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Effect of transplacental mitotic inhibitors on the fetal hamster eye.

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Effect of Transplacental Mitotic Inhibitors
on the Fetal Hamster Eye'
VERGIL H. FERM
Department of Pathology, Dartmouth Medical School,
Hanover, New Hampshire
ABSTRACT
Congenital malformations of the hamster eye, consisting of microphthalmia and anophthalmia have been noted consistently in the offspring of pregnant hamsters treated with intravenous injections of colchicine, Vincaleukoblastine
(VLB) and Vincristine (VCR) on the eighth day of gestation. In a n attempt to compare the mitotic-arresting activity of these three compounds on the lens and retina of
the fetal hamster eye, various concentrations of the drugs were injected into pregnant
hamsters on the fourteenth day of gestation and the fetuses recovered after three
hours. Mitotic counts were done on comparable sections of lens and retinas of the
fetuses. Colchicine has the most marked mitosis-inhibiting activity of these three
drugs. The most effective mitosis-inhibiting dosage of colchicine also correlates with
its most effective teratogenic dose. VLB and VCR do not inhibit mitosis in the fetal
hamster eye as markedly as colchicine and their most effective mitosis-inhibiting dosages are equal to or above levels which cause a 95-100% embryonic mortality. It is
possible that some common biological mechanism other than inhibition of mitosis may
be responsible for the similar teratogenic activity of these compounds. The natural
resistance of the golden hamster to colchicine does not appear to be manifest in the
developing embryos in utero.
Congenital malformations in hamster
fetuses following intravenous injections of
pregnant hamsters with colchicine (Ferm,
'63) and Vincaleukoblastine, (Vinblastine,
VLB) and Vincristine (VCR) (Ferm, '63a)
have been reported. These malformations,
all induced by maternal treatment on the
eighth day of gestation, have included,
among others, unilateral and bilateral
microphthalmia and anophthalmia. The
rather frequent occurrence of these types
of eye abnormalities after administration
of all three of these teratogenic agents suggests a common mode of action on the
developing eye. This teratogenic effect may
be directly attributable to the mitosis-inhibiting activity of these compounds.
The adult hamster has a marked natural
resistance to the anti-mitotic effect of
colchicine (Orsini and Pansky, '52; Turbyfill and Sodemall, '57). This resistance
apparently extends to hamster embryo
cells in uitro, for Midgley, Pierce and Dixon
('59) have shown that hamster embryo
cells grown in tissue culture demonstrate
a cellular resistance at least 100 times
greater than do human cells. VLB, on the
other hand, effectively inhibits mitosis in
the adult hamster (Cardinali, Cardinali,
Handler and Agrifiglio, '61). While the
effect of VCR on mitosis in the hamster
has not been reported, it does effectively
arrest mitosis in mouse bone marrow.
(Cardinali, Cardinali and Enein, '63).
The present study is concerned with the
problem of the relative mitosis-inhibiting
effects of these compounds on the fetal eye
tissue of the hamster. Preliminary studies
revealed an apparent increase of cells in
metaphase within embryonic tissue after
drug administration, and a more quantitative and comparative study of the effect of
these compounds seemed advisable. Since
all three agents had a similar teratogenic
effect on the embryonic eye, this tissue was
selected for study. This data is also intended to give some indication of the permeabiIity of the placenta to these agents.
MATERIALS AND METHODS
Female golden hamsters were bred in a
manner described elsewhere (Ferm, '63)
and housed in individual cages until the
fourteenth day of gestation. Under Nembutal anesthesia a small 1 cm incision was
made over the right femoral vein and various amounts of colchicine, VLB, and VCR
(table 1) , made up fresh daily in distilled
1 Supported by U. S. Public Health Research grant
GM 10210-01.
129
130
VERGIL H. FERM
TABLE 1
Effect of intravenous colchicine, wincaleukoblastine, and wincristine on mitosis
in the eye of the fetal hamsteT
Intravenous
Meta hase arrest
per Ens section
Metaphase arrest
per retina section
Colchicine
5 mgm/kg
10 mgm/kg
20 mgm/kg
50 mgm/kg
7.3 & 1.8
20.3 rt 4.6
7 . 5 2 1.3
5.7f 1.0
67.1-t 25.7
251.9k29.4
74.7-t 9.6
64.6C 4.9
Vincaleukoblastine
0.25 mgm/kg
0.5 mgm/kg
1.0mgm/kg
2.5mgm/kg
3.9 22.5
4.2C 0.3
17.0a3.1
13.323.1
46.8 r 16.7
32.62 6.1
55.5+-12.4
136.5217.3
Vincristine
0.1 mgm/kg
0.5 mgm/kg
1.0mgm/kg
2.5mgm/kg
5.3 f0.7
5.221.8
19.522.5
9.22 1.7
50.5'
3.0
66.5-t 26.6
81.2& 6.8
79.7228.0
4.02 1.7
3.92 0.7
1
.
7
'
0
.
2
35.8-t 7.3
39.22 8.5
31.52 0.6
Controls
c1
c2
c3
1 Average number of cells arrested in metaphase in each section, based on 2&50
each dose level (see text).
sections for
water, were injected directly into the fe- which did not include clear and uniform
moral vein over a one minute period. The sections through these structures comparincision was closed with two or three silk able to those in figures 3-10 were not used.
sutures. Three hours later the animals No distinction was made between right or
were sacrificed, the fetuses removed, and left eyes and both are included in the data
fixed in Bouin's fluid. Serial cross sections if usable by the above criteria. Between
were cut at 5 through the fetus at the 10 and 25 counts were made for each anilevel of the eyes and each sixth section mal in this study.
mounted on a slide, and stained with
RESULTS
H & E. Control animds were injected in
Evidence
for
the
transplacental passage
an identical manner with equal volumes
of saline, and sacrificed three hours after of the compounds under investigation, as
injection. The fourteenth day was chosen demonstrated by changes in the number of
for this study since at this time consistent mitoses per section i n hamster fetal eyes
and comparable sections of eye material three hours after injection, is summarized
could most easily be obtained. While it is in table 1 and figures 3-10. Colchicine
not possible to compare directly these stud- has a marked effect by arresting celI diviies relating to placental permeability on sion in metaphase at a level of 10 mg/kg,
the fourteenth day to the teratology stud- a level which corresponds to its most efies of the eighth day, there is no apparent fective teratogenic dose (Ferm, '63). Inreason to suspect any differential change creasing dosages of colchicine do not cause
during development in the relative sensitiv- as great an effect in arresting metaphase
ities of the eyes to these agents. This pos- but these levels equal or exceed those
which have caused an embryonic death
sibility cannot be ruled out, however.
Two animals were used for each dose rate of 95-100% on the eighth day of
level for each compound. All celIs in gestation (Ferm, '63).
VLB and VCR both cause an increase in
metaphase or anaphase in each lens and
retina were counted. Except for the con- mitotic arrest of these tissues. With these
trols, less than 1% of cells counted in compounds, however, the mitotic arrest is
this study were in anaphase. Sections not as striking as with the 10 mg/kg
131
EFFECT OF MITOTIC INHIBITORS O N THE FETAL EYE
colchicine level. There is no apparent cor- tween primary anophthalmia (agenesis), a
relation of the peak of mitotic arrest with secondary form (associated with other
the suggested teratogenic levels of these severe malformations of the neural plate),
compounds (VLB-0.25 mg/kg; VCR-0.1 and degenerative or atrophic anophthalmg/kg, Fern, ’63a). Histological examin- mia. Anophthalmia is most likely the result
ation of the fetal lens and retinas taken of an earlier or more severe insult to these
from those animals which received doses developmental mechanisms, while later
above the teratogenic levels of the com- aberrations in development lead to varying
pounds tended to show more signs of cell degrees of microphthalmia. The unilateral
degeneration : nuclear pyknosis, cell rup- expression of these malformations is diffiture and intercellular edema. In addition, cult to explain. However, in the chick,
many of these fetuses showed gross evi- where left-sided defects are more common,
dence of multiple cutaneous hemorrhages. this may be explained by a developmental
lag in the left eye as compared to the right
DISCUSSION
eye, so that an abnormal environmental
The development of the eye involves the factor would probably affect the eyes at
interaction of a number of components - different stages in their development
the lens placode, the optic vesicle, and (Gruenwald, ’44). This may also be the
mesodermal components including the vas- explanation in mammalian unilateral eye
cular supply to this region. Malformations malformations. Hereditary anophthalmia
of the eye may result from a failure of associated with albinism has been reported
any of these integral components to inter- in the hamster (Knapp and Polinvanov,
act properly at the appropriate time and ’58) although the histological structure of
may be attributed to genetic and/or exter- the eye has not been detailed.
nal teratogenic factors. Stockard (’24)
Mitotic activity in the developing retina
has stated that “the eye frequently shows is located in the outer nuclear layer and
maldevelopment in both its primary and cells then migrate internally (fig. 3 ) . Only
secondary structures, and this fact is to occasional mitotic figures are found in the
be correlated with its long period of devel- inner layers. Likewise mitotic activity in
opment.” Mann (’57) distinguishes be- the lens is distinctly localized in the exterCONTROLS
COLCHICINE
VlNCRlSTlNE
VINCALEUKOBLASTINE
C
0
._
25-
aJ
v)
v)
20-I
L
aJ
a
15
T T
-
v)
a
v)
0
+
.5
I
lo-
IT
6
h
T
5 -
I
ix
I
,
I
I
,
Saline
1
5
1
1
1
10 2 0 5 0
rngrn/kg
1
1
1
1
iI
I
I
I
0.1 0.5 1.0 2.5
0.25 0.5 1.0 2 . 5
rngrn/kg
rngrn/kg
Fig. 1 Effect of intravenous injection of test compounds on mitotic inhibition in fetal hamster
lens on the fourteenth day of gestation.
132
VERGIL H. FERM
nal cellular layer on the anterior surface
of the lens (fig. 1). Thus counts of metaphase arrest as done in these experiments
would reflect the major changes in cell
division within these tissues.
There is some correlation between the
most effective teratogenic dose and the effect on embryonic mortality of these compounds (table 1). However, there is a lack
of correlation between the peak of mitotic
inhibition and effective teratogenic dose for
VLB and VCR. VLB and VCR, which have
strikingly similar structur a1 formulae
(Neuss et al., '62) demonstrate a marked
difference in their tumor spectrum effect
both experimentally and clinically ( Johnson, Svoboda and Wright, '62; Selawry
and Hananian, '63). Clinically, the use of
busulf an and 6-mercaptopurine has produced microphthalmia on one occasion in
a human embryo. The presumed pharmacologic effect of this drug was mitotic inhibition (Diamond, Anderson, and McCreadie, '60). Busulfan has not produced
eye abnormalities in the offspring of pregnant rats or in developing chicks (Murphy,
Del Miro and Lacon, '58). Similarly, eye
defects have not been reported in malformed rat fetuses (Haskin, '48) or mal-
1
C
0
.+
CONTROLS
COLCHICINE
W
200
ACKNOWLEDGMENT
I wish to acknowledge the able technical
assistance of Miss Peggy Starusky in various aspects of this work.
LITERATURE CITED
Cardinali, G., G. Cardinali, A. H. Handler and
M. F. Agrifiglio 1961 Comparative effects of
colchicine and Vincaleukoblastine on bone marrow mitotic activity in the Syrian hamster.
Proc. SOC.Exp. Biol. and Med., 107: 891-892.
Cardinali, G., G. Cardinali and M. A. Enein 1963
Studies on the antimitotic activity of Leurocristine (Vincristine). Blood, 21: 102-110.
VlNCRlSTlNE
VINCALEUKOBLASTINE
I
250-
V
W
cn
formed mouse fetuses (Nishimura and
Takagaki, '59) following nitrogen mustard
treatment of maternal animals during pregnancy.
Colchicine appears to have a marked effect on cell division of hamster embryos
in utero. The reason for this effect as
contrasted to the relative lack of effect on
the same tissues in vitro is not apparent.
It appears then, that this problem is more
complex than a simple explanation based
on mitotic inhibition. It may well be that
some common denominator other than inhibition of cell division may be responsble
for the similar teratogenic effects of these
three compounds on the embryonic eye.
-
c
.+
0,
LT
W
a
I
I5Ot
v)
::
.-
loo-
20,
50-
W
T
4-
I
ITr f
1
1
Soline
1
I
5
t
1
1
10 20 5 0
rngm/kg
,
I
,
0.1 0.5 1.0 2.5
mgm/kg
I
,
,
,
0.25 0.5 1.0 2.5
mgm/ k g
Fig. 2 Effect of intravenous injection of test compounds on mitotic inhibition i n fetal hamster
retina on the fourteenth day of gestation.
EFFECT O F MITOTIC INHIBITORS O N THE FETAL EYE
Diamond, I., M. M. Anderson and S. R. McCreadie
1960 Transplacental transmission of busulfan
in a mother with leukemia. Production of fetal
malformation and cytomegaly. Pediatrics, 25:
85-90.
Ferm, V. H. 1963 Colchicine teratogenesis in
hamster embryos. Proc. SOC. Exp. Biol. and
Med., 112: 775-778.
1963a Congenital malformations i n hamster embryos following Vinblastine and Vincristine treatment. Science, 141: 426.
Gruenwald, P. 1944 Studies on Developmental
Pathology 11. Sporadic unilateral microphthalmia and associated malformations in chick embryos. Am. J. Anat., 74: 217-258.
Haskin, D. 1948 Some effects of nitrogen mustard on the development of external body form
in the fetal rat. Anat. Rec., 102: 493-512.
Johnson, I. V., G . H. Svoboda and H. F. Wright
1962 Experimental basis f o r clinical evaluation of two new alkaloids from Vinca rosea,
Linn. Proc. Amer. Assn. Cancer Res., 3: 331
(abst).
Knapp, B. H., and S. Polinvanov 1958 Anophthalmic albino: a new mutation i n the Syrian
hamster, Cricetus (Mesocricetus) auratus. Am.
Nat., 92: 317-318.
Mann, I. 1957 Developmental Abnormalities of
the Eye. J. B. Lippincott Company, Philadelphia, 419 pp.
133
Midgley, A. R., B. Pierce and F. Dixon 1959
Nature of colchicine resistance in golden hamster. Sci., 130: 4 0 4 1 .
Murphy, M. L., A. DelMiro and C. Lacon 1958
The comparative effects of five polyfunctional
alkylating agents in the rat fetus, with additional notes on the chick embryo. Ann. N. Y.
Acad. Sci., 68: 762-781.
Neuss, N., M. Gorman, H. E. Boaz and N. J.
Cone 1962 Vinca alkaloids. XI. Structures
of Leurocristine and Vincaleukoblastine. J.
Amer. Chem. SOC.,84: 1509-1510.
Nishimura, H., and S. Takagaki 1959 Congenital malformations i n mice induced by nitrogen
mustard. Acta Schol. Med. Univ. (Kioto), 36:
20-26.
Orsini, M. W., and B. Pansky 1952 The natural
resistance of the golden hamster to colchicine.
Sci., 115: 88-89.
Selawry, 0. S., and J. Hananian 1963 Vincristine treatment of cancer in children. J.A.M.A.,
183: 741-746.
Stockard, C. R. 1924 The structure of the vertebrate eye as a n index of developmental deficiencies: with the bearing on recent inheritance
studies. Amer. Nat., 58: 24-35.
Turbyfill, C. L., and A. L. Soderwall 1957 Sensitivity of hamster to colchicine. Sci., 226:
749.
PLATE 1
EXPLANATION O F F I G U R E S
134
3
Section of lens and retina from control hamster on fourteenth day of
gestation.
4
5 mgm/kg colchicine intravenously.
5
10 mgm/kg colchicine intravenously. Separation of retina from
choroid layer probably represents an artifact in all of these specimens.
6
20 mgm/kg colchicine.
7
0.25 mgm/kg Vincaleukoblastine.
8
2.5 mgm/kg Vincaleukoblastine.
9
0.1 mgm/kg Vincristine.
10
1.0 mgm/kg Vincristine.
EFFECT OF MITOTIC INHIBITORS ON THE FETAL EYE
Vergil H. Ferm
PLATE 1
135
PLATE 2
E X P L A N A T I O N O F FIGURES
11 Cross section through eyes of 14 day hamster fetus whose mother
received 0.25 mg/kg VLB on the eighth day of gestation. Eye on the
left is microphthalmic. Eye on right is completely absent except for
small amount of mesenchymal tissue (arrow).
12
136
Left eye of 14 day hamster fetus whose mother received 0.1 mgm/kg
VCR on the eighth day of gestation. Rudimentary lens, retina, choroid, are present but eye is markedly microphthalmic.
EFFECT OF MITOTIC INHIBITORS ON THE FETAL EYE
Vergil H. Ferm
PLATE 2
137
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