stages 1,2–3, and 7 were thus transferred after 16 and 17 days, the larvae of initial stage 10–11 after 15 days, and the larvae of initial stage 15–16 after 11–12 days. All these larvae were in an identical condition and at approximately the same developmental stage. After having been kept in water for an additional 2 days, fixation of the larvae had to be carried out.
The permanently immersed embryos of initial stage 29–30 were placed in CPZ relatively late (5 days later than those of initial stage 15–16) and after 5 days reached stage 37-38 in a good and active condition. They continued to progress slowly beyond that stage. Eight days after the beginning of the experiment it became clearly noticeable that they were microcephalie, but otherwise in good condition and of normal mobility. Ten days after the start of the experiment, signs of disintegration were noted at the ends of the gills and fins, whereupon all the larvae were transferred to water in an attempt to prevent degeneration at the earliest possible sign of damage. They were kept in water for an additional 4 days until their controls had reached stage 46. Progressive peeling of the epidermis was noted throughout, including degeneration of the gills and fins. In some, the typical haemorrhages and loss of mobility appeared.
DISCUSSION
From the experiments described the impression was gained that in all cases a sufficiently high concentration of CPZ was capable of arresting development almost immediately, followed later by typical cytolysis. Under milder CPZ treatment development was able to proceed normally for a certain period, and the subsequent grave results only became apparent at a much later stage. Our results show that after a very strong effect of CPZ (resulting from a combination of high concentrations and prolonged treatment) not followed by immediate death the embryos usually develop normally up to stage 37-38. This seems to be a critical stage at which their development ceases and degeneration sets in. Larvae which succeeded in passing beyond stage 38 were generally able to reach the terminal stage of the experiment, regardless of whether they were normal or defective. These findings have been compared with those of Løvtrup & Werdinius (1957) and Løvtrup (1959) who measured the oxygen consumption of axolotl embryos for a period of about 40 days from the beginning of their development. These authors showed that there is a general rise in oxygen consumption during the first 25 days of development at a temperature of 15–16° followed by a steep decline. They were, however, able to show that on the ascending line of the curve, two periods of constant O2 consumption could be distinguished, one starting at the end of the late neurula stage and continuing throughout most of the tail-bud stage, and the other at the larval stage. An attempt was made to ascertain whether a connexion exists between stages 36–37–38, at which the larvae in our experiment usually ceased to develop, and between Løvtrup’s oxygen consumption curves, and whether these were of physiological significance. This comparison was rendered difficult by the fact that the curves do not indicate larval stages, but merely days of development at a given temperature. The temperature at which our experiments were conducted was not constant, and on the average somewhat higher than that used by Løvtrup. We were able to calculate that in our experiments the development was roughly 5/4 times more rapid, and that stages 36–37–38 corresponded to the second ‘constant respiration’ period. During this period fat consumption is said to form the main source of energy, steeply decreasing at the end of the period when protein combustion commences to rise rapidly. The arrested development of our experimental animals may thus theoretically be linked to interference with protein consumption, an assumption which still lacks factual support. It is interesting to note that dinitro-phenol, whose teratogenic effect is generally similar to that of CPZ, seems to inhibit the penetration of basic proteins into the cell (Fischer & Wagner, 1954, quoted by Brachet, 1957), and that an effect of CPZ on the cell membrane has been suggested by Karreman et al. (1959). The arrested development at this stage might, on the other hand, be ascribed to damage to the cellular respiratory systems preventing the larvae from increasing their respiratory rate after the constant period, and thus leading to a metabolic standstill which inhibits differentiation and is followed by inevitable degeneration. In this connexion it should be mentioned that various authors have shown that CPZ has an arresting effect on the general metabolism, due to the inhibition of various enzyme systems and the uncoupling of oxidative phosphorylation (Grenell et al., 1955; Abood & Romanchek, 1957; Berger et al., 1957; Bernsohn et al., 1958).
More specifically the uncoupling of oxidative phosphorylation at the stage of ferrocytochrome-C oxidation, and the inhibition of ATP-ase and cyto-chrome-c-oxidase activity have been demonstrated (Dawkins et al., 1959, a, b;,Dawkins et al., 1960). In addition interference with mitochondrial diaphorase flavin (Low, 1959), inhibition of d-amino-acid oxidase (Yagi et al., 1960), decrease of phospholipid turnover (Ansell & Dohmen, 1956), and inhibition of cholin-esterase (Courvoisier et al., 1953) have been observed.
The action of CPZ is also assumed to be connected with its property of being a powerful electron donor with unique charge transfer properties (Karreman et al., 1959).
Among the larvae which managed to survive beyond stage 38 and continued to develop until the termination of the experiment, some appeared completely normal, while others, subjected to higher CPZ concentrations for longer periods, showed typical defects. The affected animals generally showed a tendency to microcephaly, outwardly expressed by a shortening of the anterior part of the head and a tendency to degeneration of the eyes (ranging from decreased size to almost complete absence). A similar effect has been described by several authors as a result of various substances probably acting through different cellular systems. Thus Dawson (1938) noted similar phenomena in Rana pipiens following treatment of the eggs with 2,4-dinitrophenol (at a concentration of 5·43 × 10−6 M)—a substance also known to cause uncoupling of oxidative phosphorylation, although in a slightly different way to CPZ. Similar findings were those of Deuchar who examined the effect of chloro-acetophenone (CAP) (14–20 ×10−6 M), on the development of Xenopus laevis embryos. Lack of oxygen (Rübsaamen, 1950) is a further factor known to cause microcephaly.
Deuchar stresses the specificity of the effect of CAP, which causes defects to the nervous system without damaging the mesoderm, and suggests that this effect may perhaps be characteristic of the action of SH inhibitors, of which CAP is one. Dawson, on the other hand, also obtained a clear effect on the mesoderm with 2,4-dinitrophenol, but did not regard his results as being specific. External examination of our material indicates that here, too, damage may have occurred to the mesodermal tissues, in view of the distinct tendency to curvature of the body axis in most of the affected animals. Unfortunately, our results cannot be fully compared with those of Dawson despite the fact that our lowest concentration (3·5 × 10−6 M) was even lower than his concentration of 2,4-dinitrophenol (5·43 × 10−6 M). While Dawson was able to rear the embryos constantly in the solution with good results, we were not able to do so, apparently owing to the relatively higher sensitivity of our material to CPZ. On the other hand, Dawson did not carry out parallel experiments of short-term immersion in higher concentrations or a protracted follow-up. In this respect there is a close resemblance between our experiments and those of Deuchar, in which the concentration used was roughly between that of our two lowest concentrations. Deuchar left the embryos in solution for varying periods of several hours each, at different developmental stages, and then reared them normally. Both in Deuchar’s and in our experiments the defects appeared at relatively later stages of development. Thus some embryos immersed as gastrulae for 2 hours in CAP or CPZ (concentration of 113 × 10−6 M) solution, later developed serious eye defects. Preliminary experiments not included in this work have shown that even uncleaved eggs left for a short time in the higher concentrations of CPZ develop into defective larvae. The question thus arises as to the time of action of the substances mentioned and as to whether they are stored in the eggs and assert their effect in the course of time on the development of certain organs, or whether they bring about early inhibition of one of the metabolic systems which manifests itself at a later developmental stage. Only further biochemical and metabolic experiments may elucidate this point. In this connexion the phenomenon of depigmentation might be mentioned, as this is a further late expression of the early action of the drug, apparently connected with an inhibition of melanin synthesis.
A further similarity between our observations and those of Dawson lies in the fact that in those cases in which development ceases completely at a relatively early stage, an accelerated rate of development was noted prior to such arrest. This phenomenon might also imply a similar mode of action of 2,4-dinitrophenol and CPZ.
RÉSUMÉ
Effet de la chlorpromazine sur le développement embryonnaire d’Axolotl (Ambystoma mexicanum)
Des embryons d’Axolotl ont été placés à divers stades du développement dans différentes concentrations de chlorpromazine pour des durées variables. Les embryons furent élevés par la suite jusqu’au stade 46 et ont fait l’objet d’une étude externe au stéréomicroscope.
Les effets toxiques de la CPZ sur les embryons croissent avec la durée du traitement et la concentration.
Les degrés suivants de cet effet ont été observés: (a) Arrêt presque immédiat du développement, suivi de la mort avec cytolyse. (b) Développement se poursuivant jusqu’au stade 37-38, puis s’arrêtant avec des phénomènes dégénératifs. (c) Développement jusqu’au stade de référence (46), avec microcephalie et anomalies marquées des yeux, des branchies, de la pigmentation et des organes axiaux, autant que l’étude externe permet d’en juger, (d) Développement jusqu’au stade de référence (46) avec apparence normale, sauf une tendance à présenter une certaine pauvreté en mélanine des mélanophores de certaines parties du corps (en particulier de la tête), (e) Développement apparemment normal.
Dans la discussion une relation est envisagée entre l’arrêt du développement au stade 37–38 et la courbe de la respiration de l’embryon d’Axolotl au cours du développement établi par Løvtrup et Werdinius. Il apparaît que ces stades critiques correspondent à la seconde période de consommation constante d’oxygène qui précède l’élévation rapide de la respiration. Il est supposé que l’effet de la CPZ sur les embryons se traduise par l’incapacité d’accroître leur taux respiratoire aux stades suivants, amenant un arrêt du développement suivi de phénomènes dégénératifs.
Une comparaison est établie entre les effets de la CPZ et des autres substances tératogènes, mettant en évidence les différences et les similitudes.
ACKNOWLEDGEMENTS
The authors wish to express their thanks to Specia Paris for the donation of Largactil used in this work. We also are grateful to Miss Sh. Citron for her devoted help.
REFERENCES
EXPLANATION OF PLATES
Initial stage 29–30
Fig. A. A typical larva of initial stage 29–30 after 2 hours’ treatment in CPZ concentration of 352 × 10−8 M. The head is degenerated, the epidermis disintegrated, the blastopore enlarged. The tail has undergone degeneration and only its axial organs remain.
Fig. B. Control of D.
Fig. C. The only larva which reached the end of the experiment after 5 hours’ treatment in CPZ concentration of 113 × 10−8 M.
Fig. D. The only larva which reached the end of the experiment after 24 hours’ treatment in CPZ concentration of 65 × 10−8 M.
Fig. E. Larva after 48 hours’ treatment in CPZ concentration of 34 × 10−8 M.
Fig. F. Larva after 72 hours’ treatment in CPZ concentration of 34 × 10−6 M.
Fig. G. Control of C–F.
Approximate magnification × 7.
Initial stage 29–30
Fig. A. A typical larva of initial stage 29–30 after 2 hours’ treatment in CPZ concentration of 352 × 10−8 M. The head is degenerated, the epidermis disintegrated, the blastopore enlarged. The tail has undergone degeneration and only its axial organs remain.
Fig. B. Control of D.
Fig. C. The only larva which reached the end of the experiment after 5 hours’ treatment in CPZ concentration of 113 × 10−8 M.
Fig. D. The only larva which reached the end of the experiment after 24 hours’ treatment in CPZ concentration of 65 × 10−8 M.
Fig. E. Larva after 48 hours’ treatment in CPZ concentration of 34 × 10−8 M.
Fig. F. Larva after 72 hours’ treatment in CPZ concentration of 34 × 10−6 M.
Fig. G. Control of C–F.
Approximate magnification × 7.
Fig. A. Head of control at stage 46 (end of experiment) with normal pigmentation.
Fig. B. Head of larva immersed in CPZ concentration of 3·5 × 10−6 M. for 96 hours at initial stage 2–3. Melanophore in top of head poor in pigment and showing a pale inner ‘court’.
Fig. C. Head of larva immersed in CPZ concentration of 3·5 × 10−8 M for 96 hours at initial stage 1. A typical unpigmented area is seen on the head.
Fig. D. Control showing the characteristic pigmentation of the flank (stage 46).
Fig. E. Larva immersed in CPZ concentration of 34 × 10−8 M for 48 hours at stage 29–30. Normal number of melanophores, which are, however, poorly pigmented.
The photography and printing of the above pictures were carried out under identical conditions.
Approximate magnification × 20.
Fig. A. Head of control at stage 46 (end of experiment) with normal pigmentation.
Fig. B. Head of larva immersed in CPZ concentration of 3·5 × 10−6 M. for 96 hours at initial stage 2–3. Melanophore in top of head poor in pigment and showing a pale inner ‘court’.
Fig. C. Head of larva immersed in CPZ concentration of 3·5 × 10−8 M for 96 hours at initial stage 1. A typical unpigmented area is seen on the head.
Fig. D. Control showing the characteristic pigmentation of the flank (stage 46).
Fig. E. Larva immersed in CPZ concentration of 34 × 10−8 M for 48 hours at stage 29–30. Normal number of melanophores, which are, however, poorly pigmented.
The photography and printing of the above pictures were carried out under identical conditions.
Approximate magnification × 20.