/. Embryol. exp. Morph. Vol. 22, 1, pp. 115-25, August 1969
Printed in Great Britain
Effects of trypan blue
and Niagara blue 2 B on the in vitro absorption
of ions by the rat visceral yolk sac
By MARTEN M. KERNIS 1 and E. MARSHALL JOHNSON 1
Department of Anatomical Sciences, University of Florida
Prior to 1927, when Brunschwig proposed that the rat visceral yolk sac is
functionally a placenta, it was customary to consider the chorio-allantoic
placenta as the primary organ for bringing nourishment to and taking waste
from the developing embryo and growing fetus. Eight years later, Everett (1935)
predicted that the visceral yolk sac is functionally at least as important as the
chorio-allantoic placenta, while Noer & Mossman (1947) suggested that the
yolk sac functions differently from the placenta and is therefore complementary
in its role. More recently, Halliday (1955) indicated that the proximal yolk sac
of the rat is capable of absorbing antibodies by day 17 of gestation. This was
confirmed by Brambell & Halliday (1956), who were able to demonstrate that
the vitelline epithelium and its underlying vascular system are partly responsible
for antibodies penetrating into the embryo. Padykula, Deren & Wilson (1966)
noted that the rat yolk sac has the ability to absorb vitamin B12 and that this
ability is stimulated by the presence of intrinsic factor. The yolk sac has also
been shown to be capable of actively transporting certain amino acids (Deren,
Padykula & Wilson, 1966) and is able to accumulate ferritin by means of
pinocytosis (Lambson, 1966).
In addition to being a physiologically dynamic organ, the yolk sac also
undergoes biochemical changes during gestation. Padykula (1958) and Johnson
& Spinuzzi (1966, 1968) have demonstrated that the activities of a variety of
histochemically and electrophoretically distinguishable enzymes in the yolk sac
change with increasing gestational age. In addition, the latter investigators have
been able to show that the repertory of multiple molecular forms of certain
enzymes is altered in the presence of a potent teratogenic agent.
Since most investigations of mammalian placental function have been limited
to studies in late fetal or term placentae, the fact that the rat visceral yolk sac
also serves a placental function affords the opportunity to study the relationships
and exchange mechanisms operating between the embryo and a placental organ
1
Authors' address: Department of Anatomy, College of Medicine, University of Illinois,
P.O. Box 6998, Chicago, Illinois 60680.
116
M. M. KERNIS AND E. M. JOHNSON
during the critical periods of embryonic morphogenesis. The understanding of
exchange during embryogenesis is not only of considerable value in analysing
normal development, but may also lead to knowledge concerning the mechanisms of teratogen-induced congenital malformation. It follows that the effect
of a teratogen on the ability of the yolk sac to absorb and possibly transfer
material to the embryo is one of the first problems to be studied.
Trypan blue was chosen as the teratogen because it is accumulated in the
vitelline epithelium (Goldmann, 1909) and because its administration to pregnant rats results in a high incidence of congenitally malformed fetuses (Gillman,
Gilbert, Gillman & Spence, 1948). Niagara blue 2B, an azo dye similar in
molecular structure to trypan blue but not effective as a teratogen at the same
dose (Beaudoin & Pickering, 1960), was also used to correlate any drug-induced
alterations in ion absorption with the incidence of congenital malformation.
METHODS
To determine the incidence of congenital malformation caused by trypan blue
and Niagara blue 2 B, proestrous black-hooded Long-Evans rats were mated with
males of the same strain. The day on which spermatozoa were found in the
contents of a vaginal smear was designated as day zero of gestation. On day 8
of gestation, females were given a single, subcutaneous injection of a 1-8 %
aqueous solution of either trypan blue or Niagara blue 2B at a dose of 167 mg/
kg maternal body weight. (Trypan blue was purified and donated through the
courtesy of Mr Floyd Greene of the Matheson, Coleman and Bell Company,
Norwood, Ohio; Niagara blue 2B was obtained in unpurified form from the
Hartman-Leddon Co., Philadelphia, Pennsylvania.) Control pregnant animals
were left untreated. At day 20 (1 day before parturition), the females were killed
by cervical dislocation. The fetuses were removed, examined for gross malformations and fixed in Bouin's fluid for later free-hand sectioning (Wilson, 1965)
to detect any gross internal malformations.
To measure the absorption of radioactively labeled ions, control and azo
dye-treated rats were killed by cervical dislocation on either day 12, 13 or 14 of
gestation. Each uterus was removed in toto and placed in a Petri dish containing
warmed (38 °C) culture medium consisting of bovine serum, chicken-embryo
extract ultrafiltrate and phosphate-Ringer buffer in a ratio of 3:1:1 (Netzloff,
Chepenik, Johnson & Kaplan, 1968). One implantation site at a time was
separated from the remaining intact uterus and transferred to another Petri
dish which contained warmed culture medium. The uterine muscle was opened
along the antimesometrial border and the decidua capsularis and parietal yolk
sac were dissected free of the chorio-allantoic placenta and visceral yolk sac.
A silk ligature was tied around the umbilical vessels at the hilus of the chorioallantoic placenta. The placenta and uterus were separated from the ligated
visceral yolk sac which remained as a complete and vascularized membrane
In vitro absorption of ions
117
surrounding the embryo (see Netzloff, Johnson & Kaplan, 1968, for photographs of the preparation).
The visceral yolk sac, vitelline vessels and embryo were examined to be certain
that (1) the yolk sac was not punctured, (2) none of the vitelline vessels were
ruptured, and (3) the embryo had a beating heart which perfused the vitelline
vessels with blood. If the preparation did not meet this last criterion at the end
of its incubation period it was discarded. No more than six embryos were used
from any one pregnancy.
The preparations, handled by the loose ends of the ligature, were transferred
to disposable 30 ml beakers containing warmed culture medium to which either
45
CaCl2, Na235SO4 or 22NaCl had been added. The final activities of ions were
0-210 //c/ml for calcium, 0-072/tc/ml for sulfate and 0-019 /tc/ml for sodium.
After 60 min of incubation at 38 °C the preparations were removed from the
medium, examined for the presence of a beating heart and rinsed several times
in 0-9 % saline. The visceral yolk sacs were separated from the embryos and
both were rinsed 5 times in saline. The tissues were transferred to individual test
tubes containing 0-25 ml of concentrated nitric acid and dissolved over a low
flame. Each resulting solution was placed on a tared, stainless steel, ringed
planchet, dried for 24 h at 70 °C and for another 24 h at 120 °C. The planchets
were weighed and then counted for radioactivity with a thin-window, halogenquenched, Geiger-Muller tube. Corrections were made for the decay rates of
the isotopes and the results were expressed in terms of cpm/preparation and
cpm/mg dry weight. A Student's f-test was employed to detect differences
between experimental and control means, and a P value of 0-05 or less was
considered significant.
RESULTS
The administration of a single, subcutaneous injection of trypan blue at a
dosage of 167 mg/kg maternal body weight on day 8 of gestation in the rat
resulted in a 62 % incidence of living, malformed fetuses (Table 1). Although
the same treatment with Niagara blue 2B induced only a 2 % malformation
rate, this was still significantly greater than the spontaneous incidence in normal
control animals. The most common malformations induced by treatment with
either trypan blue or Niagara blue 2B were of the central nervous system and
the eye and included such defects as anencephaly, exencephaly, meningocele,
meningomyelocele, anophthalmia and microphthalmia. In many cases, the
young bore multiple congenital abnormalities of varying severity.
The effects of both azo dyes on the general growth of yolk sacs and embryos
are summarized in Table 2. The presence of trypan blue resulted in a significant
decrease both in yolk sac weight on days 12 and 13 and in embryonic weight on
day 13. Niagara blue 2B treatment did not change the dry weight of the tissues
except on day 14, when the embryos were significantly heavier than the corresponding controls. By day 14, treated yolk sacs had weights similar to controls.
118
M. M. KERNIS AND E. M. JOHNSON
Where the presence of either of the azo dyes caused significant alterations in
the absorption (cpm/preparation) or specific activity (cpm/mg dry weight) of
any of the ions studied, the tissues from dye-treated mothers tended to accumulate more radioactivity than their corresponding controls. The absorption and
specific activities of 45Ca2+, 35SO42~, and 22Na+ are presented in Tables 3-5.
Although the absorption of ions in terms of cpm/preparation seemed to
Table 1. Incidence of malformation^
Control!
Trypan
blue§
Niagara
blue 2B||
—
12
136
167
22
240
167
12
144
134(99%)
2 (1 %)
130(54%)
110(46%)
134(93%)
Dose (mg/kg)
No. of females
No. of sites
No. live
No. dead
Survivors
Normal
Abnormal
134(100%)
0
49(38%)
81 (62%)
10(7%)
131(98%)
3(2%)
t Animals were sacrificed on day 20 of gestation.
% Maternal animals left untreated.
§ A single subcutaneous injection of a 1-8 % aqueous solution of the dye administered on
day 8 of gestation.
II Ibid.
Table 2. Dry weight of yolk sacs and embryos^
Treatment
groupsj
Yolk sac
Control
Trypan blue
Niagara blue 2B
Embryo
Control
Trypan blue
Niagara blue 2B
Day 12
Day 13
1-21 ±008
(62/11)§
0-94 ±005*
(59/12)
108 + 009
(40/7)
1-81 ±006
(67/12)
1-55 + 005*
(54/11)
1 66 ±006
3 04 + 009
(61/H)
5-96 ± 0 1 7
(65/12)
5-32±018*
(52/11)
5-73±O-13**
(36/6)
2-89±0-10
(58/12)
314±010
(40/7)
(35/6)
Day 14
2-91 ± 0 0 9
(61/11)
2-73 + 008
(41/11)
2-92 + 0-08
(34/6)
10-99 ±0-38
(61/11)
10-91 ±0-38
(40/11)
12-50±0-31*
(34/6)
* Significant difference (P < 005) between experimental and control tissues.
** Significant difference (P < 0-05) between tissues treated with trypan blue and Niagara
blue 2B.
t Dry weight in milligrams, mean ± standard error.
% See legend in Table 1 for details of treatment.
§ (Number of preparations/number of mothers.)
In vitro absorption of ions
119
increase as a function of gestational age and tissue dry weight, the control
specific activities of the ions did not seem to follow a general pattern. In
addition, the significant increases of specific activities of the dye-treated tissues
were not limited to any particular gestational age. Trypan blue caused an
increase in 45Ca2+ specific activity in yolk sacs at day 13 only but no statistically
significant differences in the embryos at any day of gestation (Table 3). Sulfate
Table 3. Uptake ofi5Ca2+-\
(cpm
Absorption
Ca2+/preparation)
45
Specific activity
(cpm 45Ca2+/mg dry weight)
A
Day 12
Yolk sac
Embryo
Day 13
Yolk sac
Embryo
Day 14
Yolk sac
Embryo
Control
Trypan blue
Control
Trypan blue
121 ±6
(16/3)
23±4
(16/3)
120 ±9
(14/3)
10 + 6
(13/3)
136±6
(16/3)
7±1
(16/3)
144 ±9
(14/3)
3±2
(13/3)
200+10
(18/3)
59±4
(16/3)
223 ±12
(15/3)
62± 11
(14/3)
87±4
(18/3)
9±1
(16/3)
126±5*
(15/3)
410 + 43
(20/4)
150 + 29
(19/4)
352 ±39
(10/3)
132 + 34
(10/3)
174 ±12
(20/4)
16±3
(19/4)
132± 13*
(10/3)
14 + 3
(10/3)
11 ±2
(14/3)
* Significant difference (P < 005) between experimental and control tissues.
f See text and Table 1 for details of treatment. Data expressed as mean ± standard error.
Figures in parentheses indicate: (number of preparations/number of mothers).
specific activity was greater in yolk sacs treated with trypan blue on all days
studied, while yolk sacs treated with Niagara blue 2B seemed to be essentially
normal by day 13 (Table 4). No significant differences in sulfate uptake between
control and treated embryos were apparent. Yolk sacs after either dye treatment
had greater 22 Na + specific activities than controls at days 12 and 14, while day 13
embryos after Niagara blue 2B treatment accumulated more ion than controls
(Table 5).
DISCUSSION
The incidence of congenital malformation induced by either trypan blue or
Niagara blue2Bfound in this study agrees with those reported by others (Wilson,
1955; Wilson, Beaudoin & Free, 1959; Beaudoin & Pickering, 1960; Beaudoin,
1962; Beaudoin & Kahkonen, 1963; Lloyd & Beck, 1966). The small variations
between the present and previous results are probably related to several factors.
120
M. M. KERNIS AND E. M. JOHNSON
These are: (1) the strain of rats studied; (2) the time, dose and route of administration of the dye; (3) the purity of the dye. Judged by the malformation rate, it
follows that at least 3 out of 5 of the preparations treated with trypan blue
incubated in vitro for the ion uptake studies were destined to have at least one
abnormality, while about 1 out of 50 embryos treated with Niagara blue 2B
would be malformed.
Table 4. Uptake of*6SOA2-\
Absorption
(cpm 35SO|~/preparation)
Niagara
blue 2B
Control
53±4*
21 ±2
(24/4)
22±3
(24/4)
152± 11
(25/5)
137± 12
(25/5)
194±12* 187 ±22
(20/4)
(17/3)
110±13** 176 ±25
(20/4)
(18/3)
354 ±29
(20/4)
356 ±51
(21/4)
411 ±28
(13/4)
352 ±61
(12/4)
Control
Day 12
Yolk sac
Embryo
Day 13
Yolk sac
Embryo
Day 14
Yolk sac
Embryo
Specific activity
(cpm : i5SO!-/mg dry >weight)
38 + 3
(24/4)
60 ±6
(24/4)
Trypan
blue
43 ±5
(19/4)
48±3**
(19/4)
(17/3)
77 ±10
(17/3)
347 ± 56
(18/3)
326 ±49
(18/3)
Trypan
blue
Niagara
blue 2B
(19/4)
18 + 2
(19/4)
33±4*
34±3*
(17/3)
24±4
(17/3)
112 + 6
(25/5)
29 ±3
(25/5)
163±18*
(20/4)
24±3
(20/4)
118 + 13
(17/3)
33 ±5
(18/3)
116±9
(20/4)
35 ±5
(21/4)
166± 13*
(13/4)
36±6
(12/4)
118± 16**
(18/3)
27 ±4
(18/3)
* Significant difference (P < 005) between experimental and control tissues.
** Significant difference (P < 005) between tissues treated with trypan blue and Niagara
blue 2B.
f See text and Table 1 for details of treatment. Data expressed as mean ± standard error.
Figures in parentheses indicate: (number of preparations/number of mothers).
Since Gillman et ah (1948) first demonstrated the teratogenicity of trypan blue
in the rat, its site and mechanism of action have been enigmas. It is still unclear
whether the dye causes malformations by affecting some maternal physiological
process necessary to normal embryonic development, by altering the function
of the placenta or yolk sac, or by directly affecting the embryo. Because trypan
blue had never been seen to penetrate the rat embryo and because it is a potent
teratogen in chicks (suggesting that no maternal influence is involved), Beck,
Lloyd & Griffiths (1967) concluded that the most reasonable site of action for
trypan blue in the rat is the visceral yolk sac. Indeed, under in vitro circumstances
they have demonstrated that increasing concentrations of trypan blue are able
to inhibit the activities of certain enzymes isolated from the lysosomes of visceral
yolk sacs near term. They therefore suggested that the inhibition of those lysosomal enzymes results in the inability on the part of the visceral endoderm to
In vitro absorption of ions
121
digest absorbed material. Any barrier to these large undigested molecules
would result in a lack of transfer of nutritional elements to the embryo. Although
this hypothesis is quite attractive, judgement should be reserved until it is substantially shown that the enzyme inhibition occurs in vivo and also that such
inhibition occurs in yolk sacs from earlier stages in gestation, particularly at
that critical time in development when trypan blue is most effective in producing
malformations.
Table 5. Uptake o
(cpm
Embryo
Day 13
Yolk sac
Embryo
Day 14
Yolk sac
Embryo
(cpm
Specific activity
Na+/mg dry weight)
22
Trypan
blue
Niagara
blue 2B
Control
Trypan
blue
Niagara
blue 2B
(21/4)
71 ±5
(26/5)
95 + 7
(26/5)
73 + 5*
(23/4)
122±13*
(23/4)
81 ±5
(22/4)
29 ±3
(21/4)
105 ±7*
(26/5)
35 ±3
(26/5)
116 + 8*
(23/4)
43 + 5
(23/4)
171 ±9
(24/4)
153 ±13
(24/4)
162+10
(19/4)
199±43
(18/4)
167 ±9
(18/3)
189 + 21
(18/3)
95 ±6
(24/4)
22 + 2
(24/4)
105 ±8
(19/4)
30 + 5
(18/4)
102±6
(18/3)
33±4*
(18/3)
462 + 33
(18/3)
785 + 141
(18/3)
633 + 55* 570 ±47
(16/3)
(18/4)
703 ±48** 1040+145
(18/4)
(16/3)
144 ±10
(18/3)
58 ±10
219±18*
(18/4)
55 + 4
(18/4)
186 ±14*
(16/3)
80± 11
(16/3)
Control
Day 12
Yolk sac
Absorption
Na+/preparation)
22
58±4
(22/4)
85 ±9
08/3)
* Significant difference (P < 005) between experimental and control tissues.
** Significant difference (P < 005) between tissues treated with trypan blue and Niagara
blue 2B.
t See text and Table 1 for details of treatment. Data expressed as mean ± standard error.
Figures in parentheses indicate: (number of preparations/number of mothers).
The yolk sac, however, may not be the only site of trypan blue action. In
contradiction to many previous reports, Davis & Gunberg (1968) have been
able to visualize trypan blue in both stained and unstained 11-, 12- and 13-day
rat embryos with both low- and high-power light microscopy. Apparently, the
dye accumulated in the epithelium of the embryonic gut and possibly in some
of the surrounding mesenchymal cells. This finding may indicate that the dye
has a direct effect on embryonic tissue. The amount of dye in the embryo,
however, appears to be quite small and its distribution is somewhat remote
from the sites of malformation. Thus, the ability of the yolk sac to absorb ions
and the effect of trypan blue on this function is still of considerable import.
These results show that, with the exception of the 45Ca2+ activities of day 14
yolk sacs, wherever there was a significant difference between trypan blue-
122
M. M. KERNIS AND E. M. JOHNSON
treated and control yolk sacs or embryos, the specific activity of the dye-treated
tissue was always greater. Since day 12 and 13 trypan blue-treated yolk sacs
weighed significantly less than the corresponding controls, the increased specific
activities indicate that either a smaller amount of protein or a fewer number of
cells (or both) was capable of absorbing the same or greater amounts of ion.
Although Niagara blue 2B treatment did not result in reduced tissue weights,
where statistical differences did exist, the dye-treated tissues also had greater
specific activities than controls. This phenomenon would tend to suggest that
the machinery used by the yolk sac to absorb ions is altered by some interaction
with Niagara blue 2B. This interaction is as yet unidentified.
Although the absorption of ions by embryos generally paralleled the uptake
by yolk sacs as gestation proceeded, there appeared to be no consistency in the
relative amounts of ions taken up when the yolk sacs were compared to similarly treated embryos at the same day of development. For example, the presence
of trypan blue in the yolk sac at day 13 increased the 35SOf~ uptake, but the
increase was not reflected in the embryo (Table 4). However, the presence of
Niagara blue 2B on day 13 had no effect on the yolk sac's ability to absorb
sulfate, while it may have caused an increase in the amount of ion passing into
the embryo. With regard to 22 Na + (Table 5), no differences were seen in day 13
yolk sacs, while treatment with Niagara blue 2B caused an increased amount of
label to penetrate into the embryo. Since the presence of trypan blue in the yolk
sac was never correlated with a concomitant significant increase of ions in the
embryo, there is the possibility that the dye may prohibit the passage of these
particular ions at these particular stages of development. Whether the same
holds true for other stages of development and for other ions or organic molecules has not as yet been determined.
The relationship between treated and control tissues may also change from
day to day. For example, trypan blue-treated yolk sacs had significantly greater
35
SOfr specific activities than controls at each day of gestation (Table 4). The
day 12 Niagara blue 2B-treated yolk sacs also absorbed more sulfate than
controls. The change occurs at day 13, when there was no difference between
Niagara blue 2B and control yolk sacs, thus indicating a possible recovery.
Conceivably, recovery from Niagara blue 2 B treatment could occur more rapidly
than from treatment with trypan blue, for the former appears to be excreted
from the maternal tissues and proximal yolk sac at a greater rate than the latter
(Lloyd & Beck, 1966).
Recovery from a teratogenic insult is not a new concept. Johnson (1965) and
Johnson & Spinuzzi (1966, 1968) have described recovery of enzyme forms after
an initial alteration caused by a folic acid deficiency. Two of the enzymes discussed were glucose-6-phosphate dehydrogenase and alkaline phosphatase,
both of which having been previously implicated in transport mechanisms (Moog
& Wenger, 1952; Karnovsky, 1962). In addition, Netzloff et al (1968) have
shown that folic acid deficient, abnormally developing embryos consume
In vitro absorption of ions
123
oxygen at a greater rate than controls. Also, recovery, or normal oxygen uptake,
was found 24-48 h after the pregnant female ceased eating the diet deficient in
folic acid and began ingesting the vitamin-supplemented ration.
The fact that there were changes in the specific activities or amounts of different ions absorbed by control and experimental tissues on varying days of
gestation is not surprising. Embryonic and yolk sac tissues were undergoing a
rapid and extensive biochemical and morphological differentiation during this
period of gestation. As a result, it is likely that as the tissues differentiated, their
ionic and nutritional requirements were altered with their particular needs at
distinct stages of development. More important, however, is the finding that
the presence of azo dyes could affect the ability of mammalian yolk sacs to
absorb and possibly transfer certain ions. Indeed, Grabowski (1963) has already
shown that the presence of trypan blue in the yolk of developing chickens caused
significant imbalances in embryonic serum electrolytes.
That Niagara blue 2B resulted in changes in the absorption of ions similar to
those produced by trypan blue would indicate at least two possibilities. First,
differences in ionic uptake as seen in these experiments were not causally related
to mechanisms of malformation, as Niagara blue 2B at the dosage employed
is only about 3 % as potent a teratogen as trypan blue. Secondly, since both
agents are most effective as teratogens when administered at day 8 of gestation,
changes in ionic uptake as late as day 12 may simply reflect the presence of the
dyes within the yolk sac, and indeed may have no relationship to causes of
malformation. In order to be able to study the effects of a teratogen on yolk sac
function, another agent which is capable of causing a high percentage of malformations when administered at a later stage in gestation must be utilized. In
this manner, alterations in yolk sac function may be monitored before, during
and after the teratogenic insult.
Thus, the complex relationship between the embryo and its yolk sac should
be studied further. Experiments utilizing the in vitro embryonic system described above might indeed show that the yolk sac plays an important role in
normal embryonic differentiation and that alterations in yolk sac function and
the resulting changes in the embryonic microenvironment could be involved as
a mechanism of teratogenesis.
SUMMARY
1. When injected into pregnant rats on day 8 of gestation, trypan blue is a
potent teratogen. Niagara blue 2B at the same dose is considerably less effective
as a teratogen, but still results in a rate of malformation which is significantly
greater than the spontaneous incidence of malformation for the Long-Evans
black-hooded strain of rats. Both dyes result in the same syndrome of malformations, affecting the central nervous system and special sense organs primarily.
2. On days 12, 13 and 14 of gestation, both trypan blue and Niagara blue
2 B cause a significant increase in the absorption of 45Ca2+, 35SO|~ and 22 Na +
124
M. M. KERNIS AND E. M. JOHNSON
by yolk sacs and, in some cases, developing embryos. These changes in the
specific activities of yolk sacs suggest that both dyes have an effect on normal
yolk sac function and indicate that alterations in the function of the yolk sac
may bear a direct relationship to the induction of congenital abnormalities.
Future studies, however, must utilize a teratogen which is effective at later days
of gestation, so that transfer phenomena may be studied before, during and
after the teratogenic insult.
RESUME
Ejfets du bleu try pan et du bleu Niagara 2B
sur Vabsorption d'ions in vitro par le sac vitellin visceral du rat
1. Le bleu trypan est un agent teratogene puissant quand on Finjecte a des
rattes gestantes le 8e jour de la gestation. A la meme dose, le bleu Niagara 2B
est beaucoup moins efficace comme teratogene, mais provoque encore des
malformations dont le taux est plus eleve, de maniere significative, que l'apparition de malformations spontanees dans la lignee de rats Long-Evans, a 'capuchon noir'. Les deux colorants provoquent le meme syndrome de malformations,
affectant en premier lieu le systeme nerveux central et certains organes des sens.
2. Les 12e, 13e et 14e jours de la gestation, le bleu trypan et le bleu Niagara
2B provoquent tous deux un accroissement significatif de l'absorption de
45
Ca2+, 35SOfr, et 22Na+ par les sacs vitellins et, dans certains cas, les embryons
en cours de developpement. Ces modifications dans les activites specifiques des
sacs vitellins suggerent que les deux colorants ont un effet sur les fonctions normales du sac vitellin et indiquent que des alterations de ces fonctions peuvent
se trouver en relation directe avec l'induction d'anomalies congenitales. Neanmoins, des recherches ulterieures devront utiliser un agent teratogene qui soit
efficace a une periode plus tardive de la gestation, de sorte que les phenomenes
de transport puissent etre etudies avant, pendant et apres l'agression teratogene.
The authors wish to express their gratitude to Dr Stanley Kaplan for reviewing this
manuscript. Acknowledgment for technical assistance is given to Mrs Barbara McGuire,
Mrs Dorothy Ramsey, Mrs Minnie Smith and Miss Stella MacAshan.
This study is a portion of a dissertation presented to the Graduate School of the University
of Florida as partial fulfilment of the requirements for the degree of Doctor of Philosophy.
The investigation was supported by Public Health Service Training Grant 3T1 GM 579
and Research Grant HD 00109.
REFERENCES
A. R. (1962). Interference of Niagara blue 2B with the teratogenic action of
trypan blue. Proc. Soc. exp. Biol. Med. 109, 709-11.
BEAUDOIN, A. R. & KAHKONEN, D. (1963). The effect of trypan blue on the serum proteins
of the fetal rat. Anat. Rec. 147, 387-95.
BEAUDOIN, A. R. & PICKERING, M. J. (1960). Teratogenic activity of several synthetic compounds structurally related to trypan blue. Anat. Rec. 137, 297-305.
BECK, F., LLOYD, J. B. & GRIFFITHS, A. (1967). Lysosomal enzyme inhibition by trypan blue:
A theory of teratogenesis. Science, N.Y. 157, 1180-2.
BEAUDOIN,
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125
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(Manuscript received 30 July 1968, revised 10 December 1968)