/ . Embryol. exp. Morph. Vol. 20, 3, pp. 261-84, November 1968
Printed in Great Britain
261
Teratological studies with sulphonamides
and their implications
By WALTER LANDAUER 1 AND NOBORU WAKASUGP
From the Nuffield Institute of Comparative Medicine,
The Zoological Society of London,
and Department of Animal Genetics, University College London
It is one of the principal aims of teratological research to trace the manner in
which particular chemical compounds give rise to abnormal phenotypes by
interfering with well-defined metabolic events. Problems that seemed to lend
themselves to investigations of this nature arose with the discovery that malformations can be produced by sulphonamides, which in clinical practice are
or have been used as inhibitors of carbonic anhydrase and the activity of which
was considered to be quite specific in that respect. We had earlier reported on
the effects which one such compound has on developing chicken embryos and
shall now describe results obtained with three further sulphonamides. We shall
then attempt to discuss the meaning of these and related data in terms of general
teratological problems.
Lay ton & Hallesy (1965) were the first to observe that acetazolamide (2-acetylamino-l,3,4-thiadiazole-5-sulphonamide), when given orally to pregnant rats or
mice, was responsible, among the newborn young, for a certain incidence of
peculiar and interesting abnormalities of the forelimbs. Wilson, Maren &
Takano (1966) confirmed these findings in rats. Three additional sulphonamides
produced similar symptoms in rats or mice (Wilson et al. 1966; Wilson, Maren,
Takano & Ellison, 1968; Layton, as quoted by Maren, 1967) and reference to
this work will be made later on.
The results of our earlier experiments with acetazolamide (Landauer &
Wakasugi, 1967) had questioned the likelihood that the teratogenic effects of
the compound can be accounted for by its role as an inhibitor of carbonic
anhydrase. These observations suggested, on the contrary, interference with
NAD functions as the intervening agency. Another sulphonamide compound,
namely sulphanilamide (p-aminobenzenesulphonamide), had much earlier been
reported as teratogenic for chicken embryos (Ancel, 1945; Zwilling & DeBell,
1
Author's address: Department of Animal Genetics, University College London, Wolfson
House, 4 Euston Buildings, London, N.W.I, England.
2
Author's address: Department of Animal Husbandry, Faculty of Agriculture, Nagoya
University, Chikusa, Nagoya, Japan.
262
W. LANDAUER & N. WAKASUGI
1950; Landauer & Clark, 1964a). The experimental evidence has pointed to
similar metabolic disturbances of chick development after treatment with either
acetazolamide or sulphanilamide, and the fact that sulphanilamide is known to
be only a weak inhibitor of carbonic anhydrase (for which, in fact, neither its
in vitro nor its in vivo activity had ever been considered specific) lent impetus to
an inquiry into the teratogenic nature of additional 'carbonic-anhydrase
inhibiting' sulphonamides.
The present report will deal with the results of experiments in which developing chicken embryos were treated with dichlorphenamide (4,5-dichloro-rabenzenedisulphonamide), methazolamide (5-acetylimino-4-methy 1-A2-1,3,4- thiadiazoline-2-sulphonamide) and j?-sulphamoylbenzoic acid (/7-carboxybenzenesulphonamide). In addition to using these compounds singly, we injected them in
combination with various metabolites, and we performed, in addition, new
tests with acetazolamide and sulphanilamide.
MATERIALS AND METHODS
All eggs used in our work came from an interline cross of White Leghorn
fowl bred by Thornbers (Berkshire) Ltd. in Twyford, Berkshire. The majority
of tests were done after 96 h of incubation, but some were made after 24 h.
In all experiments we injected the compounds to be tested into the yolk sac and
the amount injected was generally 0-05 ml. Dichlorphenamide and sulphanilamide were dissolved in propylene glycol (propane- 1,2-diol). Methazolamide was
converted to its sodium salt with 1 N-NaOH and adjusted to pH 10. Other
compounds were dissolved in demineralized water with 0-25 % phenol added.
All solutions, except those of sodium folate and ADP, were tyndallized. Sodium
folate was injected as a sterile Folvite (Lederle) solution; ADP was dissolved in
sterile water and adjusted to pH 6-8 with phosphate buffer and was then used
within 2-3 h.
The compounds with which we worked and their sources were as follows:
dichlorphenamide (Merck Sharp and Dohme Ltd.), methazolamide ('Neptazane', Cyanamid International), sodium acetazolamide ('Diamox', Lederle
Laboratories), sulphanilamide and 3-acetylpyridine (Sigma Chemical Co.),
/7-sulphamoylbenzoic acid ('Dirnate', Aldrich Chemical Co.), ADP (C. F.
Boehringer Soehne), sodium folate ('Folvite', Lederle Laboratories), nicotinamide (Hopkins and Williams Ltd.), ethyl carbamate and propylene glycol
(British Drug Houses).
The methods of incubation and of treatment with the various compounds
were the same as in our previous work (Landauer & Wakasugi, 1967). The
number of fertile eggs used in our tests, the post-operative mortality, the number
of embryos surviving the 18th day of incubation and the incidence of any gross
malformations found among the latter are recorded in the tables.
Sulphonamide teratogens
263
RESULTS
Dichlorphenamide. Treatment of chicken embryos at 96 h with dichlorphenamide led to two types of abnormalities, namely malformations of the maxilla
and syndactylism (Table 1). The defects of the maxilla were in most instances
confined to a shortening of the upper beak, varying from slight to extreme, but
at times the upper beak deviated from the median plane (cross-beak). The extent
of syndactylism was also variable; its expression, as in the corresponding
mutant phenotype, concerned in the first place the relation between the third
and fourth toes, but in more extreme cases involved the entire foot. We used
dosages of 0-5,1 and 2 mg/egg. Even the smallest amount of the drug was highly
toxic, but the curve of embryo mortality did not rise steeply beyond treatment
Table 1. The effects of dichlorphenamide injected at 96 h of incubation
Dosage in mg (/*M)
0-5
(1-64)
10
(3-28)
20
(6-55)
154
Treated . . .
Mortality to 18 days (%)
351
Survivors
100
Normal (%)
650
Short or abnormal upper beak (%)
60
Syndactylism (%)
300
Miscellaneous defects (%)
00
249
50-2
124
43-5
12-9
52-4
0-8
452
52-4
215
36-3
18-1
591
1-9
with 1 mg. A somewhat similar dosage/response effect was seen in the incidence
of malformations: response rose directly with dosage from 0-5 to 1 mg, but
became less proportional between dosages of 1 and 2 mg. This was true for
beak defects as well as for syndactylism. The interference with development
produced by treatment with dichlorphenamide was for all tested dosages much
greater for the primordia of the feet than for those of the maxilla.
When tested at 24 h of incubation (Table 2) dichlorphenamide was extremely
toxic. The results with dosages of 0-4 and 0-75 mg/egg demonstrated clearly,
however, that the teratogenic responses were at this stage qualitatively the same
as those obtaining at 96 h.
Methazolamide. Chicken embryos which at the end of 96 h of incubation had
been treated with sodium methazolamide (Table 3) showed developmental
reactions very similar to those produced by dichlorphenamide and, as will be
discussed later, by acetazolamide. Most of the deformed embryos exhibited
maxillary abnormalities and syndactylism. The upper beak tended to be shorter
than normal, with an occasional asymmetry (cross-beak). In rare instances tibia
and fibula had sharp bends of the shaft either unilaterally or bilaterally. The
incidence of abnormalities was a similar one for maxilla and feet. Between
264
W. LANDAUER & N. WAKASUGI
0-5 and 2 mg/egg the malformations of the maxilla showed an approximately
linear dosage/effect relationship. No syndactylism was produced by treatment
with 0-5 mg, but in the range of 1 and 2 mg the response resembled in incidence
that observed for the maxilla. The occurrence of malformations of tibia-fibula,
rare at first, rose sharply when the dosage of sodium methazolamide was increased to 2-5 mg/egg (8-4 % among a total of 179 survivors as shown in Table 6).
Table 2. The effects of dichlorphenamide injected at 24 h of incubation
Dosage in mg
0-4
(1-31)
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal upper beak (%)
Syndactylism (%)
Miscellaneous defects (%)
0-75
(2-46)
165
861
23
17-4
47-8
73-9
4-3
161
86-3
22
40-9
22-7
54-5
4-5
Table 3. The effects of sodium methazolamide injected at 96 h of incubation
Dosage in mg (JAM)
0-5
(2-12
114
Treated . . .
18-4
Mortality to 18 days (%)
93
Survivors
96-8
Normal (%)
3-2
Short or abnormal upper beak (%)
00
Syndactylism (%)
00
Tibia-fibula bent (%)
00
Miscellaneous defects (%)
10
(4-23)
113
310
78
75-6
10-3
12-8
51
00
20
(8-46)
172
33-7
114
60-5
22-8
26-3
3-5
0-9
When injected after 24 h of incubation (Table 4) sodium methazolamide in
dosages of 0-5 and 1 mg/egg, though highly toxic, showed among the survivors
of the 13th and 18th day of incubation only a low incidence of abnormalities.
The few instances of recorded malformation were of the same type as those more
commonly encountered at the later stage of development, defects of the upper
beak and syndactylism.
Acetazolamide. In our earlier publication (Landauer & Wakasugi, 1967) we
had neglected to record the occurrence of syndactylism as a result of treatment
with acetazolamide. In testing the effects of 5 and 10 mg/egg acetazolamide,
injected after 96 h of incubation, we find now that the incidence of syndactylism
Sulphonamide
teratogens
265
was 23-6 and 43-1 %, respectively, among 72 and 51 embryos surviving the 18th
day.
p-Sulphamoylbenzoic acid. We inquired into the teratogenic nature of j?-sulphamoylbenzoic acid by injecting dosages of 1-5, 3, 5, 10, 20 and 40 mg/egg at
96 h of incubation and of 1-5, 3, 5 and 10 mg/egg at 24 h. Toxicity increased in
both stages with dosage, reaching 97 % with the largest amount at 96 h. In the
tests with 96 h embryos all survivors of the 13th day of incubation were normal.
A low incidence of malformed embryos was found among those that had been
treated at 24 h and had survived the 13th day, but these gave no indication of a
teratogen-specific effect.
Table 4. The effects of sodium methazolamide injected at 24 h of incubation
Dosage in mg OM)
0-5
10
(212)
(4-23)
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal upper beak (%)
Syndactylism (%)
Miscellaneous defects (%)
158
34-8
103
961
2-9
10
1-9
156
49-4
79
96-2
2-5
00
10
Metabolic protection. In a search for the steps by which dichlorphenamide and
sodium methazolamide produce abnormalities of the developing chicken embryo we made tests in which the two teratogens were given in combination with
a number of compounds which are know to play an important role in metabolic
functions and which in earlier work had been found to lessen the interference of
certain compounds with normal development. Such experiments were now
made at the 96 h stage by the simultaneous injection of 1-5 mg dichlorphenamide
and 5mg 1-tryptophan hydrochloride, 1-5 or 2mg dichlorphenamide and
0-25 mg nicotinamide; 2 mg dichlorphenamide and 0-75 mg sodium folate; and,
finally, of 2-5 mg sodium methazolamide and either 0-25 mg nicotinamide or
0-75 mg sodium folate. None of these combinations reduced the teratogenic
effect of either dichlorphenamide or sodium methazolamide. The combination
of 10 mg acetazolamide and 0-75 mg sodium folate was also without avail.
When, however, an injection of ADP was given immediately preceding treatment at 96 h with either one of the two teratogens the beneficial results were
unequivocal and striking. The combination of 3 mg ADP and 2 mg dichlorphenamide (Table 5) led among the survivors of the 18th day of incubation, as
compared with dichlorphenamide alone, to an increase in the incidence of
normal appearing embryos from 37-8 % to 61-9 % (P < 0-001), to a lowering
in the occurrence of maxilla defects from 23-7 % to 13-1 % (P = 0-05) and to a
266
W. LANDAUER & N. WAKASUGI
reduction in the frequency of syndactylism from 49- 4 % to 27-4 % (P < 0-001).
When treatment with sodium methazolamide was similarly supplemented with
ADP (Table 6) the incidence of embryos escaping any gross defects rose, in
comparison with methazolamide alone, from 44-1 % to 75-6 % (P < 0-0001);
the occurrence of maxilla malformations declined from 35-2 % to 14-6 %
(P < 0-001); the frequency of syndactylism was reduced from 26-3 % to 9-8 %
(P < 0-01) and that of abnormalities of the tibia-fibula declined from 8-4 % to
Table 5. The effects of dichlorphenamide (2 mgjegg) alone or
preceded by ADP (3 mg/egg) at 96 h of incubation
Dichlorphenamide ADP and
alone
dichlorphenamide
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal upper beak (%)
Syndactylism (%)
Miscellaneous defects (%)
332
530
156
37-8
23-7
49-4
1-3
161
47-8
84
61-9
131
27-4
1-2
Table 6. The effects of sodium methazolamide (2-5 mgjegg) alone or
preceded by ADP (3 mg/egg) injected at 96 h of incubation
Methazolamide
ADP and
alone
methazolamide
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal upper beak (%)
Syndactylism (%)
Tibia-fibula bent (%)
Miscellaneous defects (%)
310
42-3
179
441
35-2
26-3
8-4
00
169
51-5
82
75-6
14-6
9-8
2-4
1-2
2-4 % {P about 0-07). Except for the incidence of malformations of the tibiafibula (the least common among the abnormalities produced by methazolamide)
supplementation of dichlorphenamide and of methazolamide with ADP thus
led to a significant, and in most instances very highly significant, lowering in the
teratogenic effects of these compounds and a corresponding increase in normally
developing embryos. Embryo mortality was not significantly altered. The antiteratogenic effectiveness of ADP was tested only with the dosage of 3 mg/egg.
Supplementation with smaller or larger amounts might well have given even
more favourable results.
Interactions of sulphanilamide with dichlorphenamide and methazolamide. In
earlier experiments we had obtained results of considerable interest by treating
chicken embryos simultaneously with acetazolamide and sulphanilamide
Sulphonamide teratogens
267
(Landauer & Wakasugi, 1967). We decided now on corresponding tests in which
embryos were, after 96 h of incubation, exposed to combinations of 2 mg
dichlorphenamide and 1 mg sulphanilamide and of 2-5 mg sodium methazolamide and 1 mg sulphanilamide. The results are presented in Tables 7 and 8. In
Table 7. The effects of dichlorphenamide (2 mgjegg) and sulphanilamide
(1 mgjegg) injected alone or in combination at 96 h of incubation
Dichlorphenamide
Dichlorphenamide
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal upper beak (%)
Syndactylism (%)
Micromelia (%)
Parrot and/or short lower beak (%)
Miscellaneous defects (%)
120
and
sulphanilamide
117
Sulphanilamide
118
50-8
55-6
24-6
59
52
89
32-2
18-6
46-2
51-7
9-6
661
00
51-9
11
2-2
00
00
00
00
1-9
47-2
22-5
2-2
Table 8. The effects of sodium methazolamide (2-5 mgjegg) and sulphanilamide
(1 mg/egg) injected alone' or in combination at 96 h of incubation
Methazolamide
and
Methazolamide
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal upper beak (%)
Syndactylism (%)
Tibia-fibula bent (%)
Micromelia (%)
Parrot and/or short lower beak (%)
Miscellaneous defects (%)
105
sulphanilamide
106
Sulphanilamide
118
47-6
53-8
24-6
55
49
89
25-7
40-0
28-3
22-4
26-5
51-7
291
3-6
00
20
00
00
00
00
00
11
2-2
00
47-2
22-5
2-2
both situations it was found that the teratogenic consequences of treatment with
sulphanilamide were completely abolished in the presence of the second teratogen. There was suggestive evidence for the conclusion that inversely the
teratogenic effects of dichlorphenamide and methazolamide were reduced in
the presence of sulphanilamide, but the only difference that was close to statistical significance was that for abnormalities of the upper beak in the tests with
methazolamide (P about 0-05).
Sulphanilamide and sodium folate. In an attempt to learn more about the
metabolic pathway by which sulphanilamide produces its teratogenic effects on
268
W. LANDAUER & N. WAKASUGI
chicken embryos we decided to determine if folic acid would afford protection
against injected sulphanilamide. The results of our relevant tests are given in
Table 9. One mg of sulphanilamide and 0-75 mg sodium folate were injected
simultaneously into eggs that had been incubated for 96 h. The double injections
led to a rise in embryo mortality between 5 and 18 days (P about 0-02). Many
more embryos, however, developed normally (P < 00001) when sodium folate
was added to treatment with sulphanilamide; the incidence of micromelia was
reduced from 44-9 % to 21-9 % (P < 0-0001) and the frequency of beak defects
Table 9. The effects of sulphanilamide (1 mgjegg) and sodium folate
(0-75 mg/egg) injected alone or in combination at 96 h of incubation
Sulphanilamide
and sodium
Sulphanilamide
folate
Treated . . .
211
16-6
Mortality to 18 days (%)
176
Survivors
54-5
Normal (%)
Parrot and/or short lower beak (%) 27-3
Micromelia (%)
44-9
0-6
Miscellaneous defects (%)
211
26-5
155
77-4
14-2
21-9
0-6
was also lowered to less than half of what it was in the presence of sulphanilamide alone (P < 0-01). The protection which folic acid conferred would be very
highly significant even if the incidence of malformations were based on the same
survival as in the controls and can surely not be in doubt. The amount of folic
acid used in these experiments was dictated by the concentration of commercially
available 'Folvite' (Lederle) solution. The beneficial effects on development
might have been greater still in other dosage combinations.
Interactions of dichlorphenamide or methazolamide with 3-acetylpyridine. We
wish, finally, to record the results of treating chicken embryos at 96 h of incubation with a combination of either dichlorphenamide and 3-acetylpyridine or
sodium methazolamide and 3-acetylpyridine (Table 10). When embryos are
treated with 3-acetylpyridine alone the resulting malformations include muscular
hypoplasia as the principal symptom, a shortened maxilla as a less common
defect and, among the most deformed variants, a 'crooked neck' (to be discussed
later on). When dichlorphenamide and 3-acetylpyridine were given simultaneously, all the skeletal abnormalities were grossly exaggerated in incidence.
It should be noted that the combined treatment of the two teratogens leads to
synergism of the cervical malformation, although dichlorphenamide when given
alone does not produce it, and, similarly, that the incidence of dichlorphenamide-induced syndactylism is exaggerated in the presence of 3-acetylpyridine,
though it is not found among embryos treated with 3-acetylpyridine alone.
A somewhat similar situation was found when sodium methazolamide and
Sulphonamide teratogens
269
3-acetylpyridine were given in combination, although only synergism of
syndactylism had statistical significance in these tests. The incidence of muscular
hypoplasia, the only non-skeletal defect, was in both combinations reduced
below that due to 3-acetylpyridine alone, but the difference was significant only
when methazolamide had been added to treatment with 3-acetylpyridine.
Table 10. The effects of treatment with dichlorphenamide and 3-acetylpyridine or
with sodium methazolamide and 3-acetylpyridine, separately or combined, at 96 h
of incubation (dosages in mgjegg)
Dichlorphenamide
3-acetylpyridine
Methazolamide
Treated . . .
Mortality to 18 days (%)
Survivors
Normal (%)
Short or abnormal
upper beak (%)
Crooked neck (%)
Syndactylism (%)
Tibia-fibula bent (%)
Muscular hypoplasia (%)
Miscellaneous defects (%)
*
t
X
§
||
Difference from
Difference from
Difference from
Difference from
Difference from
10
00
00
10
0-5
00
00
0-5
00
00
0-5
20
00
00
20
144
500
72
9-7
41-7
172
33-7
114
60-5
22-8
249
50-2
124
43-5
12-9
139
59-7
56
00
57-1*
121
20-7
96
10
13 5
00
52-4
00
00
0-8
25-Of
98-2J
00
92-9
00
2-1
00
00
990
00
8-3
69-4§
00
81-9H
00
00
26-3
3-5
00
0-9
sum of dichlorphenamide and methazolamide: P = 0-001.
methazolamide: P < 00001.
dichlorphenamide: P < 00001.
methazolamide: P <^ 00001.
3-acetylpyridine: P < 00001.
DISCUSSION
We have now, together with our earlier report (Landauer & Wakasugi, 1967),
gathered information on the teratogenic activity in chicken embryos of four
sulphonamide compounds which are known as specific inhibitors of carbonic
anhydrase, namely acetazolamide, dichlorphenamide, methazolamide and
/7-sulphamoylbenzoic acid. When tested after 96 h of incubation, three of these
compounds were responsible for identical malformations of the facial skeleton
and the feet, and one (p-sulphamoylbenzoic acid) appeared to be non-teratogenic. A shortened upper beak was the principal abnormality of the facial
skeleton and syndactylism that of the feet, both with considerable variance in
expression. These two symptoms were the only ones for which acetazolamide
and dichlorphenamide were responsible at this developmental stage; they were
by far the most common ones following the injection of methazolamide, but
treatment with the latter compound led in rare cases also to a sharp bent of the
tibia-fibula shaft.
The same three sulphonamide compounds gave less uniform responses when
18
JEEM2O
270
W. LANDAUER & N. WAKASUGI
injected earlier in development, after 24 h of incubation. Among survivors of
treatment with methazolamide a few had a shortened upper beak and one
was syndactylous, but no other malformations were found and it would seem
that the compound has little teratogenicity at this stage. Dichlorphenamide, on
the other hand, yielded the same specific response, and with an even higher
incidence, after treatment at 24 h than after 96 h, and as at the later stage did
not interfere with other parts. Embryos exposed to acetazolamide at the 24 h
stage also tended to show the typical abnormalities of upper beak and toes, but
gave, in addition, a significant incidence of rumplessness and one embryo had
bent tibiae. Methazolamide and dichlorphenamide thus showed in frequency of
response of the two principal affected sites a noteworthy dissimilarity of stageresponse relationship, if treatment at 24 and 96 h is compared, suggesting that
the two drugs have quite distinct receptor ' affinities' in early stages, but similar
ones later.
Malformations other than those that have just been described were rare and,
as judged by extensive control material, were clearly unrelated to treatment with
the particular sulphonamide compounds and the developmental stages and
dosages at which they had been used.
We have reported earlier (Landauer & Wakasugi, 1967) that the beak abnormalities produced by acetazolamide either at 24 or 96 h of incubation follow in
their incidence a linear dosage-effect relationship. The same appears to be true
for syndactylism at 96 h, for which we have now gathered data. Treatment with
methazolamide at 96 h of incubation apparently also led in beak defects and
syndactylism to a linear increase of incidence with dosages between 0-5 and
2 mg. After dichlorphenamide injected at 96 h, on the other hand, the dosageresponse curves for beak defect as for syndactylism flattened strikingly above a
dosage of 1 mg/egg.
We have noted above interesting differences in receptor affinity for methazolamide and dichlorphenamide following treatment at 24 and 96 h of incubation.
The two compounds yielded equally interesting differences in 'efficacy' (Stephenson, 1956) if the principal sites responding to the teratogens are contrasted at
one and the same stage. Such a comparison will show that dichlorphenamide
had much greater efficacy in producing syndactylism than in leading to abnormalities of the maxilla. This was true at both developmental stages and for all
dosages that were tested. With methazolamide, on the contrary, the efficacy was
closely similar in the two sites when three dosages were used at 96 h and the
same was probably true for our admittedly inadequate tests at 24 h.
The manner in which 'carbonic-anhydrase inhibiting' sulphonamides and
certain other teratogens interfere with development of chicken embryos will be
discussed below in greater detail. Suffice it for now to establish the following
points. (1) There are sulphonamides that inhibit carbonic anhydrase without
producing teratogen-specific malformations of chicken embryos. This was
demonstrated by our data on j?-sulphamoylbenzoic acid. (2) Sulphanilamide and
Sulphonamide teratogens
271
related bacteriostatic sulphonamides produce different and more extreme
abnormalities of chick development than do the' carbonic-anhydrase inhibiting'
compounds which are the subject of the present report; yet it is known that
sulphanilamide also acts as an inhibitor of carbonic anhydrase, if a weak one.
Another bacterisotatic sulphonamide which produces in chicken embryos
malformations similar to those of sulphanilamide, namely A^-sulphamyl-A^-wbutylcarbamid, has, in addition, hypoglycaemic properties. Sulphonamides with
pronounced hypoglycaemic activity have not found detailed use in teratological
work, but we have extensive, unpublished data on the effect of l-butyl-3ptolylsulphonylurea (Tolbutamid, Orinase) in chicken embryos after treatment at
24 or 96 h of incubation. The compound had very low teratogenic activity, but produced after high dosages (given at 96 h) a low incidence of cleft palate with crossbeak, abnormalities of the tibiotarsal-tartsometatarsal joints and micromelia.
There is good evidence, as will be discussed below, that the teratogenic
activity of sulphanilamide and of allied compounds interferes with NAD
functions and that this interference is completely overcome by providing
adequate amounts of nicotinamide. (3) Our present data have proven satisfactorily that the teratogenic effects of acetazolamide, dichlorphenamide and
methazolamide on chick development can be greatly lessened or prevented
altogether in the presence of ADP, thus demonstrating once again that disturbances of NAD functions are paramount in producing the discussed deviations from normal development.
Teratogenicity of a sulphonamide compound, specifically known as inhibitor
of carbonic anhydrase, was first reported by Layton & Hallesy (1965) from
experiments in which they had treated pregnant rats and mice with acetazolamide
and had found among some of the newborn young skeletal defects of forearm
and hand. Subsequent investigations demonstrated that very similar results can
be produced with dichlorphenamide, ethoxyzolamide (6-ethoxybenzolthiazolo2-sulphonamide) and methazolamide (Wilson et al. 1966; Layton, as quoted by
Maren, 1967). The remarkable uniformity of response which these four sulphonamides call forth in mammalian embryos thus parallels the similar uniformity of
abnormalities in chick development produced by three of them (acetazolamide,
dichlorphenamide and methazolamide), even though the sites of deviation from
normal morphogenesis are very different ones.
The aetiological interpretations of the sulphonamide experiments with rats
and mice have had a curious history.1 Layton & Hallesy (1965) made the
1
It seems to the senior author that work pursued during recent years in the field of mammalian teratology has been much less productive than it needed to be. The peculiarities of
uterine implantation and of placentation seem to have acted as bogeys intimidating many
investigators from using techniques or material far better suited to causal analysis than is the
study of young treated in utero. The excellent review by Beck & Lloyd (1966) on experiments
with trypan blue should do much to drive home this point. The use of organ culture, histochemistry or many other in vitro techniques with mammalian material—at a pinch even of
chicken embryos—would surely have brought greater profits in terms of essential principles.
18-2
272
W. LANDAUER & N. WAKASUGI
cautious observation that 'the only pharmacological activity ascribed to
acetazolamide has been carbonic anhydrase inhibition', but that they are 'not
aware of any postulated role of carbonic anhydrase in limb development'. In a
more positive statement Maren (1965) asserted that the data of Layton &
Hallesy on acetazolamide 'have certain features which lead me to believe that
carbonic anhydrase inhibition is not involved in the teratogenic effects' and
that 'indeed the effects may not be those of acetazolamide itself'. Yet, more
recently, Maren (1967, quoting from Wilson et al. 1968) expressed it as his
conviction that 'the question of whether specific carbonic anhydrase inhibition
is involved is probably answered by the fact that ethoxzolamide, dichlorphenamide and methazolamide all yield the abnormality'. The facts alluded to suggest
obviously at best that chemically related compounds are likely to produce
related effects. Maren continues by saying that 'we currently believe that the
lesion is secondary to carbonic anhydrase inhibition in the mother or embryonic
membranes, since drug restricted to days 10-11 of pregnancy elicit the lesion,
but enzyme does not appear in the embryo until day 13'. It is, of course, exactly
this quandary that made us doubt, as it clearly did Maren in 1965, that carbonicanhydrase inhibition plays any role whatever in the origin of sulphonamideinduced malformations whether of rats, mice or chicks. Hence we searched for
another explanation that would account for the teratogenicity of these 'carbonic-anhydrase inhibiting' sulphonamides. We believe that we have found it
in the fact that, for chicken embryos at any rate, ADP provides very efficient
protection against the teratogenic activity of acetazolamide, dichlorphenamide
and methazolamide.
The conclusions arrived at from the data on supplementation with ADP find
notable support from the results of experiments with sulphanilamide and
related sulphonamides. Ancel (1945) first reported that various sulphonamides
with bacteriostatic activity, including sulphanilamide, sulphapyridine and
iV-sulphanilylacetamide, uniformly lead to a syndrome of micromeha, syndactylism and parrot beak. This was confirmed by Zwilling & DeBell (1950) and
extended to A^-sulphanyl-iVa-H-butylcarbamid (Nadisan) by Tisna-Amidjaja
(1958) and to 6-sulphanilamido-2,4-dimethylpyrimidine (Aristamid) by TisnaAmidjaja (1958) and Erhard (1961). Zwilling & DeBell have shown that the
teratogenic effects of sulphanilamide can be entirely precluded by providing
supplementary nicotinamide. A similarly beneficial effect of nicotinamide on the
teratogenic activity of 6-sulphanilamido-2,4-dimethylpyrimidine is suggested by
the observations of Tisna-Amidjaja and the same may well hold for all the
sulphonamides which are responsible for the micromelia syndrome of chicken
embryos. Since the principal metabolic effect of sulphanilamide is that of analog
and competitor of j^-aminobenzoic acid and since the latter is chiefly concerned
in the synthesis of folic acid, it seemed important to determine if folic acid would
also counteract the teratogenic effects of sulphanilamide. Our tests have shown
that this is, indeed, true. It was pointed out by Henry (1944) that following
Sulphonamide teratogens
273
sulphanilamide treatment 'it is not inhibition of the overall total respiration of
a cell which is significant in inhibition of cell division, but rather the inhibition
of that fraction which is specifically concerned with providing the energy for cell
division'. Experimentation with analogs of folic acid has provided elegant proof
for this conclusion (Grant, 1960). It would seem most likely that sources of
energy production are the common denominator of nicotinamide and folic acid
in their protective role against the teratogenicity of sulphanilamide. The role
played in chick development by sulphanilamide as a teratogen may be that of
interfering with purine synthesis or with functions of NAD-linked dehydrogenases, resulting in local scarcity of folic acid.
O,NH2
Y!
N
Sulphanilamide
N
Acetazolamide
Dichlorphenamide
COOH
i
T TT
N
NN
SO2NH2
Methazolamide
Ethoxyzolamide
/>Sulphamoylbenzoic acid
Fig. 1. The structural formulae of sulphanilamide and of sulphonamides
which inhibit carbonic anhydrase.
A comparison of the 'carbonic-anhydrase inhibiting' sulphonamides, including /7-sulphamoylbenzoic acid and also sulphanilamide, shows (Fig. 1) that
all six compounds contain the sulphamyl moiety SO2NH2, substitution of which
is known to be incompatible with the pharmacodynamic properties of sulphonamides. The fact that/7-sulphamoylbenzoic acid is virtually non-teratogenic
to chicken embryos shows, however, that it is not the sulphamyl group per se
which is the cause of malformations, although it is presumably an important
link. The dissimilarity in symptoms of treated chicken embryos between sulphanilamide and the three 'carbonic-anhydrase inhibiting' compounds used by
us is presumably an expression of predilection for different sites of attachment;
yet the appreciable differences in chemical constitution between the three (or,
in the mammalian tests, four) 'carbonic-anhydrase inhibiting' compounds did
274
W. LANDAUER & N. WAKASUGI
not prevent involvement of identical sites and production of remarkably similar
results.
A comparison of our observations on sulphonamides with our earlier results
(Landauer, 1957; Landauer & Wakasugi, 1967) from experiments with analogs
of nicotinamide (Fig. 2) shows interesting similarities and differences. Nicotinamide itself, the prototype compound that plays an important role in cellular
metabolism, is teratogenic when given in excess by producing a shortening of
the maxilla. There is every reason to believe that this effect is due to inhibition
of nucleotide-linked dehydrogenases (Alivisatos & Denstedt, 1952). The several
o
o
f^r-t
f^r\
LJ
k J
\H,
Nicotinamide
f^Y\
CH3 H . N - L J
3-Acetylpyridine
o
NH.C2H5
A'-Ethylnicotinamide
^ / ^
NH2
6-Aminonicotinamide
o
p
r^^n—c
x
c
^
o
N(C2H3)2.
^Af-Diethylnicotinamide
(Nikethamide, Coramin)
U
^
N
/ >
NH.CH 3
A'-Methylnicotinamide
Fig. 2. The structural formulae of nicotinamide and analogs of nicotinamide.
analogs of nicotinamide showed great diversity in their effects on developing
chicken embryos: 3-acetylpyridine produced muscular hypoplasia, shortness of
upper beak and a crooked neck; 6-aminonicotinamide led to micromelia and
parrot (short lower) beak, as does an analog of tryptophan; iV-ethylnicotinamide
brought about abnormalities of the upper beak and defective development of
tibiotarsus and fibula with secondary luxation of the feet; nikethamide and Nmethylnicotinamide had only slight teratogenic qualities. Malformations produced in chicken embryos by the sulphonamides and the analogs of nicotinamide
all concerned the facial and appendicular skeleton, the only additional defects
being those due to treatment with 3-acetylpyridine (muscular hypoplasia and
crooked neck). The incidence of any of these abnormalities, whether due to
sulphonamides or analogs of nicotinamide, could, without exception, be greatly
reduced or prevented altogether by providing nicotinamide in some and ADP in
the remaining ones as protecting supplement. The relatively slight differences in
teratogenic effect which exist between the several ' carbonic-anhydrase inhibiting' sulphonamides may be due to chemical dissimilarities between them, but
the differences in effect between analogs of nicotinamide are clearly a result of
distinctions between sites of attachment. The importance of nicotinamide and
Sulphonamide teratogens
275
ADP in providing protection against all these teratogens is testimony to the
catholicity of their effectiveness.
The protection against teratogenic interference with morphogenesis can take
various forms and these, as far as they apply to our material, should be discussed
briefly. To start with, the activity of a teratogen can become frustrated if its
anchoring groups are blocked by formation of an addition compound. This is
presumably what happened in all those instances in which, in our experiments,
sulphanilamide lost its teratogenic effectiveness when it was administered in
combination with iV-ethylnicotinamide, acetazolamide, dichlorphenamide and
methazolamide (Landauer & Wakasugi, 1967, and present data), and also after
joint treatment of sulphanilamide with ethyl carbamate (unpublished data).
This last result is of interest since it had been shown by Johnson, Eyring &
Kearns (1943) that sulphanilamide and ethyl carbamate combine in solution
and that (at low temperature) the presence of the ethyl carbamate causes strong
antagonism to sulphanilamide inhibition. Again, in earlier work we could
ascribe to complexing of boric acid with riboflavin reduced teratogenicity of the
complex compared to boric acid alone (Landauer, 1952, 1953). It seems very
likely that in all such instances inability of the complex to attach itself to sites
that would otherwise be responsible for interference with steps of morphogenesis
accounted for 'protection'.
Competition for sites of attachment is clearly another important path leading
to neutralization of teratogenic activity. This occurs presumably whenever
metabolites or supplementary drugs either prevent or lower the chances of a
teratogen to reach vulnerable sites of attachment or become attached to additional receptors of these sites and thereby overcome or repair the activity of
neighbouring teratogen-occupied receptors. It is most likely that the protective
effect of normal metabolities, such as our results with tryptophan, nicotinamide
and ADP, find their explanation on this basis. Additional illustrations are
presumably provided by (1) the fact that supplementary 3-acetylpyridine, on
account of its well-established metabolic value when substituted into pyridine
nucleotide (Kaplan, Ciotti & Stolzenbach, 1956) provides partial protection
against the effects of simultaneously injected 6-aminonicotinamide (Landauer &
Clark, 1962 a) and safeguards completely against the teratogenic action of
sulphanilamide (Landauer & Clark, 1964«); (2) differential protection against
the teratogenic effects of eserine sulphate by furnishing supplements of either
tryptophan or nicotinamide.
The extent to which a particular metabolite affords protection against the
morphogenetic disturbances of teratogens is frequently far from complete. This
is often, and perhaps always, due to ignorance of the proper timing and dosage
of the protecting compound. Of more intrinsic interest is the fact that in certain
situations protection may be differential or that a metabolite which becomes
teratogenic when applied in excess (such as nicotinamide) may with lower
dosage, but in the presence of another teratogen, produce an unexpected exag-
276
W. LANDAUER & N. WAKASUGI
geration of these malformations. Interesting examples of this kind have been
found in experiments involving an abnormality of the cervical vertebrae known
as 'crooked neck', and of which a brief historical statement is required as
background.
The crooked-neck malformation was first described by Asmundson (1945) as
part of the homozygous phenotype of a recessive lethal mutation of fowl which
he called 'crooked neck dwarf (symbol en). The principal morphological features of embryos homozygous for the en gene, besides abnormality of the neck,
are muscular hypoplasia, especially along the legs, and shortness of the upper
beak. The twisted and short condition of the neck is, according to our own
(unpublished) observations, caused by defective development and fusion of
cervical vertebrae. The same is true for the homologous condition which we
obtained after treatment of embryos with nicotine sulphate, eserine and 3-acetylpyridine. Nicotine sulphate produced all the symptoms of the en/en phenotype,
and we could demonstrate that their incidence was significantly greater in cn\ +
embryos than in their + / 4- sibs (Landauer, 1960). This was accepted as evidence
of phenocopy production, i.e. of the existence of certain shared pathways in
development of mutant and homologous, non-genetic condition. Similar proof
for the phenocopy nature of induced abnormalities was obtained in experiments
with 6-aminonicotinamide (Landauer, 1965).
An example of differential protection was found in an experiment in which,
after 96 h of incubation, either nicotinamide (5 mg/egg) or tryptophan hydrochloride (5 mg/egg) was used as supplement to treatment with 0-5 mg/egg
eserine sulphate (Landauer, 1957). The following percentage frequencies of
malformations were found among survivors of the eighteenth day of incubation:
Micromelia
Parrot beak
Crooked neck
Eserine
alone
Eserine and
tryptophan
Eserine and
nicotinamide
96-0%
95-0%
93-3%
0-0%
1-8%
93-6%
0-0%
00%
800%
The entire syndrome of parrot beak, crooked neck and micromelia occurred
when eserine alone was given. Supplementation with either tryptophan or
nicotinamide led to virtually complete protection against interference with beak
and leg development, but reduced abnormality of the cervical vertebrae little, if
at all. The causes of such differential protection (or lack thereof) remain
unknown, even if, as suggested above, competition for sites probably plays a
role.
An example of extreme exaggeration of the crooked-neck condition was
observed (Landauer & Clark, 1964a) when treatment with sulphanilamide
(1 mg/egg) and 3-acetylpyridine (0-5 mg/egg) was combined. The crooked-neck
abnormality, in our experience, never occurred after injection of sulphanilamide
Sulphonamide teratogens
277
alone; after treatment with 0-5 mg 3-acetylpyridine we found it only among
those embryos that showed the most extreme deviation from normality. The
effect of combined treatment is illustrated by the following percentage figures:
Stage of treatment
Sulphanilamide
alone
All at 96h
Sulphanilamide at 96 h
3-acetylpyridine
at 120 h
Sulphanilamide and
3-acetylpyridine
3-acetylpyridine
alone
00%
16-7%
3-2%
00%
62-8%
5-7%
The same kind of synergism was found when embryos were treated with
dichlorphenamide and 3-acetylpyridine (Table 10). Veldstra (1956, 1963) has
emphasized that synergism is most commonly brought about by 'sites of loss'
being occupied by an associated compound, i.e. by a compound which, without
having specific pharmacodynamic properties, enhances the effect of a drug by
replacing normal metabolites. It seems most likely that such was the origin of
synergism in our material.
In our studies on the experimental production of malformations in chicken
embryos it was observed early that the effects of many and important teratogens
could be forestalled by providing supplementary nicotinamide and this led us,
on the basis of much additional information, to the conclusion that the morphogenetic disturbances produced by these compounds arose via diphosphopyridine-mediated pathways, interference with dehydrogenation and with synthesis
of ATP (Landauer, 1948a, 1954; Landauer & Clark 19626, 1963). These conclusions have found crucial support in our proof that the teratogenic activity of
all three sulphonamides used in the current experiments was greatly reduced in
the presence of ADP. The nature and extent of these defence mechanisms varies
with the chemical constitution of particular teratogens, but also with factors of
the internal environment of embryos, such as age-determined dissimilarities of
response to one and the same teratogenic ompound. Examples can be seen, on
the one hand, in the fact that at the same developmental stage tryptophan and
nicotinamide will be helpful supplements with some teratogens and ADP with
different ones, and, on the other hand, by such observations as that in early
embryonic stages ADP will provide protection from insulin teratogenicity,
while nicotinamide does not (Barron & McKenzie, 1962), but that in later stages
both will be effective.
The importance of pyridine nucleotides as carriers of hydrogen atoms, as
shown in the classic publication of Warburg & Christian (1936), and the
elucidation of the mechanism of ATP production via oxidation of Krebs-cycle
intermediates opened the way to an understanding of the need for and use of
energy production in biological functions. A beginning has now been made in
studying events which by interfering with the availability of energy resources
278
W. LANDAUER & N. WAKASUGI
produce abnormality of embryonic development. Thus the knowledge that
3-acetylpyridine and 6-aminonicotinamide can be substituted into pyridine
nucleotide led Coper & Herken (1963), Coper & Neubert (1963) and Herken
1965, 1966) to experiments in which they could show that the presence of
3-APNAD is by extra-mitochondrial events, namely by processes occurring in
the microsomes, responsible for reduced production of ATP, while the presence
of 6-aminonicotinamide-substituted pyridine nucleotide causes its cessation.
Our own observations have, on the other hand, provided proof (Landauer, 1957)
that the malformations induced by treatment with 3-acetylpyridine or 6-aminonicotinamide can be prevented altogether by offering supplementary nicotinamide in adequate amounts.
Morphogentic abnormality of chick development may occur in yet another
way of interference with ATP production, namely by the uncoupling of oxidative phosphorylation. Uncoupling compounds include dinitrophenol, arsenate,
antimycin A, thyroxin, probably insulin, and many others; the majority of
those that were tested were found to be teratogenic (Ancel, 1950; Landauer &
Clark, 1963, 19646; Ernster, 1965; Stoll and Maraud, 1965; Reporter & Ebert,
1965). In his fascinating discussion on bionergetics Szent-Gyorgyi (1957)
suggested in regard to the uncoupling reaction of dinitrophenol that it ' is not
involved in a definite chemical reaction, but acts rather through some physical
principle, as in the quenching of E*', i.e. of 'excitational energy', as in bioluminescence. Since sulphanilamide is a known quencher of bioluminescence
(Johnson & Chase, 1942; Johnson, Eyring & Williams, 1942), such a physical
factor needs to be considered as one of the steps in a reaction chain that leads
to teratogenicity. Another, perhaps related, physical agency in the origin of
abnormality is suggested by work of Lee, Dobson &van Rooyen(1966), who, from
in vitro observations of the effects which 6-aminonicotinamide has on chickembryo fibroblasts, came to believe that the compound lowered the capacity
of these cells to take up glucose. Such a membrane effect would, if confirmed,
account directly for reduced production of ATP as a cause of abnormal
development.
We believe that the facts which have been presented demonstrate that the
teratogens under consideration exert their effects by starving specific sites of
developing embryos of energy required for normal functions. It is to be expected
that sites so affected would do worse still if, in addition to site-specific treatment,
the organism as a whole were denied adequate nutrition. That this is indeed so
has been brought out convincingly by experiments in which teratogen treatment
was combined with fasting (Runner & Miller, 1956; Kalter, 1960; Dagg &
Kallio, 1962; Smithberg & Runner, 1963; Runner, 1967). Such combinations of
specific and systemic 'starvation' presumably result in differential lowering in
rates of cell division and in cell death. There is much evidence for the important
role of differential cell death in normal and abnormal morphogenesis (e.g.
Saunders & Fallon, 1966; Zwilling 1942, 1959). Recent work by Blake, Blake,
Sulphonamide teratogens
279
Loh & Kun (1967) has brought out that there is 'apparent tissue specificity of
NAD precursors causing markedly different rates of NAD accumulation in
various tissues', a conclusion that strongly supports the interpretation of our
data.
The phenocopy problem and the genotypic responsiveness of embryos to
teratogens are not part of the present discussion, but it is of great interest to
note that cytoplasmic factors play a role in the biochemical changes which occur
in the origin of malformations. This was first brought out in experiments in
which the production of rumplessness by treatment with insulin was studied on
embryos of White Leghorn and Jungle fowl stock and on embryos from reciprocal crosses between the two breeds. The parent stocks differed greatly in response
to insulin and the progenies of the two crosses behaved in incidence closely
similar to that of the respective maternal stock (Landauer, 1948b). Analogous
observations have been reported for mice by Goldstein, Pinsky & Fraser (1963)
and by Verrusio, Fraser & Pollard (1967). Two different types of cellular
organelles have been implicated as site of these cytoplasmic events, the microsomes and the mitochondria (general reviews by Siekevitz, 1965; Ernster, 1965).
We have referred already to the fact that the effects of 3-acetylpyridine are
generated by the microsomes rather than the mitochondria. It has been shown,
on the other hand, that uncouplers of oxidative phosphorylation intervene in
the electron-transport chain of the mitochondria. For one such compound,
Janus Green B, for which in vitro and in vivo inhibition of oxidative phosphorylation has been demonstrated (Dianzani & Scuro, 1956), Braun (1954,
1964, 1966) has described in detail his beautiful studies on its teratogenic effects
in developing chicken embryos and the role played in them by the mitochondria.
Yerrusio et al. (1967) also suggested that mitochondrial activity played a role in
their material.
It seems altogether likely that functional modifications of dehydrogenation
and ATP production, originating with the microsomes or mitochondria, are one
of the important causative agencies by which teratogens intervene in normal
development. In a study on oxidative phosphorylation in mitochondria isolated
from Minute mutants of Drosophila, Farnsworth (1965) came to the conclusion
that: ' In all Minutes, oxidative phosphorylation was found to be abnormal in
that phosphorylation was uncoupled from oxygen uptake to a greater extent as
compared to controls. The results strengthen the hypothesis that lesions in
oxidative phosphorylation are associated with the Minute phenotype and
demonstrate genetic control over this basic cellular process.'
All in all it would therefore seem justified on current evidence to assume that
microsomes and mitochondria are an important ground on which the activities
of genes and of environmental forces meet and on which by interaction phenocopies may be produced.
280
W. LANDAUER & N. WAKASUGI
POSTSCRIPT
Experiments in which chicken embryos of stages 4 and 5 (HamburgerHamilton) were explanted to a culture medium into which thalidomide, with
or without the addition of ATP, had been incorporated, have been reported
recently by Ruano Gil (1967). He found that among thirty-four control embryos
23-5 % showed platyneuria; in the presence of thalidomide the same arrest in
closure of the neural tube rose to 82-2 %(N = 73), but fell to the control level
(24-2 % of 34) when thalidomide and ATP were present simultaneously. In light
of our own results it seems likely that thalidomide, at least in this material, led
to localized interference with sources of cellular energy and that ATP helped to
overcome this interference. A report by Dyban & Akimova (1966), seen only in
abstract, seems to point to similar conclusions. A paper by Hallesy & Layton
(1967) was received too late to be discussed, but the title was added to our list of
references.
SUMMARY
Experiments performed on chicken embryos gave the following results.
1. Treatment with dichlorphenamide and methazolamide at 96 h of incubation
led to shortening of the maxilla and syndactylism; methazolamide was, in
relatively rare cases, responsible also for bending the tibia-fibula shaft. Treatment with acetazolamide, for which a short maxilla had been reported previously, in addition causes syndactylism. The same symptoms as at 96 h were
produced after 24 h of incubation by dichlorphenamide, and with even higher
incidence; but only as a rare event by methazolamide. Another sulphonamide,
^-sulphamoylbenzoic acid, known like the first three compounds for its activity
as inhibitor of carbonic anhydrase, has little, if any, teratogenic effects.
2. ADP given before injecting one of the teratogens at 96 h greatly reduced
the teratogenic effects of dichlorphenamide and methazolamide, as it had done
with acetazolamide in earlier tests.
3. The teratogenic effectiveness of sulphanilamide is abolished in the presence
of either dichlorphenamide, methazolamide or ethyl carbamate, probably by
formation of addition compounds.
4. Sodium folate was found, in tests at 96 h, greatly to lessen the incidence of
malformations produced by sulphanilamide.
5. When treatment with dichlorphenamide and 3-acetylpyridine was combined at 96 h a high degree of synergism occurred in incidence of beak defects,
crooked neck and syndactylism; similar, if less extreme, exaggeration occurred
when methazolamide and 3-acetylpyridine were combined.
6. There is no evidence that the teratogenic effects of any of the sulphonamides were caused by inhibition of carbonic anhydrase; there is much evidence
that the effects were due to interference with NAD functions.
The implications of our results are discussed in relation to their significance
for general problems of experimentally induced malformations.
Sulphonamide teratogens
281
RESUME
Etudes teratologiques avec les sulfonamides et lews implications
Les experiences realisees sur l'embryon de Poulet ont donne les resultats
suivants:
1. Le traitement avec la dichlorphenamide et la methazolamide a 96 h de
l'incubation determine le raccourcissement du maxillaire (superieur) et la
syndactylie; la methazolamide est dans des cas relativement rares egalement
responsable de la coudure du tibia-perone. Le traitement avec l'acetazolamide
provoque non seulement un raccourcissement du maxillaire ainsi que l'ont
montre les recherches anterieures, mais ainsi la syndactylie. Ces memes effets,
observes a 96 h, ont ete obtenus en proportions plus elevees, apres 24 h d'incubation a l'aide de dichlorphenamide mais dans quelques cas seulement avec la
methazolamide. Une autre sulfonamide, l'acide j?-sulfamoylbenzoique, connue
comme les trois premiers composes pour son activite inhibitrice de l'anhydrase
carbonique s'il y en a peu d'effets teratogenes.
2. ADP administre avant l'injection de l'une des substances teratogenes a
96 h reduit les effects teratogenes de la dichlorphenamide et de la methazolamide;
le meme effet a ete observe dans le cas de l'acetazolamide au cours de recherches
anterieures.
3. L'efficacite teratogene de la sulfanilamide est inhibee en presence soit de
dichlorphenamide, de methazolamide ou de carbonate d'ethyle, vraisemblablement par la formation de composes supplementaires.
4. Le folate de sodium applique a 96 h reduit de maniere importante la proportion de malformations obtenues avec la sulfanilamide.
5. La combinaison des deux traitements, dichlorphenamide et 3-acetylpyridine a 96 h de l'incubation produit un effet synergique eleve pour l'obtention
de malformations du bee, de torsions cervicales et de la syndactylie; une exageration similaire, mais moins intense, a ete obtenue dans le cas d'une combinaison
de methazolamide et de 3-acetylpyridine.
6. Ces recherches n'apportent pas la preuve que les effets teratogenes des
sulfonamides sont provoquees par une inhibition de l'anhydrase carbonique; il
semble plutot que les effets soient dus a une interference avec les fonctions NAD.
Les implications de nos resultats sont discutees en rapport avec leur importance par les problemes generaux concernant les malformations experimentales.
Our work was generously supported by the Association for the Aid of Crippled Children
and the Muscular Dystrophy Associations of America. We wish to express particular gratitude to Dr L. G. Goodwin, Director of the Nuffield Institute of Comparative Medicine, The
Zoological Society of London, for the gracious liberality with which he offered us the hospitality of the Institute's laboratories, and to Dr Robert J. Slater, Director of the Association
for the Aid of Crippled Children, for his sympathetic interest in our work.
We are grateful to Mr A. J. Lee for Figs. 1 and 2, to Dr David C. P. Brown and Merck
Sharp and Dohme Ltd. (Hoddesdon, Hertfordshire) for supplies of dichlorphenamide and
to Dr W. Boehm and Cyanamid International (Pearl River, New York) for a gift of Neptazane
(methazolamide).
282
W. LANDAUER & N. WAKASUGI
REFERENCES
S. G. A. & DENSTEDT, O. F. (1952). Nicotinamide inhibition of tri- and diphosphopyridine nucleotide-linked dehydrogenases. / . Biol. Chem. 199, 493-504.
ANCEL, P. (1945). L'achondroplasie. Sa realisation expeiimentale — sa pathogenic Ann.
Endocrin. 6, 1-24.
ANCEL, P. (1950). La Chimioteratogenese. Paris: G. Doin and Cie.
ASMUNDSON, V. S. (1945). Crooked neck dwarf in the domestic fowl. /. Hered. 36, 173-76.
BARRON, P. & MCKENZIE, J. (1962). The inhibitory action of insulin in the early chick
embryo. /. Embryol. exp. Morph. 10, 88-98.
BECK, F. & LLOYD, J. B. (1966). The teratogenic effects of azo dyes. Adv. Teratol. 1, 131-93.
BLAKE, R. L., BLAKE, S. L., LOH, H. H. & KUN, E. (1967). The effect of nicotinamide and
homologs on the activity of inducible enzymes and NAD content of the rat liver. Molec.
Pharmacol 3, 412-22.
BRAUN, S. (1954). Janus Green B teratological action in embryonated hen's eggs and embryogenetic and carcinogenetic bearings of its mechanism of action. Ada Morph. Acad. Sci.
Hung. 4, 61-79.
BRAUN, S. (1964). Current problems of experimental teratogenesis. Ada Morph. Acad. Sci.
Hung. Suppl. 13, 1-14.
BRAUN, S. (1966). The role of mitochondria in the early morphogenesis of chick embryos.
Ada Morph. Acad. Sci. Hung. 14, 51-8.
COPER, H. & HERKEN, H. (1963). Schadigung des Zentralnervensystems durch Antimetaboliten
des Nikotinsaureamids. Dt. med. Wschr. 88, 2025-36.
COPER, H. & NEUBERT, D. (1963). Reaktionsgeschwindigkeit NADP-abhangiger Oxydoreduktasen aus Rattengehirn mit 3-Acetylpyridin-Adenin-Dinucleotid-Phosphat. J. Neurochem.
10, 513-22.
DAGG, C. P. & KALLIO, E. (1962). Teratogenic interaction of fluorodeoxyuridine and thymidine (Abstract). Anat. Rec. 142, 301-2.
DIANZANI, M. U. & SCURO, S. (1956). The effects of some inhibitors of oxidative phosphorylation on the morphology and enzymatic activities of mitochondria. Biochem. J. 62,
205-15.
DYBAN, A. P. & AKIMOVA, I. M. (1966). The significance of B vitamins and genetic factors in
the response to thalidomide of rat embryos. [In Russian.] Arkh.Anat. Gistol Embriol. 51,
3-17.
ERHARD, R. (1961). Untersuchungen iiber die Wirkung von Aristamid auf Hiihnerembryonen.
Arch. EntwMech. Org. 153, 217-35.
ERNSTER, L. (1965). Control of cell metabolism at the mitchondrial level. Fedn. Proc. 24,
1222-36.
FARNSWORTH, M. W. (1965). Oxidative phosphorylation in the Minute mutants of Drosophila.
J. exp. Zool. 160, 363-8.
GOLDSTEIN, M., PINSKY, M. F. & FRASER, F. C. (1963). Genetically determined organ specific
responses to the teratogenic action of 6-aminonicotinamide in the mouse. Genet. Res. 4,
258-65.
GRANT, P. (1960). The influence of folic acid analogs on development and nucleic acid
metabolism in Ranapipiens embryos. Devi. Biol. 2, 197-251.
HALLESY, D. W. & LAYTON, W. M. J. (1967). Forelimb deformity of offspring of rats given
dichlorphenamide during pregnancy. Proc. Soc. exp. Biol. Med. 126, 6-8.
HENRY, R. J. (1944). The mode of action of sulfonamides. Josiah Macy Jr. Foundation
Review Series 2, no. 1.
HERKEN, H. (1965). Pharmakologische Untersuchungen zum Problem der selektiven Vulnerabilitat des Gehirns. Arzneimittel-Forsch. 15, 707-18.
HERKEN, H. (1966). Die Wirkung von Antimetaboliten auf den Zellstoffwechsel des Warmbluters. Der Internist 7, 375-91.
JOHNSON, F. H. & CHASE, A. M. (1942). The sulfonamide and urethane inhibition of Cypridina
luminescenence in vitro. J. cell. comp. Physiol. 19, 151-61.
ALIVISATOS,
Sulphonamide teratogens
283
F. H., EYRING, H. & KEARNS, W. (1943). A quantitative theory of synergism and
antagonism among diverse inhibitors, with special reference to sulfanilamide and urethane.
Arc/is. Biochem. 3, 1-31.
JOHNSON, F. H., EYRING, H. & WILLIAMS, R. W. (1942). The nature of enzyme inhibitions in
bacterial luminescence: sulfanilamide, urethane, temperature and pressure. /. cell. comp.
Physiol. 20, 247-68.
KALTER, H. (1960). Teratogenic action of a hypocaloric diet and small doses of cortisone.
Proc. Soc. exp. Biol. Med. 104, 518-20.
KAPLAN, N. O., CIOTTI, M. M. & STOLZENBACH, F. E. (1956). Reaction of pyridine nucleotide
analogues with dehydrogenases. /. biol. Chem. 221, 833-44.
LANDAUER, W. (1948 a). The effect of nicotinamide and a-ketoglutaric acid on the teratogenic
action of insulin. / . exp. Zool. 109, 283-90.
LANDAUER, W. (19486). Hereditary abnormalities and their chemically-induced phenocopies.
Growth 12, 171-200.
LANDAUER, W. (1952). Malformations of chicken embryos produced by boric acid and the
probable role of riboflavin in their origin. /. exp. Zool. 120, 469-508.
LANDAUER, W. (1953). Complex formation and chemical specificity of boric acid in production of chicken embryo malformations. Proc. Soc. exp. biol. Med. 82, 633-6.
LANDAUER, W. (1954). On the chemical production of developmental abnormalities and of
phenocopies in chicken embryos. /. cell. comp. Physiol. 43 (Suppl. 1), 261-305.
LANDAUER, W. (1956). Hereditary and induced cross-beak of fowl. Experiments with ethyl
carbamate. /. exp. Zool. 132, 25-38.
LANDAUER, W. (1957). Niacin antagonists and chick development. /. exp. Zool. 136,
509-30.
LANDAUER, W. (1960). Nicotin-induced malformations of chicken embryos and their bearing
on the phenocopy problem. /. exp. Zool. 143, 107-22.
LANDAUER, W. (1965). Gene and phenocopy: selection experiments and tests with 6-aminonicotinamide. / . exp. Zool. 160, 345-54.
LANDAUER, W. & E. M. CLARK (1962a). The interaction in teratogenic activity of the two
niacin analogs 3-acetylpyridine and 6-aminonicotinamide. /. exp. Zool. 151, 253-8.
LANDAUER, W. & CLARK, E. M. (19626). Teratogenic interaction of insulin and 2-deoxy-Dglucose in chick development. J. exp. Zool. 151, 245-52.
LANDAUER, W. & CLARK, E. M. (1963). Interaction of insulin and chlorpromazine in teratogenesis. Nature, Lond. 198, 215-16.
LANDAUER, W. & CLARK, E. M. (1964 a). On the teratogenic nature of sulfanilamide and
3-acetylpyridine in chick development. /. exp. Zool. 156, 313-22.
LANDAUER, W. & CLARK, E. M. (19646). Uncouplers of oxidative phosphorylation and
teratogenic action of insulin. Nature, Lond. 204, 285-6.
LANDAUER, W. & WAKASUGI, N. (1967). Problems of acetazolamide and iV-ethylnicotinamide
as teratogens. /. Exp. Zool. 164, 499-516.
LAYTON, W. M. JR. & HALLESY, D. W. (1965). Deformity of forelimbs in rats: association
with high doses of acetazolamide. Science, N.Y. 149, 306-8.
LEE, S. H. S., DOBSON, P. R. & VAN ROOYEN, C. E. (1966). Antiviral substances: 6-aminonicotinamide and 1-adamantanamine hydrochloride. Chemotherap. 11, 163-77.
MAREN, T. H. (1965). Forelimb deformity in rats: association with acetazolamide. Science,
N.Y. 150, 79.
MAREN, T. H. (1967). Carbonic anhydrase: chemistry, physiology and inhibition. Physiol.
Rev. 47, 595-781.
REPORTER, M. C. & EBERT, J. D. (1965). A mitochondrial factor that prevents the effects of
antimycin A on myogenesis. Devi Biol. 12, 154-84.
RUANO GIL, D. (1967). The influence of thalidomide on the development of chick embryos
cultivated in vitro. Acta anatomica 66, 226-37.
RUNNER, M. N. (1967). Comparative pharmacology in relation to teratogenesis. Fedn Proc.
26, 1131-6.
RUNNER, M. N. & MILLER, J. B. (1956). Congenital deformity in the mouse as a consequence
of fasting. Anat. Rec. Y2A, 437-8.
JOHNSON,
284
W. LANDAUER & N. W A K A S U G I
J. W., JR. & FALLON, J. F. (1966). Cell death in morphogenesis. In Major Problems
in Developmental Biology (ed. M. Locke), pp. 289-314. New York and London: Academic
Press.
SIEKEVITZ, P. (1965). Origin and functional nature of microsomes. Fedn Proc. 24, 1153-5.
SMITHBERG, M. & RUNNER, M. N. (1963). Teratogenic effects of hypoglycemic treatments in
inbred strains of mice. Am. J. Anat. 113, 479-89.
STEPHENSON, R. P. (1956). A modification of receptor theory. Brit. Jour. Pharmac. Chemother
11, 379-93.
STOLL, R. & MARAUD, R. (1965). Introduction a Vetudedes malformations. Paris: GauthierVillars.
SZENT-GYORGYI, A. (1957). Bioenergetics. New York: Academic Press.
TISNA-AMIDJAJA, D. A. (1958). Wachstumsbeeinflussung durch Acetylcholin, Histamin,
Alloxan, Nadisan, Aristamid, Nicotinsaureamid undp-Aminobenzoesaure beim Hiihnerembryo. Arch. EntwMech. Org. 150, 655-88.
VELDSTRA, H. (1956). Synergism and potentiation with special reference to the combination
of structural analogues. Pharmac. Rev. 8, 339-87.
VELDSTRA, H. (1963). Synergism of drugs. Proc. Third. Int. Cong. Chemother., Stuttgart, 1962,
pp. 42-51.
VERRUSIO, C. A., FRASER, F. C. & POLLARD, D. R. (1967). Cytoplasmic inheritance of a
difference in teratogenic response to 6-aminonicotinamide in the mouse. Abstracts, The
Teratology Society Seventh Annual Meeting, p. 45.
WARBURG, O. & CHRISTIAN, W. (1936). Pyridin, der Wasserstoffiibertragende Bestandteil
von Garungsfermenten (Pyridin-Nucleotide). Biochem. Z. 287, 291-328.
SAUNDERS,
WILSON, J. G., MAREN, T. H. & TAKANO, K. (1966). Teratogenicity of carbonic anhydrase
inhibitors in the rat. Abstracts, The Teratology Society Sixth Annual Meeting, pp. 30-1.
J. G., MAREN, T. H., TAKANO, K. & ELLISON, A. C. (1968). Teratogenic action of
carbonic anhydrase inhibitors in the rat. Teratol. (in the Press; quoted from Maren, 1967).
ZWILLING, E. (1942). The development of dominant rumplessness in chick embryos. Genetics
27, 641-56.
ZWILLING, E. (1959). Micromelia as a direct effect of insulin. Evidence from in vitro and in
vivo experiments. / . Morph. 104, 159-79.
ZWILLING, E. & DEBELL, J. T. (1950). Micromelia and growth retardation as independent
effects of sulfanilamide in chick embryos. /. exp. Zool. 115, 59-81.
WILSON,
{Manuscript received 13 February 1968)