/. Embryol. exp. Morph. Vol. 22, 1, pp. 107-113, August 1969
Printed in Great Britain
107
The permeability of the hamster placenta to
radioactive cadmium
By VERGIL H. FERM, 1 DAVID P. HANLON 2
and JOHN URBAN 1
From the Departments of Anatomy /Cytology and Biochemistry,
Dartmouth Medical School
Studies on the permeability of the mammalian placenta to teratogenic agents
during the early critical stages of embryogenesis have been relatively few. Most
of these studies have been done in the pregnant rabbit, in which the unusually
large size of the blastocyst makes it possible to obtain samples of blastocyst
fluid for analysis. Thus, investigations have been done on the permeability of
the rabbit blastocyst wall to trypan blue and other teratogenic azo dyes (Ferm,
1956), changes in blastocyst sugar content following maternal administration
of insulin (Curry & Ferm, 1962; Lutwak-Mann, 1962) and the effect of thalidomide and other agents on the implanting rabbit blastocyst (Lutwak-Mann &
Hay, 1962; Hay, 1964; Fabro, Smith & Williams, 1965; Keberle et al. 1965;
Fabro, Hague & Smith, 1967).
However, the much smaller size of the rat, mouse and hamster blastocysts
makes comparable studies in these species extremely difficult and most attempts
to investigate the placental permeability to teratogens in these animals in the
early critical stages of embryogenesis have been directed toward histological,
histochemical and radioautographic techniques.
The site-specific teratogenic effect of cadmium upon hamster embryos and
the inhibition of this effect by the simultaneous administration of zinc has been
described (Ferm & Carpenter, 1968). Essentially, cadmium injected intravenously into pregnant hamsters on the eighth day of gestation causes a high
incidence of facial abnormalities ranging from simple unilateral cleft lips to
massive destruction of facial architecture. The simultaneous administration of
an equivalent amount of zinc, which in itself is only mildly teratogenic, markedly
inhibits the teratogenic effect of cadmium. While it is likely that the teratogenicity of cadmium is due to a direct effect upon the embryonic tissues, little
data is available on the rate or degree of permeability of the rodent placenta to
1
Author's address: Department of Anatomy/Cytology, Dartmouth Medical School,
Hanover,
New Hampshire, 03755, U.S.A.
2
Author's address: Department of Biochemistry, Dartmouth Medical School, Hanover,
New Hampshire, 03755, U.S.A.
108
V. H. FERM, D. P. HANLON AND J. URBAN
either cadmium or zinc. The teratogenic effect of cadmium and other heavy
metals makes possible studies on the permeability of the placenta during early
stages of embryonic development utilizing radioisotopes of these metals.
The demonstration that cadmium is teratogenic in the golden hamster,
together with the development of techniques for the dissection of 9-day-old
hamster embryos from gestation sacs (Ferm, 1965) prompted us to investigate
the permeability of the early hamster placenta to radioactive cadmium. In
addition, since zinc protects the hamster embryo from the teratogenic effect
of cadmium, these studies were extended to include the effect of zinc on the
placental permeability to radioactive cadmium.
MATERIALS AND METHODS
Timed matings of virgin female hamsters were obtained in a manner previously
described (Ferm, 1967). Sufficient carrier free 109CdCl2 (Isoserve) was added to
1-50 x 10~3M CdSO4 in water to yield a solution which contained 6-6 microcuries/ml. In some cases the l-50x 10~3M isotopic cadmium solution was also
made 1-50 x 10~ 3 M in ZnSO4. On the eighth day of gestation the cadmium salt
solution, with or without zinc (Table 1) was injected into the sublingual vein of
the pregnant hamsters. In most cases doses of 0-5 ml./100 g. of maternal body
weight were administered. In a few cases the volume injected was less, but never
less than 0-3 ml./lOO g. of body weight.
On the ninth or twelfth day of gestation the maternal animals were killed by
ether anesthesia. Samples were taken of maternal whole blood, maternal liver,
uterus, placenta, embryos and, for 12-day pregnant animals only, the yolk sac
placentas were also dissected separately. The embryos from the 9-day pregnant
animals were separately dissected but were pooled for counting because of their
small size. Four 12-day-old embryos were counted together, as were their
specific chorio-allantoic and yolk sac placentas and the corresponding uterine
segment. The tissues were weighed in previously tared plastic tubes and the
radioactivity of each sample was determined with a Nuclear Chicago Radiation
Analyzer system employing a well counter. The counting efficiency was 20 %.
With each sample assayed for radioactivity the total number of counts accumulated was such that the probable error in counting was always less than I %.
RESULTS
Our findings on the distribution of 109Cd in pregnant hamsters are presented in
Table 1. Detectable counts in the embryonic tissue at 9 days and 12 days of
pregnancy indicate that the placenta is permeable to cadmium. One can also
ascertain that zinc in equimolar amounts has no significant effect on the total
cadmium content of placenta and embryos at 9 days. However, relative changes
in the concentration of cadmium in the various tissues assayed do occur between
the ninth and twelfth days of pregnancy. These differences are better seen as
Placental permeability
109
to cadmium
im
Table 1. Distribution of Cd 24-96 h after intravenous injection of
the pregnant hamster on the eighth day of gestation
Day
sacrificed
9
9
9
9
9
9
9
9
9
9
9
12
12
12
12
12
12
12
12
Metal
ions
injected
Cd
Cd
Cd
Cd
Cd
Cd
Cd +
Cd +
Cd +
Cd +
Hamster
number
Zn
Zn
Zn
Zn
Cd + Zn
Cd
Cd
Cd
Cd
Cd
Cd
Cd + Zn
C d + Zn
3
3
3
3
3
3
3
3
3
3
3
551*
552
553
668
670
671
618
619
620
704
707
3
3
3
3
3
3
3
3
622
623
624
645
649
652
705
706
Counts per minute per gram of tissue
MicroK
curies f
10D
EmCd Maternal Maternal
liver
injected blood
Uterus Placenta bryo
506
164 300
—
337
—
189
2-64
445
3-33
185 649
326
3-33
148 255
115 441
349
2-64
163 698
427
3-33
128
97 168
200
182 220
3-33
496
200
149
50 377
3-33
495
105 124
3-33
406
210 863
214 811
541
3-30
534
224 851
3-30
244 721
599
3-30
680
243 331
3-30
343
163 815
200
68 902
283
3-30
3-30
364
72 325
* Non-pregnant animal
3-33
3-33
14 780
5 920
2 690
6 601
5 345
4 587
6 588
3 244
7 165
2 155
4 345
3 571
3 533
4710
4 976
4 709
3 420
1 968
2 507
—
4 120
2110
4 100
4 126
4 070
5 754
2 701
5 763
1 845
3 723
990
1090
1 295
1 938
1714
1 981
390
784
—
768
623
375
556
531
545
275
628
881
Yolk
sac
—
—
—
—
—
—
—
—
—
—
—
—
—
—
570
11
12
14
25 1401
849
16
20 1 108
8-8 306
116 717
Table 2. Ratios of imCd in whole blood, liver, uterus, placenta,
embryos and yolk sacs normalized to maternal blood
Day
sacrificed
9
9
9
9
9
9
9
9
9
9
12
12
12
12
12
12
12
12
Metal
ions
injected
Cd
Cd
Cd
Cd
Cd
Cd + Zn
Cd + Zn
Cd + Zn
Cd + Zn
Cd + Zn
Cd
Cd
Cd
Cd
Cd
Cd
Cd + Zn
Cd + Zn
MaHamster
number
3552
3553
3668
3 670
3 671
3618
3619
3620
3704
3707
3622
3 623
3624
3645
3649
3652
3 705
3706
ternal Maternal
liver
blood
1
1
1
1
1
1
1
1
—
—
417
455
331
383
759
367
1
338
1
212
1
1
1
1
1
1
1
1
519
397
421
409
358
478
244
199
Yolk
Uterus
Placenta
Embryo
17-5
14-2
14-8
16-4
131
15-4
25-3
14-4
14-5
8-78
8-80
6-53
8-82
8-31
6-93
9-97
6-95
6-89
12-2
11-2
9-21
12-7
117
13-5
211
11-6
2-28
3-30
0-843
1-71
1-52
1-28
12-4
7-52
2-44
202
2-43
3-34
2-52
5-78
1-38
215
215
1-27
5-91
115
00246
00222
00262
00417
00236
00583
00318
00329
Sac
—
—
—
—
—
—
—
—
—
—
—
—
2-34
1-25
3-23
108
1-97
110
V. H. FERM, D. P. HANLON AND J. URBAN
ratios of cpm/g for the tissues of individual hamsters. Values which have been
normalized to blood levels of radioactivity are presented in Table 2. A statistical
evaluation of the derived data in Table 2 is presented in Table 3.
Table 3. Statistical evaluation of ratios of imCd distribution in hamster tissues
relative to blood levels of imCd following intravenous injection of
the mother on the eighth day of gestation
Cadmium
Cadmium + zinc
A
Mean
Liver
Uterus
Placenta
Embryo
Liver
Uterus
Placenta
Embryo
S.D.
A
S.E.
Ninth day
±36-7
±0-77
±0-58
±0-31
401
15-20
11 40
1 93
±63-6
±1-72
±1-32
±0 69
430
8-23
309
00328
Twelfth day
±58-3
±25-4
±1-29
±0-53
± 1-31
±0-53
±00144
± 00059
Mean
S.D.
S.E.
401
15-68
13-22
2-35
±206
±5-92
±4-85
±0-64
±92
±2-60
±2-16
±0-29
222
6-92
1-77
00323
±31
±004
±017
± 00078
±22
±003
±012
±00055
DISCUSSION
Teratogenic effects of specific chemical agents in mammalian development
might be attributed to one of three general possibilities, or perhaps a combination of these possibilities. First, an alteration of some factor in the maternal
system which secondarily affects embryonic differentiation. An example of this
possibility as it relates to the teratogenicity of cadmium might be altered protein
structure as reported by Kench & Sutherland (1966) in cases of human cadmium
intoxication. Second, cadmium teratogenicity may be related to blocking placental transfer of some essential material necessary for normal embryonic differentiation. Third, the teratogenic effect of cadmium may be due to a direct effect of
this metal upon specific embryonic tissues.
In the present study the permeability of the hamster placenta to cadmium
injected on the eighth day of embryonic development has been demonstrated.
Interestingly, studies of pregnant mice in late gestation (Berlin & Ullberg, 1963)
showed no evidence of placental transfer of radioactive cadmium from mother
to fetus as measured by radioautographic techniques. Although the present
experiments show cadmium transfer across the hamster placental membrane,
examination of the data in Table 1 indicates considerable variation in the concentration of cadmium in different individuals. This could result from variable
success in injecting radiocadmium, and, of course, be partly due to innate
biological variations. However, the ratio of the distribution of isotopic cadmium
in the different body tissues of each animal shows consistent patterns.
Placentalpermeability to cadmium
111
Even though different animals were used for the ninth- and twelfth-day
experiments, the concentration of cadmium in whole blood is relatively constant
over the 3-day period. It is probable, therefore, that valid comparisons can be
made of cadmium distribution ratios not only between individual hamsters
sacrificed on the same day, but also between ninth-day data and twelfth-day
data based on values normalized for whole blood.
At the ninth day the liver, uterus, placenta and embryonic tissues contain
concentrations of cadmium above that of blood. Obviously much of the injected
cadmium resides in the maternal liver at 9 days. In fact, if it is assumed that the
liver is approximately 6 % of the total body weight, the cadmium content of this
organ accounts for 40 % of the cadmium injected. As is the case with blood,
the liver concentration of cadmium is essentially the same at 9 and 12 days. The
apparent steady state observed for maternal blood and liver does not hold for
uterus, placenta and embryo. At 12 days the cadmium level in these tissues has
decreased respectively to one-half, one fourth and one sixtieth that observed at
9 days. A likely mode of cadmium excretion would be through the gut wall
(Berlin & Ullberg, 1963). The mechanisms responsible for the loss of cadmium
from the uterus and placenta are unknown. However, it is relevant to note that
9-day embryos weigh an average of 5 mg and 12-day embryos weigh an average
of 150 mg. Thus, the 60-fold decrease in the cadmium content between 9-and
12-day embryos could in part be the result of a dilution of the cadmium present
on the ninth day. Some active removal of cadmium would be required to account
for the total decrease in concentration. Simple dilution of cadmium in the
embryonic tissues would require the existence of a cadmium barrier after
the ninth day of gestation. Alternatively, all the decrease in cadmium could
result from active removal via the developing yolk sac in the later stages of
gestation.
While the teratogenic effect of cadmium has been clearly demonstrated, the
actual mechanism of teratogenesis is unknown. It is known, however, that
cadmium exerts its teratogenic effect by the ninth day of gestation (Ferm &
Carpenter, 1968). The means by which zinc protects against teratogenesis cannot
be related to a block in the transfer of cadmium across the placenta since the
present data show that the simultaneous injection of an amount of zinc equimolar to that of cadmium does not affect the amount of cadmium transferred
from maternal to embryonic tissue between the eighth and ninth days, although
the teratogenic effects of cadmium are negated. Since only two individuals given
cadmium and zinc injections were killed on the twelfth day, the significance of
the lower concentration of liver cadmium in these cases could be questioned. It
is possible that the zinc replaces cadmium at certain embryonic sites. Thus,
although cadmium transfer is not impeded, those sites specifically related to the
production of a teratogenic lesion by cadmium could be occupied by zinc, cadmium being bound elsewhere in the embryo at non-specific sites. The complete
obviation of a cadmium effect by equimolar zinc is interesting in that many
112
V. H. FERM, D. P. HANLON AND J. URBAN
biological complexes and chelates of zinc and cadmium are of the same stability
(Bjerrum, Schwarzenbach & Sillen, 1957).
Several interesting possibilities are suggested for further investigation. The
teratogenic effect of a zinc deficiency in rats has been demonstrated by Hurley
(1968), and it may be that those results can be explained by a relative cadmium
excess. The antagonistic effect of cadmium on zinc metalloenzymes such as
carbonic anhydrase (Lindskog & Malmstrom, 1962) and alkaline phosphatase
(Plocke & Vallee, 1962) could provide a lead as to the nature of the teratogenic
sites. But our preliminary studies on the effect of eighth-day cadmium injection
on levels of embryonic alkaline phosphatase in the hamster do not show any
dramatic effect on the activity of this enzyme at subsequent stages of gestation.
Also, the site-specific effect of cadmium in the induction of cleft lips and palates
suggests a localization of cadmium on these receptor sites in the developing
visceral arch system. A detailed study of the distribution of cadmium in the
embryo might show localization of cadmium in this target area. Finally, our
data indicate that, while the placenta is permeable to cadmium injected on the
eighth day, perhaps the placental membranes slow down or block the transfer
of cadmium as these tissues differentiate and become more mature. The development and functional protective activity of the hamster yolk sac placenta during
later stages of gestation suggest a possible site for the apparent block of cadmium
transfer (Carpenter & Ferm, 1969).
SUMMARY
1. When pregnant hamsters are injected intravenously with radioactive cadmium on the eighth day of gestation, significant amounts of this isotope can
be detected in the embryos 24 h later. This interval corresponds to the period
during which cadmium is teratogenic for the hamster embryo.
2. Further, although it has been shown that the simultaneous administration
of zinc will prevent the teratogenic effect of cadmium, in these experiments zinc
does not prevent the placental transfer of radioactive cadmium.
3. The relative decrease in embryonic concentration of radioactive cadmium
on the twelfth day of gestation, 96 h after injection, may be accounted for on the
basis of a dilution effect or an active block in placental transport, perhaps in
the developing yolk sac placenta.
RESUME
La permeabilite du placenta de hamster au cadmium radioactif
1. Apres injection intraveineuse de cadmium radioactif a des hamsters au
huitieme jour de la gestation, on peut deceler des quantites significatives de cet
isotope dans des embryons preleves 24 h apres cette injection.
2. De plus, alors qu'il avait ete demontre que 1'injection simultanee de zinc
Placental permeability to cadmium
113
empeche l'effet teratogenetique du cadmium, dans de telles experiences, le
zinc n'inhibe pas le transfert du cadmium radioactif a travers le placenta.
3. La decroissance relative de la concentration embryonnaire en cadmium
radioactif au 12e jour de la gestation, 96 h apres l'injection, peut etre interpretee
sur les bases d'un effet de dilution ou peut-etre d'un bloquage actif dans
le transport transplacentaire au niveau du placenta ombilical en voie de
developpement.
This research was supported by USPHS grants HD-02616, GM-10210, and GM-15549.
John Urban was supported by a Summer Student Research Fellowship (USPHS grant 5 SOI
FR 05392-06).
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(Manuscript received 9 December 1968)
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