/ . Embryol. exp. Morph. Vol. 39, pp. 203-220, 1977
Printed in Great Britain
203
Effects of prenatal administration of
hadacidin., a cancer chemotherapeutic agent, on the
development of hamster fetuses
ByRAVINDRA M. SHAH1
From the Departments of Oral Biology and Pathology,
Faculties of Dentistry and Medicine, University of British
Columbia, Vancouver, Canada
SUMMARY
A single intraperitoneal injection of 1-2-5 g/kg body weight of hadacidin was teratogenic
when administered to pregnant hamsters between days 8 and 11 of gestation. Both the
frequency of malformation and resorption were related to the dose and time of hadacidin
administration. The drug produced both gross and microscopic malformation in the fetus and
impaired its general growth. The malformation involved craniofacial structures, central
nervous system, respiratory, digestive and urinary systems, limbs and tail.
Morphologically different types of cleft lip and cleft palate were related to the dose and
time of hadacidin treatment. An association was observed between cleft lip, cleft palate
and micrognathia on one hand, and between cleft palate and micrognathia on the other.
Significance of association between the lip, the palate and the mandibular malformation was
discussed and the hypothesis that human cases of Pierre Robin syndrome may result from an
environmental assault was supported. It was suggested that the fetal weight and the microscopic analysis should be included in the criteria for the teratological drug safety evaluation
procedures.
INTRODUCTION
In 1962, Kaczka, Gitterman, Dulaney & Folkers synthesized a new cancer
chemotherapeutic chemical compound, JV-Formyl hydroxyaminoacetic acid,
from broth culture of Penicillium frequentans. The compound was found to
interdict the growth of human intestinal adenocarcinoma and bronchiogenic
carcinoma (White, 1962; Gitterman et al. 1962). Ellison (1962) has applied the
drug with some success also in leukaemia patients. Shigura & Gordon (1962 a, b)
studied the biochemical mechanism of hadacidin action and suggested that the
agent inhibits a specific enzyme, adenyl succinate synthatase, involved in purine
biosynthesis.
The teratogenic effect of hadacidin was noted, only in rats, by Chaube &
Murphy (1963) and Roux & Horvath (1970). Lejour-Jeanty (1966, 1970)
studied in vivo lip development, and Fairbanks & Kollar (1974) studied in vitro
1
Author's address: Faculty of Dentistry, University of British Columbia, Vancouver, B.C.,
V6T 1W5, Canada.
204
R. M. SHAH
palatal development following hadacidin treatment. None of these studies,
however, analyzed different types of malformation that were produced in the
offsprings.
The study of Walker & Fraser (1956), Kalter (1969), Dostal & Jelinek (1970,
1971), Walker (1967, 1971), Shah & Travill (1976) and Shah & Kilistoff (1976)
have demonstrated the importance of species and strain in determining the
outcome of teratological experiments. Vichi (1969), Noel (1973) and others have
suggested that when a drug is found to be potentially teratogenic in one animal
species, other species should also be investigated. Golden hamster has been
found to be suitable for teratological studies (Ferm, 1967; Shah & Chaudhry,
1974). The present study deals with the effect of prenatal administration of
hadacidin on the development of hamster fetuses.
MATERIALS AND METHODS
Male and female Golden Syrian hamsters weighing approximately 85 ± 5 g
were maintained under controlled environmental conditions for minimum of
1 week as described earlier by Shah & Chaudhry (1973). The animals were caged
singly, fed on a diet of Purina Chow and allowed drinking water ad libitum.
On the appropriate day of the estrous cycle the animals were mated from 19-00
to 21-00 h and the midpoint of the mating period (20-00 h) was considered day 0
of pregnancy.
Each pregnant hamster was given a single intraperitoneal injection of aqueous
solution of either 100, 125, 150, 175, 200 or 250 mg sodium hadacidin. The
injection was made on days 8, 9, 10 or 11 of gestation. A group of pregnant
animals were similarly treated with comparable amount of water at each time.
Both the experimental and controls were killed on day 15 of gestation. The
uteri were exposed and examined for total implantation sites and fetal resorption.
The viable fetuses were weighed and examined for external malformations.
Several fetuses from both the experimental and control groups were fixed in
Bouin's fluid and processed for serial histological sectioning.
Data on external malformations were analysed on an IBM computer (370/168)
using the University of British Columbia's modified version of Goodman's
(1971) program of Normit Analysis. Subsequent statistical evaluation of the
associations between different malformations was carried out by Dr M. Greig
(personal communication).
RESULTS
External examination
Hadacidin was teratogenic in hamster fetuses when 100-250 mg was given,
intraperitoneally, to pregnant mothers between days 8 and 11 of gestation
(Table 1). The malformation rate varied with the hadacidin dose and time of its
administration. On day 8 the malformation rate decreased as the dose was
Teratogenicity ofhadacidin
205
Table 1. Teratogenicity of single intraperitoneal
injection ofhadacidin in hamster
No. of
Gestational day
No. of No. of live No. of malformed Mean fetal weight
(g)f + S.D.
of injection Dose (mg) animals fetuses resorptions* fetuses
8
10
11
100
125
150
175
200
250
100
125
150
175
200
250
125
150
175
200
250
150
175
200
250
3
3
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
36
30
24
14
20
1
34
32
24
23
23
27
29
34
29
22
20
24
25
22
32
1
4
6
14
11
18
3
0
1
4
6
6
0
0
1
6
12
2
2
1
0
3
19
17
11
18
1
34
32
24
23
28
27
1
24
29
22
20
0
0
1
8
1-6 ±0-21
1-4 ±0-28
1-4 + 018
1-4 ±019
1-3 ±0-24
10± —
l-4±015
l-3±019
l-5±0-ll
1-5 ±0-28
l-5±O-33
l-2±0-21
1-8 ±0-26
J-4 ±0-25
l-3±0-35
1-3 ±0-23
1-3 ±0-20
l-6±0-ll
l-6±014
1-4 ±0-23
l-5±0-18
* Frequency of resorption in control animals: 0-10 °/(
t Mean weight of control fetuses at term: 20 g.
reduced from 250 to 100 mg. The same dose range, however, on day 9 produced
abnormalities in all fetuses, indicating day 9 to be the most critical time to
produce malformed fetuses. The rate was considerably lower on day 11, than on
days 8-10, when only 200-250 mg showed malformed fetuses.
The resorption rate varied with the dose and time of hadacidin administration (Table 1). On day 8, the resorption rate was higher than controls (P > 0-05)
following administration of 150-250 mg hadacidin. On days 9 and 10, however,
it was significantly high only with 200-250 mg hadacidin. On day 11 the resorption rate was comparable to controls.
Fetuses, following hadacidin administration to pregnant hamsters, weighed
significantly lower than controls (P > 0-05) (Table 1), indicating a generalized
growth retardation. The weight reduction, however, was not related to the dose
and time of drug administration, as was the case in malformation and resorption
rates. On day 11, although there were no malformed fetuses and the resorption
rate was normal with 150-175 mg, the average fetal weight was significantly
lower than controls (P > 0-05).
14
EMB 39
206
R. M. SHAH
FIGURES 1-4
Hadacidin-treated hamster fetuses at term.
Fig. 1. Open eye, limb anomalies, kinky tail and micrognathia in a hamster fetus
following 250 mg hadacidin on day 9 of gestation, x 2-5.
Fig. 2. Exencephaly and cleft lip in a hamster fetus following 200 mg hadacidin on day
8 of gestation, x 2-5.
Fig. 3. Microcephaly, open eye, limb anomaly, retarded mid-facial growth and
generalized edema in a hamster fetus following 175 mg hadacidin on day 9 of
gestation, x 2-5.
Fig. 4. Herniated gut and cleft lip in a hamster fetus following 200 mg hadacidin on
day 8 of gestation, x 2-5.
Teratogenicity of hadacidin
207
Table 2. Incidence of external malformation following different
doses of hadacidin injected at various times during gestation
No. of fetus with developmental defect of
day of
injection
8
9
10
11
Dose (mg)
of live
fetuses
100
125
150
175
200
250
100
125
150
175
200
250
125
150
175
200
250
150
175
200
250
36
30
24
14
20
1
34
32
24
23
28
27
29
34
29
22
20
24
25
22
32
malformed
fetuses Brain Eye
3
19
17
11
18
1
34
32
24
23
28
27
1
24
29
22
20
0
0
1
8
0
1
8
6
6
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
4
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
GI Lower
Lip Palate Limb tract jaw Tail
2
15
10
6
14
0
0
1
4
3
7
23
0
0
0
0
0
0
0
0
0
3
15
4
8
14
0
30
31
22
20
28
27
0
10
14
9
16
0
0
1
8
0
1
6
2
3
0
6
14
23
10
27
26
1
24
29
21
20
0
0
0
1
0
1
1
5
8
0
1
1
1
1
4
1
0
0
0
0
0
0
0
0
0
1
2
2
2
7
0
0
7
0
5
11
23
0
2
4
0
5
0
0
0
0
The external malformation included exencephaly, microcephaly, open eyelids,
cleft lip, cleft palate, herniated gut, kinky or aplastic tail, micrognathia and limb
anomalies such as amelia, micromelia, phocomelia, adactyly, ectrosyndactyly,
syndactyly and polydactyly (Figs. 1-4). In Table 2, various malformations are
analyzed in relation to the dose and time of hadacidin treatment. The malformation observed corresponded to their period of rapid organogenesis (Boyers,
1948). Following hadacidin treatment on day 8 of gestation deviant development
was observed in lip, palate, brain, tail, gastro-intestinal tract, lower jaw, limb
and eye. The pattern, however, changed on day 9 when the most common
malformations were that of the tail, palate, limbs, lower jaw and lip, in that
order. On day 10, the major external malformations were that of limbs and
palate and on day 11 of palate only.
The palatal cleft varied in its morphology: some being complete and others
incomplete or partial. The complete cleft palate involved the entire length of
the secondary palate (Fig. 5). The incomplete or partial cleft involved either the
anterior third (Fig. 6) or the posterior third of the secondary palate, or a combination of both, with fusion in the middle (Fig. 7).
14-2
0
9
3
0
8
0
34
32
24
23
28
27
0
3
0
16
8
0
0
0
0
208
R. M. SHAH
FIGURES 5-8
Ventral view of hadacidin-induced malformations. Both the lower
jaw and the tongue have been removed.
Fig. 5. Complete cleft of the secondary palate following 175 mg hadacidin on day 9 of
gestation, x 8.
Fig. 6. Partial cleft of the secondary palate following 125 mg hadacidin on day 9 of
gestation. The cleft is present only in the anterior third of the palate (arrow), x 8.
Fig. 7. Partial cleft of the secondary palate following 150 mg hadacidin on day 9 of
gestation. The cleft is present both in the anterior and the posterior thirds of the
palate with fusion in the middle, x 8.
Fig. 8. Unilateral cleft lip, partial cleft in the anterior third of the palate and cleft
of the alveolar ridge in a hamster fetus following 150 mg hadacidin on day 10 of gestation. x8.
Teratogenicity ofhadacidin
FIGURES
209
9-10
Ventral view of hadacidin-induced malformations. Both the lower jaw and the
tongue have been removed.
Fig. 9. Bilateral cleft lip and complete cleft of the secondary palate in a hamster
embryo following 200 mg hadacidin on day of gestation, x 8.
Fig. 10. Median cleft lip and complete cleft of the secondary palate in a hamster
embryo following 150 mg hadacidin on day 9 of gestation, x 8.
The lip cleft was limited only to the upper one and varied in its morphology.
Some cleft lip were unilateral whereas others were bilateral and median. The
unilateral cleft lip occurred at the junction of the median nasal process and one
of the maxillary process, either right or left (Fig. 8). The bilateral cleft lip
involved the median nasal process along with both the maxillary processes
(Fig. 9). The median cleft lip appeared to be due to deficiency of the median
nasal process only (Fig. 10).
An analysis of data from Table 2 indicated that the malformation frequency
of palate, lower jaw and lip are related to the dose of hadacidin and time of
its administration. Because of spatial and temporal relationship between palate,
mandible and lip during embryonic development, their malformations were
analyzed further.
An analysis of different types of cleft lip and associated cleft palate is presented
in Table 3. One may deduce that there is an overall association between induction
of cleft lip and cleft palate in hamster fetuses (x2 29-26; P > 0-2) and that the
association is more pronounced on day 9 (x2 1-78; P > 0-5) than on day 8 of
gestation (x2 14-59; P > 0-5). Furthermore, the frequency of bilateral and
median cleft lip (which are the more severe form of cleft lip than unilateral ones)
are directly related to the dose of hadacidin.
210
R. M. SHAH
Table 3. Incidence of different types of induced fetal cleft lip and associated cleft palate
following administration of different doses of sodium hadacidin to pregnant hamsters during
gestation
Types of clefit
Gestational
A
day
No. of
fetuses
Unilateral
wim
injection Dose (mg) cleft lip Total
8
9
100
125
150
175
200
250
125
150
175
200
250
2
15
10
6
14
0
1
Types of associated[
cleft palate
Bilateral
C
oi
lip
A
1
7
8
2
5
0
0
0
1
0
0
Left
Right
0
3
3
4
2
5
0
0
0
1
5
0
0
0
0
II1CUI an
{
Total
CP*
0
0
0
2
2
15
4
2
12
1
5
0
0
12
1
1
4
2
7
23
5
8
0
1
3
r
1
8
2
2
9
0
0
0
0
0
4
0
0
2
2
0
4
6
0
3
0
19
0
4
23
* Cleft of the complete secondary palate.
f Cleft of the anterior and posterior part of the secondary palate.
X Cleft of the anterior part of the secondary palate.
4
3
7
23
1
A
APf
At
0
1
0
0
0
0
2
3
0
4
0
C
0
1
0
0
0
0
Unilateral cleft lip was associated with cleft palate in only 25 % cases (4/16).
The cleft palate in such instance, was of incomplete type (3/4), involving only
the anterior part of the secondary palate. Bilateral and median cleft lip, on the
other hand, were associated with complete cleft of the secondary palate in 99 %
cases (50-51). One may infer that severe forms of cleft lip are generally associated with the extensive cleft of the secondary palate.
The data presented in Tables 1, 2 and 4 were pooled and it was observed that
both the frequency and severity of cleft palate and micrognathia (x2 22-00;
P > 0-01) was more pronounced on day 9 than any other day of gestation.
The cleft palate in such instance was of complete type (57/69). Incomplete cleft
palate, on the other hand, was not associated with retardation of mandibular
development.
Since on day 9 of gestation, a strong association was observed between cleft
lip and cleft palate on one hand and cleft palate and micrognathia on the other,
the data related to these three defects were pooled and analyzed. It was noted
that there is an overall association between cleft lip, cleft palate and micrognathia (x214-70; P > 0-0001) following administration of hadacidin on day 9 of
gestation.
Microscopic examination
Hadacidin treatment on day 8 of gestation. A fetus, at term, may show one
or more of the following malformations: retardation in the development of
0
0
1
0
Teratogenicity of hadacidin
211
Table 4. Incidence of different types of induced fetal cleft palate and associated micrognathia
following different doses of sodium hadacidin to pregnant hamsters during gestation
Micrognathia associated
with different
types of cleft ]palate
Gestational
No. of fetus
day of
with cleft
injection
palate
Dose (mg)
CP*
APf
At
P§
8
100
125
150
175
200
250
3
15
4
8
14
0
2
12
1
4
8
0
0
1
0
0
0
0
1
2
3
4
6
0
0
0
0
0
0
0
9
100
125
150
175
200
250
30
31
22
20
28
27
0
8
6
6
13
26
1
1
1
0
2
0
29
22
15
14
13
1
10
150
175
200
250
10
14
9
16
0
7
4
11
0
2
2
1
11
150
175
200
250
0
0
1
8
0
0
0
3
0
0
0
0
*
f
t
§
Cleft
Cleft
Cleft
Cleft
Type of cleft palate
^
A
r
CP
AP
A
1
2
1
1
7
0
1
2
0
1
5
0
0
0
0
0
0
0
0
0
1
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7
0
5
11
23
0
5
0
4
10
23
0
1
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
10
5
3
4
0
0
0
0
2
4
0
5
0
1
0
5
0
1
0
0
2
2
0
0
0
0
0
0
0
0
1
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Total
of the complete secondary palate.
of the anterior and posterior part of the secondary palate.
of the anterior part of the secondary palate.
of the posterior part of the secondary palate.
bone, cartilage and muscles (Fig. 11); disorganization of cellular arrangement
in the brain and intracranial edema and hemorrhage (Fig. 12); absent, flattened
and partly ossified nasal septum (Figs. 13, 14); oronasal and nasofacial fistula
(Fig. 15); absence of epithelial stratification and degenerative changes in the
epithelial cells on the dorsal surface of the tongue (Fig. 16); hypoplasia of the
submandibular, sublingual and parotid salivary glands; aplasia of salivary
glands in the cheek and soft palate; failure of the palatal processes to develop
(Fig. 17); fusion of the palate with tongue, floor of the mouth and mandible
(Figs. 12, 18); fusion between the mandible and cheek (Fig. 19); abnormalities
of the odontogenesis such as persistence of dental lamina (Fig. 20), absence of
one or both maxillary incisors, separation of dental papillae from the enamel organ (Fig. 21); hemorrhage in the lung and derangement in the lung
p
212
R. M. SHAH
12
14
FIGURES
11-16
Hematoxylene and eosin-stained frontal sections of hamster fetuses at term,
following hadacidin administration on day 8 of gestation.
Fig. 11. Retardation in muscle development following 175 mg hadacidin. The fetus
also shows intracranial edema, x 2-5.
Fig. 12. Cellular de-arrangement in the brain, intracranial hemorrhage and palatomandibular fusion (arrow) following 200 mg hadacidin. x 4.
Fig. 13. Missing nasal septum following 200 mg hadacidin. The nasal cavities are
moved laterally, x 4.
Fig. 14. Partly ossifying nasal septum following 125 mg hadacidin. x 8.
Fig. 15. Nasofacial fistula (arrow) following 200 mg hadacidin. x 8.
Fig. 16. Lack of stratification and cellular degeneration in the epithelium of the
dorsal surface of the tongue (arrows) following 175 mg hadacidin. x 8.
Teratogenicity ofhadacidin
22
FIGURES
17-22
Hematoxylene and eosin-stained frontal section of hamster fetuses at
term following hadacidin administration on day 8 of gestation.
Fig. 17. Absent palatine processes following 200 mg hadacidin. x 4.
Fig. 18. Fusion of the palatal shelf (P) with the tongue and floor of the mouth
following 175 mg hadacidin. x4.
Fig. 19. Fusion between the mandible and the cheek (arrow) following 200 mg
hadacidin. x4.
Fig. 20. Persistence of the dental lamina (arrow) following 150 mg hadacidin. x 12.
Fig. 21. Separation of the dental papilla from the enamel organ following 200 mg
hadacidin. x4.
Fig. 22. Hemorrhage and collapse of lung following 125 mg hadacidin. x 4.
214
R. M. SHAH
FIGURES
23-26
Hematoxylene and eosin-stained frontal section of hamster fetuses at term.
Fig. 23. De-arrangement in the lung parenchyme following 125 mg hadacidin
administration on day 8 of gestation, x 4.
Fig. 24. Abnormal differentiation of the renal cortex and medulla following 200 mg
hadacidin on day 8 of gestation, x 4.
Fig. 25. Large cystic space in the kidney following 150 mg hadacidin on day 8 of gestation. x4.
Fig. 26. Communication (arrow) between pleural and peritoneal cavity through
diaphragm following 200 mg hadacidin on day 9 of gestation, x 4.
parechyme (Figs. 22, 23); abnormal differentiation of the renal cortex and
medulla (Fig. 24) and large cystic spaces in the kidney (Fig. 25).
Hadacidin treatment on day 9 of gestation. With the exception of anomalies of
the tongue and salivary glands, a fetus may show malformation of the brain,
orofacial structures, kidney, lung and mesenchymal derivatives (bone, muscles,
cartilage) similar to those observed following treatment on day 8. In addition, a
fetus may show communication between the pleural and peritoneal cavity
through diaphragm (Fig. 26). In such a fetus, the anatomical relationship
between the thoracic and abdominal viscera is generally changed.
Hadacidin treatment on day 10 of gestation. Apart from abnormalities of
palate and lung, as noted following treatment on day 8 and 9, a fetus may show
intracranial edema similar to one on day 8 (Fig. 11). Anomalies of the teeth
Teratogenicity of hadacidin
215
were less severe and included only disarray in the dental papilla. Malformations
of the nasal cavity, salivary glands, tongue, kidney and mesenchymal derivatives
were absent.
Hadacidin treatment on day 11 of gestation. With the exception of malformation of palate, lung and intracranial edema, all the other malformations described following treatment on day 8 were absent following treatment on day 11.
DISCUSSION
Doses of hadacidin employed during present study were not toxic to pregnant
hamsters but showed teratogenic" and lethal effect on the fetuses. The latter
observation agrees with Chaube & Murphy (1963) and Roux & Horvath
(1970) results from rat. The observations of current study further endorses the
principles of experimental teratology, discussed explicitly by Fraser (1960),
Saxen & Rapola (1969) and Wilson (1973) that production of malformation with
an environmental agent depends not only on the nature of the teratogen, the
dose, and the mode and time of administration but also on the species. The
malformation rate observed following hadacidin treatment, during present
study, was dose and time dependant. The results, however, were different from
those of Chaube & Murphy (1963). For example, these authors needed a single
intraperitoneal injection of 3-5 g/kg body weight of hadacidin to produce high
frequency of malformed fetuses. In hamster, maximum malformation rate was
achieved with 1-2-5 g/kg body weight of hadacidin. The dose range tested in the
present study is of significance since it is considerably lower than the recommended therapeutic dose (White, 1962; Murphy, 1965). There were, however,
two major differences between the present study and Chaube & Murphy's study:
the species and the type of solvent used. In our study aqueous solution of hadacidin was administered to hamster whereas Chaube & Murphy used carboxymethyl cellulose as a solvent for hadacidin treatment of rat.
In most existing guidelines for drug safety, including the World Health
Organization document of 1967, prenatal growth retardation is rarely given
enough significance even though such retardation, in both human and laboratory animals, may have implications in the postnatal life (Gruenwald, 1961;
McLaren & Michie, 1963; Jensh & Brent, 1967; Hurley, 1968; Winick, 1969,
1971; Widdowson, 1971; Crichton et al. 1972; Wilson, 1973). In the present
study, mean fetal weight following hadacidin treatment was significantly reduced,
except with 125 mg on day 10, indicating a generalized growth retardation of the
fetus. Furthermore, hadacidin treatment on day 11 produced no external malformation although the fetal weight was significantly lower as compared to
controls. These observations, thus, lend further support to the proposition
that the growth retardation, following an environmental assault, may occur even
after the period of rapid organogenesis is over (Hurley, 1968; Wilson, 1973). The
present study also endorses the suggestion of Gruenwald (1961), McLaren &
216
R. M. SHAH
Michie (1963), and Jensh & Brent (1967) that prenatal fetal weight reduction,
following an environmental assault, may perhaps be one of the mildest forms of
teratological expression and failure to recover from such reduction should be
considered as a developmental defect (Wilson, 1973). Brent (1972) noted that
'drug-induced fetal growth reduction may be a more sensitive parameter to
measure (teratogenicity) than malformation and lethality indices'.
Giroud & Martinet (1956), Buresh & Urban (1964), Dostal & Jelinke (1971),
Walker (1967), Shah & Travill (1976) and Shah &Kilistoff (1976) have noted
morphological variation of the cleft palate during experiments on rodents. In
humans also cleft palate varies in its morphology (Smiley, 1972). Results of the
present study confirm the observations of Shah & Travill (1976) and Shah &
Kilistoff (1976) that different types of cleft palate are related to the dose and
time of teratogen administration. Our results, however, differ from the latter
studies in that cleft of the anterior and posterior part of the secondary palate
was not induced with high reliability. Varied suggestions have been discussed
in earlier reports (Shah & Travill, 1976; Shah & Kilistoff, 1976) to explain
different types of cleft palate and are, therefore, not discussed in the present
communication. It must be noted, however, that the types of induced cleft may
also depend on the nature of the drug and the species used. For example,
Giroud & Martinet (1956) used high doses of vitamin A in rats; Buresh &
Urban (1964) used cholesterol in rats; Dostal & Jelinek (1971) used steroid
hormones in mice; and studies reported from our laboratory (Shah & Travill,
1976; Shah & Kilistoff, 1976), including the present one, used glucocorticoids
and a cancer chemotherapeutic agent in hamster.
Micrognathia and cleft palate have been described as prominent features in
numerous syndromes (Gorlin, Pindborg & Cohen, 1976). In cases of Pierre
Robin syndrome (PRS) it has been assumed that micrognathia is a contributory
cause to the cleft palate (Robin, 1923, 1934; Eley & Farber, 1930; Wesemen,
1959; Latham, 1966; Cocke, 1966; Burdi et al. 1972). Many of these cases of
PRS were thought to be genetic in origin. In many animal experimental studies
simultaneous occurrence of micrognathia and cleft palate have been recorded
but an association between them is not discussed (Trasler, Walker & Fraser,
1956; Nelson, 1957; Walker, 1959; Deuschle & Kalter, 1962; Harris, 1967).
Poswillo (1965,1966) and Cocke (1966) discussed such an association and noted
that experimentally induced conditions are identical to those observed in humans.
In the present study an association was observed between micrognathia and
complete cleft palate following an appropriate combination of the dose and
time of hadacidin treatment. It was, however, not possible to establish a
cause and effect relationship from post-hoc observations, since all the craniofacial structures grow simultaneously as shown by Diewert (1974) from her
studies on rat. The fetus with cleft palate and micrognathia also showed several
other malformations. Weisengreen & Sorsky (1940), Canick (1954), Routledge
(1960), Youmans (1960), Smith & Stowe (1961), Kraus, Kitamura & Ooe
Teratogenicity ofhadacidin
111
(1963) and Kitamura & Kraus (1964) have observed malformations of various
organs and systems in cases of PR syndrome. Thus based on the aforementioned
clinical and experimental observations, along with results of the present study,
one may support Poswillo's (1965) and Cocke's (1966) proposition that Pierre
Robin syndrome may result from an environmental assault on the embryo.
Cohen (1976), in a recent review, noted that PRS can be induced in developing
human fetus by such drugs as alcohol, hydantoin and trimethadione.
Fogh-Anderson (1942), Glass (1955) and Poswillo (1974) classified human
palatal clefts into two groups. The first group includes those cases of cleft
palate which occur in association with cleft lip (CLP). The second one includes
cases of isolated cleft palate (CP). It is generally agreed that etiologically these
two groups are distinct entities (Fogh-Anderson, 1942; Glass, 1955; Burston,
1959; Smiley, 1972; Poswillo, 1974). Literature abounds in human cases of
cleft lip and cleft palate occurring concurrently. In normal A/J mouse, 10 %
offspring usually have CLP (Kalter, 1969). On the other hand, extensive embryological experimentation on hamster in our laboratory during the past 7 years
have not shown a single case of spontaneous CLP. During present study, CP
and CLP were induced following different combinations of the dose and time of
hadacidin administration (Table 3). Hamster thus appear to be a satisfactory
biological system for the study of etiology and pathogenesis of CP and CLP.
Using different teratological procedures, several investigators have produced
CLP and the work is, therefore, not reviewed here. Poswillo (1974) has suggested
that micrognathia is not a common accompaniment of CLP in human, although
observations in the present report are contrary to Poswillo's suggestion.
In most teratological evaluation studies, observations have been restricted
to the external malformations of the fetus. Such studies generally do not reflect
aberrations at the tissue level. Studies by Warkany & Deuschle (1955), Auerbach
& Barrow (1972), Warkany & Patering (1972), Poswillo (1973) and others have,
however, observed several malformations at the microscopic level of the fetal
tissues following drug treatment of the pregnant mother. Results of the present
study clearly indicate that administration of drug to pregnant female may have
far-reaching implication at the organ and tissue level in the developing fetus.
The anomalies of such tissues and organs may not elicit signs and symptoms
until functional disabilities can be assessed. The 'free hand razor blade sectioning' method proposed by Wilson (1964) is limited in its usefulness since it would
not allow one to recognize malformations at the tissue level. We recommend
that microscopic examination of vital organs be made part of the drug safety
evaluation procedures. Potentially such information would then allow one to
rationally interfere with internal milieu of the fetus to break teratologic sequence
and thereby prevent the defective development.
In brief, foregoing discussion clearly indicates that hadacidin is teratogenic
when administered, even in subclinical doses, to pregnant hamster, and its
therapeutic use during human pregnancy should be carefully guarded. The drug
218
R. M. SHAH
produces both gross and microscopic malformations in the fetus and impairs
its general growth. The fetal weight and microscopic analysis should, therefore,
be included in the criteria for the drug safety evaluation.
The author remains grateful to Dr M. Greig of The University of British Columbia's
computing service for statistical analysis of the data and to Mrs J. Galbraith-Hamilton, Miss
V. Beretanos, Mr R. Paton and Mrs L. Lee for various assistance. The research was supported
by the Natural, Applied and Health Sciences grant of the University of British Columbia.
Grateful thanks are also extended to Dr W. D. Dorian, executive director of medical research, Merck Frosst Laboratories, Montreal, Canada, for his generous supply of hadacidin.
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(Received 29 November 1976, revised 17 January 1977)