Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Murine Heart Development: Alteration in Fetal and Postnatal Cardiac Growth, and Postnatal C
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《毒物学科学杂志》
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
ABSTRACT
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 μg 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 μg TCDD/kg. In addition, cardiocyte proliferation was reduced in GD 17.5 fetal hearts at the 6.0-μg 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 μg 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 induced 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; Ivnitski-Steele 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 -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 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 μg TCDD/kg in corn oil via oral gavage (5.0 μl/g body weight). On GD 17.5 one group of pregnant dams were sacrificed (control, n = 21 litters; 1.5, n = 6; 3.0, n = 4; 6.0, n = 20; 12, n = 8; 24, n = 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. Additionally, for characterization of the postnatal effects of TCDD, a second group of pregnant dams from the control (n = 9 litters) and 6.0-μg TCDD/kg (n = 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 –70°C 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 μm, 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 = 4 litters; TCDD, n = 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 μg 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 μm. 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 = 4 litters; TCDD, n = 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 Q-Gene software (Simon, 2003), and expressed as a percent of control values (control, n = 6 litters; TCDD, n = 6; one pup analyzed per litter).
Electrocardiograms.
The P 21 mice were anesthetized with 2.5% avertin (20 μl/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 μl 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 = 5 litters; TCDD, n = 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 one-way analysis of variance (ANOVA) was used for dose- and time-related comparisons with post hoc comparisons made using the Holm-Sidak test. Two-way 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-μg 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 μg TCDD/kg (Fig. 1). This decrease in heart-to-body weight ratio appeared to plateau at >6.0 μg 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 μg 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 μg 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 heart-to-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 -myosin heavy chain (MHC) mRNA in these hearts, using real-time PCR. We found that pups from 6.0 μg TCDD/kg litters showed a significant increase in the cardiac expression of ANF mRNA (Fig. 4), while MHC 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 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.
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 -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 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.
In this study, we found that a maternal dose as low as 3.0 μg and 6.0 μg 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 μg TCDD/kg on GD 14 induces a 54% incidence of hydronephrosis, while doses of 12 and 24 μg/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-μg/kg maternal dose and increased further at 12 and 24 μg/kg. And, while TCDD doses of 3–24 μg/kg fail to induce cleft palate on GD 14, doses of 12 and 24 μg/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 (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 μg 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 μg 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 -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 cardiac hypertrophy develops, even in the absence of changes in MHC 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 -adrenergic 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 -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 -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 μg 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 μg 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
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 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.
ACKNOWLEDGMENTS
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.
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Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
ABSTRACT
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 μg 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 μg TCDD/kg. In addition, cardiocyte proliferation was reduced in GD 17.5 fetal hearts at the 6.0-μg 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 μg 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 induced 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; Ivnitski-Steele 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 -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 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 μg TCDD/kg in corn oil via oral gavage (5.0 μl/g body weight). On GD 17.5 one group of pregnant dams were sacrificed (control, n = 21 litters; 1.5, n = 6; 3.0, n = 4; 6.0, n = 20; 12, n = 8; 24, n = 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. Additionally, for characterization of the postnatal effects of TCDD, a second group of pregnant dams from the control (n = 9 litters) and 6.0-μg TCDD/kg (n = 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 –70°C 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 μm, 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 = 4 litters; TCDD, n = 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 μg 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 μm. 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 = 4 litters; TCDD, n = 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 Q-Gene software (Simon, 2003), and expressed as a percent of control values (control, n = 6 litters; TCDD, n = 6; one pup analyzed per litter).
Electrocardiograms.
The P 21 mice were anesthetized with 2.5% avertin (20 μl/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 μl 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 = 5 litters; TCDD, n = 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 one-way analysis of variance (ANOVA) was used for dose- and time-related comparisons with post hoc comparisons made using the Holm-Sidak test. Two-way 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-μg 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 μg TCDD/kg (Fig. 1). This decrease in heart-to-body weight ratio appeared to plateau at >6.0 μg 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 μg 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 μg 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 heart-to-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 -myosin heavy chain (MHC) mRNA in these hearts, using real-time PCR. We found that pups from 6.0 μg TCDD/kg litters showed a significant increase in the cardiac expression of ANF mRNA (Fig. 4), while MHC 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 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.
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 -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 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.
In this study, we found that a maternal dose as low as 3.0 μg and 6.0 μg 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 μg TCDD/kg on GD 14 induces a 54% incidence of hydronephrosis, while doses of 12 and 24 μg/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-μg/kg maternal dose and increased further at 12 and 24 μg/kg. And, while TCDD doses of 3–24 μg/kg fail to induce cleft palate on GD 14, doses of 12 and 24 μg/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 (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 μg 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 μg 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 -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 cardiac hypertrophy develops, even in the absence of changes in MHC 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 -adrenergic 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 -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 -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 μg 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 μg 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
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 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.
ACKNOWLEDGMENTS
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.
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