COMPARISON OF TERATOGENIC CHEMICALS IN THE RAT AND CHICK EMBRYOS An Exhibit with Additions for Publication By M. Lois Murphy, M.D., C. P. Dagg, Ph.D., and David A. Karnofsky, M.D. Embryology Section, Divixion of Experimental Chemotherapy, Sloan-Kettering Institute f or Cancer Research, New York Presented at the Annual Meeting, October 8-11, 1956. This research was supported, in part, by grants from the American Cancer Society, Inc., the Albert and Mary Lasker Foundation, and grant C-675 from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service. ADDRESS: (M.L.M.) 444 East 68th Street, New York, New York. 701 T HE CHICK EMBRYO 15 used widely in studying drugs for teratogenic activity. Chemicals, which have been injected at the same period of incubation of the chick eml)ryo, may produce separate and char- acteristic patterns of developmental ab- normalities. The effects of many drugs, such as insulin, azasenine, sulfanilamide, 4-aminopteroyl glutamic acid, 8-azaguanine, physostigmine, thallium, lead, boric acid and cortisone, have been neviewed.1 The chick embryo in the egg is an isolated and independent system, whereas mammalian embryos are usually intimately involved with the maternal host which may detoxify, excrete or otherwise protect the fetus against noxious chemicals. It was, therefore, of interest to determine the effects of drugs, teratogenic in the chick embryo, on the mammalian fetus. The rat was selected because of its availability and be- cause there is considerable information on rat embryology and teratology.’’ Because some drugs, apparently inactive in the chick embryo, have produced consistent de- velopmental abnormalities in the rat fetus, the pregnant rat also has been used for the initial study of selected compounds. The objectives of these studies were: 1) to determine and compare the terato- genie action of drugs on the chick and rat embryos; 2) to determine the consistency and specificity of action of each drug; 3) to determine the relation between the time during gestation when a drug is intro- duced and the occurrence of specific ab- normalities; 4) to determine the relation- ship of the dose of a drug toxic to tile mother, the dose toxic to the embryo, and the dose producing consistent develop- mental defects, and 5) to detect compounds which protect the embryo from the terato- genie action of a chemical. Observations on five distinctive com- pounds are presented in this exhibit. MATERIALS AND METHODS White Leghorn eggs were obtained from a commercial source and incubated at 38#{176}C. The test chemicals, at various dose levels, were routinely injected into the yolk sac of the 4- day-old embryos. The embryos surviving the treatment were killed at 18 days of incuba- tion, or 14 days after the injection of the drug. Previously unmated female rats, Wistar strain, were placed in cages with males and watched for 12 hours. Those observed in copu- lation were isolated and regarded as zero days pregnant. At various times, ranging from 7 to 16 days of gestation, groups of rats received a single intraperitoneal injection at various dose levels of the test drugs. The pregnant rats were sacrificed on the twenty-first day of ges- tation, the uteri examined, the implantation sites counted, and the surviving fetuses re- moved for study. The chick embryos and rat fetuses were cx- amined grossly, weighed and then cleared and the skeleton stained with alizarin so that it could be examined macroscopically in the whole animal. The test chemicals were dissolved in saline or suspended in carboxymethylcellulose (CMC) immediately before injection. by guest on June 2, 2018 www.aappublications.org/news Downloaded from
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COMPARISON OF TERATOGENIC CHEMICALS INTHE RAT AND CHICK EMBRYOS
An Exhibit with Additions for Publication
By M. Lois Murphy, M.D., C. P. Dagg, Ph.D., and David A. Karnofsky, M.D.Embryology Section, Divixion of Experimental Chemotherapy, Sloan-Kettering Institute
f or Cancer Research, New York
Presented at the Annual Meeting, October 8-11, 1956.This research was supported, in part, by grants from the American Cancer Society, Inc., the Albert
and Mary Lasker Foundation, and grant C-675 from the National Cancer Institute of the National
Institutes of Health, U. S. Public Health Service.
ADDRESS: (M.L.M.) 444 East 68th Street, New York, New York.
701
T HE CHICK EMBRYO 15 used widely in
studying drugs for teratogenic activity.
Chemicals, which have been injected at the
same period of incubation of the chick
eml)ryo, may produce separate and char-
acteristic patterns of developmental ab-
normalities. The effects of many drugs,
such as insulin, azasenine, sulfanilamide,
4-aminopteroyl glutamic acid, 8-azaguanine,
physostigmine, thallium, lead, boric acid
and cortisone, have been neviewed.1�
The chick embryo in the egg is an
isolated and independent system, whereas
mammalian embryos are usually intimately
involved with the maternal host which may
detoxify, excrete or otherwise protect the
fetus against noxious chemicals. It was,
therefore, of interest to determine the effects
of drugs, teratogenic in the chick embryo,
on the mammalian fetus. The rat was
selected because of its availability and be-
cause there is considerable information on
rat embryology and teratology.’’ Because
some drugs, apparently inactive in the chick
embryo, have produced consistent de-
velopmental abnormalities in the rat fetus,
the pregnant rat also has been used for
the initial study of selected compounds.
The objectives of these studies were:
1) to determine and compare the terato-
genie action of drugs on the chick and rat
embryos; 2) to determine the consistency
and specificity of action of each drug; 3) to
determine the relation between the time
during gestation when a drug is intro-
duced and the occurrence of specific ab-
normalities; 4) to determine the relation-
ship of the dose of a drug toxic to tile
mother, the dose toxic to the embryo, and
the dose producing consistent develop-
mental defects, and 5) to detect compounds
which protect the embryo from the terato-
genie action of a chemical.
Observations on five distinctive com-
pounds are presented in this exhibit.
MATERIALS AND METHODS
White Leghorn eggs were obtained from a
commercial source and incubated at 38#{176}C.The
test chemicals, at various dose levels, were
routinely injected into the yolk sac of the 4-
day-old embryos. The embryos surviving the
treatment were killed at 18 days of incuba-
tion, or 14 days after the injection of the drug.
Previously unmated female rats, Wistar
strain, were placed in cages with males and
watched for 12 hours. Those observed in copu-
lation were isolated and regarded as zero days
pregnant. At various times, ranging from 7 to
16 days of gestation, groups of rats received asingle intraperitoneal injection at various dose
levels of the test drugs. The pregnant ratswere sacrificed on the twenty-first day of ges-
tation, the uteri examined, the implantation
sites counted, and the surviving fetuses re-
moved for study.
The chick embryos and rat fetuses were cx-
amined grossly, weighed and then cleared
and the skeleton stained with alizarin so that
it could be examined macroscopically in the
whole animal.
The test chemicals were dissolved in saline
or suspended in carboxymethylcellulose (CMC)immediately before injection.
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702 �vlURPHY - COMPARISON OF TERATOGENIC CHEMICALS
FIG. 1.
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AMERICAN ACADEMY OF PEDIATRICS - PROCEEDINGS 703
DEVELOPMENT OF THE CHICK AND
RAT EMBRYOS (FIG. I)
CmcK E�fBYnos:’ ‘#{176} The incubation
period of the cilick embryo is 21 days.
Growth and differentiation proceeds nap-
idly, and many detailed descriptions are
available. Tile classical monograph on chick
embryology is by Lillie and Hamilton.”
Hamburger and ‘ 2 have prepared
an atlas of the stages of embryonic de-
velopment, and related them to incubation
time. By 18 to 24 hours the primitive streak
has appeared, heart beat begins about the
end of second day (9 somite stage), the tail-
bud appears at about 33� days of incubation,
and the initiation of endocrine function and
the end of metamorphosis” begins about
the eigllth to ninth days.1’ Cartilage ap-
pears at tile fifth day, and active calcifica-
tion of bone begins about the eleventh day.
When the chick hatches it weighs about
30 gin, has feathers and is able to feed
itself.
RAT E�IBRYos:’4” Tile rat, from the time
of fertilization, has a gestation period of
about 22 days. Implantation begins about
the seventh day, and embryonic develop-
ment is, in general, about 6 to 8 days be-
ilind that of tile chick embryo. Thus, tile
primitive streak appears at 83� to 9 days,
the heart begms to beat about the ninth
daY, tile tailbud appears at 113k days, and
the “end of metamorphosis” appears at 16
to 17 days.13 Ossification begins on the
sixteenth day. Thus, the 4-day-old chick
embryo is at about the same stage as the
11 to 12-day-old rat fetus, and the 8-day
chick embryo is equivalent in development
to tile 15 to 16-day rat fetus. These com-
parative periods are important, because it
is noted that teratogenic effects to be de-
scribed are most commonly produced in
the chick embryo treated on the fourth day,
and the rat fetus on the tenth to twelfth
days. At birth the rat fetus weighs 4 to
5 gm which is equal to the 12-day chick
embryo, and its development is far less
advanced than the chick.
NITROGEN MUSTARD (FIG. 2)
Methyl-bis (�-chloroethyl)amine hydro-
chloride (HN2), a nitrogen mustard, is a
crystalline substance, soluble in water.
Closely related structurally and pharmaco-
logically to the war-gas, mustard gas, it was
intensively studied during World War II
as a potential chemical warfare �
In the past 10 years it has been widely used
in the treatment of neoplastic disease in
man; principally in tumors of the lymph
nodes and blood forming organs.’#{176} HN2
has wide and clearly defined patterns of
biologic effects, which have been reviewed
on several occasions.”” It is carcinogenic,
mutagenic, and in mammals toxic doses
produce severe depression of the bone mar-
row, with decrease in the formed elements
of the blood, involution of lymphatic tissue,
and injury to the intestinal mucosa; these
effects are similar to those produced by total
body irradiation. HN2 inhibits the growth
of transplantable tumors, and produces
characteristic histologic changes in mouse
tumors, and in mouse and human tumors
growing on the chonioallantoic membrane
(CAM) of the chick embryo.’2 Teratogenic
effects in tile rat fetus have been described
by Haskins.”
Teratogenic Effects
CHIcic EMBRYO: The LD,0 of HN2 is
variable and doses in the range of 0.01
mg/egg may be lethal for tile 4-day em-
bryo. Death usually occurs within 1 to 5
days after injection, and those surviving
in the dose range of the LD,0 are usually
normal. An occasional embryo, however,
shows weight inhibition, crossed beak, and
thin legs.
RAT EMBRYO: The LD,0 of single doses
of HN2 given intrapenitoneally to the preg-
nant rat is in the range of 2.0 mg/kg; this
is in tile same range as the LD,0 in the
nonpnegnant nat (1.9 mg/kg by the sub-
cutaneous route and 1.1 mg/kg intrave-
nously). Pregnant rats were given single in-
jections of HN2 intraperitoneally ranging
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AMERICAN ACADEMY OF PEDIATRICS - PROCEEDINGS 713
consisted principally of shortening of the
tansometatarsals with ectrodactyly ranging
from tile loss of one to all four toes. An
analogue of azasenine, 6-diazo-5-oxo-1-
norleucine (DON) gave approximately the
same effects but is about 50 times more
active by weight.
RAT EMBRYOS:36 Tile LD,0 of azasenine
in tile pregnant rat ranges between 75 and
100 mg/kg by the intrapenitoneal route.
Pregnant rats received single doses of 2.5
mg/kg of azaserine from the seventh to
fourteenth day of gestation. Those treated
on tile seventh and fourteenth days de-
livered normally.
Pregnant rats treated on the ninth day
Silowed 100% resorption of the implanta-
tiOll sites. Rats treated on days 10, 11
and 12 showed approximately 50% ne-
sorption with 80% of the surviving embryos
showing abnormalities, although weight in-
hibition was not marked. Embryos treated
on the tenth day occasionally showed cleft
palate and harelips. The cleaned skeleton
revealed fused or branched ribs, abnonmali-
ties of the lower tiloracic vertebral bodies
and arches manifested as cephalocaudal
fusions of the bodies on hemivertebrae.
Tile 11-day-treated embryos showed cleft
palate, bifid or fused sternebrae, small or
absent radii, and vertebral abnormalities
similar to the tenth day, but involving the
lumbar instead of the thoracic vertebrae.
In those treated at 12 days, the skeletal ab-
Ilornlalities were similar to those at ii days;
l)ut included small on absent pelvic bones,
femur or fibula. The extremities showed
syndactyly on absent toes.
DON was far more active by weight than
azaserine; pregnant rats treated on the tenth
day with 0.2 mg/kg intraperitoneally
showed complete fetal resorption, and 50%
resorption occurred at 0.1 mg/kg. Surviving
animals did not show the incidence of de-
velopmental abnormalities produced by
azasenine.
Discussion
Azasenine, a llighly specific antimetabo-
lite, produced severe developmental ab-
normalities in the chick embryo and rat
fetus. The abnormalities of the appendicu-
lan skeleton in the rat fetus and chick em-
bryo appeared to be related.
ACKNOWLEDGMENTS
We are pleased to acknowledge the fol-
lowing sources of compounds used in this
study:
Merck & Co., Inc., methyl, bis(�-chloro-
etyl)amine; Chemical-Biological Coordina-
tion Center, National Research Council,
and Dr. C. S. Rondestvedt, Jr., Univen-
sity of Michigan, tniazene, 3,3-dimethyl-l-
phenyl-; Dr. J. M. Ruegseggen, Ledenle Lab-
oratories, American Cyanamid Company,
i,3,4-thiadiazole,2-ethylamino-; Squibb In-
stitute for Medical Research, nicotinamide,
6-amino; Parke, Davis and Company, azas-
enine and DON; Dr. Earl Balis, Sloan-
Kettering Institute for Cancer Research,
imidazole, 4-amino-5-carboxamido-.
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1957;19;701Pediatrics M. Lois Murphy, C. P. Dagg and David A. Karnofsky
EMBRYOS: An Exhibit with Additions for PublicationCOMPARISON OF TERATOGENIC CHEMICALS IN THE RAT AND CHICK
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