/ . Embryol exp. Morph., Vol. 17, 1, pp. 229-237, February 1967
With 4 plates
Printed in Great Britain
•
229
The effect of mitomycin C on developing
chicken embryos
By GEORGE KURY 1 & JOHN M. CRAIG 1
From the Department of Pathology, Boston Hospital for Women,
Lying-in Division, and Harvard Medical School
INTRODUCTION
The mitomycins were isolated from the broth of Streptomyces caespitosus by
Hata, Shimada & Ishu (1957). Mitomycin C, one of a series of chemically
related substances, has been most frequently used in experimental and clinical
studies. It was found to have a marked antibacterial, antiviral and antitumor
activity in animals and man. In humans, mitomycin C has been used with
varying success in the palliative treatment of osteogenic sarcoma, carcinoma of
stomach, chronic myelogenous leukemia, reticulum cell sarcoma and other
malignancies (Cancer Chemotherapy. Rep., 1959; Frank & Osterberg, 1960;
Evans, 1961). The teratogenic activity of mitomycin C has been only briefly noted
(Yamura, 1961; Takaya, 1965).
The purpose of these experiments is to study the effects of mitomycin C in
chicken embryos after yolk-sac injection during an early period of development.
To study the teratogenic activity of drugs in chicken embryos, the optimal times
of yolk-sac injection are the 3rd and 4th days of development. If the drugs are
injected during the first 48 h of development, the teratogenic and lethal doses
are so similar that the embryos usually die shortly after injection. By the 3rd and
4th days, however, when development is more advanced, but still in the active
phase of organogenesis, a number of affected embryos may survive longer or
until hatching. By the end of the 4th day the organ systems are well established;
although the action of many drugs can be demonstrated up to the 8th day of
development or later, the chances of producing malformations are reduced.
MATERIALS AND METHODS
Mitomycin C was dissolved in saline by shaking, and was stored at 4 °C and
protected from light. No solutions older than 6 days were used. Fertilized White
Rock eggs were obtained commercially and incubated at 37-2-39-5 °C. Between
48 and 90 h of incubation, mitomycin C was injected into the yolk sac of eggs.
1
Authors' address: The Department of Pathology, Boston Hospital for Women, Lying-in
Division, 221 Longwood Avenue, Boston, Mass. 02115, U.S.A.
230
G. KURY & J. M. CRAIG
Eggs used for control were injected with 0-1 ml of saline at the same time. The
injections were done_under sterile conditions using tuberculin syringes and 25 G/
5/8 in. needles. The puncture hole was sealed by melted paraffin. A number of
uninjected eggs were also incubated and used for control. The eggs were candled
each day for viability and the dead embryos were examined. A number of
embryos were removed from viable eggs injected either by mitomycin C or saline
on the 6th to 9th day of incubation and were immediately fixed in 4 % neutral
formaldehyde. A selected number of these were serially sectioned and studied
histologically. The remainder of the viable eggs were opened on the 19th-20th
day of incubation. After weighing and inspection the specimens were fixed in
4 % neutral formaldehyde. Following fixation, the brain and viscera were inspected and dissected under the microscope, and a selected number of mitomycin C treated, and control tissues were further processed for histological
studies. The malformed and normal embryos selected for skeletal studies were
eviscerated, fixed in 95 % alcohol, cleared in acetone, 95 % alcohol and 1 %
aqueous KOH solutions. They were then placed in 0-5 % aqueous KOH solution
to which a few drops of aqueous alizarin red solution was added. The specimens
were dehydrated in increasing concentrations of glycerin in water and stored in
pure glycerine.
RESULTS
The optimal single teratogenic dose range for mitomycin C in these experiments was 2-16 ^g/egg on the third day and 2-28 /*g/egg on the 4th day.
Tables 1 and 2 summarize the results. Of 600 eggs used, 361 died within
10 days after injection; these had no gross abnormalities. Forty-five embryos
treated by mitomycin C were sacrificed between the 6th and 9th days of development. Thirty of those were injected on the 3rd and 15 on the 4th day of development. Eighteen embryos injected on the 3rd day had gross abnormalities
including bilateral microphthalmia (14), stunting (17) (Platel) and cerebral hemorrhage (3). The embryos injected on the 4th day did not have gross abnormalities.
Thirteen embryos (7 microphthalmic injected on the 3rd day and 6 injected on
the 4th day) were selected for serial sectioning and histological studies. Histological examination of microphthalmic embryos showed a small retina with
arrest of development and degeneration at an early stage (Plate 2). The cavity
of the optic vesicle remained opened in several specimens. At 9 days of development there was a vitreal hemorrhage and ingrowth of periocular mesenchyme
into the vitreal space. No rosette formation or coloboma was seen in the
retina. Two of these embryos had necrosis of lens epithelium and coagulation
of fibers consistent with cataract formation. The walls of cerebral vesicles and
spinal cord contained extensive cellular necrosis, rosette formation and
hemorrhage in both the 3rd and 4th day injected specimens (Plate 3). The
necrosis of nervous elements was so massive in some specimens that only
scattered islands of viable cells were seen around the ventricular cavity. There
/. Embryoi. exp. Morph., Vol. 17, Part 1
PLATE 1
Viable embryos sacrificed on the 9th day of development. Left, control. Middle, bilateral
microphthalmia and retardation of growth; treatment, 4 fig of mitomycin C on the 3rd day.
Right, retardation of growth; treatment, 4 /tg of mitomycin C on the 3rd day. x 2.
G. KURY & J. M. CRAIG
facing p. 230
/ . Embryo I. exp. Morph., Vol. 17, Part 1
PLATE 2
*iv
Microphthalmia in a 9-day-old embryo treated by mitomycin C on the 3rd day. Arrest of
development and differentiation of retina, x 52. Both photomicrographs from hematoxylin
and eosin preparations.
G. KURY & J. M. CRAIG
Necrosis, hemorrhage, rosette formation in the wall of cerebral vesicle. Intraventricular hemorrhage.
Nine-day-old embryo. Treatment: 4 /*g of mitomycin C on the 3rd day. x 132.
s
/. Embryo!, exp. Morph., Vol. 17, Part 1
PLATE 4
Fig. A
Fig. B
G. KURY & J. M. CRAIG
facing p. 231
Mitomycin C and chick embryos
231
were also foci of necrosis and recent hemorrhage in the head mesenchyme.
Cardiac abnormalities were found in two specimens, both of which were injected
on the 3rd day. One embryo had imperfect development of the endocardial
cushion of great vessels. There was myocardial necrosis involving the region of
atrioventricular groove, the walls of atria and left ventricle in another specimen.
Table 1. Effect of mitomycin C on developing White Rock embryos
after yolk-sac injection on the third day of development
Number of eggs injected
Number of dead embryos (grossly normal): died within
10 days after injection
Number of grossly normal embryos sacrificed on 6th-9th days
Number of grossly normal embryos sacrified on 19th-20th days
Number of grossly malformed embryos sacrificed on 6th-9th
days
Type of abnormality:
Bilateral microphthalmia
Stunting
Cerebral hemorrhage
Number of grossly malformed embryos sacrified at 19th-20th
days
Type of abnormality:
Stunting
Malformation of lower extremities
Short lower beak
Defect of calvari
Marked edema
260
160
12
60
18
14
17
3
10
7
7
2
1
1
Among the 260 eggs injected on the 3rd day, 70 viable embryos were examined
at the 19th-20th day of development. Ten of these had gross abnormalities;
60 were grossly unremarkable. Three hundred and forty eggs were injected on
the 4th day. Among 124 viable embryos examined on the 19th-20th day, 90 were
grossly unremarkable and 34 had gross abnormalities. The type of abnormalities
obtained on both days are tabulated in Tables 1 and 2. Beside the listed abnormalities, 15 % of treated viable embryos sacrificed on the 19th-20th day had
enlargement and brown-red discoloration of liver and kidneys.
Thirty-five treated, grossly abnormal and five control embryos (all sacrificed
on the 19th-20th day of development) were cleared and the skeletons stained
with alizarin red. The type and number of skeletal anomalies found, and the
frequency of involvement of the bones of extremities, head and trunk are listed
PLATE 4
Fig. A. Marked shortening and curvature deformity of tibias and metatarsals, hypophalanges
of toes. Embryos cleared and bones stained with alizarin red. Treatment: 15 /tg of mitomycin C on the 4th day. x 1-3.
Fig. B. Control, x 1-3.
232
G. KURY & J. M. CRAIG
in Table 3. It is interesting to note that the most frequently involved bone was
the tibia, which was shortened or curved or both (Plate 4). Absence of individual
bones was less frequent. In four specimens the metatarsal bones did not unite
as in normal embryos, but remained separate. Some of the separated metatarsals had curvature deformity. The maldevelopment of the bones of the upper
Table 2. Effect of mitomycin C on developing White Rock embryos
after yolk-sac injection on the fourth day of development
Number of eggs injected
Number of dead embryos (grossly normal) (died within
10 days after injection)
Number of grossly normal embryos sacrified at 6th to 9th days
Number of grossly normal embryos sacrificed at 19th to
20th days
Number of grossly malformed embryos sacrificed at 19th to
20th days
Type of abnormality:
Stunting
Defect of calvarium
Malformation of lower extremities
Defect of abdominal wall
Abnormality of beak
340
201
15
90
34
10
6
12
8
15
Table 3. List of skeletal malformations produced by yolk-sac injection of mitomycin C in 9 embryos on day 3 and in 26 embryos on day 4 of development
First number represents embryos injected on day 3. Number in parentheses represents embryos injected on day 4. Right and left sides of body were counted independently.
Absence
Calvarium
Ribs
Vertebrae
Pubis
Radius
'Elbow joint'
Femur
Tibia
Fibula
Metatarsals
Toes
Failure of
unification
Shortening
Curvature
deformity
Fusion
3(4)
-
—
—
2(2)
(1)
2(4)
—
1
—
2(3)
-
(4)
—
—
—
(2)
4(2)
1
-
1
2(6)
6(4)
6(10)
4(9)
2(6)
3(3)
(4)
6(16)
(4)
(6)
—
extremity, axial skeleton, toes and joints was infrequent compared to those
of the tibia.
Thirty-eight embryonic tissues removed from 19- to 20-day-old treated
embryos were processed for histological studies. Among these, histological
examination of grossly normal brains, eyes, lungs, pancreas, gastro-intestinal
tracts and endocrine organs revealed no abnormalities. The livers and kidneys,
Mitomycin C and chick embryos
233
which showed enlargement and brown-red discoloration grossly, had marked
extramedullary hematopoiesis. There was a decreased number of lymphoid
follicles in the spleen and decrease of lymphoid tissue in the bursa of Fabricius.
The lower extremities showed a marked reduction of hematopoietic activity in
the bone marrow. The number of osteoblasts was also markedly reduced.
The spontaneous mortality among 300 uninjected eggs used for control was
9-10 %. The mortality among the 300 eggs injected with 0-1 ml of saline on the
3rd day was 35-45 %, and on the 4th day 20-30 %, varying with individual
groups. One malformation (cyclopia) was found among the saline-injected
embryos. Forty injected control embryos were examined on the 6th-9th day of
development, five of which were also examined histologically. The remainder
of viable saline control embryos were examined at the 19th-20th day of development. With the exception of the cyclopic specimen, all dead and viable control
embryos examined grossly and histologically were unremarkable.
According to the research department of the poultry farm from which our
fertilized eggs were obtained, the incidence of spontaneous malformations is
1 in 3000 eggs. The malformations observed are those of eyes, beak and spinal
column (spina bifida). One example of an embryo with supernumarary lower
extremity (three legs) was found in approximately 500000 eggs. With the aid of
pedigree breeding, in which each egg is labelled with the serial number of mother
hen and the father is known, stocks carrying a higher incidence of spontaneous
malformations, infections, or those of low vitality, are quickly eliminated. To
maintain the viability of stocks used, each flock is slaughtered after a 10-month
period of egg laying. The cyclopic embryo found among our injected specimens
may represent a spontaneous malformation. The increased rate of mortality of
injected control embryos is probably due to mechanical trauma directly involving the embryo or major vitelline blood vessels.
DISCUSSION
The teratogenic effect of mitomycin C has been previously studied by Takaya
in albino rats (Takaya, 1965) and Yamura in mice (Yamura, 1961). Takaya
injected mitomycin C subcutaneously from the 6th to 10th or from the 10th to
14th days of development. The embryos were examined on the 21st day of
gestation. When mitomycin C (0-4-0-8 mg/kg) was injected from the 6th to
10th day of gestation of the treated embryos, 24-5 % had developmental
anomalies, 62-3 % were normal and 13-2 % died without detectable gross
malformations. The 60 malformed embryos had the following anomalies:
microphthalmia (25 specimens), hydronephrosis (39 specimens), exencephaly
(1 specimen), and defect of abdominal wall (1 specimen). The number of malformed embryos markedly decreased (9-4 %) if mitomycin C was administered
from the 10th to 14th day of gestation. The only abnormality detected then was
hydronephrosis.
234
G. KURY & J. M. CRAIG
According to Takaya, Yamura obtained similar results by treating pregnant
mice with mitomycin C. Karnofsky, Lacon & Lowe (1963) examined the antitumor activity of mitomycin C against sarcoma 180 and Ridgway osteogenic
sarcoma explanted on the 8th day of incubation to the chorioallantoic membrane
of chick embryos. When mitomycin C was injected on the 12th day of incubation
into the yolk sac of tumor-bearing eggs, the injected embryos were smaller than
the controls, and five of them had thin legs, edema, feather inhibition and
clubbing. The total number of eggs injected with mitomycin C was 47. The
effect of mitomycin C has not been previously examined in chicken embryos at
the time of early development. Although the malformations produced by drugs
may vary from species to species, it is interesting to note that there is some
similarity in the type of malformations produced by mitomycin C in chickens,
rats and mice. There is a high incidence of microphthalmia and growth retardation and a low incidence of exencephaly and abdominal-wall defect in all three.
However, hydronephrosis, a frequent finding in rats and mice, is not present in
chicken embryos; there are no anomalies of extremities in rats.
The types of malformations produced by mitomycin C in chickens are not
specific to this compound alone, if they are considered separately. Retardation
of growth, defects of calvarium and abdominal wall, microphthalmia and rosette
formation were observed in embryos of chickens and several other species after
radiation and administration of many other compounds. For example, microphthalmia and curvature deformity of tibia and metatarsals were also observed
in chicken embryos after yolk-sac injection of fluorinatedpyrimidines (Karnofsky,
1965; Kury & Craig, 1966). However, when the effects of different compounds
are compared after studying a large number of embryos grossly, histologically
and following skeletal staining, an individual pattern may be demonstrated for
each compound. For example, fluorinated pyrimidines more often affect the
bones of upper extremities, toes and joints and cause absence of individual bones
than does mitomycin C. Microphthalmia can also be produced by both, but
there were no corneal cysts, eyelid defects or malformation of the pecten in our
present experiments in contrast to embryos treated with fluorinated pyrimidines
(Kury & Craig, 1966). Quaternary ammonium compounds and physostigmine
(Karnofsky, Ross & Leavitt, 1954) affect the vertebrae much more often than
mitomyein C or the fluorinated. pyrimidines. Why the development of certain
bones is more influenced by one drug than another is still unknown.
Toxicity studies of mitomycin C in adult animals have shown gastro-intestinal
hemorrhages, a reduction of all hematopoietic cells in the bone marrow (rats,
dogs, rhesus monkeys), nuclear atypia and swelling of gastro-intestinal epithelium (rats and rhesus monkeys), a decrease in size of thymus, spleen and
lymph nodes (rats), and necrotizing nephrosis (rhesus monkeys). There were
also petechial and diffuse hemorrhages in the tonsils, lungs, heart, gall bladder
and adrenal cortex (dogs and rhesus monkeys) (Cancer Chemother. Rep., 1959).
The depression of hematopoietic activity was also striking in our chickens, but
Mitomycin C and chick embryos
235
the lymphoid tissue in the spleen and bursa of Fabricius was only slightly involved. The gastro-intestinal tract and kidney were unremarkable.
The site of action of chemotherapeutic agents in living cells might be on
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid or protein
synthesis. There is a great deal of variation in the mechanism of action of compounds in each of these general categories. Mitomycin C, like alkylating agents,
actinomycin D and certain 5-halogenated pyrimidines, acts primarily on DNA
synthesis. In intact cells, mitomycin C inhibits DNA synthesis from DNA
precursors without much initial effect on RNA or protein synthesis. Depolymerization of DNA is a secondary phenomenon (Schwartz, Sternberg & Philips,
1963; Szybalsky & Iyer, 1964). The exact mechanism of its action is still not
clear. Actinomycin D inhibits DNA-dependent RNA synthesis. Both mitomycin C and actinomycin D inhibit thymidine uptake and mitosis in proliferating tissues, but in contrast to the prompt action of mitomycin C, actinomycin D
acts after a relatively long latent period (Schwartz et al. 1963). Other compounds
like puromycin interfere with protein synthesis on the ribosomes. FUDR
(5-fluoro-2'-deoxyuridine) interferes with thymidine synthesis by blocking the
formation of thymidilic acid.
It is reasonable to assume that the inhibition of DNA synthesis in rapidly proliferating embryonal tissues is an important initiating factor in the pathomechanism of malformations produced by mitomycin C in rats, mice and chickens.
However, there is a combination of other factors to take into account, such as
time of injury, stage of proliferation and regenerative ability of a given tissue,
interruption of important inductive processes, local metabolic pathways, etc.
The mesenchyme, retina and the neuroblasts in the cerebral ventricles are
rapidly proliferating in chicken embryos during the 3rd and 4th days of development. If the injury of these tissues is not too extensive, complete restitution
might come from adjacent proliferating areas. But if the tissue damage is more
extensive and the mitotic activity in the area subsides, this reduces the chances
of regeneration; malformation of extremities, cranial vault, eyes and brain
(rosette formation) is more likely to occur. Although the mechanism of rosette
formation is still not well understood, some investigators believe that mechanical
factors may be important, such as the disturbance of the balance of ventricular
and tissue pressure exerted on the periventricular neuroectoderm, secondary
to the partial destruction of this layer.
The differences in susceptibility of embryonic tissues to mitomycin C in
chickens, rats and mice and the variability of malformations obtained are
attributable to the known differences in genetic background, in local developmental pathways and tissue metabolism.
236
G. KURY & J. M. CRAIG
SUMMARY
1. The effect of mitomycin C before completion of organogenesis was studied
in chicken embryos. The injection of mitomycin C into the yolk sac produced
developmental anomalies which varied with the time of injection. Embryos
treated on the 3rd day of development (2-16 /*g/egg) had retardation of growth,
bilateral microphthalmia, cerebral hemorrhage, malformations of lower extremities, lower beak and defect of calvarium. Specimens treated on the 4th day
of development (2-28 /tg/egg) had retardation of growth, abnormalities of beak,
lower extremities and calvarium. Histological examination of viable embryos
sacrificed 2-6 days after injection revealed necrotic changes involving the
developing retina, lens, cerebral vesicles, mesenchyme and heart. Histological
examination of 19- to 20-day-old treated embryos showed depression of
hematopoietic and osteoblastic tissues.
2. The skeletal abnormalities produced included shortening and curvature,
deformity of the bones of lower extremities, especially the tibia. Absence of
other bones of the lower extremities was also encountered.
RESUME
Action de la mitomycine C sur le developpement de Vembryon de poulet
1. On a etudie, sur des embryons de poulet, Faction de la mitomycine C avant
l'achevement de l'organogenese. L'injection de mitomycine dans le sac vitellin
a produit des anomalies du developpement, variables selon le moment de
l'injection. Des embryons traites le 3eme jour du developpement (2 a 16 jug par
ceuf) presentaient un retard de croissance, de la microphtalmie bilaterale, des
hemorragies cerebrales, des malformations des extremites inferieures, de la
partie inferieure du bee et l'absence de calvarium. Des individus traites le 4eme
jour du developpement (2 a 28 /ig par ceuf) presentaient un retard de croissance,
des anomalies du bee, des extremites inferieures et du calvarium. L'examen
histologique d'embryons viables sacrifies 2 a 6 jours apres l'injection a revele
des modifications necrotiques interessant la retine, le cristallin, les vesicules
cerebrales, le mesenchyme et le coeur. L'examen histologique d'embryons traites
ages de 19 a 20 jours a revele une diminution des tissus hematopoietique et
osteoblastique.
2. Les anomalies du squelette obtenues comprenaient le raccourcissement et
l'inflechissement des os des extremites inferieures, en particulier le tibia. On
a egalement observe l'absence d'autres os des extremites inferieures.
Dr Alexander Gourevits, Director, Microbiological Research, Bristol Laboratories,
Syracuse, New York, and Dr Charlotte Maddock, Children's Cancer Research Foundation,
35 Binney Street, Boston, Massachusetts, kindly provided the mitomycin C used in these
experiments. Mr John Hedges provided the technical assistance in histology. This work was
aided by grants HD-00144 and 1 SOI FR-05481-03-5 USPHS.
Mitomycin C and chick embryos
237
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