Download Effect of 2,3,7,8-Tetrachlorodibenzo-p

TOXICOLOGICAL SCIENCES 88(1), 242–249 (2005)
doi:10.1093/toxsci/kfi302
Advance Access publication August 24, 2005
Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Murine Heart
Development: Alteration in Fetal and Postnatal Cardiac Growth, and
Postnatal Cardiac Chronotropy
E. A. Thackaberry,* B. A. Nunez,* I. D. Ivnitski-Steele,* M. Friggins,* and M. K. Walker*,†,1
*College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; †Department of Cell Biology and
Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Received July 21, 2005; accepted August 22, 2005
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals are potent cardiovascular teratogens in developing piscine
and avian species. In the present study we investigated the effects
of TCDD on murine cardiovascular development. Pregnant mice
(C57Bl6N) were dosed with 1.5–24 mg TCDD/kg on gestation day
(GD) 14.5. At GD 17.5, fetal mice exhibited a dose-related
decrease in heart-to-body weight ratio that was significantly
reduced at a maternal dose as low as 3.0 mg TCDD/kg. In
addition, cardiocyte proliferation was reduced in GD 17.5 fetal
hearts at the 6.0-mg TCDD/kg maternal dose. To determine if this
reduction in cardiac weight was transient, or if it continued after
birth, dams treated with control or 6.0 mg TCDD/kg were allowed
to deliver, and heart weight of offspring was determined on
postnatal days (P) 7 and 21. While no difference was seen on P 7,
on P 21 pups from TCDD-treated litters showed an increase in
heart-to-body weight ratio and in expression of the cardiac
hypertrophy marker atrial natriuretic factor. Additionally, electrocardiograms of P 21 offspring showed that the combination of
in utero and lactational TCDD exposure reduced postnatal heart
rate but did not alter cardiac responsiveness to isoproterenol
stimulation of heart rate. These results demonstrate that the fetal
murine heart is a sensitive target of TCDD-induced teratogenicity,
resembling many of TCDD-induced effects observed in fish and
avian embryos, including reduced cardiocyte proliferation and
altered fetal heart size. Furthermore, the combination of in utero
and lactational TCDD exposure can induce cardiac hypertrophy
and bradycardia postnatally, which could increase the risk of
cardiovascular disease development.
Key Words: TCDD; aryl hydrocarbon receptor; cardiomyocyte
proliferation; cardiac hypertrophy; ECG; bradycardia.
INTRODUCTION
The developing piscine and avian cardiovascular systems are
especially sensitive to structural and functional changes in1
To whom correspondence should be addressed at College of Pharmacy,
University of New Mexico, 2502 Marble NE, Albuquerque, NM 87131. Fax:
(505) 272-0704. E-mail: [email protected].
duced by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) and related chemicals. In both fish and bird embryos,
TCDD increases vascular permeability, as evidenced by
pericardial and subcutaneous edema, and the presence of
edema precedes embryo mortality (Guiney et al., 2000; Rifkind
et al., 1985). More detailed analysis in fish embryos has shown
that edema is preceded by an inhibition of vascular remodeling,
reduced blood flow, and circulatory failure (Bello et al., 2004;
Hornung et al., 1999). Similarly, in avian embryos, TCDD
inhibits blood vessel formation (Ivnitski et al., 2001), and this
inhibition is associated with reduced expression and responsiveness to the angiogenic cytokine vascular endothelial growth
factor A (Ivnitski-Steele et al., 2005; Ivnitski-Steele and
Walker, 2003). It is notable that this same pattern of toxicity
is observed in fish and bird embryos when TCDD exposure
occurs shortly after fertilization or when exposure occurs after
organogenesis is largely complete (Belair et al., 2001; IvnitskiSteele et al., 2005).
In addition to the alterations in vascular structure and
permeability, changes in cardiac structure and function also
have been described in piscine and avian embryos exposed to
TCDD. In the piscine embryo, TCDD reduces heart size, and
this is associated with a significant reduction in cardiomyocyte
proliferation (Antkiewicz et al., 2005; Hornung et al., 1999).
Additionally, these structural defects are associated with
functional deficits, including reduced cardiac output and,
eventually, ventricular standstill (Antkiewicz et al., 2005),
suggesting that cardiac conduction may be disrupted. Similarly,
in the chick embryo, TCDD induces ventricular cavity dilation
associated with thinner ventricle walls and induces a significant
reduction in cardiomyocyte proliferation (Ivnitski-Steele and
Walker, 2003; Walker et al., 1997; Walker and Catron, 2000),
while functional deficits include cardiac arrhythmias and
reduced responsiveness to b-adrenergic stimulation (Fan
et al., 2000; Sommer et al., 2005; Walker and Catron, 2000).
The importance of the timing of TCDD exposure to cardiac
teratogenic end points has not been studied in detail; however,
chick embryos exposed to TCDD after organogenesis exhibit
less severe cardiac structural or functional deficits than when
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TCDD AND MURINE HEART DEVELOPMENT
they are exposed shortly after fertilization (Ivnitski-Steele
et al., 2005; Sommer et al., 2005).
In contrast to the responses of piscine and avian embryos to
TCDD-induced cardiovascular teratogenicity, there is only
limited evidence that the cardiovascular system also is a target
of TCDD in the developing mammalian embryo. One common
feature shared among these vertebrate species to TCDD during
development is edema and hemorrhage. Subcutaneous edema
and intestinal hemorrhages have been observed in rat and
hamster fetuses after exposure to TCDD in utero (Olson et al.,
1990), suggesting that TCDD-induced alterations in vascular
structure and function may occur during mammalian development. It is noteworthy, however, that in both piscine and avian
embryos exposed to TCDD, the presence of cardiac structural
defects and functional deficits occurs prior to the appearance of
edema (Antkiewicz et al., 2005; Walker and Catron, 2000).
TCDD-induced cardiac structural defects and/or functional
deficits have not been reported in the mammalian fetus, and the
reasons for these species differences are not known. It is
possible that TCDD fails to induce cardiac teratogenicity during
mammalian development or, alternatively, that the effects are
subtle in nature and have not been studied in sufficient detail to
identify their occurrence. Thus, in this study we tested the
hypothesis that two of the most sensitive effects of TCDD on the
developing piscine and avian heart, altered heart size and
reduced cardiac proliferation, would also be observed after
in utero exposure of the fetal mouse. We further hypothesized
that these effects would not be associated with edema or
mortality, but rather would be associated with altered cardiac
development after birth. To test these hypotheses, first we exposed pregnant mice to graded doses of TCDD during a developmental window when cardiomyocyte proliferation peaks,
and we assessed fetal heart weight and cardiac proliferative
index. Second, we investigated the consequences of the combination of in utero and lactational TCDD exposure on postnatal
cardiac development by assessing postnatal heart weight and
cardiac conduction. Our results demonstrate that the fetal
murine heart is a sensitive target of TCDD-induced teratogenicity and that subtle, but measurable, effects on the fetal heart
are associated with altered cardiac development postnatally.
MATERIALS AND METHODS
Animals. Six-to-eight-week-old C57Bl6N mice (Harlan, Indianapolis, IN)
were maintained on a 12-h light/dark cycle and given food and water ad libitum.
All experiments were approved by the University of New Mexico IACUC.
Nulliparous female mice were mated, and the morning a plug was observed was
designated GD 0.5. On GD 14.5 pregnant mice were dosed with corn
oil (control), or 1.5, 3.0, 6.0, 12, or 24 lg TCDD/kg in corn oil via oral gavage
(5.0 ll/g body weight). On GD 17.5 one group of pregnant dams were sacrificed
(control, n ¼ 21 litters; 1.5, n ¼ 6; 3.0, n ¼ 4; 6.0, n ¼ 20; 12, n ¼ 8; 24, n ¼ 8),
and maternal body and liver weights were recorded, fetuses were dissected free
and weighed, and embryonic hearts were isolated, washed briefly in PBS,
weighed, and randomly assigned to be frozen for mRNA analysis or fixed for
immunohistochemistry. There was no attempt to identify the embryonic gender.
243
Additionally, for characterization of the postnatal effects of TCDD, a second
group of pregnant dams from the control (n ¼ 9 litters) and 6.0-lg TCDD/kg
(n ¼ 10) treatment groups were allowed to deliver. Offspring were euthanized
on postnatal day (P) 7 or 21, heart and body weights were recorded, and hearts
were frozen at 70C for mRNA analysis. Values from all offspring within
a litter were averaged and not segregated by gender.
Immunohistochemistry. Two immunohistochemical methods were used to
assess proliferative index of the fetal murine heart: detection of proliferating
cell nuclear antigen (PCNA) and incorporation of 5#-bromo-2#-deoxyuridine
(BrdU, Sigma Chemical, St. Louis, MO). For PCNA analysis, fetal hearts were
fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at
7.0 lm, stained with anti-PCNA antibody (BD Biosciences, San Diego, CA)
followed by goat antimouse-IgG conjugated to horseradish peroxidase, and
color developed using 3,3#diaminobenzidine as described elsewhere (Ivnitski
et al., 2001). Sections were then stained with propidium iodide to visualize all
nuclei (control, n ¼ 4 litters; TCDD, n ¼ 4; 2–4 hearts/litter). Identical
photographs of 4–6 fields per heart were captured under brightfield or
fluoresence illumination with a Texas Red filter, and PCNA positive nuclei
and total nuclei, respectively, were counted in each field, using ImagePro Plus
software. Results were expressed as percent PCNA-positive nuclei. For BrdU
incorporation experiments, pregnant mice were injected intraperitoneally (ip)
with 50 lg BrdU/kg in sterile saline 2 h prior to the GD 17.5 collection. Fetal
hearts were fixed in 30% glacial acetic acid in ethanol, embedded in paraffin,
and sectioned at 7.0 lm. Sections were stained with anti-BrdU antibody
(Development Studies Hybridoma Bank, The University of Iowa, Iowa City,
IA) followed by goat antimouse-IgG conjugated to horseradish peroxidase, and
color developed using 3,3#diaminobenzidine as described elsewhere (Ivnitski
et al., 2001). Sections were then stained with propidium iodide, and 4–6 fields
per heart were analyzed as described for PCNA (control, n ¼ 4 litters; TCDD,
n ¼ 4; 2–4 hearts/litter). Neither of these methods distinguished the type of cell
that was staining positive for the proliferation markers and thus, we report the
cardiac proliferative index for all cardiac cells or cardiocytes on the section.
Real-Time PCR. To quantify the expression of cardiac hypertrophy
markers, total RNA was isolated from neonatal hearts on P 21 using Trizol
reagent (Invitrogen, Carlsbad, CA). cDNA was generated using reverse
transcriptase (Promega, Madison, WI) with oligo dT and an 18sRT primer to
amplify the 18S control rRNA. The sequences of the primers used have been
previously reported (Lund et al., 2003, 2005). mRNA expression was then
quantified using an I-Cycler (BioRad, Hercules, CA). The efficiency of each
primer set was determined from a standard curve with known quantities of
cDNA, and all sets used were >90% efficient. Real-time polymerase chain
reactions (PCR) reactions were run in triplicate for the genes of interest and 18 s
control simultaneously, and the difference between the CT values was
determined. Values were then converted to mean relative expression using QGene software (Simon, 2003), and expressed as a percent of control values
(control, n ¼ 6 litters; TCDD, n ¼ 6; one pup analyzed per litter).
Electrocardiograms. The P 21 mice were anesthetized with 2.5% avertin
(20 ll/g) and a three-lead surface electrocardiogram (ECG) was recorded for
5 min from subcutaneous 26-gauge needles using a PM-1000 high performance
transducer amplifier, DI-720 data acquisition unit, and WinDaq waveform
software (DATAQ Instruments, Akron, OH). Mice were then injected ip with
8.0 ng isoproterenol (ISO, in 100 ll sterile saline), ECGs were recorded for
5 min, the animals were injected with a second, 160-ng, dose of ISO, and ECGs
were again recorded for 5 min (control, n ¼ 5 litters; TCDD, n ¼ 4; 3–5 pups
randomly selected and analyzed per litter). The ECGs were analyzed using
Advance Codas software (DATAQ Instruments, Akron, OH) to calculate RR,
PQ, QRS, and QT intervals. Heart rate was determined from the RR interval,
and QT corrected for heart rate (QTc) was calculated as described previously
(Mitchell et al., 1998).
Statistics. Student’s t-test was used for individual comparisons, and oneway analysis of variance (ANOVA) was used for dose- and time-related
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comparisons with post hoc comparisons made using the Holm-Sidak test. Twoway repeated measures ANOVA was used to compared ISO-related changes in
heart rate. A p < 0.05 was considered statistically significant in all cases.
RESULTS
TCDD Did Not Cause Overt Maternal or Fetal Toxicity
None of the TCDD doses significantly altered maternal body
weight, maternal weight gain, litter size, or fetal weight at GD
17.5 (Table 1). In addition, survival of pups to P 21 from the
control or 6.0-lg TCDD/kg treatment groups was not significantly different (data not shown). Maternal and fetal liver
weight was significantly increased, which is an effect of TCDD
that has been reported previously (Peterson et al., 1993).
TCDD Reduced Fetal Heart-to-Body Weight Ratio
Although TCDD did not induce overt maternal or fetal
toxicity, a subtle change in heart development was noted. On
GD 17.5 the fetal heart-to-body weight ratio was significantly
decreased at maternal doses <3.0 lg TCDD/kg (Fig. 1). This
decrease in heart-to-body weight ratio appeared to plateau at
>6.0 lg TCDD/kg.
TCDD Reduced Fetal Cardiocyte Proliferation
To determine if the reduction in heart weight at GD 17.5 was
associated with a decrease in cardiac proliferation, we stained
fetal heart sections for the presence of PCNA, a nuclear protein
marker of S-phase, or for BrdU, a thymidine analog incorporated into DNA during S-phase. Fetal heart sections from dams
treated with 6.0 lg TCDD/kg exhibited a significant reduction
in both PCNA-positive and BrdU-positive cells (Fig. 2). This
reduction in cardiocyte proliferation was observed throughout
the developing heart, but was most evident in the interventricular septum (data not shown).
In Utero and Lactational TCDD Exposure Increased
Postnatal Heart-to-Body Weight Ratio
To determine if the reduced heart weight observed in GD
17.5 fetuses after in utero TCDD exposure persisted after birth,
pregnant dams treated with control or 6.0 lg TCDD/kg were
allowed to deliver, and the offspring were examined at P 7 and
P 21. At P 7, the heart-to-body weight ratio of TCDD-exposed
pups tended to be smaller, but it was not significantly different
from control pups at p < 0.05. However, P 21 pups from
TCDD-treated litters exhibited a significant increase in heartto-body-weight ratio (Fig. 3).
In Utero and Lactational TCDD Exposure Increased
Postnatal Cardiac Expression of Atrial Natriuretic
Factor (ANF)
To determine if the cardiac enlargement seen at P 21 in pups
from TCDD-treated litters was associated with the upregulation of pathological markers of cardiac hypertrophy, we
quantified ANF and b-myosin heavy chain (bMHC) mRNA
in these hearts, using real-time PCR. We found that pups from
6.0 lg TCDD/kg litters showed a significant increase in the
cardiac expression of ANF mRNA (Fig. 4), while bMHC
mRNA was undetectable in all samples.
In Utero and Lactational TCDD Exposure Induced
Postnatal Bradycardia
To determine if the cardiac hypertrophy observed in pups
from TCDD-treated litters was associated with altered cardiac
conduction or the presence of cardiac arrhythmias, ECGs were
recorded from anesthetized pups on P 21. Surface lead II ECGs
failed to show any evidence of cardiac arrhythmias from P 21
pups from either control or TCDD-treated dams (Fig. 5) and
this was further reflected by an absence of any differences in
ECG wave intervals, including PQ, QRS, QT, or QT corrected
for heart rate (QTc) (Table 2). In contrast, however, P 21 pups
TABLE 1
Effects of In Utero TCDD Exposure on Maternal and Fetal Liver and Body Weight
Maternal
Treatment group
N
Weight (g)a
Control
1.5 lg/kg
3.0 lg/kg
6.0 lg/kg
12 lg/kg
24 lg/kg
21
6
4
20
8
8
39.3
36.1
39.1
39.6
38.8
38.4
a
±
±
±
±
±
±
0.7
0.7
1.6
0.6
1.1
0.4
Weight gain (g)b
5.9
6.1
6.9
5.8
6.5
6.7
±
±
±
±
±
±
0.3
0.3
1.2
0.3
0.6
0.7
Maternal weight at the time of sacrifice (GD 17.5).
Maternal weight gain between GD 14.5 (dosing) and sacrifice (GD 17.5).
*p < 0.05.
b
Fetal
Liver/BW (%)
5.2
6.1
6.7
5.9
5.9
5.7
±
±
±
±
±
±
0.1
0.3*
0.4*
0.1*
0.2*
0.1*
Litter size
8.9
7.7
8.5
8.5
9.1
8.8
±
±
±
±
±
±
0.4
0.5
0.1
0.4
0.5
0.3
Body weight (g)
0.96
0.90
0.92
0.97
0.95
0.97
±
±
±
±
±
±
0.05
0.01
0.02
0.02
0.01
0.03
Liver/BW (%)
5.75
7.37
7.46
6.91
6.95
7.08
±
±
±
±
±
±
0.19
0.20*
0.29*
0.17*
0.09*
0.17*
TCDD AND MURINE HEART DEVELOPMENT
FIG. 1. Fetal heart-to-body weight ratio on GD 17.5 from dams treated
with control or TCDD on GD 14.5. Pregnant dams were treated with control
(corn oil), 1.5, 3, 6, 12, or 24 lg TCDD/kg on GD 14.5, and fetuses were
collected and weighed on GD 17.5. The fetal heart-to-body weight ratio was
expressed as a percent of control. Sample size (n ¼ litter number): control, 21;
1.5 lg/kg, 6; 3.0 lg/kg, 4; 6.0 lg/kg, 20; 12 lg/kg, 8; and 24 :g/kg, 8. *p < 0.05.
exposed to TCDD in utero and via lactation exhibited
a significant reduction in heart rate (Fig. 5, Table 2). This
difference is apparent from the lower number of heart beats
present in the same ECG recording interval in Figure 5 and
from the increased average RR interval and decreased heart
rate (HR) shown in Table 2.
FIG. 2. Fetal cardiocyte proliferation on GD 17.5 from dams treated with
control or TCDD on GD 14.5. For PCNA staining, fetal hearts from control,
1.5, 3.0, and 6.0 lg TCDD/kg treated litters were collected, fixed, and stained
with an anti-PCNA antibody. For BrdU staining, pregnant dams treated with
control or 6.0 lg TCDD/kg were injected with 50 lg BrdU/kg 2 h prior to fetal
collection. Fetal hearts were then collected, fixed, and stained with anti-BrdU.
Results are expressed as proliferative index, which equals PCNA-positive or
BrdU-positive nuclei divided by total nuclei 3 100. n ¼ 4 litters per treatment
per analysis, 2–3 fetal hearts randomly selected and stained per litter. *p < 0.05.
245
FIG. 3. Fetal (GD 17.5) and neonatal (P 7 and P 21) heart-to-body weight
ratio from dams treated with control or TCDD on GD 14.5. Results are
expressed as heart-to-body weight ratio 3 100. Developmental windows of
murine cardiac hyperplasia versus binucleation/hypertrophy are indicated on
the x-axis (Soonpaa et al., 1996). Sample sizes (n ¼ litter number): control GD
17.5, P 7 and P 21: 21, 3, and 6, respectively; TCDD GD 17.5, P 7, and P 21: 20,
4, and 6. s ¼ Control, d ¼ 6.0 lg TCDD/kg. *p < 0.05; †p < 0.10.
In Utero and Lactational TCDD Exposure Does Not Alter
the Postnatal Responsiveness to Isoproterenol
(ISO)-Stimulated Heart Rate
To determine if P 21 pups from TCDD-treated litters were
responsive to b-adrenergic-stimulation of heart rate, ECGs
were recorded prior to and after injection with 8.0 and 160 ng
ISO. Although both the basal heart rate and ISO-stimulated
heart rate of P 21 pups from TCDD litters was significantly
lower than pups from control litters (Fig. 6), the relative
FIG. 4. Cardiac atrial natriuretic factor (ANF) mRNA expression in P 21
pups from dams treated with control or 6.0 lg TCDD/kg on GD 14.5. ANF
mRNA and 18S RNA expression were measured by real-time PCR from total
cardiac RNA. The DCT (cycle threshold difference) was calculated by
subtracting the CT of the 18S from the CT of ANF for each RNA sample, and
TCDD results were expressed relative to control. Sample sizes (n ¼ litter
number): control, 6; TCDD 6; one pup heart randomly selected and analyzed/
litter. *p < 0.05.
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THACKABERRY ET AL.
FIG. 5. Representative lead II surface electrocardiogram (ECG) from a
P 21 pup exposed to (A) control or (B) a single maternal dose of 6.0 lg TCDD/kg
on GD 14.5. P 21 mice were anesthetized with 2.5% avertin ip and a three-lead
ECG was recorded from subcutaneous 26-gauge needles for 5 min. Each
tracing represents a recording time of ~760 ms. The heart rate of the control pup
in (A) was 474 beats/min and that of the TCDD pup in (B) was 395 beats/min.
The P, Q, R, S, and Twaves are labeled. In the mouse ECG the T-wave is attached
to the S-wave and is often bipolar (Mitchell et al. 1998).
responsiveness to ISO-stimulation was the same. The 8.0 ng
ISO stimulated a 15.8 ± 2% and 19.9 ± 3.6% increase in heart
rate in control and TCDD pups, respectively, while the 160 ng
ISO stimulated an additional 26.7 ± 2% and 33.2 ± 6% increase
in heart rate in control and TCDD pups, respectively. These
ISO-stimulated percent increases in heart rate were not
significantly different between control and TCDD pups.
DISCUSSION
Although the effects of TCDD on the developing piscine and
avian cardiovascular systems are well-documented, relatively
little is known about the effects of TCDD on the developing
mammalian heart. Obvious cardiac morphological malformations have not been reported in mammals; however, more
subtle effects may still exist, and these would have the potential
to result in functional abnormalities later in life.
FIG. 6. Basal and isoproterenol (ISO)-stimulated heart rate in P 21 pups
from dams treated with control or TCDD on GD 14.5. P 21 mice were
anesthetized with 2.5% avertin ip and ECGs were recorded prior to (basal) and
after administration of 8.0 and 160 ng ISO. Sample sizes (n ¼ litter number):
control, 5; TCDD, 4; 3–5 pups randomly selected and analyzed/litter. *p < 0.05.
In this study, we found that a maternal dose as low as 3.0 lg
and 6.0 lg TCDD/kg on GD 14.5 significantly reduces fetal
heart-to-body weight ratio and cardiac proliferation, respectively, on GD 17.5. These doses are comparable to those that
induce some of the most sensitive teratogenic effects of TCDD
reported in the developing murine fetus. For example, a single
maternal dose of 3.0 lg TCDD/kg on GD 14 induces a 54%
incidence of hydronephrosis, while doses of 12 and 24 lg/kg
increase the incidence to 76% and 64%, respectively (Couture
et al., 1990). Similarly, the severity of hydronephrosis is
increased significantly at a 3.0-lg/kg maternal dose and
increased further at 12 and 24 lg/kg. And, while TCDD doses
of 3–24 lg/kg fail to induce cleft palate on GD 14, doses of 12
and 24 lg/kg induce 76% and 100% incidence of cleft palate,
respectively, when administered on GD 12 (Couture et al.,
1990). Thus, the fetal heart appears to be among the more
sensitive organs to TCDD-induced teratogenesis on GD 14 in
the mouse.
In this study, TCDD treatment at GD 14.5 likely avoided the
potential for major morphological cardiac defects, because
cardiac morphogenesis in the mouse is complete by this stage
of development (Vuillemin and Pexieder, 1989); however, we
specifically targeted this developmental window because it
coincides with a period of peak cardiomyocyte proliferation
TABLE 2
Effect of In Utero and Lactational TCDD Exposure on ECG Parameters of P21 Mice
Exposure groupa
RR (ms)
HR (bpm)
PQ (ms)
QRS (ms)
QT (ms)
QTcb
Control
6.0 lg TCDD/kg
140 ± 3
161 ± 5*
429 ± 10
375 ± 11*
35 ± 0.6
37 ± 1.2
9.6 ± 0.5
10.4 ± 0.5
40 ± 0.5
42 ± 1.1
26.7 ± 0.7
27.2 ± 0.2
a
Sample sizes: control, n ¼ 5 litters; TCDD, n ¼ 4 litters; 3–5 pups analyzed/litter.
Rate-corrected QT interval (QTc) was calculated according to the methods of Mitchell et al. 1998.
*p < 0.05.
b
TCDD AND MURINE HEART DEVELOPMENT
(Soonpaa et al., 1996). TCDD has been shown to decrease
cardiomyocyte proliferation during development in both the
chick embryo and the zebrafish embryo (Antkiewicz et al.,
2005; Ivnitski et al., 2001), and our data demonstrate for the
first time that in utero TCDD exposure also significantly
reduces cardiocyte proliferation in the murine fetus. Although
we did not distinguish specific cell types, cardiomyocytes
likely account for a significant proportion of the cells affected
by TCDD, as they account for the majority of cells in the
developing heart. Although the mechanism by which TCDD
induces fetal cardiocyte growth arrest is not known, the
reduction in proliferation is associated with a reduction in the
mRNA expression of cyclins A2, E1, and E2 as reported in our
companion article (Thackaberry et al., 2005). The only cyclin
to be downregulated at the mRNA level during cardiogenesis is
cyclin A2, and this downregulation is coincident with withdrawal of cardiomyocytes from the cell cycle (Chaudhry et al.,
2004). Additionally, bone morphogenetic protein 10 expression
(BMP10) is required for normal proliferation of fetal cardiomyocytes (Chen et al., 2004), and we also found that BMP10
mRNA expression was reduced in the GD 17.5 fetal heart as
reported in our companion article (Thackaberry et al., 2005).
Thus, it is plausible that one mechanism by which TCDD reduces
cardiocyte proliferation is by disrupting cell cycle regulation.
To determine if the reduced cardiac size at GD 17.5 was
transient or persisted into postnatal development, we also
determined heart weight at 7 and 21 days after birth in pups
born to dams treated with control or 6.0 lg TCDD/kg. The
mean heart/body weight ratio of TCDD-exposed pups on P 7
was reduced by 14%, compared to control pups; however, the
relatively small sample size and lack of statistical difference at
p < 0.05 precludes concluding that there is a difference in heart
weights 1 week after birth. In contrast, the heart/body weight
ratio of TCDD-exposed pups was significantly increased at
P 21 as compared to control offspring. These results parallel
those in a previous report which shows that offspring of dams
exposed to a single 5.0 lg TCDD/kg dose on GD 13 exhibit
increased heart weight postnatally (Lin et al., 2001). Cardiomyocytes exit the cell cycle prior to birth, and all DNA
synthesis in cardiomyocytes after birth contributes solely to
binucleation (Soonpaa et al., 1996). Thus, all cardiac growth
that occurs postnatally in mice is due to hypertrophy. The
enlargement of hearts in P 21 pups from TCDD litters suggests
that inappropriate cardiac hypertrophy is occurring. This
explanation is supported by the fact that P 21 pups also exhibit
increased expression of the cardiac hypertrophy marker gene,
ANF. ANF, and b-MHC are normally expressed in the heart
during fetal development and subsequently downregulate after
birth (Mercadier et al., 1989; Morkin, 2000). However, their
expression upregulates under conditions of pathological cardiac hypertrophy. Induction of ANF mRNA, in particular, is an
early response of cardiac myocytes to stretch that occurs during
load-induced hypertrophy (Torsoni et al., 2003). Furthermore,
induction of cardiac ANF mRNA occurs in neonatal mice as
247
cardiac hypertrophy develops, even in the absence of changes
in bMHC expression (Yoshimine et al., 1997). Thus, one
explanation may be that the TCDD-induced reduction in
cardiocyte proliferation prior to birth results in a smaller heart,
which then undergoes abnormal hypertrophy postnatally as
a mechanism to maintain appropriate cardiac output as body
weight increases. Future histological studies will be needed to
determine whether structural hypertrophy occurs postnatally.
To determine if the early signs of cardiac hypertrophy in P 21
pups were associated with altered cardiac conduction or
arrhythmias, we conducted ECG analysis. Our most significant
finding from these studies was that pups exposed in utero and
via lactation to TCDD exhibit bradycardia, reflected by ~12%
reduction in heart rate compared to control pups. It seems
unlikely that the bradycardia results from an effect of TCDD on
the developing sinoatrial node, because these pacemaker cells
and the entire murine cardiac conduction system are fully
differentiated and functional by GD 13 (Rentschler et al.,
2001), 1.5 days prior to TCDD exposure. Alternatively, the
TCDD-induced bradycardia may be a result of the increased
ANF expression. During early events in cardiac hypertrophy,
ANF enhances vagal activity and potentiates reflex bradycardia
(Woods, 2004).
In addition, our results, which show that TCDD reduces
basal heart rate but does not alter the responsiveness to badrenergic stimulation of heart rate, differ from those reported
in other TCDD studies. For example, overtly toxic doses of
TCDD in the adult rat reduce both basal and b-adrenergic–
stimulated heart rate (Hermansky et al., 1988; Kelling et al.,
1987), whereas TCDD exposure of the developing chick
embryo does not alter basal heart rate but does reduce the
chronotropic response to b-adrenergic stimulation (Sommer
et al., 2005; Walker and Catron, 2000). The reasons for these
differences are unclear; however, they may result from differences in the timing and dose of TCDD used and suggest that
multiple mechanisms may be involved.
Finally, the degree to which the effects of TCDD on
postnatal heart development result from in utero versus
lactational exposure is unknown. Based on toxicokinetic
models, a single maternal dose of 6.0 lg TCDD/kg would
result in a fetal TCDD concentration of approximately 84 ng
TCDD/kg, whereas this same in utero dose would lead to body
burdens as high as 2.4 lg TCDD/kg in pups after lactational
transfer (Gasiewicz et al., 1983; Nau et al., 1986; Weber and
Birnbaum, 1985). Thus, it is possible that the effects of TCDD
on heart size and cardiac chronotropy at P 21 result, in part,
from the considerably higher dose of TCDD received during
lactation. Future cross-fostering studies will need to be
conducted to determine the importance of lactational TCDD
exposure to the cardiovascular effects observed postnatally.
In conclusion, our results demonstrate that the fetal murine
heart is a sensitive target of TCDD-induced teratogenicity,
resembling many of the TCDD-induced effects observed in fish
and avian embryos, including reduced cardiocyte proliferation
248
THACKABERRY ET AL.
and altered fetal heart size. In addition, these results confirm
that in utero and lactational TCDD exposure induces postnatal
cardiac hypertrophy and demonstrate that this exposure also
induces postnatal bradycardia. More detailed assessment of the
effects of TCDD on postnatal cardiovascular physiology and
function is needed to determine the potential to which early
TCDD exposure may represent a previously unidentified risk
factor for cardiovascular disease in adulthood.
a decrease in myocyte proliferation and an increase in cardiac apoptosis.
Teratology 64, 201–212.
Ivnitski-Steele, I. D., Friggens, M., Chavez, M., and Walker, M. K. (2005).
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous
angiogenic stimuli, including vascular endothelial growth factor A (VEGFA). Birth Defects Res. A Clin. Mol. Teratol. 73, 440–446.
Ivnitski-Steele, I. D., and Walker, M. K. (2003). Vascular endothelial growth
factor rescues 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibiton of coronary
vaculogenesis. Birth Defects Res. A Clin. Mol. Teratol. 67, 496–503.
ACKNOWLEDGMENTS
Kelling, C. K., Menahan, L. A., and Peterson, R. E. (1987). Effects of 2,3,7,8tetrachlorodibenzo-p-dioxin treatment on mechanical function of the rat
heart. Toxicol. Appl. Pharmacol. 91, 497–501.
The authors thank Dr. Daniel Theele, Christie Wilcox, and Darlene
Gabaldon for their valuable assistance. This work was supported by grant
ES012335 to M.K.W., grant ES012855 to E.A.T., and by the P30 National
Institute of Environmental Health Sciences (NIEHS) Center grant ES12072.
Lin, T. M., Ko, K., Moore, R. W., Buchanan, D. L., Cooke, P. S., and Peterson,
R. E. (2001). Role of the aryl hydrocarbon receptor in the development of
control and 2,3,7,8-tetrachlorodibenzo-p-dioxin–exposed male mice. J.
Toxicol. Environ. Health 64, 327–342.
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