EPA/600/J-93/470
November 1993
Critical Reviews in Toxicology, 23(3):283-335 (1993)
Developmental and Reproductive Toxicity of
Dioxins and Related Compounds: Cross-
Species Comparisons
Richard E. Peterson,3-"' H. Michael Theobald,3 and Gary L. Kimmelc
•School of Pharmacy and "Environmental Toxicology Center, University of Wisconsin, Madison,
Wl; and "U.S. Environmental Protection Agency, Washington, D.C.
•Address all correspondence to: Dr. Richard E. Peterson, School of Pharmacy, University of Wisconsin, 425 N. Charter Street,
Madison, WI 53706
ABSTRACT: Developmental toxicity to TCDD-like congeners in fish, birds, and mammals, arid reproductive
toxicity in mammals are reviewed. In fish and bird species, the developmental lesions observed are species
dependent, but any given species responds similarly to different TCDD-like congeners. DeveloDmentaLtoxicito
in fish resembles "blue sac disease," whereas structural malformations can occur in atJtdS-i5rr?TJircLslKcies.
In mammals, developmental toxicity includes decreased growth, structural malformations, functional artSEflSanNi
and prenatal mortality. At relatively low exposure levels, structural malformations are noicnrnrmiD.irwmawfljgiiAB
species. In contrast, functional alterations are the most sensitive signs of developmental TOxicity. These include
effects on the male reproductive system and male reproductive behavior in rats, and neurobehavioral effects in
monkeys. Human infants exposed during the Yusho and Yu-Cheng episodes, and monkeys and mice exposed
perinatally to TCDD developed an ectodermal dysplasia syndrome that includes toxicity to the skin and teeth.
Toxicity to the central nervous system in monkey and human infants is a potential part of the ectodermal dysplasia
syndrome. Decreases in spermatogenesis and the ability to conceive and carry a pregnancy to term are the most
sensitive signs of reproductive toxicity in male and female mammals, respectively.
KEY WORDS: perinatal exposure, structural malformations, functional alterations, antiestrogenicity, sexual
differentiation, Ah receptor.
I. INTRODUCTION
2,3,7,8 - Tetrachlorodibenzo - p - dioxin
(TCDD) is one of 75 possible chlorinated di-
benzo-/?-dioxin (CDD) congeners. It is one of the
most potent of the CDDs..hrominated_dihenzo-
p-dioxins (BDDs), chldriii&tdC feHtKKEAurans
(CDFs), brominated diOenz'otvirans'ClDFs),
polychlorinated biphenyte" ^PCffil/ThBdiDoly-
brominated biphenyls (PBBS^/ Ailuil.WCDD
serves as the prototype congener for investigating
the toxicity elicited by these classes of chemicals.
Developmental and reproductive toxicity is gen-
erally believed to be caused by the parent com-
pound. There is no evidence that TCDD metab-
olites are involved. The toxic potency of TCDD
is due to the number and position of chlorine
substitutions on the dibenzo-p-dioxin molecule.
CDD congeners with decreased lateral (2,3,7,
and 8) or increased nonlateral chlorine and bro-
mine substituents are less potent than TCDD;
however, most of these congeners will produce
toxicity, and the pattern of responses within an-
imals of the same species, strain, sex, and age
Printed on Recycled Paper
283
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will generally be similar to that of TCDD.2 3 PCB
congeners with zero or one ortho chlorine, two
para chlorines, and at least two meta chlorines
can assume a coplanar conformation sterically
similar to TCDD and also produce a pattern of
toxic responses similar to that of TCDD. In con-
trast, PCB congeners with two or more ortho
chlorines cannot assume a coplanar conformation
and do not resemble TCDD in toxicity.1'3
CDD and CDF congeners chlorinated in the
lateral positions, as compared with those lacking
chlorines in the 2,3,7, and 8 positions, are pref-
erentially bioaccumulated by fish, reptiles, birds,
and mammals.4"6 Furthermore, coplanar PCBs
and/or monoorf/io-chlorine-substituted analogs
of the coplanar PCBs bioaccumulate in fish,
wildlife, and humans.7"11 This is of concern be-
cause the combined effects of the lateral-substi-
tuted CDD, BDD, CDF, BDF, PCB, and PBB
congeners that act through an aryl hydrocarbon
(Ah) receptor mechanism have the potential to
decrease feral fish and wildlife populations sec-
ondary to developmental and reproductive tox-
jcjtv 5,12-14 Humans are not exempt from the de-
velopmental and reproductive effects of complex
halogenated aromatic hydrocarbon mixtures. Such
mixtures that contain both TCDD-like congeners
and nonTCDD-like congeners have been impli-
cated in causing the developmental and repro-
ductive toxicity in the Yusho and Yu-Cheng poi-
soning incidents in Japan and Taiwan.15"17 Thus,
exposure to TCDD-like congeners is a health con-
cern for humans, domesticated animals, fish, and
wildlife, although the relative contributions of
TCDD- and nonTCDD-like congeners are not
known in some exposure situations.
A mechanism of action that CDD, BDD,
CDF, BDF, PCB, and PBB congeners substituted
in the lateral positions have in common is that
they bind to the Ah receptor, which then binds
to a translocating protein that carries the activated
TCDD receptor complex into the nucleus. These
activated TCDD receptor complexes bind to spe-
cific sequences of DNA referred to as dioxin-
response elements (DREs), resulting in altera-
tions in gene transcription. There is evidence that
this Ah receptor mechanism is involved in the
antiestrogenic action of TCDD and in its ability
to produce the structural malformations cleft pal-
ate and hydronephrosis in mice. However, its role
in producing other signs of developmental and
reproductive toxicity is less firmly established or
not established. This leaves open the possibility
for some of these TCDD effects to not be Ah
receptor mediated.
The information in this review has been or-
ganized into sections on developmental toxicity,
reproductive toxicity in males, and reproductive
toxicity in females. However, it is important to
emphasize that developmental and reproductive
toxicity, particularly in females, can be interre-
lated. Therefore, the reader should view the sec-
tion subheadings as topic indexes that indicate
where most of the information on a particular
endpoint is concentrated. The endpoints de-
scribed within each subheading are not intended
to be thought of as being independent of end-
points described in other sections. For example,
the effects of TCDD on the actions of reproduc-
tive hormones, peptides, and steroids can be in-
volved in reproductive dysfunction as well as
developmental toxicity.
II. DEVELOPMENTAL TOXICITY
The manifestations of developmental toxicity
to TCDD have been divided into three categories
for convenience in assessing the data base with
respect to an Ah receptor-mediated response.
These categories include death/growth/clinical
signs, structural malformations, and functional
alterations. Exposure-related effects on death/
growth/clinical signs are described for fish, birds,
laboratory mammals, and humans along with
structure activity results that are consistent with,
but do not prove, an Ah receptor-mediated mech-
anism. Structural malformations, particularly cleft
palate formation and hydronephrosis, occur in
mice. However, in other mammalian species,
postnatal functional alterations, some of which
may be irreversible, are the most sensitive ad-
verse developmental effects of TCDD-like con-
geners. These include effects on the male repro-
ductive system of rats and object-learning
behavior in monkeys.
284
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DATE DUE
A. Death/Growth/Clinical Signs
1. Fish
Early life stages of fish appear to be more
sensitive to TCDD-induced mortality than adults.
This is suggested by the LD50 of TCDD in rain-
bow trout sac fry (0.4 |Ag/kg egg weight) being
25 times less than that in juvenile rainbow trout
(10 |xg/kg body weight).13'18 The significance of
this finding is that early life stage mortality caused
by high concentrations of TCDD-Iike congeners
in fish eggs may pose the greatest risk to feral
fish populations.5-13 Cooper19 reviewed the de-
velopmental toxicity of CDDs and CDFs in fish
and Cook et al.5 discussed components of an
aquatic ecological risk assessment for TCDD in
fish. The reader is referred to this literature for
more in-depth coverage than is presented here.
TCDD is directly toxic to early life stages of
fish. This has been demonstrated for Japanese
medaka, pike, rainbow trout, and lake trout ex-
posed as fertilized eggs to graded concentrations
of waterborne TCDD. In these species, TCDD
causes an overt toxicity syndrome characterized
by edema, hemorrhages, and arrested growth and
development culminating in death. 13>14'20~23 His-
topathological evaluation of lake trout embryos
and sac fry has shown this syndrome to be es-
sentially identical to that of blue sac disease.21'23
Following egg exposure to TCDD, signs of tox-
icity are not detected in medaka until after the
liver rudiment forms,22 and in lake trout, toxicity
is first detected approximately 1 week prior to
hatching, but becomes fully manifest during the
sac fry stage.14-23 Among all fish species inves-
tigated thus far, lake trout are the most sensitive
to TCDD developmental toxicity. Following ex-
posure of fertilized lake trout eggs to graded wa-
terborne concentrations of TCDD, the NOAEL
for sac fry mortality is 34 pg TCDD/g egg, the
LOAEL is 55 pg TCDD/g egg, and the egg TCDD
concentration that causes 50% mortality above
control at swim up (LD50) is 65 pg TCDD/g egg.14
Thus, TCDD is a potent developmental toxicant
in fish and the effect is not secondary to maternal
toxicity.
The Ah receptor has not been identified in
early life stages of fish; however, it is assumed
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Bird embryos also are more sensitive to
TCDD toxicity than adults. The LD50 of TCDD
in the chicken embryo (0.25 (xg/kg egg weight)
is 100 to 200 times less than the TCDD dose that
causes mortality in adult chickens (25 to 50 jig/
kg body weight).29-30 The LD50 of TCDD injected
into fertilized ring-necked pheasant eggs (1.1 to
1.8 (xg/kg egg weight) is 14 to 23 times less than
the TCDD dose that causes 75% mortality in ring-
necked hen pheasants (25 (J-g/kg body weight).31
Among bird species, most of the develop-
mental toxicity research has been done on chick-
ens. Injection of TCDD or its approximate iso-
stereomers into fertilized chicken eggs causes a
toxicity syndrome in the embryo characterized
by pericardial and subcutaneous edema, liver le-
sions, inhibition of lymphoid development in the
thymus and bursa of Fabricius, microophthalmia,
beak deformities, cardiovascular malformations,
and mortality.32"40 On the other hand, injection
of a coplanar PCB into fertilized turkey eggs at
a dose high enough to cause microophthalmia,
beak deformities, and embryo mortality did not
285
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produce liver lesions, edema, or thymic hypo-
plasia, all hallmark signs of TCDD toxicity in
the chicken embryo.36 This disparity in signs of
TCDD embryotoxicity among bird species is not
unique to the turkey and chicken. In fertilized
eggs of ring-necked pheasants and eastern blue-
birds, injection of TCDD produces embryo mor-
tality, but all of the other signs of toxicity seen
in the chicken embryo are absent, including car-
diovascular malformations3MM2 Thus, in bird
embryos, the signs of toxicity elicited by TCDD
and its approximate isostereomers are highly spe-
cies dependent; the only toxic effect common to
all bird species is embryo mortality.
There is evidence in chicken embryos that
the Ah receptor may be involved in producing
developmental toxicity. The Ah receptor has been
detected in chicken embryos36'43 and the rank or-
der potency of PCB congeners for producing
chicken embryo mortality is 3,3',4,4',5-PCB >
3,3',4,4'-TCB > 3,3',4,4',5,5'-HCB >
2,3,3',4,4'-PCB > 2,3,4,4',5-PCB, with
2,2',4,5'-TCB, 2,2',4,4',5,5'-HCB and
2,2',3,3',6,6'-HCB being inactive, and this rank
order is similar to that for a classic Ah receptor-
mediated response in the chicken embryo, i.e.,
cytochrome P-4501A1 induction.35-37-44 Inas-
much as the nonsteroidal anti-inflammatory drug
benoxoprofen suppresses 3,3',4,4'-TCB-in-
duced toxicity in the chicken embryo without
altering its ability to induce microsomal enzyme
activity,45 induction of cytochrome P-4501A1 and
toxicity are part of a pleiotropic response linked
to the Ah receptor, but these effects are not other-
wise causally related. Also, for 3,3',4,4'-TCB,
3,3',4,4',5,5'-HCB, and TCDD, there is a
marked dissociation of the dose-response rela-
tionship for lethality and enzyme induction in the
chicken embryo.35
A decrease in activity of uroporphyrinogen
decarboxylase (URO-D) and an increase in ac-
cumulation of uroporphyrins are effects that are
readily produced by exposure of cultured chicken
embryo liver cells to TCDD, 3,3' ,4,4'-TCB, and
other PCBs.46-^8 Coplanar PCB congeners are
more potent inhibitors of URO-D activity in cul-
tured chicken embryo liver cells than are non-
coplanar PCB congeners,49 suggesting an Ah re-
ceptor-mediated mechanism. Unlike the results
in cultured cells, however, a lethal dose of TCDD
(6 nmol/egg) does not affect URO-D activity or
cause any increase in accumulation of uropor-
phyrins in chicken embryos.35 Thus, TCDD-in-
duced lethality in chicken embryos is not asso-
ciated with effects of TCDD on URO-D activity,
even though a decrease in URO-D activity might
be expected to occur if a sufficient dose of TCDD
could be reached without being lethal.
The chicken embryo heart is a target organ
for TCDD and other halogenated aromatic hydro-
carbons that act via an Ah receptor mechanism.
The classic sign of chick embryo toxicity in-
volving the heart is pericardial edema. However,
TCDD has other effects on the chick embryo
heart that are less well known. These include its
ability to produce cardiovascular malformations
and to increase cardiac release of arachidonic acid
metabolites. When fertilized chicken eggs are in-
jected with graded doses of TCDD, cardiovas-
cular malformations are produced, including ven-
tricular septal defects, aortic arch anomalies, and
conotruncal malformations. Approximately 1.6
pmol TCDD/egg (9 ng/kg egg, assuming a 55-g
egg weight) causes cardiovascular malformations
in 46% of treated embryos vs. 29% of control
embryos.3233 The cardiovascular malformation
response may be unique to the chicken embryo
because in fertilized ring-necked pheasant and
eastern bluebird eggs injected with TCDD the
incidence of such malformations is not in-
creased.31-41-42
In the chicken embryo heart, arachidonic acid
metabolism is stimulated by TCDD, resulting in
increased formation of prostaglandins.50 Dose-
response relationships for the release of immu-
noreactive PGE2, PGF2a, and TXB2 from chick
embryonic heart are biphasic, with an apparent
maximally effective dose of 100 pmol TCDD/
egg. When the egg TCDD dose is increased fur-
ther, release of these prostaglandins tends to de-
cline toward levels in control hearts. Biphasic
dose-response curves for cardiac PGE2 release
also were obtained with 3,3',4,4'-TCB and
3,3',4,4',5,5'-HCB.50 The thymus and bursa of
Fabricius are other TCDD target organs in the
chicken embryo. TCDD, 3,3',4,4'-TCB, and
3,3',4,4'-TCAOB cause dose-related decreases
in lymphoid development of both of these im-
mune system organs.38"40 Cultured thymus an-
lage from chick embryos are 100 times more
286
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sensitive to the inhibitory effect of TCDD on
lymphoid development than cultured thymus an-
lage from turkey and duck embryos.38 This sug-
gests that the reason thymic atrophy was not seen
in turkey embryos at egg doses of 3,3',4,4'-TCB
that were overtly toxic36 was not because the
turkey embryo thymus was incapable of respond-
ing to 3,3',4,4'-TCB, rather turkey embryos ap-
pear to be more sensitive to the lethal rather than
the immunotoxic effect of this coplanar PCB.
Within the same bird species, the signs of
developmental toxicity elicited by TCDD and its
approximate isostereomers are similar. In the
chicken embryo, TCDD, 3,3',4,4',5-PCB,
3,3',4,4'-TCB, and 3,3',4,4',5,5'-HCB all cause
pericardial and subcutaneous edema, liver le-
sions, microopthalmia, beak deformities, and
mortality; and TCDD, 3,3',4,4'-TCB and
3,3',4,4'-TCAOB inhibit lymphoid develop-
ment.32-37"39 In pheasant embryos, an altogether
different pattern of responses is seen. Neverthe-
less, TCDD-like congeners, TCDD, and
3,3',4,4'-TCB, injected into fertilized pheasant
eggs, produce the same pheasant embryo-specific
pattern. This pattern consists of embryo mortality
in the absence of ederna, liver lesions, thymic
hypoplasia, and structural malformations.31-51
The lethal potency of TCDD and its approx-
imate isostereomers in embryos of different bird
species varies widely. The chicken embryo is an
outlier in that it is by far the most sensitive of
all bird species to TCDD. Turkey, ring-necked
pheasant, mallard duck, domestic duck, domestic
goose, golden-eye, herring gull, black-headed
gull, and eastern bluebird embryos are consid-
erably less sensitive to the embryo-lethal effect
of TCDD and TCDD-like congeners.31-36-41-42-51-52
TCDD is 4 to 7 times more potent in causing
embryo mortality in chicken than pheasant em-
bryos, and 3,3',4,4'-TCB is 20 to 100 times more
potent in chicken than in turkey embryos.30-31-36
In chicken embryos, an egg dose of 4 jig/kg
3,3',4,4'-TCB increased embryo mortality,
whereas an egg dose of 100 jig/kg of the same
coplanar PCB had no embryotoxic effect in
pheasants and mallard ducks, and a dose of 1000
(xg/kg egg had no effect on embryo mortality in
domestic ducks, domestic geese, golden eye, her-
ring gulls, and black-headed gulls.51-53 In contrast
to the above-mentioned species differences, the
potency of 3,3',4,4'-TCB in causing embryo
mortality among different strains of chickens is
quite similar with the LD50 in six different strains,
varying less than fourfold.53
Graded doses of TCDD have been admin-
istered to fertilized eastern bluebird and ring-
necked pheasant eggs for the purpose of deter-
mining the LOAEL and NOAEL for embryo-
toxicity. Mortality was the most sensitive
embryotoxic effect in both species. For eastern
bluebirds, the LOAEL was 10,000 pg TCDD/g
egg and the NOAEL was 1000 pg TCDD/g egg.42
For ring-necked pheasants, the LOAEL was 1000
pg TCDD/g egg and the NOAEL was 100 pg
TCDD/g egg. The LD50 for embryo mortality in
the ring-necked pheasant is 1354 pg TCDD/g egg
when the dose is injected into the egg albumin
and 2182 pg TCDD/g egg when the dose is in-
jected into the egg yolk.31 In contrast, for chick-
ens, the LD50 for embryo mortality is 240 pg
TCDD/g egg.30
3. Laboratory Mammals
When exposed to TCDD during adulthood,
laboratory mammals display wide differences in
the LD50 of TCDD. It is interesting to note, how-
ever, that when exposure occurs during prenatal
development, the potency of TCDD tends to be
similar across species. The LD50 of TCDD in
adult hamsters, 1157 to 5051 (xg/kg, makes adult
hamsters three orders of magnitude more resistant
to TCDD-induced lethality than are adult guinea
pigs.54,55 yet, a maternal dose of 18 jig TCDD/
kg can increase the incidence of prenatal mor-
tality in the hamster embryo/fetus. Because this
dose is only 12-fold larger than the 1.5 jig TCDD/
kg dose that increases the incidence of prenatal
mortality in the guinea pig, the hamster embryo/
fetus approaches other rodent species in its sen-
sitivity to TCDD-induced lethality.56-57 Thus, the
magnitude of the species differences in lethal
potency of TCDD is affected by the timing of
TCDD exposure during the life history of the
animal.
Exposure to TCDD during pregnancy causes
prenatal mortality in the monkey, guinea pig,
rabbit, rat, hamster, and mouse (Table 1). Given
a particular dosage regimen, the response is dose
287
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TABLE 1
Relationship between Maternal Toxicity and Prenatal Mortality in Laboratory
Mammals Exposed to TCDD during Gestation
Species/strain
Monkey/rhesus
Guinea pig/Hartley
Rabbit/New Zealand
Rat/Wistar
Rat/Sprague-Dawley
Hamster/Golden Syrian
Mouse/CD-1
Daily
TCDD dose
(,tg/kg/day)
O1
0.1
0.25
0.5
1
0'
0.125
0.25
0.5
1
1
2
4
0'
0.03
0.125
0.5
2
8
O1
25
50
100
200
400
Cumulative TCDD
dose (jig/kg)
0°
0.2
1
5
0'
0.15
1.5
0
1
2.5
5
10
0
1.25
2.5
5
10
10
20
40
0
0.3
1.25
5
20
80
Oh
1.5
3
6
18
0
250
500
1000
2000
4000
Overt maternal
toxicity
Percent
prenatal
mortality"
25
25
81
100
7
12
42
22
100
3
1
2
9
8
36=
Ref.
74
57
79
78
25
21
15
41 *
95=
100=
58
7
6
13
14
87
97
62
57
76
• Decreased body weight gain or marked edema compared to vehicle-dosed controls. A (+) or (-)
indicates the presence or absence of an effect.
b Percentage of absorptions plus late gestational deaths relative to all implantations. A (+) or (-) is
given to indicate the presence of absence of an effect.
0 TCDD administered in a single or divided doses between gestational days 20 and 40.
" Effects include thickening and reddening of the eyelids, weight loss, dryness and granularity of the
skin, loss of hair, and, in some cases, anemia, purpura, and bleeding from the nose and mouth.
• Single dose of TCDD administered on gestational day 14.
' TCDD administered daily on days 6-15 of gestation.
= Significant at p <0.05.
h Single dose of TCDD administered on gestational day 7 or 9.
' TCDD administered daily on days 7-16 of gestation.
From Couture, L. A., Abbott, B. D., and Birnbaum, L. S., Teratology, 42, 619, 1990.
288
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related, and there appear to be species and/or
strain differences in susceptibility to TCDD-in-
duced prenatal mortality. The rank order of sus-
ceptibility from the most sensitive to least sen-
sitive species would appear to be monkey =
guinea pig > rabbit = rat = hamster > mouse.
However, an important caveat must be applied
to the information presented in Table 1. This is
that the time period during which exposure of
the embryo/fetus to TCDD occurs is just as im-
portant a determinant of prenatal mortality as is
the dose of TCDD administered. This point is
illustrated in the text that follows when prenatal
mortality is described for different strains of mice.
It is important to note that the concept of a
critical time period for exposure makes the anal-
ysis of lethality data in the embryo/fetus quali-
tatively different from that which might be ap-
plied to similar data in adult animals. For example,
a common dosing regimen used in mice, rats,
and rabbits (Table 1) is to administer 10 daily
doses of TCDD to the pregnant dam on days ~6
to 15 of gestation. This dosing regimen is ex-
pected to cover the critical period of early de-
velopment that results in the greatest incidence
of prenatal toxicity. However, in nearly all spe-
cies of adult laboratory mammals, a single lethal
dose of TCDD would be expected to produce a
similar delayed onset death regardless of the age
of the adult animal. Susceptibility to TCDD-in-
duced prenatal mortality, in contrast, may be
greatly dependent on the age of the embryo/fetus.
In this case, multiple doses of TCDD that cover
this critical period may result in prenatal mor-
tality, whereas a single dose may miss the critical
time and not result in prenatal mortality.
The following paragraphs illustrate a type of
analysis using an index of cumulative maternal
dose similar to the type of analysis that might be
applied to lethality data resulting from multiple
dosing of adult animals. After presenting the re-
sults of applying this type of analysis to prenatal
mortality data from different species, the caveat
of critical time dependence is applied to the data
obtained by using different strains of mice. This
then illustrates the importance of considering
dosage regimen when evaluating published pre-
natal mortality data. In regard to prenatal mor-
tality, a difference of one gestational day may be
critically important. We show that the form of
analysis using cumulative maternal dose gives the
greatest possible degree of species variation. In
turn, different species seem to actually be more
similar with respect to susceptibility to prenatal
mortality than would be superficially suggested
by this type of analysis.
Using the cumulative dose data that are given
in Table 1, there appears to be a 10- to 20-fold
difference in the fetolethal potency of TCDD when
the monkey/guinea pig is compared to the rabbit/
rat/hamster. In the CD-I mouse administered cu-
mulative doses of TCDD on gestational days 7
to 16, not including day 6, it appears to require
a daily dose of 200 u>g TCDD/kg to significantly
increase prenatal mortality. Given a TCDD half-
life of approximately 5.5 days in the pregnant
dam,58 the pregnant CD-I mouse would be ex-
posed to a maximal accumulated dose of ap-
proximately 1200 (Jig TCDD/kg by the lowest
dosage regimen that significantly increased pre-
natal mortality. Therefore, by using the index of
cumulative dose, the CD-I mouse would appear
to be ~ 1200-fold less sensitive than the monkey/
guinea pig for TCDD-induced prenatal mortality.
However, in NMRI mice administered TCDD
only on day 6 of gestation, prenatal mortality
begins to increase after a single dose of 45 |Jig
TCDD/kg.59 The NMRI embryo/fetus is less sus-
ceptible to TCDD-induced prenatal mortality
when the TCDD is administered on later gesta-
tional days up to day 15. Thus, there appears to
be only about a 45-fold difference between the
monkey/guinea pig and the NMRI mouse when
the NMRI embryo/fetus is exposed specifically
on day 6. In C57BL/6 mice, prenatal mortality
is significantly increased after a single maternal
dose of 24 (xg TCDD/kg given on gestational day
6.60 This mouse strain therefore is about 20- to
30-fold less sensitive to TCDD-induced prenatal
mortality than is the monkey/guinea pig when
exposed specifically on day 6. As with the NMRI
mouse, there was little or no increase in prenatal
mortality for the C57BL/6 strain when TCDD
was administered to the pregnant dam on ges-
tational days 8, 10, 12, or 14.
The concept of a critical window for TCDD-
induced lethality in the embryo/fetus suggests an
explanation for the apparent insensitivity of the
CD-I embryo/fetus exposed to cumulative doses
of TCDD. It could very well be that the critical
289
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window for prenatal mortality in the mouse oc-
curs on or before gestational day 6. If the embryo/
fetus is not exposed to TCDD by gestational day
6, much larger doses of TCDD are required to
produce prenatal mortality. Given that exposure
of the pregnant CD-I dams did not begin until
gestational day 7, this interpretation is consistent
with the ability of a single 24 jig TCDD/kg dose
to increase the incidence of prenatal mortality
when administered to pregnant C57BL/6 mice on
gestational day 6, but not when administered on
gestational days 8, 10, 12, or 14.60 Similarly,
Neubert and Dillman59 found that the largest in-
crease in prenatal mortality occurred when a sin-
gle dose of TCDD was given on gestational day
6 compared to when the TCDD dose was ad-
ministered on one of the gestational days 7 to 15.
In addition, this would suggest that the CD-I
embryo/fetus does not have quite the relative in-
sensitivity to the lethal effects of TCDD com-
pared to the embryo/fetus of other species that
would be indicated by using the cumulative ma-
ternal dose as the index of exposure.
Human pregnancies also are affected by crit-
ical periods during which the human embryo/
fetus is susceptible to particular forms of chem-
ical-induced developmental toxicity. In the first
2 weeks of human pregnancy, the predominant
adverse developmental response is prenatal mor-
tality, although other manifestations of devel-
opmental toxicity may occur during this period.
The gestational period between weeks 2 through
8 is when the human embryo/fetus is most sus-
ceptible to the occurrence of structural malfor-
mations. Minor structural defects and postnatal
functional alterations are the dominant adverse
developmental effects that occur after 8 weeks
of pregnancy.
It should be noted that the concept of a critical
window for prenatal mortality could potentially
alter all of the species comparisons made pre-
viously that were based on the cumulative ma-
ternal doses shown in Table 1. If this turned out
to be the case, then the true differences between
species with respect to their susceptibility to
TCDD-induced prenatal mortality could be sub-
stantially less than those indicated by using the
cumulative maternal dose. This, of course, would
involve a comparison between species using only
single doses of TCDD given during the critical
time period for each species. At the present time,
it does not seem possible to make such a com-
parison from the information available in the lit-
erature.
Similar to fish and birds, the mammalian em-
bryo/fetus is more sensitive to the lethal action
of TCDD than is the adult. The maternal dose of
TCDD that causes 58% fetal mortality in ham-
sters is 64 to 280 times less than the LD50 of
TCDD in adult hamsters.54'55-61 In Sprague-Daw-
ley rats, the cumulative maternal dose of TCDD
that causes 41 % prenatal mortality is 5 to 10 times
less than the approximate LD50 of TCDD in adult
rats of the same strain.62'63 In rhesus monkeys,
the cumulative maternal TCDD dose that causes
81% prenatal mortality is 6 and 25 times less,
respectively, than the lowest TCDD dose re-
ported to cause mortality in 1-year-old and adult
rhesus monkeys.64"66
A general finding in all nonprimate labora-
tory mammals, with the possible exception of the
hamster, is that TCDD-induced prenatal mortal-
ity is most commonly associated with maternal
toxicity that is not severe enough to result in
maternal lethality. This is seen in Table 1 for the
guinea pig, rabbit, rat, and mouse. In each spe-
cies, the dose-response relationship for maternal
toxicity, indicated by decreased maternal weight
gain and/or marked subcutaneous edema of the
dam, is essentially the same as that for increased
prenatal mortality. What this means is that there
may be an association between the fetolethal ef-
fect of TCDD and maternal toxicity in all of these
species. Even in the hamster, where maternal
toxicity is far less severe, fetuses exhibit in-
creases in neutrophilic metamylocytes and bands,
whereas increases in leukocyte number and bands
also are found in maternal blood.56>S7 More re-
cently, in mice, it has been shown that TCDD
exposure causes rupture of the embryo-maternal
vascular barrier, which results in hemorrhage of
fetal blood into the maternal circulation.67 It is
not known whether these extraembryonic he-
matologic changes are contributory to or coin-
cidental with developmental toxicity in these spe-
cies. However, their occurrence reinforces the
concept that prenatal mortality can be associated
with maternal toxicity.
In rhesus monkeys, on the other hand, fewer
data are available to make the association be-
290
-------
tween prenatal mortality and maternal toxicity.
While only small numbers of monkeys have been
studied to date, the results following dietary
exposure to 25 ppt TCDD6869 and 50 ppt
TCDD70"73 before and during pregnancy suggest
that TCDD-induced prenatal mortality can occur
in monkeys in the absence of overt toxic effects
on the mother (see Section III. A. 1). In other stud-
ies, developmental toxicity in monkeys exposed
to a total cumulative maternal dose of 1 (xg
TCDD/kg administered during the first trimester
indicated a high incidence of prenatal mortal-
ity.65'74 However, maternal toxicity occurred in
some but not all of the mothers exposed. In these
monkeys, 13 of 16 pregnancies resulted in pre-
natal mortality. Within 20 to 147 days after abort-
ing, 8 of the 13 females that had aborted showed
signs of maternal toxicity and 3 of these monkeys
died. Thus, the remaining 5 of 13 instances of
prenatal mortality apparently occurred in the ab-
sence of overt maternal toxicity. The results of
these studies indicate that some levels of TCDD
exposure can result in prenatal mortality in mon-
keys even though overt toxicity seems absent in
the mother. As is described in Section III.A.I,
however, only limited attention has been given
to female reproductive toxicity in general, and to
the effects of maternal toxicity during pregnancy
on fetal development in particular. Therefore, the
relationship between maternal toxicity and pre-
natal mortality in the monkey is not well charac-
terized. The integrity of the embryo-maternal
vascular barrier, for example, has not been evalu-
ated after TCDD exposure. It is possible that
TCDD-induced developmental toxicity results
from actions exerted on the mother, embryo/
fetus, placenta, or any combination of these sites.
In most laboratory mammals, gestational ex-
posure to TCDD produces a characteristic pattern
of fetotoxic responses that consist of thymic hy-
poplasia, subcutaneous edema, decreased fetal
growth, and prenatal mortality. In addition to
these common fetotoxic effects are other effects
of TCDD that are highly species specific. Ex-
amples of the latter are cleft palate formation in
the mouse and intestinal hemorrhage in the rat.
Table 2 shows those maternal and fetal toxic re-
sponses that are produced by gestational exposure
to TCDD in various species of laboratory mam-
mals. In the mouse, hydronephrosis is the most
sensitive sign of prenatal toxicity, followed by
cleft palate formation and atrophy of the thymus
at higher doses, and by subcutaneous edema and
mortality at maternally toxic doses.59'75"77 In the
rat, TCDD prenatal toxicity is manifested by in-
testinal hemorrhage, subcutaneous edema, de-
creased fetal growth, and mortality.62-78 Struc-
tural abnormalities do occur in the rat but only
at relatively large doses.75 In the hamster fetus,
hydronephrosis and renal congestion are the most
sensitive effects, followed by subcutaneous edema
and mortality at fetolethal doses.56-57 In the rab-
bit, an increased incidence of extra ribs and pre-
natal mortality is found,79 whereas in the guinea
pig and rhesus monkey, prenatal mortality is
seen.57-74
4. Structure-Activity Relationships in
Laboratory Mammals
The structure-activity relationship for devel-
opmental toxicity in laboratory mammals is gen-
erally similar to that for Ah receptor binding.
Gestational treatment of rats with CDD conge-
ners that do not bind the Ah receptor, 2-MCDD,
2,7-DCDD, 2,3-DCDD, or 1,2,3,4-TCDD, does
not cause TCDD-like fetotoxic effects.78 On the
other hand, hexachlorodibenzo-p-dioxin, which
has intrinsic Ah receptor activity, produces fe-
totoxic responses in rats that are essentially iden-
tical to those of TCDD.80 Similarly, when ad-
ministered to pregnant rhesus monkeys or CD-I
mice, PCB congeners that act via an Ah receptor-
mediated mechanism, 3,3',4,4'-TCB and
3,3',4,4',5,5'-HCB, cause the same type of
fetotoxic effects as TCDD. In contrast,
4,4'-DCB, 3,3',5,5'-TCB, 2,2',4,4',5,5'-HCB,
2,2',4,4',6,6'-HCB, and 2,2',3,3',5,5'-HCB,
which essentially have no or very weak affinity
for the Ah receptor, do not produce a TCDD-
like pattern of prenatal toxicity in mice.65-81"83
Thus, most structure-activity results for overt
fetotoxic effects of the halogenated aromatic hy-
drocarbons are consistent with an Ah receptor-
mediated mechanism. Nevertheless, one finding
that stands out as being inconsistent is that
2,2',3,3',4,4'-HCB, which has a very weak, if
any, affinity for binding to the Ah receptor, causes
the same pattern of fetotoxic effects in mice as
does TCDD.81
291
-------
TABLE 2
Developmental Toxicity Following Gestational Exposure to 2,3,7,8-TCDD
Treatment Sacrifice
Species/strain
Mice/C57BL/6N
Mice/C57BL76N
Mice/C57BL76N
Mice/C57BL76N
Mice/C57BL/6N
Mice/C57BL/6J
Mice/C57BL/6J
Dally dose*
0, 1 , or 3 (ig/kg
0, 12, 17, or 22 ^g/kg
0,3, or 12 (ig/kg
0 or 3 (ig/kg
0, 6, 9, 12, 15, or
18 M-9/kg
0 or 3 M-g/kg
(subcutaneous)
20 jig/kg
days"
10, 10-13
10
11,10-13
10-13
10, 12
6-15
10
da
18
18
18
18
18
18
17
Maternal effects0
Mice/C57BL76J
Mice/NMR
NR
0, 0.5, 1, 2, or 4 y-g/kg
6-15
0.3, 3, 4, 5, or 9 ng/kg 6-15
18
18
Increase in liver-to-body weight
ratio
Increase in liver-to-body weight
ratio
Increase in liver-to-body weight
ratio
Increase in liver-to-body weight
ratio and weight gain
Increase in liver-to-body weight
ratio
Increase in liver-to-body weight
ratio
Increase in liver-to-body weight
ratio
NR
Mice/CF-1
Mice/DBA
Mice/DBA
Mice/CD-1
Mice/CD-1
Rats/CD
Rats Sprague-Dawley
Rats/Sprague-Dawley
0, 0.001, 0.01, 0.1, or
1 .3 (ig/kg
0 or 3 M.g/kg
(subcutaneous)
0, 0.5, 2, 4, or 8 ng'kg
0, 1 , or 3 (ig/kg
(subcutaneous)
0, 25, 50, 100, 200, or
400 M-g/kg
0 or 0.5 M-g/kg
2 M-g/kg
(subcutaneous)
0, 0.125, 0.5, or 2 jig/kg
0.03, 0.125, 0.5, 2, or
6-15
6-15
6-15
6-15
6-15
6-15
9-10 or
13-14
0-2
6-15
NR
18
18
17
17
20
20
21
None
Increase in liver-to-body weight
ratio
Increase in liver-to-body weight
ratio; decrease in thymus-to-
body weight ratio
None
Increase in liver-to-body weight
ratio
None at 0.5 M.Q kg
NR at 2 ng/kg
Decrease in weight gain
Decrease in weight gain; toxic-
Rats/Wistar
0,0.125,0.25,0.5, 1,2,
4, 8, or 16 M-g/kg
ity
5-14 21 Toxicity
Guinea pigs/Hartley 0, 0.15, or 1.5 jig/kg 14
Hamsters/Golden 0, 1.5, 3, 6, or 18 mj/kg 7, 9
Syrian
58 Increase in mortality; toxicity
15 Increase in liver-to-body weight
ratio
Rabbits/New Zealand 0, 0.1, 0.25, 0.5, or
1 tig/kg
6-15 28 Decrease in weight gain;
toxicity
Embryo/fetal effects0 Ref.
Increase in cleft palate 144
and hydronephrosis
Increase in cleft palate 102
and hydronephrosis
Increase in cleft palate 145
and hydronephrosis
Increase in hydrone- 277
phrosis
Increase in cleft palate 278
and hydronephrosis
Increase in cleft palate 77
and kidney anomaly
Increase in cleft palate, 273
hydronephrosis, and
fetal body weight
Increase in cleft palate, 149
hydronephrosis, and
fetal body weight
Increase in cleft palate 59
and fetal mortality; de-
crease in fetal body
weight
Increase in cleft palate 276
and hydronephrosis
Increase in cleft palate 77
and kidney anomaly
Increase in cleft palate 149
and hydronephrosis
Increase in cleft palate 77
and kidney anomaly
Increase in cleft palate, 76
hydronephrosis, and
fetal mortality
— 77
Increase in kidney
anomaly
Decrease in fetal body 274
weight
Increase in fetal mortal- 62
ity, resorptions,
edema, and gastroin-
testinal hemmorrhage
Increase in fetal mortal- 78
ity, edema, and gas-
trointestinal hemor-
rhage; decrease in
fetal weight
Increase in fetal mortal- 56
ity
Increased fetal mortality, 56
hydronephrosis, and
renal congestion; de-
creased thymus size
Increase in fetal mortal- 79
ity and resorptions;
extra ribs
292
-------
TABLE 2 (continued)
Developmental Toxicity Following Gestational Exposure to 2,3,7,8-TCDD
Species/strain
Rhesus monkeys
Monkeys/rhesus
Dally dose*
0, 5, 25, 50, or 500 ppt
7 months before and
during pregnancy
0,0.2," 1,d 1,»or
5" j
Treatment
days"
Chronic
20-40
Sacrifice
day*
Maternal effects'
Increase in mortality; toxicity
Increase in mortality; toxicity
Note: NR = Not reported.
Embryo/fetal effects' Ret.
Increase in fetal mortal- 71,
ity 223
Increase in fetal mortal- 65
ity
a Oral exposure unless otherwise noted.
6 All days adjusted to reflect plug day — gestational day 0.
c Effects reported are only those that were statistically significant.
d Cummulative dose divided into nine oral doses administered between days 20 and 40 of gestation; two to four monkeys/dose.
8 Three animals given single oral dose on either gestational days 25, 30, 35, or 40; 12 monkeys total.
From Couture, L. A., Abbott, B. D., and Birnbaum, L. S., Teratology, 42, 619, 1990.
5. Humans
In the Yusho and Yu-Cheng poisoning epi-
sodes, developmental toxicity was reported in
babies born to affected mothers who had con-
sumed rice oil contaminated with PCBs, CDFs,
and PCQs.15~17-84 In these incidents, it is essen-
tially impossible to determine the contribution of
TCDD-like vs. nonTCDD-like congeners to the
fetal/neonatal toxicity. Nevertheless, high peri-
natal mortality was observed among hyperpig-
mented infants born to affected Yu-Cheng
women, who themselves did not experience in-
creased mortality.16 Thus, in humans, the devel-
oping embryo/fetus may be more sensitive than
the intoxicated mother to mortality caused by
halogenated aromatic hydrocarbons.
In most cases, women who had affected chil-
dren in the Yusho and Yu-Cheng episodes had
chloracne themselves.85 Based on this evidence,
Rogan suggested that "exposure to amounts in-
sufficient to produce some effect on the mother
probably lessens the chance of telepathy consid-
erably."85 In support of this interpretation, overt
signs of halogenated aromatic hydrocarbon tox-
icity were not observed in infants born to appar-
ently unaffected mothers in the Seveso, Italy, and
Times Beach, MO, TCDD incidents.86-87
In laboratory mammals, the studies sum-
marized previously in Table 1 indicate an appar-
ent association between prenatal mortality and
maternal toxicity in nonprimate species. How-
ever, some TCDD-exposed rhesus monkeys were
not able to carry their pregnancies to term, even
in the absence of any overt signs of maternal
toxicity. This result in monkeys indicates that the
relationship between maternal toxicity and any
prenatal toxic effects on the human embryo/fetus
must be cautiously defined. More data may be
required to determine whether or not there is any
association between overt maternal toxicity and
embryo/fetal toxicity both in monkeys and hu-
mans.
Effects of chemical exposure on normal de-
velopment of the human fetus can have four out-
comes, depending on the dose and time during
gestation when exposure occurs: (1) fetal death,
(2) structural malformations, (3) organ system
dysfunction, and (4) growth retardation. In the
Yusho and/or Yu-Cheng incidents, all of these
outcomes were found.15-17'84 Increased prenatal
mortality and low birth weight suggesting fetal
growth retardation were observed in affected
Yusho and Yu-Cheng women. 16-84-88~92 A struc-
tural malformation, rocker bottom heel, was ob-
served in Yusho infants.84 Organ dysfunctions
involving the central nervous system (CNS),
which were characterized by delays in attaining
developmental milestones and neurobehavioral
abnormalities, were reported in Yu-Cheng chil-
dren exposed transplacentally.92-93
293
-------
Organs and tissues that originate from embry-
onic ectoderm are well-known targets for toxicity
following exposure to TCDD-like halogenated
aromatic hydrocarbons. For example, treatment
of adult monkeys with TCDD results in effects
involving the skin, meibomian glands, and nails.70
Similarly, a hallmark sign of fetal/neonatal tox-
icity in the Yusho and Yu-Cheng episodes was
an ectodermal dysplasia syndrome, which is
characterized by hyperpigmentation of the skin
and mucous membranes, hyperpigmentation and
deformation of finger and toe nails, hypersecre-
tion of the meibomian glands, conjunctivitis, gin-
gival hyperplasia, presence of erupted teeth in
newborn infants, and altered eruption of per-
manent teeth, missing permanent teeth, and ab-
normally shaped tooth roots.15-17'84-88'91-92'94-96
Accelerated tooth eruption has been observed in
newborn mice exposed to TCDD by lactation,97
as well as in the human infants mentioned pre-
viously. In addition, other effects have been re-
ported in the Yusho and Yu-Cheng exposed in-
fants that resemble effects observed following
TCDD exposure in adult monkeys. These include
subcutaneous edema of the face and eye-
lids.70-84-89'98 Also, larger and wider fontanels,
and abnormal lung auscultation were found in the
human infants.84'89'92 The similarities between
certain effects reported in human infants exposed
during the Yusho and Yu-Cheng incidents, as
well as adult monkeys and neonatal mice exposed
to TCDD, enhance the probability that certain
effects reported in the human infants were caused
by the TCDD-like PCB and CDF congeners in
the contaminated rice oil ingested by the mothers
of these infants.
Chloracne is the most often cited effect of
TCDD exposure involving the skin in adult hu-
mans. This effect has an animal correlate in the
hairless mouse and can be studied by using a
mouse teratoma cell line in tissue culture.3 Never-
theless, it has rarely been explicitly recognized
in the TCDD literature that the nervous system,
like the skin, is derived from embryonic ecto-
derm.99 As is described in Section II.C.2, neuro-
behavioral effects occur following transplacental
and neonatal exposure to TCDD-like congeners
in mice, as well as transplacental exposure to
TCDD in monkeys. In addition, some of the Yu-
Cheng children that were exposed transplacen-
tally to PCBs, PCDFs, and PCQs have affected
a clinical impression of developmental delay or
psychomotor delay including impairment of in-
tellectual development.92-93 Because there is a
clustering of effects due to TCDD-induced tox-
icity in organs derived from ectoderm, it is rea-
sonable to speculate that direct effects of TCDD-
like congeners on the CNS are responsible for
some of the neurobehavioral effects observed in
these children. Effects of TCDD on EOF recep-
tors are associated with certain aspects of the
ectodermal dysplasia syndrome, such as hyper-
keratinization of the skin100 and accelerated tooth
eruption.97 Decreased autophosphorylation of the
EOF receptor in human placentas is associated
with decreased birth weight in infants born to
exposed mothers 4 years after the initial Yu-Cheng
exposure incident.101 This last result supports the
earlier conclusion that careful study is needed to
define the relationship between maternal toxicity,
placental toxicity, and developmental toxicity in
humans. In addition, further research is needed
to characterize and elucidate the mechanisms by
which TCDD affects the nervous system.
B. Structural Malformations
Developmental effects consisting of cleft pal-
ate, hydronephrosis, and thymic hypoplasia are
produced in mice following in utero exposure to
halogenated dibenzo-/?-dioxin, dibenzofuran, and
biphenyl and naphthalene congeners, which bind
stereospecifically to the Ah receptor.102-105 Of
these effects in the mouse, cleft palate is less
responsive than hydronephrosis inasmuch as the
latter is induced in the absence of cleft palate.60
Both responses can be induced at TCDD doses
that are not otherwise overtly toxic.75 The po-
tency of TCDD for producing teratogenesis in
the mouse is clearly evident when one considers
that only 0.0003% of a maternally administered
dose can be isolated from the fetal palatal shelves
or kidneys. More specifically, a maternal TCDD
dose of 30 M-g/kg administered on gestational day
11 results in a tissue concentration of 0.65 pg
TCDD/mg in the palatal shelves 3 days after dos-
ing, and the same tissue concentration of TCDD
is present in the kidneys at that time.106
294
-------
Susceptibility to the developmental toxicity
of TCDD in mice depends on two factors: the
genotype of the fetus and the stage of develop-
ment at the time of exposure. Because mouse
strains that produce Ah receptors with relatively
high affinity for TCDD respond to lower doses
of TCDD than mouse strains that produce rela-
tively low-affinity Ah receptors,107'108 the Ah re-
ceptor is thought to mediate the developmental
effects of TCDD.3 Thus, one genetically encoded
parameter that determines the responsiveness of
different mouse strains is the Ah receptor protein
itself.
The differences that exist between mouse
strains with respect to developmental respon-
siveness to these chemicals are not absolute be-
cause all strains, including those with Ah recep-
tors of relatively low affinity, respond when
exposed to sufficiently large doses during the
critical period of organogenesis.109 In the mouse,
the peak times of fetal sensitivity vary slightly
depending on which developmental effect is used
as the endpoint. However, exposure between days
6 and 15 of gestation will produce teratogene-
60,75
In inbred strains of mice, the developmental
response, characterized by altered cellular pro-
liferation, metaplasia, and modified terminal dif-
ferentiation of epithelial tissues,3 is extremely
organ specific, occurring only in the palate, kid-
ney, and thymus.109 Pharmacokinetic differences
are not responsible for this high degree of tissue
specificity, and Ah receptors are not found ex-
clusively in the affected organs.110-111 Therefore,
other factors intrinsic to the palate, kidney, and
thymus appear to play a role along with the Ah
receptors in these tissues in producing the struc-
tural malformations. For various developmental
effects, the time at which exposure occurs is im-
portant and defines the critical period during
which the toxicant must be present in order to
produce the effect. This critical period differs
among organs and tissues within organs.
Differences exist between mammalian spe-
cies with respect to susceptibility to the devel-
opmental effects of TCDD. Although genetic dif-
ferences between species or strains may affect
absorption, biotransformation, and/or elimina-
tion of TCDD by the maternal system and its
transport across the placenta, such species dif-
ferences do not account for the lack of cleft palate
formation in species other than mice.109 Rather,
the species differences in susceptibility to cleft
palate formation appear to be due to differences
in the interaction between TCDD and the devel-
oping palatal shelves themselves. This is dem-
onstrated by the occurrence of similar responses
when palatal shelves from different species are
exposed to TCDD in organ culture.106-112'113 The
key difference is that, relative to the concentra-
tion of TCDD that affects murine palatal shelves
in culture, much higher concentrations of TCDD
are required to elicit the same effects in cultured
palatal shelves from other species (Table 3). As
a result, it appears that differences in the res-
ponsivity of palatal tissue to the effects of TCDD
explain the absence of cleft palate in nonmurine
species except at maternal doses that are fetotoxic
and maternally toxic.75'109
In mice and hamsters, hydronephrosis can be
elicited at TCDD doses that are neither fetotoxic
nor maternally toxic,57 whereas thymic hypopla-
sia is a fetal response to TCDD observed in vir-
tually all laboratory mammalian species tested.114
Studies in humans have not clearly identified an
association between TCDD exposure and struc-
tural malformations.86-115"117
1. Cleft Palate
a. Characterization of TCDD Effect
Palatal shelves in the mouse originate as out-
growths of the maxillary process. Eventually, they
come to lie vertically within the oral cavity on
both sides of the tongue. In order to form the
barrier between the oral and nasal cavities, the
shelves in the mouse must reorient themselves
from a vertical direction to a horizontal direction.
Once they come together horizontally, their me-
dial aspects bring apposing epithelia into close
contact.118-119 At this stage, the apposing medial
edge epithelia of the separate palatal shelves each
consist of an outer layer of periderm that overlays
a strata of cuboidal shaped basal cells. These
basal cells, in turn, rest on top of a continuous
basal lamina. There is a sloughing of the outer
periderm cells followed by the formation of junc-
tions between the newly apposing basal epithelial
295
-------
TABLE 3
TCDD Responsiveness of Palatal Shelves from the Mouse,
Rat, and Human in Organ Culture
Molar concentration of TCDD
Species
Mouse
Rat"
Human"
Prevention of the epithelium-to-
mesenchyme transformation process
LOEL EC100
1 x10-'3
1 x10-'°
5x1Q-11
5x10-11
1x10-a
1x10-"
Cytotoxicity
1x10-10
1x10-7
1x10-7
• At the highest concentration tested, 60% of the palatal shelves failed to
undergo the transformation process.
b One of four shelves responded by failing to undergo the transformation
process at 5x10-" M.
From Birnbaum, L S., Biological Basis lor Risk Assessment of Dioxins and
Related Compounds, Gallo, M. A., Scheuplein, R. J., and van der Heijden,
C. A., Eds., Branbury Report 35, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, 1991, 51.
cells that are left within the seam. The midline
seam thus formed consists of two healthy-ap-
pearing layers of basal cells (one on each side of
the seam), whereas the outer periderm cells have
already been shed. As the palatal shelf continues
to grow, the bilayer seam, which itself grows at
a slower rate, turns into a single layer of cells,
and then breaks up into small islands of cells.
Eventually, the basal lamina disappears, and the
elongating former basal cells within the small
islands extend filopodia into the adjacent con-
nective tissue. During this process, the former
basal cells lose epithelial characteristics and gain
fibroblast-like features. Essentially, the above-
described process makes the medial edge epithe-
lium an ectoderm that retains the ability to trans-
form into mesenchyme. Upon completion of this
epithelial to mesenchyme transformation, the once
separate and apposing palatal shelves are fused
so that a single continuous tissue is formed.120'121
Cleft palate can result from a failure of the
shelves to grow and come together, or a failure
of the shelves to fuse once they are in close ap-
position.122 TCDD and other Ah receptor agonists
act by allowing the shelves to grow and make
contact, but the subsequent process involving the
epithelial-to-mesenchyme transformation does not
occur. Therefore, a cleft is formed as the palatal
shelves continue to grow without fusing. When
TCDD is administered to pregnant mice on ges-
tational days 6 to 12, the incidence of cleft palate
formation increases with time. However, day 12
is a critical window, after which the incidence of
cleft palate formation decreases. No cleft palates
are formed when TCDD is administered on day
1460
Palatal shelves of the mouse, rat, and human
can be removed from the fetus and placed into
organ culture. Under these conditions, when the
separate shelves are placed in an apposing con-
dition in vitro, sloughing periderm cells are trap-
ped within the seam. 12° Thus, due to the presence
of these trapped dead cells, the fusion process
was originally described as a process of pro-
grammed cell death. 118'119>123 However, the newer
idea, which presumes transformation of the basal
epithelial cells into mesenchyme rather than their
death, is believed to be valid under explant con-
ditions in vitro, as well as in vivo.120 When ex-
posed to TCDD as explants in vitro, the palatal
shelves of the mouse, rat, and human all respond
to TCDD in a similar way by not completing the
fusion process. 106'112>113'124 There is death of the
outer peridermal cells, after which, the epithelial-
to-mesenchyme transformation of the basal epi-
thelial cells does not occur. Instead, there is a
differentiation of these basal cells into a stratified
squamous epithelium such that they eventually
296
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resemble the squamous keratinizing oral cells
within the tissue.125
Table 3 shows the lowest TCDD concentra-
tion that prevents the epithelial-to-mesenchyme
transformation process in isolated palatal shelves
(LOEL), the TCDD concentration that produces
a 100% maximal response (EC100), and the lowest
concentration of TCDD that produces cytotox-
icity. Palatal shelves of rats and humans respond
to TCDD in a manner identical to the mouse;
however, higher concentrations of TCDD are re-
quired to prevent the epithelial-to-mesenchyme
transformation process. The relative insensitivity
of rat palatal shelves may explain the lack of cleft
palates when fetal rats are exposed to nonmater-
nally toxic doses of TCDD. Sensitivity of human
palatal shelves to TCDD in vitro is similar to the
rat. This suggests that exposure to maternally
toxic and fetotoxic doses of TCDD would be
required to cause cleft palate formation in hu-
mans.
A disruption in the normal spatial and tem-
poral expression of EGF, TGF-a, TGF-pl, and
TGF-fJ2 correlates with altered proliferation and
differentiation in the medial region of the de-
veloping palate resulting in a palatal cleft. Thus,
the abnormal proliferation and differentiation of
TCDD-exposed medial cells may be related to
reduced expression of EGF and TGF-a. Also,
decreased levels of immunohistochemically de-
tectable TGF-pl could contribute to the contin-
ued proliferation and altered differentiation of
medial cells.126 It is important to note that EGF
and TGF-a both exert their actions by binding to
EGF receptors. The differentiation of basal cells
to a stratified squamous epithelium, which re-
sembles the keratinizing oral epithelium within
the developing palate mentioned above, is similar
to certain effects of TCDD that can be studied
in cultured human keratinocytes. These effects
in cultured human keratinocytes involve altered
EGF binding to those cells. In addition, the Ah
receptor is implicated in producing this re-
sponse.100 Thus, the mechanisms by which TCDD
produces a palatal cleft in the mouse may have
similarities to the mechanisms by which TCDD
produces other effects that are part of the ecto-
dermal dysplasia syndrome. This is consistent
with the description given by Fitchett and Hay,120
in that the medial edge epithelium within the
developing palate is essentially an ectoderm that
retains the ability to transform into mesenchymal
cells.
b. Evidence for an Ah Receptor Mechanism
i. Genetic
When wild-type C57BL/6 (AhbAhb) mice are
crossed with DBA/2 (AhdAhd) mice that contain
a mutation at the Ah locus, all of the heterozy-
gous B6D2F1 progeny (AhbAhd) resemble the
wild-type parent in that arylhydrocarbon hydrox-
ylase (AHH) activity is inducible by TCDD and
other halogenated aromatic hydrocarbons.127 Test
crosses between the B6D2F1 progeny and each
original parent strain, and other B6D2F1 progeny
mice demonstrate that in the C57BL/6 and DBA/
2 strains susceptibility to AHH induction segre-
gates as a simple dominant trait in the backcross
and F2 progeny. Thus, the trait of AHH induci-
bility is expressed in progeny that contain the
AhbAhb and AhbAhd genotypes, but is not ex-
pressed in the AhdAhd progeny from these crosses.
Certain other effects of TCDD, such as its bind-
ing affinity for the hepatic Ah receptor,128 thymic
atrophy,107 hepatic porphyria,129 and immuno-
suppressive effects,130-131 have been shown in
similar genetic crosses and test crosses to seg-
regate with the Ah locus, which permits AHH
induction. Thus, for these effects of TCDD, ge-
netic evidence demonstrates an involvement of
the Ah locus.3
Nebert's group was the first to relate devel-
opmental toxicity to the Ah locus in mice.132-133
Subsequently, Poland and Glover107 administered
a single 30-jig TCDD/kg dose to pregnant mice
on gestational day 10. It was found that there
was a 54% incidence of cleft palate in homo-
zygous C57BL/6 (AhbAhb) fetuses, a 13% inci-
dence in heterozygous B6D2F1 (C57BL/6 and
DBA/2 hybrid, AhbAhd) fetuses, and only a 2%
incidence in homozygous DBA/2 (AhdAhd) fe-
tuses. This pattern of inheritance, in which the
incidence of developmental toxicity in the het-
erozygous Fl generation is intermediate between
that of the homozygous parental strains, is con-
sistent with the autosomal dominant pattern of
inheritance described for AHH inducibility and
297
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the Ah locus,127 even if dominance is incomplete
in the case of developmental toxicity. However,
the pattern of inheritance for developmental tox-
icity described when Poland and Glover107 crossed
C57BL/6 and DBA/2 mice is not proof positive
that the Ah locus is the only genetic locus that
controls susceptibility to TCDD-induced devel-
opmental toxicity in these mouse strains.
To provide such proof, it is necessary to show
genetic linkage between the susceptibility for de-
velopmental toxicity and the Ah locus. The stan-
dard of proof would be that developmental tox-
icity and a particular allele at the Ah locus must
always segregate together in genetic crosses be-
cause if the loci are the same there can be no
recombination between the loci. This is generally
accomplished by demonstrating cosegregation
between the two loci not only in crosses between
the two homozygous parental strains, which in
and of itself is insufficient proof of genetic link-
age, but also in test crosses or backcrosses be-
tween the heterozygous Fl hybrids with each
homozygous parental strain.
It was stated earlier in this section that certain
effects of TCDD are well known to segregate
with the Ah locus due to the results of appropriate
crosses and backcrosses between responsive and
nonresponsive mouse strains and their hybrid Fl
progeny. With this standard of proof in mind,
the evidence that specifically links develop-
mental toxicity with the Ah locus is now de-
scribed. It is intended that this information con-
tain a considerable degree of detail. This is done
so that the reader can independently determine
whether or not the standard of proof has been
satisfied by the evidence available.
In order to strengthen their conclusion based
on the results of simple crosses between C57BL/6
and DBA/2 mice, Poland and Glover107 planned
to perform a backcross between the hybrid
B6D2F1 and DBA/2. However, the low inci-
dence of cleft palate in B6D2F1 mice would have
required characterizing and phenotyping a pro-
hibitively large number of fetuses. Alternatively,
the backcross between B6D2F1 and C57BL/6 was
considered in which AhbAhb and AhbAhd progeny
would have been distinguished by the amount of
high-affinity specific binding for TCDD in fetal
liver. In this case, however, overlap between in-
dividual mice would have made the results un-
certain in some of the progeny. Therefore, it was
not possible to obtain satisfactory results from
either backcross.
Instead, Poland and Glover107 examined the
incidence of cleft palate in ten inbred strains of
mice exposed to TCDD: five strains with high-
affinity Ah receptors and five strains with low-
affinity receptors. In the five latter strains, there
was only a 0 to 3% incidence of cleft palate
formation, whereas four of the five strains with
high-affinity Ah receptors developed a 3=50%
incidence. The one strain with high-affinity Ah
receptors that did not follow the pattern, the CBA
strain, also is resistant to cleft palate formation
induced by glucocorticoids and may therefore be
generally resistant to chemically induced cleft
palate formation. Overall, these results indicate
that cleft palate formation probably segregates
with the Ah locus.
The incidence of cleft palate formation was
studied in fetuses from a cross between C57BL/6
and AKR/NBom mice administered 3,3',4,4'-
TCAOB on gestational day 12.134 Although
C57BL/6 mice are responsive for AHH induction
and cleft palate formation, AKR mice are less
responsive, requiring higher doses for both ef-
fects. In a manner unlike the result of a cross
between C57BL/6 and DBA/2, the incidence of
cleft palate formation in the B6AKF1 progeny
was <2%, showing that nonresponsiveness seg-
regates as the dominant trait when C57BL/6 mice
are crossed with AKR mice. Similarly, cleft pal-
ate formation was virtually absent in the progeny
of a backcross between AKR/NBom and
B6AKF1, demonstrating dominance of the non-
inducible trait. Although Ah phenotyping of the
backcross progeny was not performed in this par-
ticular study, Robinson et al.135 had previously
evaluated segregation of the Ah locus in back-
crosses between C57BL/6 and AKR/N mice. They
found in these two strains that noninducibility for
AHH activity segregates as the dominant trait.
Thus, inducibility for cleft palate formation and
AHH activity both segregate as dominant traits
when C57BL/6 mice are crossed with DBA/2,
but noninducibility is dominant for both traits
when C57BL/6 mice are crossed with AKR/N.
These results are consistent with the interpreta-
tion that cleft palate induction probably segre-
gates with the Ah locus.
298
-------
Like Poland and Glover,107 Hassoun et al.108
were unable to determine whether or not cleft
palate formation segregates with the Ah locus in
C57BL/6 and DBA/2 mice by performing simple
backcrosses. Instead, they evaluated cosegrega-
tion of the Ah locus and 2,3,7,8-TCDF-induced
cleft palate formation using a series of recom-
binant strains called BXD mice. These strains are
fixed recombinants produced from an original
cross between the two parental strains C57BL/6J
and DBA/2J. Hybrid B6D2F1 mice were crossed
to produce F2 progeny and these were strictly
inbred by sister and brother matings into several
parallel strains. The mice used in this study were
from the F42 and F58 generations of inbreeding.
It was found that the incidence of TCDF-induced
cleft palate formation after matings within eight
different BXD strains with high-affinity Ah re-
ceptors is >85%. After similar matings with eight
different BXD strains with low-affinity Ah re-
ceptors, the incidence of TCDF-induced cleft pal-
ate formation is <2%. These results of Hassoun
et al.108 corroborate those of Poland and Glover107
and provide the best evidence currently available
that cleft palate formation segregates with the Ah
locus. Accordingly, the Ah locus and the Ah
receptor are involved in the formation of palatal
clefts that are induced by TCDD-like congeners.
As additional evidence, stereospecific, high-
affinity Ah receptors can be isolated from cytosol
fractions prepared from embryonic palatal
shelves. These receptors are present in palatal
shelves of AhbAhb, C57BL/6 fetuses, but are not
detectable in similar tissue from AhdAhd, AKR/J
fetuses.136 However, the significance of this find-
ing may be mitigated to some extent by the fol-
lowing observation. In cytosols prepared from
homogenates of whole embryo/fetal tissue (mi-
nus head, limbs, tail, and viscera), the concen-
tration of specific-binding TCDD receptors is 256
fmol/mg protein in C57BL/6 mice compared to
a concentration of 21 fmol/mg protein in the less
responsive DBA/2 strain, 15 fmol/mg protein in
the less responsive AKR/J strain, and 19 fmol/
mg protein in the less responsive SWR/J strain.
However, when embryonic tissue is cultured, the
differences between the strains in receptor num-
bers are less pronounced; and in the receptors
isolated from cultured embryonic cells of differ-
ent strains, there is only about a twofold differ-
ence in the relative binding affinity for 3H-TCDD.
The mechanistic reasons for the diminished de-
gree of difference between responsive and less
responsive mouse strains during embryonic cell
culture are not known.137
The possible influence of maternal toxicity
on cleft palate formation was evaluated by per-
forming reciprocal blastocyst transfer experi-
ments using the high-affinity Ah receptor NMRI
and lower affinity Ah receptor DBA strains of
mice.138 After administration of 30 fig TCDD/kg
or 8 mg TCAOB/kg to pregnant dams on ges-
tational day 12, 75 to 100% of all NMRI fetuses
developed cleft palates. This was true whether
the fetuses remained within the uterus of their
natural mother or were transferred into the uterus
of a DBA mouse. Under the same conditions,
none of the 24 DBA fetuses transferred into an
NMRI mother developed a cleft palate, even
though 89% of their NMRI littermates were af-
fected. Thus, these results, along with the pres-
ence of Ah receptors in palatal shelves and re-
sponsiveness of palatal shelves in organ culture
to TCDD, indicate that cleft palate formation in
mice is due to the direct effect of TCDD on the
palatal shelf itself and is not secondary to ma-
ternal toxicity.
ii. Structure Activity
Because the genetic evidence in mice indi-
cates that the Ah receptor mediates TCDD-in-
duced cleft palate formation and hydronephrosis
(see Sections II.B.l.b.i and II.B.2.b.i), struc-
ture-activity requirements based on Ah receptor-
binding characteristics should predict the relative
potencies of different agonists for producing cleft
palate and hydronephrosis. Of the halogenated
aromatic hydrocarbons, TCDD has the greatest
affinity for binding to the Ah receptor and it is
the most potent teratogen in inbred mouse strains.
Table 4 shows the relative potencies for cleft
palate induction and hydronephrosis in C57BL/6
mice for a number of TCDD-like congeners. Be-
cause TCDD is the most potent, it is assigned a
value of 1.000. When examined using probit
analysis, the dose-response curve of each con-
gener, compared to all of the others, did not
deviate from parellelism. Therefore, the relative
299
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TABLE 4
Relative Teratogenic Potency of Halogenated
Aromatic Hydrocarbon Congeners in C57BL/6
Mice
Relative potency
, TCDD/EDa, Congener)
1.000
0.235
0.100
0.095
0.049
0.026
0.010
0.005 .
0.004
0.0000287
1.000
0.444
0.333
0.057
0.021
0.074
0.049
0.009
0.018
0.0000894
Congener Cleft palate Hydronephrosis
2,3,7,8-TCDD
2,3,7,8-TBDD
2,3,7,8-TBDF
2,3,4,7,8-PeCDF
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
1,2,3,4,7,8-HxCDF
2,3,4,7,8-PeBDF
1,2,3,7,8-PeBDF
2,3,4,5,3',4'-HxCB
From References 102-104 and 109.
potencies of the congeners are valid for any given
incidence of cleft palate formation or hydrone-
phrosis. The main finding, however, is that the
rank order potency of the various congeners for
producing these two developmental effects is
generally similar to that for binding to the Ah
receptor (Table 4), with the notable exception
that the apparent binding affinities for the brom-
inated dibenzofurans have not yet been reported.
There are additional ligands for the Ah receptor
that cause cleft palate formation in C57BL/6 mice
at nonmaternally toxic doses, but they are not
listed in the table. These include 3,3',4,4'-
TCAOB,108 3,3',4,4'-tetrachlorobiphenyl,83
3,3',4,4',5,5'-hexachlorobiphenyl,82 and a mix-
ture that contained 1,2,3,4,6,7- and 2,3,4,5,6,7-
hexabromonaphthalenes.139
Also consistent with the structure-activity re-
lationships for binding to the Ah receptor is the
finding that a number of hexachlorobiphenyls do
not induce cleft palate formation. These conge-
ners either lack sufficient lateral substitution or
are substituted in such a manner that they cannot
achieve a planar conformation. Included in this
category are the diortho- and tetraoAtfco-chlorine-
substituted 2,2',3,3',5,5'-, 2,2',3,3',6,6'-,
2,2',4,4',5,5'-, and 2,2',4,4',6,6'-hexachloro-
biphenyls.81 In addition, it is consistent with the
structure-activity relationships that monoorffto-
chlorine-substituted 2,3,4,5,3',4'-HCB is a weak
teratogen. Its potency relative to that of TCDD
varies from 3 x 10"5 to 9 x 10~5 for cleft palate
formation, AHH induction, and hydronephrosis
(Table 4).8
A result that would not be expected according
to the structure-activity relationships for binding
to the Ah receptor is that dio/t/zo-chlorine-sub-
stituted 2,2',3,3',4,4'-hexachlorobiphenyl causes
cleft palate formation and hydronephrosis in
mice.81 However, another diorr/zo-chlorine-sub-
stituted PCB congener, 2,2',4,4',5,5'-hexach-
lorobiphenyl, also can cause hydronephrosis and
is a very weak inducer of 7-ethoxyresorufin-O-
deethylase (EROD) activity.140-141 It is consistent
with the interpretation that 2,2',4,4',5,5'-hex-
achlorbiphenyl is a partial Ah receptor agonist,
that it can competitively displace TCDD from the
murine hepatic cytosolic receptor, and, at large
enough doses, that it can inhibit TCDD-induced
cleft palate formation and immunotoxicity in
C57BL/6 mice.140'141 These results suggest that
PCB congeners do not have to be in a strictly
planar configuration to cause teratogenesis.
c. Species Differences
Cleft palate is induced in rats only at mater-
nally toxic TCDD doses that are associated with
a high incidence of fetal lethality. Schwetz et al.80
reported an increased incidence of cleft palate
after maternal administration of 100 (jug hexa-
chlorodibenzo-/7-dioxin/kg/day on days 6 to 15
of gestation to Sprague-Dawley rats. Couture et
al.142 also observed an increased incidence of cleft
palate formation after a single dose of 300 jxg/
kg of 2,3,4,7,8-pentachlorodibenzofuran given
to Fisher 344 rats. Similarly, cleft palate can be
produced in fetal hamsters following maternally
toxic and fetotoxic doses of TCDD.61
In monkeys, bifid uvula143 and bony defects
in the hard palate65 were reported, but there were
no corresponding soft tissue defects or clefts of
the secondary palate. Cleft palates have not been
reported in human fetuses of mothers accidentally
exposed to TCDD or mixtures of PCBs and
CDFs.17-115-117 Thus, sensitivity of the palate in
mice to TCDD is unique. In other species, in-
cluding humans, other forms of fetal toxicity oc-
cur at doses lower than those required for cleft
palate formation.
300
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2. Hydronephrosis
a. Characterization of TCDD Effect
Hydronephrosis is the most sensitive devel-
opmental response elicited by TCDD in mice. It
is produced by maternal doses of TCDD too low
to cause palatal clefting and is characterized as
a progressive hydronephrosis preferentially oc-
curring in the right kidney, which can be accom-
panied by hydroureter and/or abnormal nephron
development.77-102-144-147 Hyperplasia of the ur-
eteric lumenal epithelium results in ureteric ob-
struction. Therefore, the TCDD-induced kidney
malformation in the mouse is a true hydrone-
phrosis in that blockage of urine flow results in
back pressure damaging or destroying the renal
papilla.146
When dissected on gestational day 12 from
control embryos, isolated ureters exposed to
1 x 10~10 M TCDD in vitro display evidence of
epithelial cell hyperplasia.l48 This is significant
in that it shows that the hydronephrosis response
is due to the direct effect of TCDD on the ureteric
epithelium. Embryonic cell proliferation within
the ureter may be regulated by the actions of
growth factors, including EOF.148 In control ur-
eteric epithelia, the expression of EOF receptors
decreases with advancing development, whereas
after TCDD exposure, the rate of 3H-thymidine
incorporation and EGF receptor numbers do not
decline. Therefore, in TCDD-treated mice, there
is a correlation between excessive proliferation
of ureteric epithelial cells and increased expres-
sion of EGF receptors.
Other effects of TCDD on the developing
kidney involve changes in extracellular matrix
components and basal lamina.147 In TCDD-ex-
posed fetal kidneys, extracellular matrix fibers
are of a diameter consistent with type III collagen
similar to such fibers in unexposed fetal kidneys.
However, the abundance of these type III col-
lagen fibers is reduced by TCDD treatment. In
the developing kidney, these collagen fibers are
associated with undifferentiated mesenchymal
cells. Similarly, the expression of fibronectin,
which also is associated with undifferentiated
mesenchymal cells, is decreased by TCDD ex-
posure. In the glomerular basement membrane,
the distribution of laminin and type IV collagen
is altered by TCDD exposure. These changes in
the glomerular basement membrane may affect
the functional integrity of the filtration barrier
and could exacerbate the hydronephrosis and
hydroureter. The proteins within the extracellular
matrix and basal lamina that are altered by TCDD
exposure, i.e., laminin, fibronectin, and colla-
gen, are considered markers of a commitment to
differentiate into epithelial structures. In the
mouse embryo/fetus, TCDD exposure also blocks
differentiation within the epithelium of the de-
veloping palate. Although there are effects of
TCDD exposure on EGF in the developing ureter,
as well as the developing palate, the urinary sys-
tem, unlike parts of the soft palate, is derived
from mesoderm. Thus, it is important to note that
the ectodermal dysplasia syndrome is intended to
denote a clustering of effects that appear to in-
volve ectoderm-derived organs. It is not intended
to imply that all TCDD-induced developmental
toxicity involves organs derived from ectoderm.
b. Evidence for an Ah Receptor Mechanism
\. Genetic
With respect to involvement of the Ah locus
in TCDD-induced hydronephrosis, very few ge-
netic studies have been done. Prior to the dis-
covery of the Ah locus, Courtney and Moore77
reported a 62% incidence of hydronephrosis in
C57BL/6 mice exposed to a maternal TCDD dose
of 3 (jLg/kg/day on days 6 to 15 of gestation, whereas
the incidence in similarly exposed DBA/2 mice was
only 26%. More recently, Silkworth et al.149 re-
ported that when TCDD is administered on ges-
tational days 6 to 15 the incidence of hydrone-
phrosis is dose related. As the maternal dose of
TCDD is increased from 0.5 to 4 (xg/kg/day, the
incidence of hydronephrosis in C57BL/6 mice
increases from 31 to 92%; in DBA/2 mice, the
incidence varies from 5 to 37% over the same
dose range. In DBA/2 mice, the incidence of
hydronephrosis increases to 60% when the largest
dose of TCDD administered is doubled to 8 jxg/
kg/day (but does not reach the 92% level seen in
C57BL/6 mice at 4 ^.g TCDD/kg). Thus, the
incidence of hydronephrosis is higher in the mouse
strain that produces high-affinity Ah receptors
301
-------
(C57BL/6) compared to the strain (DBA/2) that
produces Ah receptors having lower ligand-bind-
ing affinity.150 The largest dose of TCDD used
in these experiments resulted in hydronephrosis
of the fetus without affecting the mean body
weight or body weight gain of the dam. In the
BXD strains,108 the incidence of 2,3,7,8,-TCDF-
induced hydronephrosis is 34 to 48% in eight
strains with high-affinity Ah receptors and 3 to
4% in eight strains with low-affinity Ah recep-
tors. These results obtained in the BXD strains
of mice provide the best evidence currently avail-
able of an association between the ability of
TCDD-like congeners to induce hydronephrosis
and the wild-type Ahb allele. Accordingly, the
Ah locus and the Ah receptor are involved in the
hydronephrosis that is induced by TCDD-like
congeners.
ii. Structure Activity
The rank order of potencies for various hal-
ogenated aromatic hydrocarbon congeners to
cause hydronephrosis in mice is consistent with
the structure-activity requirements for binding to
the Ah receptor (Table 4). This provides further
evidence that the Ah receptor mediates the effects
of these TCDD-like congeners on the developing
mouse kidney.
c. Species Differences
Hydronephrosis has been reported after ad-
ministration of low maternal doses of TCDD to
rats and hamsters. Possibly due to the small num-
bers of fetuses examined, the observed inci-
dences of hydronephrosis in rats after exposure
to cumulative maternal doses <2 (xg TCDD/kg
have not been statistically significant.77-151 On the
other hand, following a 1.5-^g TCDD/kg dose
administered on gestational days 7 and 9, the
incidence of hydronephrosis in hamster fetuses
was 11 and 4.2%, respectively. This is in contrast
to an incidence of <1% in control hamster fe-
tuses. Therefore, hydronephrosis is one of the
most sensitive indicators of prenatal toxicity57 in
hamsters and mice.
C. Postnatal Effects
1. Male Reproductive System of Rats
Because TCDD can decrease plasma andro-
gen concentrations and be transferred from mother
to young in utero and during lactation,152'153 it is
expected to have a great impact on the male re-
productive system during early development.154
Testosterone and/or its metabolite DHT are es-
sential prenatally and/or early postnatally for im-
printing and development of accessory sex
organs155-157 and for initiation of spermatogene-
sis.158 In addition, aromatization of testosterone
to 17p-estradiol within the CNS is required peri-
natally for the imprinting of typical adult male
patterns of reproductive behavior159 and LH se-
cretion.160 Thus, normal development of male
reproductive organs and imprinting of typical adult
sexual behavior patterns require sufficient tes-
tosterone be secreted by the fetal and neonatal
testis at critical times in early development before
and shortly after birth.161'162 If perinatal imprint-
ing fails to occur in the accessory sex organs of
a neonatal male rat, the results may be that these
organs will not develop a normal trophic response
to androgenic stimulation and will not function
normally as the animal becomes sexually mature.
a. Perinatal Androgen Deficiency
To determine if in utero and lactational ex-
posure to TCDD produces a perinatal androgenic
deficiency, Mably et al.154J63 dosed pregnant rats
with 1.0 (xg TCDD/kg on day 15 of gestation.
Plasma testosterone concentrations were greater
in control male than in control female fetuses on
days 17 to 21 of gestation, particularly during
the prenatal testosterone surge (days 17 to 19).
On days 18 to 21 of gestation, TCDD exposure
reduced the magnitude of this sex-based differ-
ence. Postnatally, plasma testosterone concentra-
tions peaked 2 h after birth in control males,
whereas in TCDD-exposed males, the peak did
not occur until 4 h after birth and was only half
as large. Thus, in male rats, perinatal exposure
to TCDD can produce both prenatal and early
postnatal androgenic deficiencies.
302
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b. Overt Toxicity Assessment
To determine how the male reproductive sys-
tem is affected by in utero and lactational TCDD
exposure. Mably et al.154'163-165 treated pregnant
rats with a single oral dose of TCDD (0.064,
0.16, 0.4, or 1.0 (xg/kg) or vehicle on day 15 of
gestation (day 0 = sperm positive). Day 15 was
chosen because most organogenesis in the fetus
is complete by this time and the hypothalamic/
pituitary/testis axis is just beginning to func-
tion.166"168 The pups were weaned 21 days after
birth, and the consequences of this single, ma-
ternal TCDD exposure for the male offspring
were characterized at various stages of postnatal
sexual development.
Mably et al.163 found that TCDD treatment
had no effect on daily feed intake during preg-
nancy and the first 10 days after delivery, nor
did it have any effect on the body weight of dams
on day 20 of gestation or on days 1, 7, 14, or
21 postpartum. Treating dams with graded doses
of TCDD on day 15 of gestation had no effect
on gestation index, length of gestation, or litter
size. Except for an 8% decrease at the highest
maternal dose, TCDD had no effect on live birth
index. Neither the 4-day nor 21-day survival in-
dex was significantly affected by TCDD. In all
dosage groups, the number of dead offspring was
equally distributed between males and females,
and of the females that failed to deliver litters,
none were pregnant. Signs of overt toxicity among
the offspring were limited to the above-men-
tioned 8% decrease in live birth index (highest
dose only), the initial 10 to 15% decreases in
body weight (two highest doses), and the initial
10 to 20% decreases in feed intake (measured for
males only, two highest doses). The latter two
effects disappeared by early adulthood, after
which the body weights of the maternally ex-
posed and nonexposed rats were similar. No male
or female offspring with gross external malfor-
mations were found.
c. Androgenic Status
Androgenic status of the male offspring,
which includes such parameters as plasma an-
drogen concentrations and androgen-dependent
structures and functions, was reduced by a single
maternal TCDD dose as low as 0.16 (xg/kg. Ano-
genital distance, which is dependent both on cir-
culating androgen concentrations and androgenic
responsiveness,169 was reduced in 1- and 4-day-
old male pups, even when slight decreases in
body length were considered. Testis descent, an
androgen-mediated development event that nor-
mally occurs in rats between 20 and 25 days of
age,170 was delayed =sl.7 days.
For accessory sex organs of an adult male
rat to grow normally and respond fully to andro-
gens, there is a critical period that starts before
birth and lasts until sexual maturity during which
adequate concentrations of androgens are nec-
essary.
155-157,171,172
To determine if perinatal
TCDD exposure affects postnatal growth of the
accessory sex organs, one rat from each litter
was sacrificed at 32, 49, 63, and 120 days of
age, corresponding to juvenile, pubertal, post-
pubertal, and mature stages of sexual develop-
ment, respectively. At each developmental stage,
dose-related decreases in seminal vesicle and
ventral prostate weights were found. These de-
creases could not be explained by decreases in
body weight.
There were trends (although not statistically
significant) for plasma testosterone and DHT
concentrations to be decreased at these times,
whereas plasma LH concentrations were gener-
ally unaffected. An exception was a 95% de-
crease in plasma LH concentration on postnatal
day 32 caused by a maternal TCDD dose of 1.0
M-g/kg. The lowest maternal TCDD dose to affect
a parameter of androgenic status was the lowest
dose tested — 0.064 (jig/kg. This dose resulted
in a significantly depressed ventral prostate weight
at 32 days of age. When ventral prostate weight
was indexed to body weight, however, 0.16 n,g
TCDD/kg was the lowest dose that caused a sig-
nificant reduction in relative ventral prostate
weight. Although there was no effect on the
body weight of male pups at this dose, 0.16 jig
TCDD/kg caused a consistent pattern of effects
that indicated a depression of androgenic status.
The reductions in seminal vesicle and ventral
prostate weights may be due to modest reductions
in plasma androgen concentrations and/or andro-
gen responsiveness caused by incomplete peri-
natal imprinting of the accessory sex organs.163
303
-------
Collectively, these results demonstrate that in
utero and lactational TCDD exposure decreases
androgenic status of male rats from the fetal stage
into adulthood. Table 5 summarizes these ef-
fects.154-163
d. Spermatogenesis
Mably et al.154-165 found that decreased sper-
matogenesis was among the most sensitive re-
sponses of the male rat reproductive system to
perinatal TCDD exposure. Testis and epididymis
weights and indices of spermatogenesis were de-
termined on postnatal days 32, 49, 63, and 120.
Perinatal TCDD exposure caused dose-related
decreases in testis and epididymis weights.
Weights of the caudal portion of the epididymis
where mature sperm are stored prior to ejacula-
tion were decreased the most, by approximately
45%. The number of sperm per cauda epididymis
was decreased by 75 and 65% on days 63 and
120, respectively, and appeared to be the most
sensitive effect of perinatal TCDD exposure on
the male reproductive system. Daily sperm pro-
duction was decreased by «43% at puberty, day
49, but the decrease was only 29% at sexual
maturity, day 120. Seminiferous tubule diameter
was decreased at all four developmental stages.
Each effect of TCDD was dose related, and in
all cases a significant decrease was seen in re-
sponse to the lowest maternal TCDD dose tested,
0.064 u,g/kg, during at least one stage of sexual
development. In general, the magnitude of the
decreases recovered with time, although not com-
pletely, thus suggesting that perinatal TCDD ex-
posure delays sexual maturation. These results
are summarized in Table 6.154'165
Severe pre- and/or postweaning undernutri-
tion can affect the reproductive system of adult
male rodents, including decreased spermatoge-
nesis.173-176 At the two highest maternal TCDD
doses, the feed consumption and body weight of
male offspring were decreased; even so, the
weight decreases did not exceed 21% of the con-
trol values.163 However, reductions in sex organ
weights, epididymal sperm reserves, and sper-
matogenesis occurred at the two lowest maternal
TCDD doses, neither of which reduced feed in-
take or body weight of the male offspring. Thus,
TABLE 5
Effects of In Utero and Lactational TCDD Exposure on Indices of Androgenic Status
Index
Anogenital distance
Time to testis descent
Plasma testosterone concentration
Plasma 5a-dihydrotestosterone concentration
Plasma LH concentration
Absolute seminal vesicle weight
Relative seminal vesicle weight*
Absolute ventral prostate weight
Relative ventral prostate weight0
Note: NS = not statistically significant.
Lowest effective maternal dose
TCDD/kg)-
Maximum effect"
0.1 6 (days 1 and 4)
0.16
NS
NS
1 .0 (day 32)
0.16 (days 32 and 63)
0.1 6 (day 63)
0.064 (day 32)
0.1 6 (days 32 and 63)
21 % decrease
1.7-day delay
69% decrease
59% decrease
95% decrease
56% decrease
50% decrease
60% decrease
53% decrease
(day 1)
(day 32)
(day 49)
(day 32)
(day 49)
(day 49)
(day 32)
(day 32)
8 The lowest dose of TCDD (given on day 15 of gestation) that caused a significant (p <0.05) effect in the male
offspring and the day or days at which this dose caused such an effect are shown.
b The magnitude of the greatest change seen in response to maternal dosing with 1.0 M-9/kQ TCDD and the day
at which this effect was seen are shown.
c Weight of organ divided by body weight of rat.
From Mably, T. A., Moore, R. W., Bjerke, D. L, and Peterson, R. E., Biological Basis for Risk Assessment of Dioxins
and Related Compounds, Gallo, M. A., Scheuplein, R. J., and van der Heijden, C. A., Eds., Branbury Report 35,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1991, 69.
304
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TABLE 6
Effects of In Utero and Lactational TCDD Exposure on Indices of Spermatogenic Function
and Reproductive Capability
Lowest effective maternal dose
(,ig TCDD/kg)-
0.40 (day 32)
0.064 (days 49, 120)
0.064 (days 63, 120)
0.064 (days 63, 120)
0.064 (days 63, 120)
0.064 (days 32, 49, 120)
0.40 (day 32)
NS
NS
NS
NS
Index
Testis weight
Epididymis weight
Cauda epididymis weight
Sperm per cauda epididymis
Daily sperm production rate
Seminiferous tubule diameter
Plasma FSH concentration
Leptotene spermatocyte: Sertoli cell ratio
Sperm motility; percentage abnormal sperm
Fertility
Gestation index; litter size; live birth index;
pup survival
Note: NS = not statistically significant.
• The lowest dose of TCDD (given on day 15 of gestation) that caused a significant (p <0.05) effect in the male
offspring and the day or days at which this dose caused such an effect are shown.
b The magnitude of the greatest change seen in response to maternal dosing with 1.0 jj-g/kg TCDD and the day
on which this effect was seen are shown.
From Mably, T. A., Moore, R. W., Bjerke, D. L, and Peterson, R. E., Biological Basis for Risk Assessment of
Dioxins and Related Compounds, Gallo, M. A., Scheuplein, R. J., and van der Heijden, C. A., Eds., Branbury
Report 35, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1991, 69.
Maximum effect"
17% decrease (day 32)
35% decrease (day 32)
53% decrease (day 63)
75% decrease (day 63)
43% decrease (day 49)
15% decrease (day 32)
15% decrease (day 32)
No dose-related effects
No dose-related effects
22% decrease (day 70)
No dose-related effects
undernutrition cannot account for these repro-
ductive system effects, including the decreases
in spermatogenesis observed at the lower mater-
nal TCDD doses.163-165
Since FSH and testosterone are essential for
quantitatively normal spermatogenesis,158 an al-
ternative explanation for the decreases in daily
sperm production is a decrease in FSH and/or
testosterone levels. In rats, the length of one sper-
matogenic cycle is 58 days,177"179 so the de-
creases in plasma FSH concentrations in 32-day-
old male offspring could contribute to the re-
ductions of spermatogenesis when the rats were
49 and 63 days of age. However, the modest
depressant effect of perinatal TCDD exposure on
plasma FSH concentrations was transitory; no
effect was found on plasma FSH levels when the
offspring were 49, 63, and 120 days old. It was
concluded that reduced spermatogenesis in 120-
day-old male rats, perinatally exposed to TCDD,
was not due to decreases in plasma FSH levels
when the animals were 49 to 120 days of age.165
Plasma testosterone concentrations in the
same rats were reduced =s69% by perinatal TCDD
exposure, yet intratesticular testosterone concen-
trations must be reduced by at least 80% in rats
before spermatogenesis is impaired.180 Based on
the magnitude of the reductions in plasma an-
drogen concentrations, it was concluded that cor-
responding reductions in testicular testosterone
production in perinatal TCDD-exposed offspring
would probably not be severe enough to impair
spermatogenesis.163>16S
In normal rats, daily sperm production does
not reach a maximum until 100 to 125 days of
age,181 but in rats perinatally exposed to TCDD,
it takes longer for sperm production to reach the
adult level. Furthermore, the length of the delay
is directly related to maternal TCDD dose,165 and
if the dose is high enough, the reduction in sper-
matogenesis may be permanent. This is sug-
gested by a maternal TCDD dose of 1.0 \igfkg
that decreased the daily sperm production in male
rat offspring that were 300 days of age.182 Be-
305
-------
cause the mechanism by which perinatal TCDD
exposure decreases spermatogenesis in adulthood
is unknown, it is unclear whether the irreversible
effect at the largest maternal dose, 1 M-g/kg, which
results in depressed feed consumption and de-
creased body weight, is caused by the same
mechanism as that at smaller maternal doses,
which do not result in undernutrition and from
which the male offspring may eventually re-
cover.
A key observation for postulating mecha-
nisms by which perinatal TCDD exposure re-
duces spermatogenesis in adulthood is the finding
that the ratio of leptotene spermatocytes per Ser-
toli cell in the testes of 49-, 63-, and 120-day-
old rats is not affected by in utero and lactational
TCDD exposure, even though daily sperm pro-
duction is reduced.165 Because Sertoli cells pro-
vide spermatogenic cells with functional and
structural support183 and the upper limit of daily
sperm production in adult rats is directly depen-
dent on the number of Sertoli cells per testis,184
three possible mechanisms for the decrease in
daily sperm production may be involved: TCDD
could (1) increase the degeneration of cells in-
termediate in development between leptotene
spermatocytes and terminal stage spermatids (the
cell type used to calculate daily sperm produc-
tion); (2) decrease post-leptotene spermatocyte
cell division (meiosis); and/or (3) decrease the
number of Sertoli cells per testis.185 Elucidating
the mechanism by which perinatal TCDD ex-
posure decreases spermatogenesis is important
because it is one of the most sensitive responses
of the male reproductive system to TCDD.
e. Epididymis
The epididymis has two functions: in prox-
imal regions, spermatozoa mature gaining the ca-
pacity for motility and fertility; in distal regions,
mature sperm are stored before ejaculation.186
Mably et al.154-165 found that motility and mor-
phology of sperm taken from the cauda epidi-
dymis on postnatal days 63 and 120 were unaf-
fected by perinatal TCDD exposure. Thus, no
effect of TCDD on epididymal function was de-
tected. The dose-dependent reduction in epi-
didymis and cauda epididymis weights in post-
pubertal rats 63 and 120 days of age can be
accounted for, in part, by decreased sperm pro-
duction. However, in immature males, 32 and
49 days of age, where sperm are not present in
the epididymis, the decrease in weights of epi-
didymal tissue cannot be explained by effects on
sperm production. Because epididymal growth is
androgen dependent, a TCDD-induced androgen
deficiency and/or decrease in androgren respon-
siveness of the epididymis could account for de-
creased size of the organ.187-188
f. Reproductive Capability
To assess reproductive capability, male rats
born to dams given TCDD (0.064, 0.16, 0.40,
or 1.0 (Jig/kg) or vehicle on day 15 of gestation
were mated with control virgin females when the
males were —70 days of age.154-165 The fertility
index of the males is defined as the number of
males impregnating females divided by the num-
ber of males mated. The two highest maternal
TCDD doses decreased the fertility index of the
male offspring by 11 and 22%, respectively.
However, these decreases were not statistically
significant, and at lower doses, the fertility index
was not reduced. The gestation index, defined
as the percentage of control dams mated with
TCDD-exposed males that delivered at least one
live offspring, also was not affected by perinatal
TCDD exposure. With respect to progeny of these
matings, there was no effect on litter size, live
birth index, or 21-day survival index. When peri-
natal TCDD-exposed males were mated again at
120 days of age, there was no effect on any of
these same parameters. Thus, despite pro-
nounced reductions in cauda epididymal sperm
reserves, when the TCDD-treated males were
mated, perinatal TCDD exposure had little or no
effect on fertility of male rats or on the survival
and growth of their offspring. These results are
summarized in Table 6.154-165
Because rats produce and ejaculate 10 times
more sperm than are necessary for normal fertility
and litter size,189'190 the absence of a reduction
in fertility of male rats exposed perinatally to
TCDD is not inconsistent with the substantial
reductions in testicular spermatogenesis and ep-
ididymal sperm reserves. In contrast, reproduc-
306
-------
tive efficiency in human males is very low; the
number of sperm per ejaculate is close to that
required for fertility.191 Thus, measures of fer-
tility using rats are not appropriate for low-dose
extrapolation in humans.192 A percent reduction
in daily sperm production in humans, similar in
magnitude to that observed in rats,154-165 may re-
duce fertility in men.
g. Sexual Differentiation of the CNS
Sexual differentiation of the CNS is depen-
dent on the presence of androgens during early
development. In rats, the critical period of sexual
differentiation extends from late fetal life through
the first week of postnatal life.161 In the absence
of adequate circulating levels of testicular andro-
gen during this time, adult rats display high levels
of feminine sexual behavior (e.g., lordosis), low
levels of masculine sexual behavior, and a cyclic
(i.e., feminine) pattern of LH secretion when
castrated and primed with ovarian steroids.160-194
In contrast, perinatal androgen exposure of rats
will result in the masculinization of sexually di-
morphic neural parameters, including reproduc-
tive behaviors, regulation of LH secretion, and
several morphological indices.193'194 The mech-
anism by which androgens cause sexual differ-
entiation of the CNS is not completely under-
stood. In the rat, it appears that ITp-estradiol,
formed by the aromatization of testosterone within
the CNS, is one of the principal active steroids
responsible for mediating sexual differentia-
tion;195 however, androgens also are involved.
i. Demasculinization of Sexual Behavior
Mably et al.154-164 assessed sexually dimor-
phic functions in male rats born to dams given
graded doses of TCDD or vehicle on day 15 of
gestation. Masculine sexual behavior was as-
sessed in male offspring at 60, 75, and 115 days
of age by placing a male rat in a cage with a
receptive control female and observing the first
ejaculatory series and subsequent postejaculatory
interval (Table 7). The number of mounts and
intromissions (mounts with vaginal penetration)
before ejaculation were increased by a maternal
TCDD dose of 1.0 M-g/kg. The same males ex-
hibited 12- and 11-fold increases in mount and
intromission latencies, respectively, and a 2-fold
increase in ejaculation latency. All latency effects
were dose related and significant at a maternal
TCDD dose as low as 0.064 |xg/kg (intromission
latency) and 0.16 n-g/kg (mount and ejaculation
latencies). Copulatory rates (number of mounts
+ intromissions/time from first mount to eja-
culation) were decreased to <43% of the control
rate. This effect on copulatory rates was dose
related, and a statistically significant effect was
observed at maternal TCDD doses as low as 0.16
M-g/kg. Postejaculatory intervals were increased
35% above the control interval, and a statistically
significant effect was observed at maternal doses
of TCDD as low as 0.40 |xg/kg. Collectively,
these results demonstrate that perinatal TCDD
exposure demasculinizes sexual behavior.
Since perinatal exposure to a maternal TCDD
dose of 1.0 ng/kg has no effect on the open field
locomotor activity of adult male rats,196 the in-
creased mount, intromission, and ejaculation la-
tencies appear to be specific for these masculine
sexual behaviors, not secondary to a depressant
effect of TCDD on motor activity. Postpubertal
plasma testosterone and DHT concentrations in
littermates of the rats evaluated for masculine
sexual behavior were as low as 56 and 62%,
respectively, of control.154'163 However, plasma
testosterone concentrations that were only 33%
of control were still sufficient to masculinize the
sexual behavior of adult male rats.197 Therefore,
the modest reductions in adult plasma androgen
concentrations following perinatal TCDD expo-
sure were not of sufficient magnitude to demas-
culinize sexual behavior.
Reductions in perinatal androgenic stimula-
tion can inhibit penile development and subse-
quent sensitivity to sexual stimulation in adult-
hood.198-199 Therefore, the demasculinization of
sexual behavior could, to some extent, be sec-
ondary to decreased androgen-dependent penile
development. However, perinatal TCDD expo-
sure had no effect on gross appearance of the rat
penis. In addition, TCDD-exposed males exhib-
ited deficits in such masculine sexual behaviors
as mount latency and postejaculatory interval,
which do not depend on stimulation of the penis
for expression.200 Thus, although some effects of
307
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TABLE 7
Effects of In Utero and Lactational TCDD Exposure on Indices of Sexual
Behavior and Regulation of LH Secretion in Adulthood
Index
Masculine sexual behavior
Mount latency
Intromission latency
Ejaculatory latency
Number of mounts
Number of intromissions
Copulatory rate (mounts plus
intromissions/minute)
Postejaculatory interval
Feminine sexual behavior1
Lordosis quotient8
Lordosis intensity score
Regulation of LH secretion
LH surge
Lowest effective maternal dose
(^.g/kg TCDD)'
0.16
0.064
0.16
0.064
1.0
0.16
0.40
0.16
0.40
0.40
Maximum effect"
1200%
1100%
97%
130%
38%
43%
increase
increase
increase
increase
increase
decrease
35% increase
300%
50%
increase
increase
460% increase'
• The lowest dose of TCDD (given on day 15 of gestation) that caused a significant (p <0.05)
effect in the male offspring is shown.
b The magnitude of the greatest change seen in response to maternal dosing with 1.0 |xg/
kg TCDD is shown (average of three trials for masculine behavior and two for feminine).
c Measured when the rats were ~60, 75, and 115 days of age.
" Feminine sexual behavior was measured following castration, estrogen priming, and
progesterone administration. The rats were 170-184 days old.
' Number of times lordosis was displayed in response to a mount divided by the number
of times each rat was mounted times 100.
1 Because control males do not secrete LH in response to progesterone, this percentage
was calculated by comparing peak plasma LH concentrations in TCDD-exposed rats with
plasma LH concentrations in control males at the same time after progesterone was
administered.
From Mably, T. A., Moore, R. W., Bjerke, D. L., and Peterson, R. E., Biological Basis for
Risk Assessment of Dioxins and Related Compounds, Gallo, M. A., Scheuplein, R. J., and
van der Heijden, C. A., Eds., Branbury Report 35, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1991, 69.
TCDD, such as decreased copulatory rate and
prolonged latency until ejaculation, could be due
to reduced sensitivity of the penis to sexual stim-
ulation, the 12-fold increase in mount latency and
increase in postejaculatory interval could not be
explained by this mechanism.
ii. Feminization of Sexual Behavior
Mabley et al.154-164 determined if the potential
of adult male rats to display feminine sexual be-
havior was altered by perinatal TCDD exposure.
Male offspring of dams treated on day 15 of
gestation with various doses of TCDD up to 1
(xg/kg or vehicle were castrated at ~ 120 days of
age and beginning at —160 days of age were
injected weekly for 3 weeks with 17p-estradiol
ben/oate, followed 42 h later by progesterone.
At 4 to 6 h after the progesterone injection on
weeks 2 and 3, the male was placed in a cage
with a sexually excited control stud male. The
frequency of lordosis in response to being
mounted by the stud male was increased from
18% (control) to 54% by the highest maternal
TCDD dose, 1.0 jig/kg (Table 7). Lordosis in-
tensity scored after Hardy and DeBold201 as a
(1) for light lordosis, (2) for moderate lordosis,
and (3) for a full spinal dorsoflexion was in-
creased in male rats by perinatal TCDD exposure.
308
-------
The effects on lordosis behavior in males were
dose related and significant at maternal TCDD
doses as low as 0.16 ^g/kg (increased lordotic
frequency) and 0.40 |xg/kg (increased lordotic
intensity). Together they indicate a feminization
of sexual behavior in these animals. Although
severe undernutrition from 5 to 45 days after birth
potentiates the display of lordosis behavior in
adult male rats,202 the increased frequency of lor-
dotic behavior was seen at a maternal TCDD dose
of 0.16 |xg/kg that had no effect on feed intake
or body weight. It was concluded that perinatal
TCDD exposure feminizes sexual behavior in
adult male rats independent of undernutrition.
iii. Feminization of LH Secretion Regulation
The effect of perinatal TCDD exposure on
regulation of LH secretion by ovarian steroids
was determined in male offspring at ~270 days
of age. There is normally a distinct sexual di-
morphism to this response. In rats castrated as
adults, estrogen-primed females greatly increase
their plasma LH concentrations when injected
with progesterone, whereas similarly treated
males fail to respond.203 Progesterone had little
effect on plasma LH concentrations in estrogen-
primed control males, but significant increases
were seen in males exposed to maternal TCDD
doses as low as 0.40 ng/kg. Thus, perinatal TCDD
exposure increases pituitary and/or hypothalamic
responsiveness of male rats to ovarian steroids in
adulthood, indicating that regulation of LH se-
cretion is feminized. Table 7 summarizes sexual
behavior and LH secretion results.154-164
iv. Comparison to Other Ah Receptor-
Mediated Responses
The induction of hepatic cytochrome
P-4501A1 and its associated EROD activity is an
extremely sensitive Ah receptor-mediated re-
sponse to TCDD exposure. Yet, in 120-day-old
male rats that had been exposed to TCDD peri-
natally, alterations in sexual behavior, LH secre-
tion, and spermatogenesis were observed when
induction of hepatic EROD activity could no
longer be detected. 154-163~165 These results suggest
that TCDD affects sexual behavior, gonado-
trophic function, and spermatogenesis when vir-
tually no TCDD remains in the body, and that
the demasculinization and feminization of sexual
behavior, feminization of LH secretion, and re-
duced spermatogenesis caused by in utero and
lactational exposure to TCDD is such that a con-
tinuing presence of TCDD is not required to pro-
duce these effects in adulthood.164-165
v. Possible Mechanisms and Significance
The most plausible explanation for the de-
masculinization and feminization of sexual be-
havior and feminization of LH secretion is that
perinatal exposure to TCDD impairs sexual dif-
ferentiation of the CNS. Undernutrition, altered
locomotor activity, reduced sensitivity of the penis
to sexual stimulation, and modest reductions in
adult plasma androgen concentrations of the male
offspring cannot account for these effects.164 On
the other hand, exposure of the developing brain
to testosterone, conversion of testosterone into
17(i-estradiol within the brain, and events initi-
ated by the binding of 17(3-estradiol to its recep-
tor are all critical for sexual differentiation of the
CNS. If TCDD interferes with any of these pro-
cesses during late gestation and/or early neonatal
life, it could irreversibly demasculinize and fem-
inize sexual behavior204^206 and feminize the reg-
ulation of LH secretion207-208 in male rats in adult-
hood.
Treatment of dams on day 15 of gestation
with 1.0 n-g TCDD/kg significantly decreases
plasma testosterone concentrations in male rat
fetuses on days 18 and 20 of gestation and in
male rat pups 2 h postpartum.163 Thus, the ability
of maternal TCDD exposure to reduce prenatal
and early postnatal plasma testosterone concen-
trations may account, in part, for the impaired
sexual differentiation of male rats exposed peri-
natally to TCDD. Other mechanisms that may
potentially contribute to the TCDD-induced im-
pairment in CNS sexual differentiation are a de-
crease in the formation of 17(3-estradiol from tes-
tosterone within the CNS, which is independent
of the decrease in plasma testosterone concen-
trations, and/or a reduction in responsiveness of
the CNS to estrogen during the critical period of
309
-------
sexual differentiation. The latter mechanism is
consistent with the Ah receptor-mediated anties-
trogen action of TCDD, which is described in
Section III. A.3 for rat and mouse uterus and for
estrogen-responsive MCF-7 and Hepa IclcV cells.
In utero and/or lactational exposure to TCDD
may cause similar effects in other animal species,
including nonhuman primates,209"211 in which
sexual differentiation is under androgenic con-
trol. Although social factors may account for
much of the variation in the sexually dimorphic
behavior in humans, there is evidence that pre-
natal androgenization influences both the sexual
differentiation of such behavior and the brain
hypothalamic structure.212"214
2. Neurobehavior
Because differentiated tissues derived from
ectoderm, i.e., skin, conjunctiva, nails, and teeth,
are sites of action of halogenated aromatic hy-
drocarbons in transplacentally exposed human in-
fants, another highly differentiated tissue derived
from ectoderm, the CNS, should be considered
a potential site of TCDD action. In support of
this possibility is the fact that sexual differentia-
tion of the CNS of adult male rats is irreversibly
altered in a dose-related fashion by perinatal ex-
posure to TCDD.154 164 As is shown later, in mice
transplacentally exposed to 3,3' ,4,4'-TCB, mon-
keys perinatally exposed to TCDD, and children
transplacentally exposed to a mixture of PCBs,
CDFs, and PCQs in the Yu-Cheng incident, the
CNS is affected by TCDD and other halogenated
aromatic hydrocarbons. Thus, functional CNS al-
terations, which may or may not be irreversible,
are observed following perinatal exposure to these
chemicals.
Ah receptors have been identified in rat
brain110 but may be associated with glial cells
rather than neurons.110'215 Following administra-
tion of 14C-TCDD in the rat, the highest concen-
trations TCDD-derived radioactivity are found in
the hypothalamus and pituitary. Much lower con-
centrations are found in the cerebral cortex and
cerebellum.216 In another study, the Ah receptor
was not detected in whole rat or mouse brain,
but was detected in the cerebrum of the hamster
and cerebrum and cerebellum of the guinea pig.217
Ah receptors appear to be absent in the human
frontal cortex.215
310
a. Mice
CD-I mice exposed transplacentally to
3,3',4,4'-TCB at a maternal oral dose of 32 mg/
kg administered on days 10 to 16 of gestation
exhibited neurobehavioral, neuropathological, and
neurochemical alterations in adulthood.218'220 The
neurobehavioral effects consisted of circling, head
bobbing, hyperactivity, impaired forelimb grip
strength, impaired ability to traverse a wire rod,
impaired visual placement responding, and im-
paired learning of a one-way avoidance task.218
The brain pathology in adult mice exhibiting this
syndrome consisted, in part, of alterations in syn-
apses of the nucleus accumbens.219 This sug-
gested that in utero exposure to 3,3',4,4'-TCB
may interfere with synaptogenesis of dopami-
nergic systems. In support of this possibility,
Agrawal et al.220 found that adult mice transpla-
centally exposed to 3,3',4,4'-TCB had decreased
dopamine levels and decreased dopamine recep-
tor binding in the corpus striatum, both of which
were associated with elevated levels of motor
activity. It was concluded that transplacental ex-
posure to 3,3',4,4'-TCB in mice may perma-
nently alter development of striatal synapses in
the brain.
Eriksson et al.221 examined the neurobehav-
ioral effects of 3,3',4,4'-TCB in NMRI mice
exposed to a single oral dose of 0.41 or 41 mg/
kg on postnatal day 10. Following sacrifice of
the mice on day 17, muscarinic receptor concen-
trations in the brain were significantly decreased,
at both dose levels. This effect was shown to
occur in the hippocampus but not in the cortex.
More recently, NMRI mice were exposed to the
same two doses of 3,3',4,4'-TCB similarly ad-
ministered on postnatal day 10.222 At 4 months
of age, the effects of PCB on locomotor activity
were assessed. At both dose levels, abnormal
activity patterns were exhibited in that the treated
mice were significantly less active than controls
at the onset of testing, but were more active than
controls at the end of the test period. This pattern
of effects can be interpreted as a failure to ha-
bituate to the test apparatus. In contrast to the
previous results with CD-I mice, circling or head
bobbing activities were not observed in these ani-
mals. Upon sacrifice after the activity testing was
complete, a small but statistically significant in-
crease (as opposed to the decrease found after
sacrifice on postnatal day 17) in the muscarinic
-------
receptor concentration of the hippocampus was
found in animals from the high-dose group. These
results suggest that the neurochemical effects of
3,3',4,4'-TCB are complex. Cholinergeric as well
as dopaminergic systems in the brain are in-
volved.
Of all the developmental and reproductive
endpoints reported in this section for laboratory
animals, the only ones that have not yet been
demonstrated to occur following perinatal ex-
posure to TCDD are the above-mentioned neuro-
toxic effects in mice. These have only been stud-
ied following perinatal exposure to 3,3'4,4'-TCB.
In addition, there is no evidence yet to show that
(1) among inbred mouse strains, having low- and
high-affinity Ah receptors, susceptibility to
3,3',4,4'-TCB-induced neurotoxicity segregates
with the Ah locus, or (2) the rank order binding
affinity of congeners for the Ah receptor corre-
lates with their rank order potency for causing
these neurotoxic effects in mice. The rapid me-
tabolism of 3,3',4,4'-TCB compared to the rel-
atively slow metabolism of TCDD in mice causes
some uncertainty about the potential involvement
of the Ah receptor in 3,3',4,4'-TCB-induced
neurotoxicity. Contributing to this uncertainty is
the hypothesis that 3,3',4,4'-TCB may produce
CNS effects by being converted to a hydroxylated
metabolite that is neurotoxic. While there is no
evidence for or against this hypothesis, there also
is no evidence for or against the Ah receptor-
mechanism hypothesis of 3,3',4,4'-TCB neuro-
toxicity. Further research is needed to test these
hypotheses. In so doing, it should become ap-
parent whether 3,3' ,4,4'-TCB-induced neurotox-
icity effects are relevant to TCDD-induced de-
velopmental toxicity.
b. Monkeys
Schantz and Bowman69 and Bowman et al.223
conducted a series of studies on the long-term
behavioral effects of perinatal TCDD exposure
in monkeys. Because these were the first studies
to evaluate the behavioral teratology of TCDD,
monkeys exposed to TCDD via the mother during
gestation and lactation were screened on a broad
selection of behavioral tests at various stages of
development.223 At the doses studied (5 or 25 ppt
in the maternal diet), TCDD did not affect reflex
development, visual exploration, locomotor ac-
tivity, or fine motor control in any consistent
manner.68 However, the perinatal TCDD expo-
sure did produce a specific, replicable deficit in
cognitive function.69 TCDD-exposed offspring
were impaired on object learning, but were un-
impaired on spatial learning. TCDD exposure also
produced changes in the social interactions of
mother-infant dyads.224 TCDD-exposed infants
spent more time in close physical contact with
their mothers. The pattern of effects was similar
to that seen in lead-exposed infants and suggested
that mothers were providing increased care to the
TCDD-exposed infants.224
c. Humans
The intellectual and behavioral development
of Yu-Cheng children transplacentally exposed
to PCBs, CDFs, and PCQs was studied through
1985 by Rogan et al.92 In Yu-Cheng children,
matched to unexposed children of similar age,
area of residence, and socioeconomic status, there
was a clinical impression of developmental or
psychomotor delay in 12 (10%) Yu-Cheng chil-
dren compared with 3 (3%) control children, and
of a speech problem in 8 (7%) Yu-Cheng children
vs. 3 (3%) control children. Also, except for
verbal IQ on the Wechsler Intelligence Scale for
Children, Yu-Cheng children scored lower than
control children on three developmental and cog-
nitive tests.92 Neurobehavioral data on Yu-Cheng
children obtained after 1985 show that the intel-
lectual development of these children continues
to lag somewhat behind that of matched control
children.93 As measured from 1985 through 1990,
this effect persists at least up to the age of 7
years, and it occurs in children born long after
the initial Yu-Cheng exposure.279 Also, Yu-Cheng
children are rated by their parents and teachers
to have a higher activity level, more health, habit,
and behavioral problems, and to have a temper-
amental clustering closer to that of a "difficult
child."93 It is concluded that, in humans, trans-
placental exposure to halogenated aromatic hy-
drocarbons can affect CNS function postnatally.
However, which congeners, TCDD-like vs.
nonTCDD-like, are responsible for the neurotox-
icity is unknown.
311
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Further research on the mechanism of these
postnatal neurobehavioral effects, dose-response
relationships, and reversibility of the alterations
is needed before the role of TCDD-like congeners
vs. nonTCDD-like congeners in causing this tox-
icity can be understood. Mechanisms that re-
spond uniquely to TCDD-like congeners may not
necessarily be involved inasmuch as three lightly
chlorinated, ortho-substituted PCB congeners,
2,4,4'-TCB, 2,2',4,4'-TCB, and 2,2',5,5'-TCB,
were detected in monkey brain following dietary
exposure to Aroclor 1016 and appear to be re-
sponsible for decreasing dopamine concentra-
tions in the caudate, putamen, substantia nigra,
and hypothalamus of these animals.225 These
nonplanar PCB congeners are believed to cause
these effects by acting through a mechanism that
does not involve the Ah receptor. On the other
hand, the results presented for mice and monkeys
suggest that TCDD-like congeners could be in-
volved in producing the observed postnatal neu-
robehavioral effects in humans.
D. Cross-Species Comparison of Effect
Levels
TCDD exposure levels that cause a variety
of developmental effects in different species are
summarized for fish in Table 8, birds in Table
9, and mammals in Table 10. Fertilized lake trout
eggs and Japanese medaka eggs were exposed to
different waterborne concentrations of 3H-TCDD.
Estimates of the amount of TCDD in these eggs
were then made from measurement of the TCDD-
derived radioactivity within them. Fertilized rain-
bow trout, chicken, ring-necked pheasant, and
eastern blue bird eggs were injected directly with
the indicated doses of TCDD. Thus, the doses
of TCDD given in Tables 8 and 9 for all fish and
bird species represent TCDD egg burdens in which
a significant portion of the dose may be present
within the yolk of the egg rather than the devel-
oping embryo.
Mammalian embryo/fetuses, on the other
hand, were exposed via administration of TCDD
to the pregnant female. Therefore, the doses given
in Table 10 are maternal TCDD doses in which
a significant portion of the dose may be retained
by the mother and never actually reach the em-
bryo/fetus. In some studies, pregnant rats and
rhesus monkeys were exposed to TCDD on a
chronic or subchronic basis, respectively. The
doses given in Table 10 for these particular stud-
ies represent the calculated maternal body bur-
dens at the time of conception. In rats, the du-
ration of chronic exposure was much longer than
the whole body elimination half-life for TCDD
in rats. Therefore, the body burden of TCDD
given for the rat is 92.8% of the calculated steady-
state body burden. In rhesus monkeys, the half-
life for whole body elimination of TCDD is longer
than was the duration of exposure prior to con-
ception. Therefore, the steady-state body burdens
that would be expected for rhesus monkeys ex-
posed to the different levels of dietary TCDD
TABLE 8
Cross-Species Comparison of NOAELS and LOAELS for TCDD Developmental
Toxicity in Fish
Species
Lake trout
Japanese medaka
Rainbow trout
Lake trout
Rainbow trout
Japanese medaka
Effect
Sac fry mortality
Lesions8
Sac fry mortality
Sac fry mortality
Sac fry mortality
Sac fry mortality
Lesions8
Exposure
Static waterborne
Static waterborne
Single injection
Static waterborne
Static waterborne
Single injection
Static waterborne
Egg Dose
(ng/kg)
34
<100
194
40
55
291
300
Effect
level
NOAEL
NOAEL
NOAEL
LOAEL
LOAEL
LOAEL
LOAEL
Ref.
14
22
275
23
14
275
22
Consist of a spectrum of effects, including hemorrhage in various areas, pericardial edema,
collapse of the yolk sphere, cessation of blood flow throughout the animal, and embryo mortality.
312
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TABLE 9
Cross-Species Comparison of NOAELS and LOAELS for TCDD Developmental
Toxicity in Birds
Species
Ring-necked pheasant
Eastern bluebird
Chicken
Chicken
Ring-necked pheasant
Ring-necked pheasant
Ring-necked pheasant
Eastern bluebird
Effect
Embryo mortality
Embryo mortality
Exposure
Single injection
Single injection
Egg Dose
(rig/kg)
Effect
level
Cardiac malformations Single injection
Embryo mortality Single injection
Embryo mortality Single injection
Embryo mortality Single injection
Embryo mortality Single injection
Embryo mortality Single injection
Ref.
100
1,000
9a
240
1,000
1,354"
2,182C
10,000
NOAEL
NOAEL
LOAEL
La*,
LOAEL
LD50
LD50
LOAEL
31
41
42
32
33
30
31
31
31
41
Chi-square analysis of the data in Table 1 of Cheung et al.32 demonstrated that the incidence of cardiac
malformations in all embryos examined at dose levels of 1.6 pmol/egg or greater are significantly
(p <0.05) increased compared to the incidence in the control group designated "all examined." As-
suming a 55-g egg weight, 1.6 pmol/egg corresponds to a TCDD egg burden of 9 ng/kg.
Injected into the egg albumin.
Injected into the egg yolk.
intake are approximately 3 times greater than the
maternal body burdens estimated at the time of
conception (Table 10). Both in rats and rhesus
monkeys, the maternal body burdens are calcu-
lated using a one-compartment open model, as-
suming 86.1% bioavailability for TCDD. The
bioavailability used for TCDD was determined
in rats.226 Because no estimate for TCDD bio-
availability has been reported in rhesus monkeys,
the same 86.1% value was used. The whole body
elimination half-life used for TCDD in the rat is
23.7 days.226 McNulty et al.227 estimated a half-
life of approximately 1 year for TCDD elimi-
nation from adipose tissue in the rhesus monkey;
for calculation of the body burdens estimated in
Table 9, this half-life was rounded to 400 days
for whole body elimination. The maternal body
burden given for chronic exposure in the rat was
calculated from the data of Murray et al.228 The
maternal body burdens given for subchronic ex-
posure in the rhesus monkey were calculated from
data obtained from Bowman,229 which included
the daily dietary TCDD exposure level for each
pregnant female used in the studies reported by
Bowman et al.68'223 and Schantz and Bowman.69
Bowman's results indicate that the range of TCDD
half-lives in these monkeys was 200 to 600 days,
which is consistent with the results of McNulty
et al.227 The body burdens estimated for rhesus
monkeys used in these studies are averages based
on the average daily TCDD consumption of all
pregnant females used at a particular level of
maternal TCDD exposure.
As summarized in Table 8, lake trout and
rainbow trout sac fry and Japanese medaka em-
bryos are similarly affected by a spectrum of
lesions, which includes hemorrhage, edema, col-
lapse of the yolk sac, cessation of blood flow,
and embryo mortality. Estimates of the NOAEL
and LOAEL are given in the table for the ap-
pearance of these lesions in Japanese medaka
embryos and for embryo mortality in the two
trout species. Fertilized lake trout eggs and Jap-
anese medaka eggs were exposed to various
TCDD concentrations dissolved in static water,
and fertilized rainbow trout eggs were injected
directly with TCDD; however, the egg doses given
in Table 8 represent the concentration of TCDD
within the eggs themselves. Therefore, the dif-
ferent NOAELs and LOAELs for developmental
toxicity in different fish species probably repre-
sent species differences in susceptibility to TCDD-
induced developmental toxicity rather than dif-
ferences in the method of TCDD exposure. Of
the three fish species, lake trout sac fry are the
most sensitive to TCDD-induced mortality. How-
313
-------
ever, based on the LOAELs shown in Table 8,
the difference in susceptibility between fish spe-
cies may be less than tenfold.
Based on the LOAELs shown in Table 9, the
sensitivity of different bird species to TCDD-
induced embryo mortality varies more than 40-
fold. The chicken embryo is more susceptible to
TCDD-induced mortality than are embryos of the
ring-necked pheasant and eastern blue bird. In
addition, chicken embryos are highly sensitive to
the formation of TCDD-induced structural de-
fects in the heart and aortic arch. The incidence
of cardiac malformations in the chicken embryo
is increased at an egg exposure level as low as
9 ng TCDD/kg egg. However, such cardiac mal-
formations have not been found in any other bird
species examined.
Table 10 summarizes the levels of TCDD
exposure that cause certain structural malfor-
mations, functional alterations, and prenatal mor-
tality in the embryo/fetus of different mammalian
species. Based on the LOAELs given in Table
10, functional alterations in learning behavior and
the male reproductive system occur at lower
TCDD doses than those required to produce
structural malformations. Although TCDD-in-
duced developmental toxicity has been exten-
sively studied in mice and rats, the LOAELs in
Table 10 indicate that the embryo/fetus of rodent
species is generally not as sensitive to TCDD-
TABLE 10
Cross-Species Comparison of NOAELS and LOAELS for TCDO Developmental Toxicity
in Mammals
Species
Monkey
Rat
Rat
Mouse
Mouse
Monkey
Rat
Monkey
Rabbit
Rat
Rabbit
Rat
Mouse
Guinea pig
Hamster
Mouse
Hamster
Mouse
Effect
Prenatal mortality
Prenatal mortality
Prenatal mortality
Hydronephrosis
Cleft palate
Object learning
Male reproductive
Prenatal mortality
Extra ribs
Fetal growth
Prenatal mortality
Prenatal mortality
Hydronephrosis
Prenatal mortality
Thymic hypoplasia
Cleft palate
Prenatal mortality
Prenatal mortality
Exposure
Multiple dose
1 ng/kg/day
Multiple dose
Multiple dose
Multiple dose
0.126 ng/kg/day
Single dose
0.642 ng/kg/day
Multiple dose
Multiple dose
Multiple dose
Multiple dose
10 ng/kg/day
Multiple dose
Multiple dose
Single dose
Single dose
Multiple dose
Single dose
Single dose
Maternal dose
22 ng/kg, 9 x , gd 20-40
27 ng/kg,a chronic
30 ng/kg/day, gd 6-15
100 ng/kg/day, gd 6-15
300 ng/kg/day, gd 6-15
19 ng/kg,a subchronic
64 ng/kg, gd 15
97 ng/kg,a subchronic
111 ng/kg, 9 x, gd 20-40
100 ng/kg/day, gd 6-15
125 ng/kg/day, gd 6-15
250 ng/kg/day, gd 6-15
270 ng/kg," chronic
500 ng/kg/day, gd 6-15
500 ng/kg/day, gd 6-15
1,500 ng/kg, gd 14
1,500 ng/kg, gd 7 or 9
3,000 ng/kg/day, gd 6-15
18,000 ng/kg/day, gd 7 or 9
24,000 ng/kg/day, gd 6
Effect
level
NOAEL
NOAEL
NOAEL
NOAEL
NOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
LOAEL
Ref.
74
228
62
276
59
69
163-165
69
74
79
62
79
228
62
149
57
57
77
61
60
Note: gd = gestational day.
Maternal body burdens of TCDD at the time of conception were calculated by assuming a one-compartment
open model and a half-life for whole body TCDD elimination of 400 days in the monkey227 and 23.7 days
in the rat.226 A bioavailability of 86.1% was used in the monkey and rat.226 The daily dietary exposure levels
in rhesus monkeys were approximately 5 and 25 ppt at the NOAEL and LOAEL doses, respectively. Rhesus
monkeys were exposed to these levels of TCDD for 7 months prior to conception. At this time (0.525 half-
lives), the accumulated amount of TCDD in rhesus monkeys was 30.5% of the calculated steady-state level.
Rats were exposed to the indicated daily doses of TCDD for a period of 90 days (3.8 half-lives) prior to
conception. At this time, the accumulated amount of TCDD in rats was 92.8% of the calculated steady-state
level.
314
-------
induced prenatal mortality as is the embryo/fetus
of the rhesus monkey. The sensitivity of the em-
bryo/fetus to TCDD-induced prenatal mortality
in different mammalian species varies approxi-
mately 240-fold. This is in contrast to the 1000-
to 5000-fold variation in the LD50 of TCDD when
adult animals of these same species are exposed.
The agreement between studies with respect to
the LOAEL in Table 9 for prenatal mortality in
rats and monkeys is particularly striking. The
500-ng/kg dose of TCDD on gestational days 6
to 15 that caused prenatal mortality in rats62 agrees
with the maternal TCDD body burden of 270
ng/kg calculated from the chronic exposure228 to
within a factor of 2. Similarly, the TCDD dose
of 111 ng/kg that was given to rhesus monkeys
9 times during the first trimester of pregnancy74
agrees with the maternal body burden of 97 ng/kg
that increased prenatal mortality in rhesus mon-
keys following subchronic dietary exposure.69
I. REPRODUCTIVE TOXICITY
A. Female
1. Reproductive Function/Fertility
TCDD and its approximate isostereomers
have been shown to affect female reproductive
endpoints in a variety of animal studies. Among
the effects reported are reduced fertility, reduced
litter size, and effects on the female gonads and
menstrual/estrous cycle. These studies are re-
viewed next. Other TCDD effects on pregnancy
maintenance, embryo/fetotoxicity, and postnatal
development were covered in Section II.
The study by Murray et al.228 used a multi-
generational approach to examine the reproduc-
tive effects of exposure of male and female rats
over three generations to relatively low levels of
TCDD (0, 0.001, 0.01, and 0.1 |Ag/kg/day). There
was variation in the fertility index in both the
control and the exposed groups, and a lower than
desirable number of impregnated animals in the
exposed groups. Even so, the results showed ex-
posure-related effects on fertility, an increased
time between first cohabitation and delivery, and
a decrease in litter size. The effects on fertility
and litter size were observed at 0.1 jxg/kg/day in
the F0 generation and at 0.01 jxg/kg/day in the
FI and F2 generations. Additionally, in a 13-week
exposure to 1 to 2 (xg/kg/day of TCDD in non-
pregnant female rats, Kociba et al.230 reported
anovulation and signs of ovarian dysfunction, as
well as suppression of the estrous cycle. How-
ever, at exposures of 0.001 to 0.01 |xg/kg/day in
a 2-year study, Kociba et al.231 reported no effects
on the female reproductive system.
Allen and colleagues reported on the effects
of TCDD on reproduction in the monkey.70"73 In
a series of studies, female rhesus monkeys were
fed 50 or 500 ppt TCDD for ^9 months. Females
exposed to 500 ppt showed obvious clinical signs
of TCDD toxicity and lost weight throughout the
study. Five of the eight monkeys died within 1
year after exposure was initiated. Following 7
months of exposure to 500 ppt TCDD, seven of
the eight females were bred to unexposed males.
The remaining monkey showed such severe signs
of TCDD toxicity that she was not bred due to
her debilitated state. Of the seven females that
were evaluated for their reproductive capabili-
ties, only three were able to conceive and, of
these, only one was able to carry her infant to
term.72 When females exposed to 50 ppt TCDD
in the diet were bred at 7 months, two of eight
females did not conceive and four of six that did
conceive could not carry their pregnancies to term.
One monkey delivered a stillborn infant and only
one conception resulted in a live birth.73 As de-
scribed in an abstracted summary, these results
at 50 and 500 ppt TCDD were compared to a
group of monkeys given a dietary exposure to
PBB (0.3 ppm, Firemaster FF-1) in which seven
of seven exposed females were able to conceive,
five gave birth to live, normal infants and one
gave birth to a stillborn infant.71 Although the
effects at 500 ppt TCDD may be associated with
significant maternal toxicity, this would not ap-
pear to be the case at the lower dose. After 50
ppt TCDD, there were no overt effects on ma-
ternal health, but the ability to conceive and
maintain pregnancy was reduced.71
In a similar series of experiments, female
rhesus monkeys were fed diets that contained 0,
5, and 25 ppt TCDD.68 69 Reproductive function
was not altered in the 5 ppt group since, after 7
months of dietary exposure to TCDD, seven of
315
-------
eight females mated to unexposed males were
able to conceive. Six of these females gave birth
to viable infants at term and one gave birth to a
stillborn infant. This was not significantly dif-
ferent from the results of the control group, which
was fed a normal diet that contained no TCDD.
All seven of the monkeys in this control group
were able to conceive and gave birth to viable
infants. The 25-ppt dietary exposure level, how-
ever, did affect reproductive function. Only one
of the eight females in this group that was mated
gave birth to a viable infant. As in the 50 ppt
group from earlier studies, there were no serious
health problems exhibited by any females ex-
posed to 0, 5, or 25 ppt TCDD. Therefore, the
results in the 25- and 50-ppt groups suggest that
maternal exposure to TCDD, before and during
pregnancy, can result in fetal mortality without
producing overt toxic effects in the mother.
McNulty74 examined the effect of a TCDD
exposure during the first trimester of pregnancy
(gestational age, 25 to 40 days) in the rhesus
monkey. At a total dose of 1 fxg/kg given in nine
divided doses, three of four pregnancies ended
in abortion, and two of these abortions occurred
in animals that displayed no maternal toxicity.
At a total dose of 0.2 |Ag/kg, one of four preg-
nancies ended in abortion. This did not appear
different from the control population, but the low
number of animals per group did not permit sta-
tistical analysis. McNulty74 also administered
single l-(JLg/kg doses of TCDD on gestational day
25, 30, 35, or 40. The number of animals per
group was limited to three, but it appeared that
the most sensitive periods were the earlier pe-
riods, days 25 and 30, and that both maternal
toxicity and fetotoxicity were reduced when
TCDD was given on later gestational days. For
all days at which a single l-|o,g TCDD/kg dose
was given (gestational day 25, 30, 35, or 40),
10 of 12 pregnancies terminated in abortion. Thus,
for 16 monkeys given 1 u.g TCDD/kg in single
or divided doses between days 25 and 40 of preg-
nancy, there were only 3 normal births.65-74
In conclusion, the primary effects of TCDD
on female reproduction appear to be decreased
fertility, inability to maintain pregnancy for the
full gestational period, and, in the rat, decreased
litter size. In a few studies, some signs of ovarian
dysfunction, such as anovulation and suppression
of the estrous cycle, have been reported.71-72230
Unfortunately, the amount of attention that has
been given to the female reproductive system,
especially in the nonpregnant state, has been lim-
ited. In addition, there is little information about
how TCDD toxicity involving the mother and/or
placenta may affect fetal development.
2. Alterations in Hormone Levels
The potential for TCDD to alter circulating
female hormone levels has been examined, but
only to a very limited extent. In monkeys fed a
diet that contained 500 ppt TCDD for =£9 months,
the length of the menstrual cycle and the intensity
and duration of menstruation were not appreci-
ably affected by TCDD exposure.72 However,
there was a decrease in serum estradiol and pro-
gesterone concentrations in five of the eight ex-
posed monkeys, and in two of these animals, the
reduced steroid concentrations were consistent
with anovulatory menstrual cycles. In summary
form, Allen et al.71 described the effects of di-
etary exposure of female monkeys to 50 ppt
TCDD. After 6 months of exposure to this lower
dietary level of TCDD, there was no effect on
the serum estradiol and progesterone concentra-
tions of these monkeys. Thus, the presence of
these hormonal alterations is dependent on the
level of dietary TCDD exposure. Shiverick and
Muther232 reported that there was no change in
circulating levels of estradiol in the rat after ex-
posure to 1 (Jtg/kg/day TCDD on gestational days
4 to 15. Taking all of these results together, the
effect of TCDD exposure on circulating female
hormone levels may depend both on species and
level of exposure. It appears that any significant
effect is only seen at relatively high exposure
levels, although more research needs to be done
to examine the effects of TCDD on female hor-
mones.
3. Antiestrogenic Action
a. In Vivo
Estrogens are necessary for normal uterine
development and for maintenance of the adult
316
-------
uterus. The cyclic production of estrogens par-
tially regulates the cyclic production of FSH and
LH that results in the estrous cycling of female
mammals. In addition, estrogens are necessary
for the maintenance of pregnancy. Any effect that
causes a decrease in circulating or target cell es-
trogen levels can alter normal hormonal balance
and action.
Early experimental results in rats indicated
that chronic exposure to TCDD can cause
suppression or inhibition of the estrous cycle.230
Rhesus monkeys chronically exposed to TCDD,
on the other hand, are affected by hormonal ir-
regularities in their menstrual cycles.70'72 Al-
though the authors of these studies in rats and
monkeys did not attribute the effects of TCDD
exposure to an antiestrogenic action, it seems
reasonable to posit from more recent information
that such an antiestrogenic action may have been
responsible.
In rhesus monkeys, the severity of the TCDD-
associated reproductive alterations was correlated
with decreased plasma levels of estrogen and pro-
gesterone.72 Thus, one possible mechanism for
these effects would be increased metabolism of
estrogen and progesterone due to induction by
TCDD of hepatic microsomal enzymes and/or a
decrease in the rate at which these steroids are
synthesized. On the other hand, serum concen-
trations of 17(3-estradiol are not significantly af-
fected when TCDD is administered to pregnant
rats.232 Thus, an alternative mechanism for
TCDD-associated reproductive dysfunction could
involve effects of TCDD on gonadal tissue itself,
such as a decrease in its responsiveness to estro-
gen. In support of this latter mechanism, the
administration of TCDD to CD-1 mice results in
a decreased number of cytosolic and nuclear es-
trogen receptors in hepatocytes and uterine cells.
Although TCDD treatment induces hepatic cyto-
chrome P-450 levels in these animals, it has no
effect on serum concentrations of IVfJ-estra-
diol.233 This indicates that the antiestrogenic ef-
fect of TCDD in CD-1 mice is not caused by a
decrease in circulating levels of estrogen.
Effects of estrogen on the uterus include a
cyclic increase in uterine weight, increased ac-
tivity of the enzyme peroxidase, and an increase
in the tissue concentration of progesterone re-
ceptors. Antiestrogenic effects of TCDD admin-
istration to female rats include a decrease in uter-
ine weight, a decrease in uterine peroxidase
activity, and a decrease in the concentration of
progesterone receptors in the uterus.234 In addi-
tion, when TCDD and 17(i-estradiol are coad-
ministered to the same female rat, the antiestro-
genic action of TCDD diminishes or prevents
17p-estradiol-induced increases in uterine weight,
peroxidase activity, progesterone receptor con-
centration, and expression of EGF receptor
mRNA 234,235 similarly in mice, TCDD admin-
istration decreases uterine weight and antago-
nizes the ability of 17p-estradiol to increase uter-
ine weight.236
The ability of TCDD to antagonize the effects
of exogenously administered estrogen in the rat
is dependent on the age of the animal. In 21-day-
old rats, TCDD does not affect 17(i-estradiol-
induced increases in uterine weight of proges-
terone receptor concentration. On the other hand,
in 28-day-old intact rats and 70-day-old ovariec-
tomized rats, both of these 17p-estradiol-me-
diated responses are attenuated by TCDD.234 Pre-
viously, it was reported that TCDD administration
does not alter the dose-dependent increase in uter-
ine weight due to exogenously administered es-
trone in sexually immature rats.237 The later work
by Safe et al.234 suggest that this apparent lack
of an antiestrogenic effect of TCDD may have
been due to the young age of the rats used.
b. In Vitro
Both TCDD and progesterone can cause a
decrease in the nuclear estrogen receptor con-
centration in rat uterine strips. However, the ef-
fect of progesterone is inhibited by actinomycin
D, cycloheximide, and puromycin, whereas the
effect of TCDD is inhibited only by actinomycin
D. The reasons why the TCDD-induced decrease
in nuclear estrogen receptors is blocked by a tran-
scription inhibitor, but not by protein synthesis
inhibitors, are not known. However, these results
indicate that TCDD and progesterone decrease
the nuclear estrogen receptor concentration via
different mechanisms.238 In addition, the anties-
trogenic actions of TCDD can be demonstrated
in cell culture, and two prominent mechanisms
could potentially be involved: (1) increased me-
317
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tabolism of estrogen due to Ah receptor-mediated
enzyme induction; and (2) a downregulation of
estrogen receptors within the target cell.
In MCF-7 cells, which are estrogen-respon-
sive cells derived from a human breast adeno-
carcinoma, antiestrogenic effects caused by the
addition of TCDD to the culture medium include
a reduction of the 17p-estradiol-induced secre-
tion of a 160-, 52-, and 34-kDa protein.239 The
last two proteins are believed to be procathepsin
D and cathepsin D, respectively. In addition,
treatment of MCF-7 cells with TCDD suppresses
the 17(3-estradiol-enhanced secretion of tissue
plasminogen activator (tPA) and inhibits estro-
gen-dependent postconfluent cell prolifera-
tion.240'241 Thus, cultured MCF-7 cells have sev-
eral estrogen-dependent responses that are
inhibited by TCDD; this characteristic makes them
a useful model system for studying the antiestro-
genic actions of dioxin.
In cultured MCF-7 cells, TCDD treatment in-
duces AHH activity, the hallmark response of Ah
receptor binding, and increases hydroxylation of
17p-estradiol at the C-2, C-4, C-6ct, and C-15a
positions. It turns out that the particular cyto-
chrome P-450 that catalyzes the C-2, C-15a, and
C-6a hydroxylations of 17(3-estradiol is cyto-
chrome P-4501A1, which is identical to AHH.243
TCDD treatment also results in reduced levels of
occupied nuclear estrogen receptors.244 In MCF-7
cells, these results indicate that the antiestrogenic
effect of TCDD could result from (1) the in-
creased metabolism of estrogens due to Ah re-
ceptor-mediated enzyme induction, and/or (2) a
decreased number of estrogen receptors in the
nucleus. Safe's group has published TCDD-con-
centration response information for both the
TCDD-induced decrease in occupied nuclear es-
trogen receptors245 and the induction of AHH and
EROD activities in MCF-7 cells.244 In addition,
they have reported that TCDD causes a decreased
number of cytosolic and nuclear estrogen recep-
tors in Hepa Iclc7 cells, which is a mouse hep-
atoma cell line.246 Independent analysis of the
data suggests that the EC50 values for these ef-
fects are not dissimilar enough to distinguish be-
tween the proposed mechanisms. Instead, it ap-
pears as though TCDD induces the enzymes AHH
and EROD over the same concentration range
that it causes a decreased concentration of oc-
cupied nuclear estrogen receptors in MCF-7 cells.
In Hepa Iclc7 cells, the lowest concentration
used was 10 pM. Although exposure to 10 pM
TCDD resulted in a statistically significant down-
regulation of estrogen receptors, Israel and
Whitlock247 reported that this concentration is the
approximate EC50 for the induction of cyto-
chrome P-4501A1 mRNA and enzyme activity
in these cells. Therefore, in Hepa Iclc7 cells, as
well as in MCF-7 cells, it would appear that the
TCDD concentrations required to produce en-
zyme induction and reduction in occupied nuclear
estrogen receptor levels are not dissimilar enough
to distinguish between the two proposed mech-
anisms.
More recently, Safe's group used an analog
of TCDD, i.e., MCDF, that inhibits the 17(3-
estradiol-induced secretion of the 34-, 52-, and
160-kDa proteins and downregulates estrogen re-
ceptors in MCF-7 cells. These effects occurred
at concentrations of MCDF for which there was
no detectable induction of EROD activity.248 In
addition, it has been stated that the downregu-
lation of estrogen receptors in Hepa Iclc7 cells
can be detected as early as 1 h after exposure of
the cell cultures to 10 nM TCDD.246 This time
is slightly less than the 2 h required by Israel and
Whitlock247 to detect an increase in cytochrome
P-4501A1 mRNA levels after exposure of Hepa
Iclc7 cells to 10 pM TCDD. After exposure of
Hepa Iclc7 cells to a maximally inducing con-
centration of 1 nM TCDD, however, there are
significant increases in the cellular concentration
of cytochrome P-4501A1 mRNA after 1 h,
whereas the induction of AHH activity takes
slightly longer.247
Gierthy et al.240 reported that exposure of
MCF-7 cells to 1 nM TCDD caused suppression
of the 17(3-estradiol-induced secretion of tPA.
This effect of TCDD, however, occurred in the
absence of any measurable decrease in the whole
cell concentration of estrogen receptors. Gier-
thy's group pretreated their cultures with serum-
free medium, which was done to reduce cell pro-
liferation and maximize the cellular content of
estrogen receptors. The disparity between this
result of Gierthy et al.,240 which suggests no ef-
fect of TCDD on the estrogen receptor content
of MCF-7 cells, and the contrary results of Safe's
group in this same cell line remains largely unex-
318
-------
plained. Overall, it appears as though no obvious
distinction between the two proposed mecha-
nisms can be made at the present time. Therefore,
it seems that the antiestrogenic effect of TCDD
results from both the increased metabolism of
estrogen and a decreased number of estrogen re-
ceptors. It is important to note that TCDD does
not compete with radiolabeled estrogens or pro-
gesterone for binding to estrogen or progesterone
receptors, and that these steroids do not bind to
the Ah receptor or compete with radiolabeled
TCDD for binding.238'249
c. Evidence for an Ah Receptor Mechanism
\. Ah Receptor Mutants
Although the antiestrogenic effects of TCDD
may be caused by either a decreased number of
occupied nuclear estrogen receptors and/or the
increased metabolism of estrogen, there is evi-
dence that the antiestrogenic effect of TCDD is
mediated by the Ah receptor. Thus, the anties-
trogenic effects of TCDD in cultured cells appear
to involve an Ah receptor-mediated alteration in
the transcription of genes. This is indicated by
studies using wild-type Hepa Iclc7 cells and mu-
tant Hepa IclcV cells in culture.246 In wild-type
cells, TCDD reduces the number of nuclear es-
trogen receptors and this response can be inhib-
ited by cycloheximide and actinomycin D. How-
ever, in class 1 mutants, which have relatively
low Ah receptor levels, TCDD has only a small
effect. Similarly, in class 2 mutants, which have
a defect in the accumulation of transcriptionally
active nuclear Ah receptors, there was no effect
of TCDD on the number of nuclear estrogen re-
ceptors. Taken together, these results indicate that
the downregulation of estrogen receptors in Hepa
IclcV cells involves an Ah receptor-mediated ef-
fect on gene transcription. As previously noted,
TCDD induces cytochrome P-4501A1 mRNA
transcription and enzyme activity in Hepa Iclc?
cells.247 This effect also is Ah receptor me-
diated.127
ii. Structure-Activity Relationships In Vivo
Relative potencies of halogenated aromatic
hydrocarbon congeners as inhibitors of uterine
peroxidase activity in the rat are similar to their
relative Ah receptor-binding affinities.250 Only
limited relative potency information is available
for the reduction of hepatic and uterine estrogen
receptor concentrations per se by these sub-
stances in rats. TCDD and 1,2,3,7,8-PeCDD both
exhibit high affinity for the Ah receptor. At an
80-n,g/kg dose of either of these two substances,
hepatic estrogen receptor concentrations are re-
duced 42 and 41%, whereas uterine estrogen re-
ceptor concentrations are reduced 53 and 49%
by TCDD and 1,2,3,7,8-PeCDD, respectively.
On the other hand, 1,3,7,8-TCDD and 1,2,4,7,8-
PeCDD bind less avidly to the Ah receptor. At
a 400-jig/kg dose of either of these two sub-
stances, hepatic estrogen receptor concentrations
are reduced 36 and 40%, whereas uterine estro-
gen receptor concentrations are reduced 21 and
24% by 1,3,7,8-TCDD and 1,2,4,7,8-PeCDD,
respectively.249 Because the potency of these con-
geners for reducing estrogen receptor concentra-
tions correlates with their Ah receptor-binding
affinities, these in vivo results provide evidence
that the antiestrogenic effect of TCDD is me-
diated by the Ah receptor.
iii. Genetic Evidence
Consistent with the interpretation based on
structure-activity relationships, there is a greater
reduction in the number of hepatic estrogen re-
ceptors when AhbAhb C57BL/6 mice are exposed
to TCDD than when AhdAhd DBA/2 mice are
similarly exposed.251 To date, however, the anti-
estrogenic effects have not been studied in the
progeny of test crosses between AhbAhb and
AhdAhd mouse strains that, respectively, produce
Ah receptors with high or low binding affinity
for TCDD. Therefore, the potential segregation
of the antiestrogenic effects of TCDD with the
Ah locus has not been verified by the results of
genetic crosses.
iv. Structure-Activity Relationships In Vitro
The Ah receptor is detectable in MCF-7 cells,
and AHH as well as EROD activities are both
inducible in these cells.245 The relative abilities
of TCDD and other CDD, CDF, and PCB con-
319
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curs perinatally rather than in adulthood. To il-
lustrate this sensitivity, a single maternal TCDD
dose of 0.16 (xg/kg given on day 15 of gestation
affects several endpoints in male offspring that
collectively indicate a deficit in androgenic sta-
tus.254-263 These include decreases in the weights
of accessory sex organs such as the ventral pros-
tate and epididymis and cauda epididymis, as
well as a decreased anogenital distance and in-
creased time of testis descent. In adult male rats
exposed perinatally to TCDD, the effects on sex-
ual behavior included increases in the number of
mounts preceding ejaculation and intromission
latency.254'265 These effects were produced by
single TCDD doses as low as 0.064 fig/kg given
on day 15 of gestation. When exposure to TCDD
occurs in adulthood on the other hand, relatively
large doses in the overtly toxic range are required
to cause decreases in ventral prostate and caput
epididymis weight.230-256 Kociba et al.230 reported
that accessory sex organ weights are decreased
in rats following exposure to 1 (xg TCDD/kg/
day, 5 days per week for 13 weeks. Using the
parameters for TCDD half-life and bioavailabil-
ity in the rat determined by Rose et al.,226 this
dosage regimen results in a TCDD body burden
of approximately 20 |xg/kg at the end of the dos-
ing period. This body burden is similar to the
ED50 of 15 |xg/kg determined by Moore et al.256
for some adverse effects of TCDD, including
decreased plasma androgen concentrations and
accessory sex organ weights in adult male rats.
In addition, it is at least 100 times greater than
doses of TCDD that decreased androgenic status
after perinatal exposure.
In male rats, TCDD exposure results in de-
creased spermatogenesis. This effect occurs after
exposure in adulthood to single doses of TCDD
as low as 3 jxg/kg,260 whereas male rats exposed
perinatally to only 0.064 (xg TCDD/kg given on
day 15 of gestation are similarly affected in adult-
hood.254-265 However, a comparison of the TCDD
doses in adult rats that decrease plasma testos-
terone levels and accessory sex organ weights
with that which decreases spermatogenesis (15 vs.
3 (xg/kg) suggests that decreases in plasma an-
drogen concentrations and/or androgen respon-
siveness in TCDD-treated adult male rats may
not completely explain the effects of TCDD on
spermatogenesis. Similarly, the reduction in
plasma testosterone concentration in perinatally
exposed male rats may be insufficient to explain
the effects of TCDD exposure on spermatogen-
esis.180-265
In adult rats, the most sensitive toxic re-
sponses to TCDD have been observed following
long-term, low-level exposure. In a three-gen-
eration reproduction study, Murray et al.228 re-
ported that dietary administration of TCDD at
doses as low as 0.01 |xg/kg/day significantly af-
fected reproductive capacity in female rats, with
no effects seen at 0.001 |xg/kg/day (NOAEL).
The same NOAEL was found in a 2-year chronic
toxicity and oncogenicity study in which an in-
creased incidence of certain types of neoplasms
was altered among rats given TCDD doses of
0.01 or 0.1 (xg/kg/day.231 Based on the phar-
macokinetics of TCDD in the rat,226 the steady-
state body burden of TCDD in these rats that
were chronically dosed (>90 days) with either
0.01 or 0.001 jxg TCDD/kg/day is approximately
0.29 (xg/kg (LOAEL) and 0.029 p,g/kg (NOAEL),
respectively. Yet, Mably et al.254-263-265 found that
a single TCDD dose of 0.064 (xg/kg given on
day 15 of gestation produced a number of statis-
tically significant effects on the reproductive sys-
tem of male rat offspring. Because 0.064 jxg
TCDD/kg was the lowest dose tested, a NOAEL
for developmental male reproductive toxicity, de-
fined as the lowest dose used that had no statis-
tically significant effect, could not be determined
by Mably et al.254-263-265 It is concluded that de-
velopmental effects on spermatogenesis occur at
a maternal TCDD dose that is lower than any
dose previously shown to produce toxicity in rats.
IV. SUMMARY
The potential for dioxins and related com-
pounds to cause reproductive and developmental
toxicity has been recognized for many years. Re-
cent laboratory studies have broadened our
knowledge in this area and suggest that altered
development may be among the most sensitive
TCDD endpoints. A substantial portion of the
literature reviewed was published after 1985 and
a special effort has been made to view the de-
velopmental toxicity of TCDD in light of the Ah
receptor model of TCDD. On the other hand,
322
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there is very little, if any, information available
that can be used to relate the reproductive toxicity
of TCDD to this receptor model.
We chose to structure this review into sec-
tions on developmental toxicity and male and
female reproductive toxicity, but we recognize
and want to emphasize that developmental and
reproductive events are interrelated at all levels
of biological complexity. Therefore, the reader
should not view the section subheadings within
each of these divisions as defining discrete end-
points that are exclusive of other endpoints. For
example, effects of TCDD on circulating levels
of sex hormones and/or on responsiveness to sex
hormones may be translated into reproductive
dysfunction if exposure occurs in adulthood as
well as abnormal development of sexual behavior
if exposure occurs perinatally. Likewise, even
though organ structure and growth are considered
separate manifestations in developmental toxicity
that are associated with perinatal exposure to
TCDD, the normal development of an organ is
dependent on normal growth processes, and in-
hibition of prenatal growth can significantly dis-
rupt the structural integrity of an organ system.
Given the current data base, developmental
toxicity endpoints tend to be observed at lower
TCDD exposure levels than are endpoints of male
and female reproductive toxicity. The lowest ef-
fective TCDD egg burden for causing develop-
mental toxicity in fish and birds and the lowest
effective maternal TCDD body burden for pro-
ducing a wide range of developmental responses
in mammals are summarized in Tables 8,9, and
10, respectively. These results indicate that a wide
variety of developmental events, crossing three
vertebrate classes and several species within each
class, can be perturbed, thus suggesting that
TCDD has the potential to disrupt a large number
of critical developmental events at specific de-
velopmental stages. In addition, only transient
exposure to relatively low levels of TCDD at
critical times may be all that is necessary to cause
irreversible disruptions in organ system or func-
tion. Higher TCDD exposure levels cause em-
bryo/fetal mortality, and this lethal effect of
TCDD-like congeners clearly poses a danger to
populations of the most sensitive native fish and
wildlife species.
Because developmental toxicity following
exposure to TCDD-like congeners occurs in fish,
birds, and mammals, it is likely to occur in man.
Certain effects in human infants exposed to a
complex mixture of PCBs, CDFs, and PCQs in
the Yusho and Yu-Cheng poisoning episodes were
probably caused by the combined exposure to
those PCB and CDF congeners that are Ah re-
ceptor agonists. The effects observed in human
infants perinatally exposed to this complex mix-
ture were similar to those reported in newborn
mice and adult monkeys exposed only to TCDD,
and this comparability of effects increases the
probability that the outcomes of exposure in Yusho
and Yu-Cheng children were due to the TCDD-
like congeners in the contaminated rice oil in-
gested by their mothers. Most significant is a
clustering of effects in organs derived from ec-
toderm, a syndrome referred to as ectodermal
dysplasia. Included in this syndrome are effects
on the skin, nails, and meibomian glands that
have occurred in both adult monkeys exposed to
TCDD and in the Yusho and Yu-Cheng infants
exposed transplacentally to PCB-, CDF-, and
PCQ-contaminated rice oils. In addition, accel-
erated tooth eruption has been reported both in
human infants affected by the Yusho and Yu-
Cheng exposures and in neonatal mice exposed
to TCDD. The CNS also is derived from ecto-
derm, and the occurrence of functional neuro-
toxic effects due to perinatal TCDD exposure in
monkeys and rats suggests that brain function as
a site of action of TCDD may be involved in the
ectodermal dysplasia syndrome. Yu-Cheng chil-
dren transplacentally exposed to PCB-, CDF-,
and PCQ-contaminated rice oil have produced a
clinical impression of developmental delay and
psychomotor delay during developmental and
cognitive tests, whereas monkeys perinatally ex-
posed to TCDD are affected by a deficit in cog-
nitive function. The concept that the ectodermal
dysplasia syndrome in the Yusho and Yu-Cheng
infants may be caused by the combination of PCB
and CDF congeners in the rice oil that are Ah
receptor agonists, but are less potent that TCDD,
is consistent with structure-activity results for
various developmental endpoints in different spe-
cies of fish, birds, and mammals. These studies
indicate that while there is variability between
species in the profile of developmental responses
elicited by TCDD, essentially all TCDD-like
PCB, CDD, and CDF congeners that have Ah
receptor affinity and intrinsic activity produce the
323
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same pattern of developmental effects within a
given vertebrate species if a sufficiently high dose
of the congener is given.
In mammals, postnatal functional alterations
involving learning behavior and the developing
male reproductive system appear to be the de-
velopmental events most sensitive to perinatal
dioxin exposure. A maternal TCDD body burden
of 19 ng/kg in the rhesus monkey at the time of
conception is associated with impaired object
learning in the offspring when they are tested at
approximately 14 months of age. In the rat, a
single maternal dose of TCDD as low as 64 ng/
kg on day 15 of gestation results in a reduction
in spermatogenesis and alteration in masculine
sexual behavior of the male offspring in adult-
hood. In birds and fish, structural malformations
and embryo mortality are the 'most sensitive ef-
fects observed after direct injection of TCDD into
fertilized eggs. In chicken embryos, a 17% in-
crease in cardiovascular malformations was pro-
duced by an egg TCDD dose as low as 9 ng/kg.
In lake trout sac fry, the lowest TCDD egg burden
to increase mortality was 40 and 55 ng/kg/egg.
In mammals, alterations in structural end-
points and diminished prenatal viability and
growth begin to predominate during gestation at
maternal TCDD body burdens and/or daily TCDD
doses that are above 100 ng/kg. The incidence
of a structural malformation that consists of extra
ribs in rabbits is increased at a maternal TCDD
dose during gestation of 100 ng/kg/day. Prenatal
mortality in monkeys has been observed where
the maternal body burden at the onset of preg-
nancy is estimated to be 111 ng/kg, a level that
produces no signs of overt maternal toxicity. In
rats, fetal growth is inhibited at a maternal TCDD
dose of 125 ng/kg/day during gestation. In mice,
hydronephrosis can be elicited at a maternal
TCDD dose of 500 ng/kg/day administered on
gestational days 6 to 15, and in hamsters the same
response can be caused by a single 1500 ng/kg
dose of TCDD administered to the dam on either
gestational day 7 or 9. These doses of TCDD
that cause hydronephrosis in mice and hamsters
are not maternally toxic. In the mouse, cleft pal-
ate is produced at a maternal TCDD dose of 3000
ng/kg/day administered on gestational days 6 to
15. Prenatal mortality in the mouse results from
a single maternal dose of 24,000 ng/kg admin-
istered on gestational day 6, however, larger doses
of TCDD are required to produce prenatal mor-
tality in the mouse when administered on later
gestational days. A general finding in fish, bird,
and mammalian species is that the embryo or
fetus is more sensitive to TCDD-induced mor-
tality than the adult. Thus, the timing of TCDD
exposure during the life history of an animal can
greatly influence its susceptibility to overt dioxin
toxicity.
With respect to male and female reproductive
endpoints, there are clear effects following dioxin
exposure of the adult animal. Such reproductive
effects generally occur at TCDD body burdens
that are higher than those required to cause the
more sensitive developmental endpoints. For ex-
ample, TCDD exposure of the adult male causes
reduced testis and accessory sex organ weights,
abnormal testis structure, decreased spermato-
genesis, reduced fertility, decreased testicular
testosterone synthesis, reduced plasma androgen
concentrations, and altered regulation of pituitary
LH secretion. However, these effects only appear
at TCDD exposure levels that are overtly toxic
to the animal. In the more limited studies focus-
ing on female reproduction, the primary effects
include decreased fertility, inability to maintain
pregnancy, and, in the rat, decreased litter size.
Signs of ovarian dysfunction and alterations in
hormone levels also have been reported.
Exposure of female mice and rats to TCDD
has an antiestrogenic effect. The dose of TCDD
required to produce this response is generally
higher than that needed to cause the most sen-
sitive signs of developmental toxicity in these
species. More specifically, hydronephrosis and
cleft palate in mice and reductions in spermato-
genesis in rats occur at maternal doses of TCDD
that are far less than those needed to exert an
antiestrogenic effect when young adult female
mice and rats are exposed to dioxin. The precise
mechanism of the antiestrogenic effect of TCDD
is not fully understood. It may be caused by both
a decrease in estrogen receptor number and/or by
an increase in cytochrome P-4501A1-mediated
estrogen metabolism within the target cell. The
antiestrogenic action of TCDD is significant in
that it may provide insight not only into the cause
of certain female reproductive effects, but also
developmental effects, such as the feminization
of sexual behavior in male rats.
324
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The most convincing evidence for establish-
ing a role of the Ah receptor in causing a partic-
ular TCDD endpoint is to show that genetic link-
age exists between the expression of that endpoint
and a particular allele at the Ah locus. Of all the
developmental and reproductive effects of TCDD,
such genetic linkage has been demonstrated only
for structural malformations in mice and anties-
trogenicity in Hepa IclcV cells. Structure-activ-
ity relationships can be used to support genetic
evidence where it exists, or in the absence of
genetic evidence, to indicate a probable role for
Ah receptor involvement. The very limited
amount of structure-activity type evidence avail-
able for embryo/fetal mortality due to TCDD-
like congeners in fish, birds, and mammals sug-
gests that this effect also may involve the Ah
receptor. For other developmental and reproduc-
tive effects of TCDD, there have been no genetic
assessments or relative potency evaluations of
congeners for Ah receptor involvement. Al-
though it remains possible that an Ah receptor
mechanism will eventually be demonstrated for
many more of these effects, it also is feasible
that the Ah receptor mechanism may not be in-
volved in all of them.
ACKNOWLEDGMENTS
The authors acknowledge Dr. Barbara D.
Abbott, Dr. Linda S. Birnbaum, Mr. Donald L.
Bjerke, Dr. Peter L. Defur, Dr. John F. Gierthy,
Dr. Claude L. Hughes, Dr. Robert W. Moore,
Dr. Stephen Safe, Dr. Susan L. Schantz, and Dr.
Hugh M. Tilson for reviewing an earlier version
of this publication. This work was supported in
part by NIH grant ESO1332.
DISCLAIMER
The views expressed in this paper are those
of the authors and do not necessarily reflect the
views or policies of the U.S. Environmental Pro-
tection Agency. The U.S. Government has the
right to retain a nonexclusive royalty-free license
to any copyright covering this article.
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