Cholinomimetic Teratogens: Studies with Chicken Embryos WALTER LANDAUER Depccrtment of H7rmcin G e n e t i c s u n d Bzometry, Uiniverszty College L o n d o n , London N W l 2 H E , E n g l a n d
ABSTRACT The cholinomimetic compounds carbachol, decamethonium, neostigmine, succinylcholine, trimethylphenylammonium, and others were tested for their interference with normal chick development. All these compounds led to abnormalities of the cervical vertebrae; at higher dosage interference with normal morphogenesis involved the whole vertebral column. Hypoplasia of the leg muscles occurred with lower incidence. Responses, tested with carbachol, rose from 24 to 72 and 96 h, then declined to 120 h of incubation. Two of the cholinometic compounds used in combined treatment produced a high degree of synergism. Gallamine, benzoquinomium, butyrylcholine, and bethanechol had protective effects. Acetylcholine, at high dosage, caused defects different from the above. It is suggested that the cholinomimetic teratogens interfere with normal development by displacing acetylcholine from its receptors or by forming complexes with i t .
The experimental teratologist is likely at times to encounter in his test-animals abnormalities of unrelated parts or organs the nature of which may pose problems of peculiar interest. He may well wonder if certain defects are parts of a syndrome in the sense of deriving either directly or by dependent steps from a common cause or if, alternatively, they originate from distinct chemical peculiarities of the teratogen in use to which particular tissues react dissimilarly. It was questions of this nature that arose from experiments with chicken embryos in which distinct teratogens administered at identical developmental stages produced defects which in their morphological and metabolic expressions appeared curiously unrelated. But were they? A brief historical review provides the setting for our new experiments. Hypoplasia of the leg muscles and defective formation of cervical vertebrae were found to occur in combination when chicken embryos had at a given stage been treated with 3-acetylpyridine, nicotine sulfate, or ethionine. The major effect of 3-acetylpyridine occurred as muscular hypoplasia, vertebral abnormality and shortness of the upper beak being encountered less commonly (Ackermann and Taylor, '48; Landauer, '57; Herrmann et al., '64). The principal result of treatment with nicotine sulfate, on the other hand, was a brevicollis condition in which the cervical vertebrae TERATOLOGY, 1 2 : 125-146.
were abnormal and often fused, muscular hypoplasia occurring less frequently or only with higher dosage (Landauer, '60; Strudel, '71). Exposure of embryos to ethionine, similar to 3-acetylpyridine, led to muscular hypoplasia and at a lower level of incidence to brevicollis and abnormal formation of the beak (Karnofsky et al., '55; Landauer and Salam, '74). Malformations due to 3-acetylpyridine can be prevented completely by giving suitable supplements of nicotinamide (Landauer, '57); nicotine treatment is not ameliorated by added nicotinamide, but considerable protection is afforded by supplements of certain amino acids, glucose, and sodium pyruvate; the teratogenicity of ethionine is greatly reduced in the presence of methionine. Physostigmine (eserine) differs from the preceding teratogens by producing, in addition to defective cervical vertebrae, micromelia, syndactylism, and abnormalities of the visceral skeleton and of the eyelids (Ancel, '45; Landauer, '49; Bueker and Platner, '56; Landauer and Salam, '72; Sullivan, '73). According to Bueker and Platner "reduced musculature of the legs" also occurs and this presumably following the use of relatively large amounts of the compound. Nicotinamide as supplement protects completely against physostigmine-induced micromelia and beak Received Jan. 13, '75. Accepted Apr. 25, '75.
125
126
WALTER LANDAUER
defects, but does little, if anything, in preventing the brevicollis condition. Finally there are the organophosphorous insecticides bidrin, azodrin, parathion, and others. Bidrin-induced abnormalities of chicken embryos, as far as skeletal defects are concerned, include brevi- and torticollis, often extending to other parts of the vertebral column, micromelia, parrot beak, and syndactylism. Nicotinamide and related compounds act as alleviating agents - except that they have little, if any, effect in preventing vertebral abnormality. Inhibition of acetylcholinesterase activity and that of other esterases was found not to correlate in a causal manner with the degree of teratogenesis of bidrin (Upshall et al., '68; Roger et al., '69). The teratogenic responses to parathion are morphologically quite similar to those brought about by bidrin (Iseki and Yamada, '66; Yamada, '68, '72; Khera and Bedok, '67; Lutz-Ostertag et al., '69a,b). The teratogenic effects that azodrin has on chicken embryos, much as those of bidrin, chiefly comprise maldevelopment of the spine and micromelia; in quail embryos the effect of azodrin includes in addition to a shortened and deformed vertebral column "amuscular legs," i.e., muscular hypoplasia (Schom et al., '72). A somewhat similar difference in response between chicken and quail embryos has been reported from tests with the organophosphorus insecticide dursban (Schom et al., '73). Chicken embryos exposed to malathion and malaoxon, on the other hand, react in their skeletal development with micromelia and beak defects, but without vertebral deformities or muscular abnormality. Supplements of tryptophan or nicotinamide prevent the malathion or malaoxon-induced defects, but do not counteract the inhibition of acetylcholinesterase activity (Greenberg and LaHam, '69, '70; Walker, '71 ; Wilson et al., '73). The intriguing questions raised by these observations suggested the desirability of further experimentation with compounds known for their cholinomimetic activity, i.e., substances related to and/or competing with acetylcholine (Feldberg, '57). This led to the work that is to be reported here with compounds that are among the best known inhibitors of neuromusccular transmission and of postsynaptic receptors.
METHODS A N D MATERIALS
The general methods have remained much the same as in earlier work (Landauer and Wakasugi, '67; Landauer and Sopher, '70; Landauer and Salam, '72-'74). Teratogens and supplementing compounds were injected into the yolk sac of chicken eggs, usually after 96 h of incubation, but in a few instances earlier or later. Most of the solutions were prepared in distilled water, with 0.3% phenol added; ethionine was dissolved in 5 N hydrochloric acid. The eggs were incubated in a Petersime automatic incubator and opened for inspection after 19 days. The chemicals used in our work and their sources were as follows: acetylcholine chloride, 3-acetylpyridine, butyrylcholine chloride, and tetracaine, Sigma London Chemical Co. Ltd.; atropine sulfate, carbachol, hexamethonium bromide, decamethonium iodide, and tubocurarine chloride, Koch Light Laboratories Ltd.; L-acetyl-pmethylcholine chloride (Methacholine), tetraethylammonium chloride, and tetramethylammoniumchloride, Ralph N. Emanuel Ltd. ;trimethylphenylammonium iodide, BDH Chemical Ltd.; dichlorphenamide, Merck Sharp and Dohme Ltd.; sodium acetazolamide, Lederle Laboratories; nicotine sulfate and physostigmine, Hopkin and Williams Ltd. In addition we received and used the following gifts: bethanechol chloride (Myotonine), Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey; benzoquinonium chloride, Sterling-Winthrop Research Institute, Rensselaer, New York; edrophonium chloride, Roche Products Ltd., Welwyn Garden City, Hertfordshire; gallamine triethiodide (Flaxedil), May and Baker Ltd. Medical Products Division, Dagenham, Essex; and pyridostigmine bromide (Mestinon), Hoffmann-LaRoche Inc., Nutley, New Jersey. Wilh most of these compounds we initially performed small-scale tests as guide of toxicity, subsequent experiments being numerically adjusted accordingly. Statistical analyses of our data were based on the texts of Snedecor ('48) and Bliss ('67). RESULTS
Quantitatiue observations following treatment at 96 h of incubation Data relating to experiments with carbachol chloride, decamethonium iodide, and
127
CHOLINOMIMETIC TERATOGENS
neostigmine bromide are presented in table 1 . The results with carbachol point to a gradual increase of toxicity with rising dosage, but more extensive data might have shown deviations from a smoothly mounting dosagelmortality relation. Interference of carbachol with normal chick development was twofold, viz., abnormality of the vertebral column and hypoplasia of the leg muscles. Both types of effect are dosage related in incidence and expression. Following treatment with 0.5 mglegg carbachol the great majority of embryos appeared quite normal, but on rare occasions the neck was short and the leg musculature was somewhat less well developed than in control embryos. With increasing dosage the incidence of abnormalities rose sharply, involving vertebral development of all embryos when treatment reached 3 mglegg, the great majority of embryos showing at the same time subnormal growth of the leg musculature. Furthermore, with increasing dosage of carbachol irregular morphogenesis and fusion of vertebrae tended to spread from the cervical region posteriorly, thereby causing humps of the back, compression of the trunk, and retraction of the tail vertebrae into the pelvis, the latter condition creating the impression of “rumplessness.” Major involvement of the trunk frequently was responsible for joint abnormalities of the legs. Muscular hypoplasia also became
more pronounced with increasing dosage of carbachol. The muscles of the right leg were often more markedly involved than those of the left, an asymmetry of response that occurred with higher frequency following treatment at 72 than at 96 h of incubation. In the presence of more extreme instances of muscular hypoplasia, and presumably as a secondary effect, some embryos had their toes in a curled position. Whenever dosages of carbachol between 1 and 3 mglegg were injected the incidence of muscular hypoplasia was regularly lower than that of vertebral involvement. In experiments with decamethonium and neostigmine there was no clear relation of dosage to embryo mortality. The developmental effects produced by the 2 compounds were very similar to those recorded for carbachol. In both instances the teratogenic responses of vertebrae exceeded those of the leg muscles. Observations on the effect of treating 96h chicken embryos with either succinylcholine chloride or trimethylphenylammonium iodide (TMPA) are represented by the data of table 2. The 2 compounds interfered with normal development of vertebrae in a way very similar to that shown for the first 3 teratogens. A s in the former material there was a clear dosagelresponse relation. The effects produced by succinylcholine and TMPA differed, however, from those of
TABLE 1
T h e effect on developing c h i c k e n embryos of trecitment w i t h curbcichol chloride, d e c a m e t h o n i u m iodide, a n d n e o s t i g m i n e bromide according t o dostrge i n mglegg, a t 96 h of incnbation No treated
Mortality to 18 days ( ” i )
0.5 1.0 1.5 2.0 2.5 3.0
60 79 87 74 332 57
6.7 11.4 10.3 10.8 12.7 24.6
0.5 0.8 1.5 2.5
64 34 88 51
7.8 11.8 13.6 5.9
0.1 0.2 0.4 0.6
10
10.0 0 22.9 5.3
Dose (mg)
N o of survivors
Normal ( % )
Short/crooked neck ( o i l
Muscular hypoplasia i f > )
1.8 17.1 51.3 65.2 94.8 100
I .8 7.1 16.7 28.8 55.9 86.0
50.8 63.3 100
10.2 63.3 75.8 62.6
Cnrbachol chloride 56 70 78 66 290 43
98.2 84.3 33.3 13.6 5.2 0
D e c a m e t h o n i u m iodide 59 30 76 48
49.2 26.7 0 0
100
Neostigmine b r o m i d e 10
35 38
9 10
27 36
100
80.0 33.3 2.8
0 20.0 63.0 97.2
0 0 0
47.2
128
WALTER LANDAUER TABLE 2
T h e cfSect oii deweloping c h i c l t r n emhiyo!, of tretitme7it with siiccinylcholinr c h l o r i d e ctnd trimrthylphriiyl(tmmoniitm i o d i d e crccorditzg to dosctge in m g l e g g , cit 96 h ofznc7ihation Dose ( m g )
No treated
Mortality to 18 days ( ‘ c )
No of survlvors
Normal
(‘c
1
Shortlcrooked neck (‘, )
Sitccinylc ho12ne c h l o r i d e
25 10
15 20 30 1.5 2.5 5.0 10.0
29 36 30 29 10 40
15 13
0 2.8 13.3 17.2
29 35 26 24
T r i m r t h y l p h e i z y l t c m m o ~ 7iitm i o d i d e 0 10 7.5 37 26.7 11 61.5 5
the former teratogens in that they interfered only rarely with normal muscle development. Additional data on this problem will be reported in the sequence. Experiments with tetramethylammonium chloride and pyridostigmine bromide, 2 other compounds with cholinomimetic activity, will not be reported in detail. The effects of tetramethylammonium were tested in amounts of 2.5 mgleggl96 h and over and those of pyridostigmine in dosages of 10 mg/egg or more. Both compounds were responsible for short and crooked necks and muscular hypoplasia of the legs similar to the data of table 1. The extent to which the various compounds interfered with normal vertebral morphogenesis was in increasing order approximately as follows: succinylcholine < trimethylphenylammonium < pyridostigmine < tetramethylammonium < carbachol < decamethonium < neostigmine. A similar sequence seems to be true for interference with muscle development. Size and weight of the body tended to decline as incidence and extent of malformations produced by the various teratogens rose, but metric data were not collected. Compounds that, in the given dosage ranges of mglegg at 96 h, produced no gross abnormalities were as follows: Lacetyl- p -methylcholine chloride 2.5-60 mg, benzoquinonium 0.5-5 mg, bethanechol chloride 2.5-40 mg, butyrylcholine chloride 2.5-20 mg, edrophonium chloride 1.5to 6 mg, gallamine triethiodide 1.510 mg, hexamethonium bromide 2.5-60 mg, tetraethylammonium chloride 2.5-30 mg, and
100
0
85.7 69.2 33.3
14.3 30.8 66.7
100 97.3 54.5
2.7 45.5
0
0 100
n-tubocurarine chloride 1.5-5 mg. Butyrylcholine was, however, highly toxic above 2.5 mglegg and tetraethylammonium above 20 mglegg. Effects of deuelopmental stage o n carbachol treatment The stage responses of chicken embryos to treatment with carbachol chloride were followed by injecting the compound into eggs at 24, 72, 96, and 120 h of incubation. The results of these tests are presented in table 3. The dosages were varied between 1.0 and 2.5 mglegg in order to demonstrate the effects on toxicity and morphogenesis at comparable levels. There was little difference in embryo mortality caused by treatment between 24 and 96 h, but given at 120 h of incubation the compound clearly had greater toxicity than at the earlier stages. The morphological responses to carbachol treatment were of the same type between 24 and 120 h, but incidence and extent of vertebral and muscular abnormality varied with time. There was a steep rise in teratogenicity from 24 to 72 h; a moderate lowering in incidence of malformations between 72 and 96 h (which was particularly significant at 1.5 mglegg); and a precipitous decline of teratogenic response from 96 to 120 h of incubation. Synergistic effects of cholinomimetic compounds in combination The results of previous observations had made i t evident that subtle differences occur in the specific developmental responses provoked by some of the cholinomimetic
129
CHOLINOMIMETIC T E R A T O G E N S TABLE 3
Tercttologictr I responses of chicken e m b r y o s to ctcrbtcchol chloride uccordiizg to developmentcil sttrge of tretrtment. Doscigr wus vciried between 1 .O clnd 2.5 m g . The dutctfor 96 h w e r e t o k e n f r o m tuble I Incubation stage (h)
Muscular hypoplasia
(mg)
No. treated
Mortality to 18 d a y s ( r~ 1
Normal ( % )
Abnormal neck ( ‘ c )
24
1.0 1.5
38 37
11.8 18 9
34 30
85.3 53.3
2.9 36.7
14.7 36.7
72
1.5 2.5
41 38
7.3 15.8
38 32
10.5 3.2
65.8 90.6
86.8 96.9
96
1 .0 1.5 2.5
79 87 332
11.4 10.3 12.7
70 78 290
84.3 33.3 5.2
17 1 51.3 94.8
7.1 16.7 55.9
120
1.5 2.5
34 36
29.4 33.3
24 24
87.5 25.0
12.5 75.0
0 25.0
Dose
compounds. Thus, among embryos that had been treated with carbachol or succinylcholine and of which 65.2 and 66.7% , respectively had an abnormally short neck, this particular abnormality had in nearly all of the former and in very few of the latter embryos been combined with irregular fusion of cervical vertebrae, or that treatment with carbachol, decamethonium, or neostigmine was likely to produce a “syndrome” of abnormal neck and muscular hypoplasia, but that succinylcholine and trimethylphenylammonium while causing dosage-dependent abnormality of the cervical vertebrae only rarely produced muscular defectiveness. It was differences of this order that raised the question of what would result if 2 such compounds were applied together? Would there be interaction or exaggeration of teratogenic effects? In order to obtain meaningful results it seemed desirable to combine any 2 compounds in such dosages that their teratogenicity ,when applied singly, would be at a relatively low level. The results are shown in tables 4-8. Data for carbachol and decamethonium, singly or combined, are provided in table 4. With carbachol alone 95.7% of the treated embryos were grossly normal, 32.1 % of the decamethonium-treated embryos escaped damage, but in combination only 2.3% of the surviving embryos failed to show either malformation of the neck or muscular hypoplasia or both. The teratogenic exaggeration of the combined treatment, if compared with the sum of the 2 controls, is highly significant. Body weight was greatly reduced after the combined
No. of survivors
(CC)
treatment, the vertebral column tended to be much shortened and deformed in its whole length, retraction of its posterior section giving the impression of rumplessness, and the trunk was compressed. In tests in which carbachol and succinylcholine were used singly or combined 15 mglegg succinylcholine and 0.5 or 1.0 mg carbachol were injected (table 5). With the lower dosage of carbachol, used as control, none of the surviving embryos were abnormal; when succinylcholine was injected 14.7% of the survivors had a defective neck; but in the combined treatment 62.5% of the survivors showed the abnormality. A similarly striking exaggeration in defectiveness of the cervical region occurred when 1.0 mg carbachol was combined with 15 mg succinylcholine. In none of the 3 control groups were there specimens with muscular hypoplasia, but following treatment with 1.0 mg carbachol and 15 mg succinylcholine there was 1 such case among 23 survivors. Table 6 presents information on the single or combined treatment with trimethylphenylammonium iodide (TMPA) and succinylcholine chloride at dosages of 2.5 and 15 mg, respectively. The tests with these 2 compounds are of particular interest since there were but rare instances of muscular hypoplasia in the 2 control groups. The synergistic effect of vertebral as well as muscular abnormality due to the combined treatment is evident. Especially striking results occurred in the combination of TMPA and carbachol chloride. TMPA was used at 2.5 mglegg and
1 30
WALTER LANDAUER TABLE 1
T h e effects ofcurbtrchoi a n d dectrmethoniitm srpctrrctely or in combination. Treiitment 96 h ofincirbution. Dosctgus: curbuchol 1.5, d e c a m e t h o n i z t m 0.25 mgiegg Carbachol and decamethonium combined
Carbachol
24 4.2 23 95.7 4.3
N o treated Mortality to 18 days ('X ) No of survivors Normal (clo ) Shorticrooked neck (5: ) Muscular hypoplasia ( % ) 1
Decamethonium
54 20.4 43 2.3 I 97.7 55.7
87 10.3 78 32.1 64.1 16.7
'
'
0
tct
Chi square over sum of both controls with P < 0.001
TABLE 5
The effects of'carbuchol ( C ) and s n c c i n y t c h o l m e c hloride (Sc) septtrcctely or ~n combzntrtion T r e a t m e n t u t 96 h ofincdxttzon T h e dostiges o f c n r b a c h o t w e r e 0 5 or 1 m g , t h a t of sztccznylcholtne I 5 mglegg sc sc
+
0.5 mg C
C 0.5 mg
+
sc 1 mgC
C 1 mg
~~
37 5.4 35 85.7 14.7
No. treated Mortality to 18 days (% ) No. of survivors Normal ( % 1 ShorVcrooked neck ( % ) Muscular hypoplasia ( V Misc. defects ( % ) I
0 0
TABLE 6
The effects of tnmethylphenyl~rmmo,Ililm iodzde ( T M P A ) and szrccznylcholine chlonde ( S c ) o n chick development b y t r e a t m e n t , separately or in c o m bination, at 96 h of incubation Dosage T M P A 2 5 m g , Sc 15 mglegg TMPA
No. treated Mortality to 18 days ( % ) No. of survivors Normal (%, ) Short neck ( % ) Muscular hypoplasia ( % ) M i x . defects ( % ) ~
I 2
28 14.3 24 100
0
0 0
3.1
0
31 25.8 23 21.7 1 78.3 1 4.3 0
35 17.1 23 82.6 17.4 0 0
Chi square over sum of both controls with P < O . O O l .
the combinations were with 0.5 or 1.0 mg carbachol (table 7). In the control groups 97.3% of the TMPA-treated embryos were normal and the 2 carbachol control groups included 96.9 and 90.3% normal embryos, respectively. When the teratogens were combined at the 2 different levels of carbachol, both showed a gross exaggeration in deviation from normality and this was true in defectiveness of the cervical regions as
~~
38 15.8 32 37.5 1 62.5 I
36 2.8 35 97.1 0
2.9 0
TMPA Sc
+
51 13.7 44
40.9 I 56.8 I 15.9 2.3
Sc
45 11.1 40 82.5 17.5 5.0 0
~~
Chi square aver sum of both controls with P < 0.001. Chi square over sum of both controls with P C 0.02.
well as in muscular morphogenesis. The high incidence of muscular hypoplasia in the combination of 2.5 mg TMPA and 1.0 mg carbachol was especially impressive. Earlier experiments with nicotine sulfate (Landauer, '60) had shown that its principal, and in low doses its only, effect on chicken embryos was a shortened neck with fused vertebrae. In larger dosages other parts of the vertebral column became defective and muscular hypoplasia of the legs occurred at the same time. New tests on the combined effect of nicotine and carbachol (table 8) demonstrated much the same high degree of synergism that had been found among paired combinations of other cholinomimetic teratogens.
Prevention and protection Experiments in which gallamine or benzoquinonium were used in combination with several of the cholinomimetic compounds were made on the presumption that as supplements these compounds might serve as physical hindrance to the teratogens in reaching their intended receptors, thereby failing to accomplish their noxious
131
CHOLINOMIMETIC TEKATOGENS TABLE 7
T h e effects ofcrirbtrchol (C) ttnd trimethylphenyltrmmoniiim iodide i T M P A ) on chiclc deuelopment b y hecctment, seporcitely or in combintrtion, t i t 96 h of i)icitbcition. Dostrges: ctirbtichol 0 . 5 or 1.0m y , T M P A 2 . 5 m g l c , g g TMPA
No treated Mortality to 18 days ((7 ) N o of survivors Normal ('IL ) Short/crooked neck ( 7 ) Muscular hypoplasia ('V ) Misc defects ( % ) I 2
+
TMPA 0.5 m g C ~
~
C 0.5 mg
TMPA + 1 mgC
1 mg
38 13 2 33 12 1 1 87 9 1 54 5 30
34 88 31 90 3 97 65 0
C
~
40 75 37 97 3 27 0 0
44 13 6 38 50 0 1 50 0 1 1322 0
32 0 32 96 9 31 3 1 0
'
Chi square over sum of both controls with P < O . O O l . Chi square over sum of both controls with P i O . 0 2 .
effects (tables 9, 10). The experiments were done at 96 h of incubation. Gallamine and benzoquinonium were always injected prior to the teratogen whose effectiveness was to be tested. As known from earlier experience double injections are likely to be responsible for a somewhat higher embryo mortality than single ones and this was true for some of the present tests as well. Neither gallamine nor benzoquinonium alone, in the amounts and at the developmental stages used, caused any interference with normal development. The results of supplementing carbachol with gallamine were studied at 2 dosage levels. In the first experiment we injected 1.5 mg carbachol and 10 mg gallamine. The presence of gallamine led in this combination to a great lowering in the incidence of defective cervical morphogenesis as well as of muscular hypoplasia; 81.6 rather than 32.1 % of the surviving embryos were grossly normal. In the second test the dosage of carbachol was raised to 2.5 mg/ egg, causing in the control group (carbachol alone) a corresponding increase in incidence as well as in qualitative expression of the carbachol-induced defects. Supplementation was done, as before, with 10 mg/egg gallamine, but in a separate group we added to this 0.25 mgfegg atropine sulfate. None of the embryos treated with carbachol alone escaped teratogenic effects, but with supplementation 76.7 and 83.7%, respectively, of the survivors were grossly normal; abnormality of the neck was reduced from an incidence of 100 to one of 23.3 and 16.3%, respectively; even more dramatically no instances of muscular hypoplasia were recorded in either of the 2 supplemented groups. The improvement
in the prevention of carbachol-induced defects by the addition of atropine to supplementary gallamine was very suggestive. In other tests the effects of 1.5 mg/egg decamethonium iodide either alone or in combination with 5 mg gallamine were compared. In the unsupplemented group all embryos showed developmental defects, but in the presence of gallamine 35.1% of the treated embryos were grossly normal; there was a highly significant reduction in decamethonium-induced defectiveness of the neck and a suggestive lowering in the incidence of muscular hypoplasia. Experiments with neostigmine and nicotine were done with dosage levels at which the cervical region became defective without producing gross abnormality of muscle development. In the combination of 1.5 mg/egg neostigmine bromide and 5 mg gallamine the incidence of neck defects was only 7.1% as compared with 97% among survivors that had been treated with neostigmine alone. A similar, if less extreme, benefit occurred in the combination of 2.5 mg nicotine sulfate and 5 mg gallamine. In TABLE 8
T h e effects of ctrrbtrchol t o i d nicotine siilfnte ou chick development f o l l o w i w trecitme>it, srptrrcrtely or i n combintrtion, crt 96 h of incubtition. Doscrges; ctirbtrchot I .25 mg, nicotine 1.5 m y CarbaCarba- chol Nicochol nicotine tine
+
N o treated Mortality to 18 d a y s (c4 ) N o of survivors Normal ( ( C ) Short/crooked neck ((Z 1 1
32 15 6 27 852 148
56
29 14 3 69 48 27 2081 852 7 9 2 ' 148
Chi squareover sum of both controls w ~ t hP < 0 001
132
WALTER LANDAUEK TABLE 9
The effect of gallamine triethiodide us supplement to treatment with the anticholinergic compounds cctrbachol, decccmcithonium iodide, rind nicotine sitlfutr. Dosuges given in mglegg, u t 96 h incubution No treated
Mortality to 18 d a y s ( ? < )
Curbuchol (1.5 m g ) Carbachol Carbachol t gallamine Gallamine
+ +
78
32.1
63 43
22.2 18.6
49 35
81.6 100
+
+
+
(Cr )
64.1 1
12.3 0
16.7 I
6.1 0
+ gctllumine ( 5 my) + tctropine siilfiite (0.25 mg) 0
100
22.6
31
53
18.9
43
76.7 2
23.3
1
0'
58
28.3
43
83.7
16.3
1
0'
45.2
I
+ gctllumine (5 my)
52
13.5
45
0
45
17.8
37
35.1
100 1
64.9
68.9 I
+ grtllumine (5 mg]
34
2.9
33
3.0
49
14.3
42
90.5
Nicotine sulfate (2.5 mg) Nicotine Nicotine gallamine
Muscular hypoplasia
38
Neostigmine bromide ( I .5 mg)
2
Abnormal neck ( / b )
+ gullrcmine (10 mg]
10.3
Decctmethoniitm iodide (1.5 my)
I
(c+ )
+
Decamethonium Decamethonium gallamine Neostigmine Neostigmine gallamine
Normal
87
Citrbuchol 12.5 my) Carbachol Carbachol gallanine Carbachol gallamine atropine
No of survivors
0
97.0 1
7.1
54.1
1
0
+ gallamine (5 mg)
33
33.3
22
68.2
51
49.0
26
88.4
2
31.4
0
11.5 2
0
C h i square w i t h P < O . O O l C h i square with P i O . 0 2 .
2 sets of new experiments (data not recorded in detail) 96-h embryos were treated with either 2 mg carbachol or 0.4 mg neostigmine and half of each of the 2 groups were given 5 mg gallamine at 120 h of incubation. In neither of these groups had supplementation any effect. Benzoquinonium was tested as supplement to treatment with either carbachol or decamethonium iodide (table 10). In the experiments with carbachol I wished to compare the effect of 2.5 mg/egg carbachol alone or as supplemented with the same amount of benzoquinonium either in the same vehicle with carbachol or as injected separately. The incidence of carbacholinduced abnormalities was greatly reduced by either manner of supplementation. The 2 ways of providing added benzoquinonium did not cause a significant difference in incidence of muscular hypoplasia, but as far as the neck region was concerned treatment in a single vehicle was much more
beneficial than that of injecting teratogen and benzoquinonium separately. Tests of combining decamethonium and benzoquinonium were also done in duplicate, but in this instance I wished to compare treatment with 2 different dosages of decamethonium, viz., 2.5 vs. 0.8 mglegg, each receiving the same amount of supplementation, viz., 2.5 mg benzoquinonium. The results were clear-cut (table 10). The large dose of injected decamethonium preceding supplementation with benzoquinonium could not overcome the teratogenic involvement of either neck vertebrae or leg musculature. With a lesser, though still highly teratogenic, amount of injected decamethonium the beneficial effects of benzoquinonium were spectacular. Physostigmine (eserine), well-known for its anticholinesterase activity, produces in chicken embryos teratogenic effects which, as mentioned previously, are likely to cause micromelia, beak defects, and still other
133
CHOLINOMIMETIC TERATOGENS TABLE 10 The cffect of benzoqitiiionitim chloride N S supplement to treatment with rarbitchol decamethonium iodide. Dosages given in mglegg, a t 96 h incubation No.
treated
Mortality to 18 d a y s ( 5 : )
Cnrhachol(2.5 mg) ( I ) As double injections Carbachol 52 3.8 Carbachol benzoquinonium 53 20.8 Benzoquinonium 29 13.8 (11) As single injection Carbachol benzoquinonium 50 12.0
survivors Normal
Muscular hypoplasia (r;)
+ benzoqitinonium (2.5mg)
+
50
4.0
42 25
45.3 100
44
45.5
+
Decamethoniitm ( I ) Decamethonium (2.5 mg) Decamethonium (2.5 mg) benzoquinonium (2.5 mg) (11) Decamethonium (0.8 mg) Decamethonium (0.8mg) benzoquinonium (2.5 mg) B enzoquinon i u m (2.5 mg)
Abnormal 1 neck (C;. )
No. of
01
96.0
' '
82.0
45.3 0
1
47.6 0
I
18.2
1
52.3
1
+ benrogitinoniztm
20
10.0
18
0
100
100
54
25.9
40
0
100
100
34
11.8
30
26.7
54
1.9
53
75.5
29
13.8
25
+
63.3
63.3
+
1
C h i square with P
'
18.9
I
0
100
20.8
I
0
< 0.001
malformations in addition to abnormality of the cervical vertebrae and of other sections of the vertebral column (Bueker and Platner, '56). To other compounds producing similarly complex effects, viz., organophosphorus insecticides, we shall refer in the discussion. At this point I was interested in determining if gallamine, similar to its antiteratogenic effect in combination with other cholinomimetic compounds, would reduce the teratogenicity of physostigmine. The results of injecting 5 mg gallamine together with 0.2 mg/egg physostigmine sulfate are presented in table 11. There was no lessening in incidence of micromelia or abnormality of the beak, but the occurrence of shortened and abnormal neck was reduced from 74.1 to 12.2%. Other instances of modification in the efficacy of carbachol as teratogen were encountered when butyrylcholine chloride or bethanechol chloride were used as supplements. Butyrylcholine chloride alone became toxic in amounts of more than 2.5 mg/egg/96 h, all embryos succumbing quickly following injection of 20 mg/egg.
Embryos surviving treatment with 2.5, 5, or 10 mg were, however, grossly quite normal. Bethanechol chloride became somewhat toxic above 20 mg/egg/96 h, but all survivors of treatment with up to 30 mglegg were grossly normal. The effects of the combined administration of 1.5 mg carbachol with 2.5 mg butyrylcholine and of 2.5 mg carbachol with 5 mg bethanechol are presented in tables 12 and 13. SupplemenTABLE 11 The &feet of gullamine triethtodide u s a supplement to treating chicken embryos with physoshgmine. Dostrges: physostigmine s u l f a t e 0.2 mg, gctllamine 5 mglegg, a t 96 h Physostigmine
No. treated Mortality to 18 days ( % ) No. of survivors Normal ( % ) Short neck (%, ) Micromelia (% ) Parrot beak ( % ) 1
C h i square with P
ABSTRACT The cholinomimetic compounds carbachol, decamethonium, neostigmine, succinylcholine, trimethylphenylammonium, and others were tested for their interference with normal chick development. All these compounds led to abnormalities of the cervical vertebrae; at higher dosage interference with normal morphogenesis involved the whole vertebral column. Hypoplasia of the leg muscles occurred with lower incidence. Responses, tested with carbachol, rose from 24 to 72 and 96 h, then declined to 120 h of incubation. Two of the cholinometic compounds used in combined treatment produced a high degree of synergism. Gallamine, benzoquinomium, butyrylcholine, and bethanechol had protective effects. Acetylcholine, at high dosage, caused defects different from the above. It is suggested that the cholinomimetic teratogens interfere with normal development by displacing acetylcholine from its receptors or by forming complexes with i t .
The experimental teratologist is likely at times to encounter in his test-animals abnormalities of unrelated parts or organs the nature of which may pose problems of peculiar interest. He may well wonder if certain defects are parts of a syndrome in the sense of deriving either directly or by dependent steps from a common cause or if, alternatively, they originate from distinct chemical peculiarities of the teratogen in use to which particular tissues react dissimilarly. It was questions of this nature that arose from experiments with chicken embryos in which distinct teratogens administered at identical developmental stages produced defects which in their morphological and metabolic expressions appeared curiously unrelated. But were they? A brief historical review provides the setting for our new experiments. Hypoplasia of the leg muscles and defective formation of cervical vertebrae were found to occur in combination when chicken embryos had at a given stage been treated with 3-acetylpyridine, nicotine sulfate, or ethionine. The major effect of 3-acetylpyridine occurred as muscular hypoplasia, vertebral abnormality and shortness of the upper beak being encountered less commonly (Ackermann and Taylor, '48; Landauer, '57; Herrmann et al., '64). The principal result of treatment with nicotine sulfate, on the other hand, was a brevicollis condition in which the cervical vertebrae TERATOLOGY, 1 2 : 125-146.
were abnormal and often fused, muscular hypoplasia occurring less frequently or only with higher dosage (Landauer, '60; Strudel, '71). Exposure of embryos to ethionine, similar to 3-acetylpyridine, led to muscular hypoplasia and at a lower level of incidence to brevicollis and abnormal formation of the beak (Karnofsky et al., '55; Landauer and Salam, '74). Malformations due to 3-acetylpyridine can be prevented completely by giving suitable supplements of nicotinamide (Landauer, '57); nicotine treatment is not ameliorated by added nicotinamide, but considerable protection is afforded by supplements of certain amino acids, glucose, and sodium pyruvate; the teratogenicity of ethionine is greatly reduced in the presence of methionine. Physostigmine (eserine) differs from the preceding teratogens by producing, in addition to defective cervical vertebrae, micromelia, syndactylism, and abnormalities of the visceral skeleton and of the eyelids (Ancel, '45; Landauer, '49; Bueker and Platner, '56; Landauer and Salam, '72; Sullivan, '73). According to Bueker and Platner "reduced musculature of the legs" also occurs and this presumably following the use of relatively large amounts of the compound. Nicotinamide as supplement protects completely against physostigmine-induced micromelia and beak Received Jan. 13, '75. Accepted Apr. 25, '75.
125
126
WALTER LANDAUER
defects, but does little, if anything, in preventing the brevicollis condition. Finally there are the organophosphorous insecticides bidrin, azodrin, parathion, and others. Bidrin-induced abnormalities of chicken embryos, as far as skeletal defects are concerned, include brevi- and torticollis, often extending to other parts of the vertebral column, micromelia, parrot beak, and syndactylism. Nicotinamide and related compounds act as alleviating agents - except that they have little, if any, effect in preventing vertebral abnormality. Inhibition of acetylcholinesterase activity and that of other esterases was found not to correlate in a causal manner with the degree of teratogenesis of bidrin (Upshall et al., '68; Roger et al., '69). The teratogenic responses to parathion are morphologically quite similar to those brought about by bidrin (Iseki and Yamada, '66; Yamada, '68, '72; Khera and Bedok, '67; Lutz-Ostertag et al., '69a,b). The teratogenic effects that azodrin has on chicken embryos, much as those of bidrin, chiefly comprise maldevelopment of the spine and micromelia; in quail embryos the effect of azodrin includes in addition to a shortened and deformed vertebral column "amuscular legs," i.e., muscular hypoplasia (Schom et al., '72). A somewhat similar difference in response between chicken and quail embryos has been reported from tests with the organophosphorus insecticide dursban (Schom et al., '73). Chicken embryos exposed to malathion and malaoxon, on the other hand, react in their skeletal development with micromelia and beak defects, but without vertebral deformities or muscular abnormality. Supplements of tryptophan or nicotinamide prevent the malathion or malaoxon-induced defects, but do not counteract the inhibition of acetylcholinesterase activity (Greenberg and LaHam, '69, '70; Walker, '71 ; Wilson et al., '73). The intriguing questions raised by these observations suggested the desirability of further experimentation with compounds known for their cholinomimetic activity, i.e., substances related to and/or competing with acetylcholine (Feldberg, '57). This led to the work that is to be reported here with compounds that are among the best known inhibitors of neuromusccular transmission and of postsynaptic receptors.
METHODS A N D MATERIALS
The general methods have remained much the same as in earlier work (Landauer and Wakasugi, '67; Landauer and Sopher, '70; Landauer and Salam, '72-'74). Teratogens and supplementing compounds were injected into the yolk sac of chicken eggs, usually after 96 h of incubation, but in a few instances earlier or later. Most of the solutions were prepared in distilled water, with 0.3% phenol added; ethionine was dissolved in 5 N hydrochloric acid. The eggs were incubated in a Petersime automatic incubator and opened for inspection after 19 days. The chemicals used in our work and their sources were as follows: acetylcholine chloride, 3-acetylpyridine, butyrylcholine chloride, and tetracaine, Sigma London Chemical Co. Ltd.; atropine sulfate, carbachol, hexamethonium bromide, decamethonium iodide, and tubocurarine chloride, Koch Light Laboratories Ltd.; L-acetyl-pmethylcholine chloride (Methacholine), tetraethylammonium chloride, and tetramethylammoniumchloride, Ralph N. Emanuel Ltd. ;trimethylphenylammonium iodide, BDH Chemical Ltd.; dichlorphenamide, Merck Sharp and Dohme Ltd.; sodium acetazolamide, Lederle Laboratories; nicotine sulfate and physostigmine, Hopkin and Williams Ltd. In addition we received and used the following gifts: bethanechol chloride (Myotonine), Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey; benzoquinonium chloride, Sterling-Winthrop Research Institute, Rensselaer, New York; edrophonium chloride, Roche Products Ltd., Welwyn Garden City, Hertfordshire; gallamine triethiodide (Flaxedil), May and Baker Ltd. Medical Products Division, Dagenham, Essex; and pyridostigmine bromide (Mestinon), Hoffmann-LaRoche Inc., Nutley, New Jersey. Wilh most of these compounds we initially performed small-scale tests as guide of toxicity, subsequent experiments being numerically adjusted accordingly. Statistical analyses of our data were based on the texts of Snedecor ('48) and Bliss ('67). RESULTS
Quantitatiue observations following treatment at 96 h of incubation Data relating to experiments with carbachol chloride, decamethonium iodide, and
127
CHOLINOMIMETIC TERATOGENS
neostigmine bromide are presented in table 1 . The results with carbachol point to a gradual increase of toxicity with rising dosage, but more extensive data might have shown deviations from a smoothly mounting dosagelmortality relation. Interference of carbachol with normal chick development was twofold, viz., abnormality of the vertebral column and hypoplasia of the leg muscles. Both types of effect are dosage related in incidence and expression. Following treatment with 0.5 mglegg carbachol the great majority of embryos appeared quite normal, but on rare occasions the neck was short and the leg musculature was somewhat less well developed than in control embryos. With increasing dosage the incidence of abnormalities rose sharply, involving vertebral development of all embryos when treatment reached 3 mglegg, the great majority of embryos showing at the same time subnormal growth of the leg musculature. Furthermore, with increasing dosage of carbachol irregular morphogenesis and fusion of vertebrae tended to spread from the cervical region posteriorly, thereby causing humps of the back, compression of the trunk, and retraction of the tail vertebrae into the pelvis, the latter condition creating the impression of “rumplessness.” Major involvement of the trunk frequently was responsible for joint abnormalities of the legs. Muscular hypoplasia also became
more pronounced with increasing dosage of carbachol. The muscles of the right leg were often more markedly involved than those of the left, an asymmetry of response that occurred with higher frequency following treatment at 72 than at 96 h of incubation. In the presence of more extreme instances of muscular hypoplasia, and presumably as a secondary effect, some embryos had their toes in a curled position. Whenever dosages of carbachol between 1 and 3 mglegg were injected the incidence of muscular hypoplasia was regularly lower than that of vertebral involvement. In experiments with decamethonium and neostigmine there was no clear relation of dosage to embryo mortality. The developmental effects produced by the 2 compounds were very similar to those recorded for carbachol. In both instances the teratogenic responses of vertebrae exceeded those of the leg muscles. Observations on the effect of treating 96h chicken embryos with either succinylcholine chloride or trimethylphenylammonium iodide (TMPA) are represented by the data of table 2. The 2 compounds interfered with normal development of vertebrae in a way very similar to that shown for the first 3 teratogens. A s in the former material there was a clear dosagelresponse relation. The effects produced by succinylcholine and TMPA differed, however, from those of
TABLE 1
T h e effect on developing c h i c k e n embryos of trecitment w i t h curbcichol chloride, d e c a m e t h o n i u m iodide, a n d n e o s t i g m i n e bromide according t o dostrge i n mglegg, a t 96 h of incnbation No treated
Mortality to 18 days ( ” i )
0.5 1.0 1.5 2.0 2.5 3.0
60 79 87 74 332 57
6.7 11.4 10.3 10.8 12.7 24.6
0.5 0.8 1.5 2.5
64 34 88 51
7.8 11.8 13.6 5.9
0.1 0.2 0.4 0.6
10
10.0 0 22.9 5.3
Dose (mg)
N o of survivors
Normal ( % )
Short/crooked neck ( o i l
Muscular hypoplasia i f > )
1.8 17.1 51.3 65.2 94.8 100
I .8 7.1 16.7 28.8 55.9 86.0
50.8 63.3 100
10.2 63.3 75.8 62.6
Cnrbachol chloride 56 70 78 66 290 43
98.2 84.3 33.3 13.6 5.2 0
D e c a m e t h o n i u m iodide 59 30 76 48
49.2 26.7 0 0
100
Neostigmine b r o m i d e 10
35 38
9 10
27 36
100
80.0 33.3 2.8
0 20.0 63.0 97.2
0 0 0
47.2
128
WALTER LANDAUER TABLE 2
T h e cfSect oii deweloping c h i c l t r n emhiyo!, of tretitme7it with siiccinylcholinr c h l o r i d e ctnd trimrthylphriiyl(tmmoniitm i o d i d e crccorditzg to dosctge in m g l e g g , cit 96 h ofznc7ihation Dose ( m g )
No treated
Mortality to 18 days ( ‘ c )
No of survlvors
Normal
(‘c
1
Shortlcrooked neck (‘, )
Sitccinylc ho12ne c h l o r i d e
25 10
15 20 30 1.5 2.5 5.0 10.0
29 36 30 29 10 40
15 13
0 2.8 13.3 17.2
29 35 26 24
T r i m r t h y l p h e i z y l t c m m o ~ 7iitm i o d i d e 0 10 7.5 37 26.7 11 61.5 5
the former teratogens in that they interfered only rarely with normal muscle development. Additional data on this problem will be reported in the sequence. Experiments with tetramethylammonium chloride and pyridostigmine bromide, 2 other compounds with cholinomimetic activity, will not be reported in detail. The effects of tetramethylammonium were tested in amounts of 2.5 mgleggl96 h and over and those of pyridostigmine in dosages of 10 mg/egg or more. Both compounds were responsible for short and crooked necks and muscular hypoplasia of the legs similar to the data of table 1. The extent to which the various compounds interfered with normal vertebral morphogenesis was in increasing order approximately as follows: succinylcholine < trimethylphenylammonium < pyridostigmine < tetramethylammonium < carbachol < decamethonium < neostigmine. A similar sequence seems to be true for interference with muscle development. Size and weight of the body tended to decline as incidence and extent of malformations produced by the various teratogens rose, but metric data were not collected. Compounds that, in the given dosage ranges of mglegg at 96 h, produced no gross abnormalities were as follows: Lacetyl- p -methylcholine chloride 2.5-60 mg, benzoquinonium 0.5-5 mg, bethanechol chloride 2.5-40 mg, butyrylcholine chloride 2.5-20 mg, edrophonium chloride 1.5to 6 mg, gallamine triethiodide 1.510 mg, hexamethonium bromide 2.5-60 mg, tetraethylammonium chloride 2.5-30 mg, and
100
0
85.7 69.2 33.3
14.3 30.8 66.7
100 97.3 54.5
2.7 45.5
0
0 100
n-tubocurarine chloride 1.5-5 mg. Butyrylcholine was, however, highly toxic above 2.5 mglegg and tetraethylammonium above 20 mglegg. Effects of deuelopmental stage o n carbachol treatment The stage responses of chicken embryos to treatment with carbachol chloride were followed by injecting the compound into eggs at 24, 72, 96, and 120 h of incubation. The results of these tests are presented in table 3. The dosages were varied between 1.0 and 2.5 mglegg in order to demonstrate the effects on toxicity and morphogenesis at comparable levels. There was little difference in embryo mortality caused by treatment between 24 and 96 h, but given at 120 h of incubation the compound clearly had greater toxicity than at the earlier stages. The morphological responses to carbachol treatment were of the same type between 24 and 120 h, but incidence and extent of vertebral and muscular abnormality varied with time. There was a steep rise in teratogenicity from 24 to 72 h; a moderate lowering in incidence of malformations between 72 and 96 h (which was particularly significant at 1.5 mglegg); and a precipitous decline of teratogenic response from 96 to 120 h of incubation. Synergistic effects of cholinomimetic compounds in combination The results of previous observations had made i t evident that subtle differences occur in the specific developmental responses provoked by some of the cholinomimetic
129
CHOLINOMIMETIC T E R A T O G E N S TABLE 3
Tercttologictr I responses of chicken e m b r y o s to ctcrbtcchol chloride uccordiizg to developmentcil sttrge of tretrtment. Doscigr wus vciried between 1 .O clnd 2.5 m g . The dutctfor 96 h w e r e t o k e n f r o m tuble I Incubation stage (h)
Muscular hypoplasia
(mg)
No. treated
Mortality to 18 d a y s ( r~ 1
Normal ( % )
Abnormal neck ( ‘ c )
24
1.0 1.5
38 37
11.8 18 9
34 30
85.3 53.3
2.9 36.7
14.7 36.7
72
1.5 2.5
41 38
7.3 15.8
38 32
10.5 3.2
65.8 90.6
86.8 96.9
96
1 .0 1.5 2.5
79 87 332
11.4 10.3 12.7
70 78 290
84.3 33.3 5.2
17 1 51.3 94.8
7.1 16.7 55.9
120
1.5 2.5
34 36
29.4 33.3
24 24
87.5 25.0
12.5 75.0
0 25.0
Dose
compounds. Thus, among embryos that had been treated with carbachol or succinylcholine and of which 65.2 and 66.7% , respectively had an abnormally short neck, this particular abnormality had in nearly all of the former and in very few of the latter embryos been combined with irregular fusion of cervical vertebrae, or that treatment with carbachol, decamethonium, or neostigmine was likely to produce a “syndrome” of abnormal neck and muscular hypoplasia, but that succinylcholine and trimethylphenylammonium while causing dosage-dependent abnormality of the cervical vertebrae only rarely produced muscular defectiveness. It was differences of this order that raised the question of what would result if 2 such compounds were applied together? Would there be interaction or exaggeration of teratogenic effects? In order to obtain meaningful results it seemed desirable to combine any 2 compounds in such dosages that their teratogenicity ,when applied singly, would be at a relatively low level. The results are shown in tables 4-8. Data for carbachol and decamethonium, singly or combined, are provided in table 4. With carbachol alone 95.7% of the treated embryos were grossly normal, 32.1 % of the decamethonium-treated embryos escaped damage, but in combination only 2.3% of the surviving embryos failed to show either malformation of the neck or muscular hypoplasia or both. The teratogenic exaggeration of the combined treatment, if compared with the sum of the 2 controls, is highly significant. Body weight was greatly reduced after the combined
No. of survivors
(CC)
treatment, the vertebral column tended to be much shortened and deformed in its whole length, retraction of its posterior section giving the impression of rumplessness, and the trunk was compressed. In tests in which carbachol and succinylcholine were used singly or combined 15 mglegg succinylcholine and 0.5 or 1.0 mg carbachol were injected (table 5). With the lower dosage of carbachol, used as control, none of the surviving embryos were abnormal; when succinylcholine was injected 14.7% of the survivors had a defective neck; but in the combined treatment 62.5% of the survivors showed the abnormality. A similarly striking exaggeration in defectiveness of the cervical region occurred when 1.0 mg carbachol was combined with 15 mg succinylcholine. In none of the 3 control groups were there specimens with muscular hypoplasia, but following treatment with 1.0 mg carbachol and 15 mg succinylcholine there was 1 such case among 23 survivors. Table 6 presents information on the single or combined treatment with trimethylphenylammonium iodide (TMPA) and succinylcholine chloride at dosages of 2.5 and 15 mg, respectively. The tests with these 2 compounds are of particular interest since there were but rare instances of muscular hypoplasia in the 2 control groups. The synergistic effect of vertebral as well as muscular abnormality due to the combined treatment is evident. Especially striking results occurred in the combination of TMPA and carbachol chloride. TMPA was used at 2.5 mglegg and
1 30
WALTER LANDAUER TABLE 1
T h e effects ofcurbtrchoi a n d dectrmethoniitm srpctrrctely or in combination. Treiitment 96 h ofincirbution. Dosctgus: curbuchol 1.5, d e c a m e t h o n i z t m 0.25 mgiegg Carbachol and decamethonium combined
Carbachol
24 4.2 23 95.7 4.3
N o treated Mortality to 18 days ('X ) No of survivors Normal (clo ) Shorticrooked neck (5: ) Muscular hypoplasia ( % ) 1
Decamethonium
54 20.4 43 2.3 I 97.7 55.7
87 10.3 78 32.1 64.1 16.7
'
'
0
tct
Chi square over sum of both controls with P < 0.001
TABLE 5
The effects of'carbuchol ( C ) and s n c c i n y t c h o l m e c hloride (Sc) septtrcctely or ~n combzntrtion T r e a t m e n t u t 96 h ofincdxttzon T h e dostiges o f c n r b a c h o t w e r e 0 5 or 1 m g , t h a t of sztccznylcholtne I 5 mglegg sc sc
+
0.5 mg C
C 0.5 mg
+
sc 1 mgC
C 1 mg
~~
37 5.4 35 85.7 14.7
No. treated Mortality to 18 days (% ) No. of survivors Normal ( % 1 ShorVcrooked neck ( % ) Muscular hypoplasia ( V Misc. defects ( % ) I
0 0
TABLE 6
The effects of tnmethylphenyl~rmmo,Ililm iodzde ( T M P A ) and szrccznylcholine chlonde ( S c ) o n chick development b y t r e a t m e n t , separately or in c o m bination, at 96 h of incubation Dosage T M P A 2 5 m g , Sc 15 mglegg TMPA
No. treated Mortality to 18 days ( % ) No. of survivors Normal (%, ) Short neck ( % ) Muscular hypoplasia ( % ) M i x . defects ( % ) ~
I 2
28 14.3 24 100
0
0 0
3.1
0
31 25.8 23 21.7 1 78.3 1 4.3 0
35 17.1 23 82.6 17.4 0 0
Chi square over sum of both controls with P < O . O O l .
the combinations were with 0.5 or 1.0 mg carbachol (table 7). In the control groups 97.3% of the TMPA-treated embryos were normal and the 2 carbachol control groups included 96.9 and 90.3% normal embryos, respectively. When the teratogens were combined at the 2 different levels of carbachol, both showed a gross exaggeration in deviation from normality and this was true in defectiveness of the cervical regions as
~~
38 15.8 32 37.5 1 62.5 I
36 2.8 35 97.1 0
2.9 0
TMPA Sc
+
51 13.7 44
40.9 I 56.8 I 15.9 2.3
Sc
45 11.1 40 82.5 17.5 5.0 0
~~
Chi square aver sum of both controls with P < 0.001. Chi square over sum of both controls with P C 0.02.
well as in muscular morphogenesis. The high incidence of muscular hypoplasia in the combination of 2.5 mg TMPA and 1.0 mg carbachol was especially impressive. Earlier experiments with nicotine sulfate (Landauer, '60) had shown that its principal, and in low doses its only, effect on chicken embryos was a shortened neck with fused vertebrae. In larger dosages other parts of the vertebral column became defective and muscular hypoplasia of the legs occurred at the same time. New tests on the combined effect of nicotine and carbachol (table 8) demonstrated much the same high degree of synergism that had been found among paired combinations of other cholinomimetic teratogens.
Prevention and protection Experiments in which gallamine or benzoquinonium were used in combination with several of the cholinomimetic compounds were made on the presumption that as supplements these compounds might serve as physical hindrance to the teratogens in reaching their intended receptors, thereby failing to accomplish their noxious
131
CHOLINOMIMETIC TEKATOGENS TABLE 7
T h e effects ofcrirbtrchol (C) ttnd trimethylphenyltrmmoniiim iodide i T M P A ) on chiclc deuelopment b y hecctment, seporcitely or in combintrtion, t i t 96 h of i)icitbcition. Dostrges: ctirbtichol 0 . 5 or 1.0m y , T M P A 2 . 5 m g l c , g g TMPA
No treated Mortality to 18 days ((7 ) N o of survivors Normal ('IL ) Short/crooked neck ( 7 ) Muscular hypoplasia ('V ) Misc defects ( % ) I 2
+
TMPA 0.5 m g C ~
~
C 0.5 mg
TMPA + 1 mgC
1 mg
38 13 2 33 12 1 1 87 9 1 54 5 30
34 88 31 90 3 97 65 0
C
~
40 75 37 97 3 27 0 0
44 13 6 38 50 0 1 50 0 1 1322 0
32 0 32 96 9 31 3 1 0
'
Chi square over sum of both controls with P < O . O O l . Chi square over sum of both controls with P i O . 0 2 .
effects (tables 9, 10). The experiments were done at 96 h of incubation. Gallamine and benzoquinonium were always injected prior to the teratogen whose effectiveness was to be tested. As known from earlier experience double injections are likely to be responsible for a somewhat higher embryo mortality than single ones and this was true for some of the present tests as well. Neither gallamine nor benzoquinonium alone, in the amounts and at the developmental stages used, caused any interference with normal development. The results of supplementing carbachol with gallamine were studied at 2 dosage levels. In the first experiment we injected 1.5 mg carbachol and 10 mg gallamine. The presence of gallamine led in this combination to a great lowering in the incidence of defective cervical morphogenesis as well as of muscular hypoplasia; 81.6 rather than 32.1 % of the surviving embryos were grossly normal. In the second test the dosage of carbachol was raised to 2.5 mg/ egg, causing in the control group (carbachol alone) a corresponding increase in incidence as well as in qualitative expression of the carbachol-induced defects. Supplementation was done, as before, with 10 mg/egg gallamine, but in a separate group we added to this 0.25 mgfegg atropine sulfate. None of the embryos treated with carbachol alone escaped teratogenic effects, but with supplementation 76.7 and 83.7%, respectively, of the survivors were grossly normal; abnormality of the neck was reduced from an incidence of 100 to one of 23.3 and 16.3%, respectively; even more dramatically no instances of muscular hypoplasia were recorded in either of the 2 supplemented groups. The improvement
in the prevention of carbachol-induced defects by the addition of atropine to supplementary gallamine was very suggestive. In other tests the effects of 1.5 mg/egg decamethonium iodide either alone or in combination with 5 mg gallamine were compared. In the unsupplemented group all embryos showed developmental defects, but in the presence of gallamine 35.1% of the treated embryos were grossly normal; there was a highly significant reduction in decamethonium-induced defectiveness of the neck and a suggestive lowering in the incidence of muscular hypoplasia. Experiments with neostigmine and nicotine were done with dosage levels at which the cervical region became defective without producing gross abnormality of muscle development. In the combination of 1.5 mg/egg neostigmine bromide and 5 mg gallamine the incidence of neck defects was only 7.1% as compared with 97% among survivors that had been treated with neostigmine alone. A similar, if less extreme, benefit occurred in the combination of 2.5 mg nicotine sulfate and 5 mg gallamine. In TABLE 8
T h e effects of ctrrbtrchol t o i d nicotine siilfnte ou chick development f o l l o w i w trecitme>it, srptrrcrtely or i n combintrtion, crt 96 h of incubtition. Doscrges; ctirbtrchot I .25 mg, nicotine 1.5 m y CarbaCarba- chol Nicochol nicotine tine
+
N o treated Mortality to 18 d a y s (c4 ) N o of survivors Normal ( ( C ) Short/crooked neck ((Z 1 1
32 15 6 27 852 148
56
29 14 3 69 48 27 2081 852 7 9 2 ' 148
Chi squareover sum of both controls w ~ t hP < 0 001
132
WALTER LANDAUEK TABLE 9
The effect of gallamine triethiodide us supplement to treatment with the anticholinergic compounds cctrbachol, decccmcithonium iodide, rind nicotine sitlfutr. Dosuges given in mglegg, u t 96 h incubution No treated
Mortality to 18 d a y s ( ? < )
Curbuchol (1.5 m g ) Carbachol Carbachol t gallamine Gallamine
+ +
78
32.1
63 43
22.2 18.6
49 35
81.6 100
+
+
+
(Cr )
64.1 1
12.3 0
16.7 I
6.1 0
+ gctllumine ( 5 my) + tctropine siilfiite (0.25 mg) 0
100
22.6
31
53
18.9
43
76.7 2
23.3
1
0'
58
28.3
43
83.7
16.3
1
0'
45.2
I
+ gctllumine (5 my)
52
13.5
45
0
45
17.8
37
35.1
100 1
64.9
68.9 I
+ grtllumine (5 mg]
34
2.9
33
3.0
49
14.3
42
90.5
Nicotine sulfate (2.5 mg) Nicotine Nicotine gallamine
Muscular hypoplasia
38
Neostigmine bromide ( I .5 mg)
2
Abnormal neck ( / b )
+ gullrcmine (10 mg]
10.3
Decctmethoniitm iodide (1.5 my)
I
(c+ )
+
Decamethonium Decamethonium gallamine Neostigmine Neostigmine gallamine
Normal
87
Citrbuchol 12.5 my) Carbachol Carbachol gallanine Carbachol gallamine atropine
No of survivors
0
97.0 1
7.1
54.1
1
0
+ gallamine (5 mg)
33
33.3
22
68.2
51
49.0
26
88.4
2
31.4
0
11.5 2
0
C h i square w i t h P < O . O O l C h i square with P i O . 0 2 .
2 sets of new experiments (data not recorded in detail) 96-h embryos were treated with either 2 mg carbachol or 0.4 mg neostigmine and half of each of the 2 groups were given 5 mg gallamine at 120 h of incubation. In neither of these groups had supplementation any effect. Benzoquinonium was tested as supplement to treatment with either carbachol or decamethonium iodide (table 10). In the experiments with carbachol I wished to compare the effect of 2.5 mg/egg carbachol alone or as supplemented with the same amount of benzoquinonium either in the same vehicle with carbachol or as injected separately. The incidence of carbacholinduced abnormalities was greatly reduced by either manner of supplementation. The 2 ways of providing added benzoquinonium did not cause a significant difference in incidence of muscular hypoplasia, but as far as the neck region was concerned treatment in a single vehicle was much more
beneficial than that of injecting teratogen and benzoquinonium separately. Tests of combining decamethonium and benzoquinonium were also done in duplicate, but in this instance I wished to compare treatment with 2 different dosages of decamethonium, viz., 2.5 vs. 0.8 mglegg, each receiving the same amount of supplementation, viz., 2.5 mg benzoquinonium. The results were clear-cut (table 10). The large dose of injected decamethonium preceding supplementation with benzoquinonium could not overcome the teratogenic involvement of either neck vertebrae or leg musculature. With a lesser, though still highly teratogenic, amount of injected decamethonium the beneficial effects of benzoquinonium were spectacular. Physostigmine (eserine), well-known for its anticholinesterase activity, produces in chicken embryos teratogenic effects which, as mentioned previously, are likely to cause micromelia, beak defects, and still other
133
CHOLINOMIMETIC TERATOGENS TABLE 10 The cffect of benzoqitiiionitim chloride N S supplement to treatment with rarbitchol decamethonium iodide. Dosages given in mglegg, a t 96 h incubation No.
treated
Mortality to 18 d a y s ( 5 : )
Cnrhachol(2.5 mg) ( I ) As double injections Carbachol 52 3.8 Carbachol benzoquinonium 53 20.8 Benzoquinonium 29 13.8 (11) As single injection Carbachol benzoquinonium 50 12.0
survivors Normal
Muscular hypoplasia (r;)
+ benzoqitinonium (2.5mg)
+
50
4.0
42 25
45.3 100
44
45.5
+
Decamethoniitm ( I ) Decamethonium (2.5 mg) Decamethonium (2.5 mg) benzoquinonium (2.5 mg) (11) Decamethonium (0.8 mg) Decamethonium (0.8mg) benzoquinonium (2.5 mg) B enzoquinon i u m (2.5 mg)
Abnormal 1 neck (C;. )
No. of
01
96.0
' '
82.0
45.3 0
1
47.6 0
I
18.2
1
52.3
1
+ benrogitinoniztm
20
10.0
18
0
100
100
54
25.9
40
0
100
100
34
11.8
30
26.7
54
1.9
53
75.5
29
13.8
25
+
63.3
63.3
+
1
C h i square with P
'
18.9
I
0
100
20.8
I
0
< 0.001
malformations in addition to abnormality of the cervical vertebrae and of other sections of the vertebral column (Bueker and Platner, '56). To other compounds producing similarly complex effects, viz., organophosphorus insecticides, we shall refer in the discussion. At this point I was interested in determining if gallamine, similar to its antiteratogenic effect in combination with other cholinomimetic compounds, would reduce the teratogenicity of physostigmine. The results of injecting 5 mg gallamine together with 0.2 mg/egg physostigmine sulfate are presented in table 11. There was no lessening in incidence of micromelia or abnormality of the beak, but the occurrence of shortened and abnormal neck was reduced from 74.1 to 12.2%. Other instances of modification in the efficacy of carbachol as teratogen were encountered when butyrylcholine chloride or bethanechol chloride were used as supplements. Butyrylcholine chloride alone became toxic in amounts of more than 2.5 mg/egg/96 h, all embryos succumbing quickly following injection of 20 mg/egg.
Embryos surviving treatment with 2.5, 5, or 10 mg were, however, grossly quite normal. Bethanechol chloride became somewhat toxic above 20 mg/egg/96 h, but all survivors of treatment with up to 30 mglegg were grossly normal. The effects of the combined administration of 1.5 mg carbachol with 2.5 mg butyrylcholine and of 2.5 mg carbachol with 5 mg bethanechol are presented in tables 12 and 13. SupplemenTABLE 11 The &feet of gullamine triethtodide u s a supplement to treating chicken embryos with physoshgmine. Dostrges: physostigmine s u l f a t e 0.2 mg, gctllamine 5 mglegg, a t 96 h Physostigmine
No. treated Mortality to 18 days ( % ) No. of survivors Normal ( % ) Short neck (%, ) Micromelia (% ) Parrot beak ( % ) 1
C h i square with P
