ToxSci Advance Access originally published online on August 17, 2006
Toxicological Sciences 2006 94(1):153-162; doi:10.1093/toxsci/kfl083
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Cardiogenic Effects of Trichloroethylene and Trichloroacetic Acid Following Exposure during Heart Specification of Avian Development
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* Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI 53706
Department of Cell Biology, Neurobiology and Anatomy, Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226
Department of Pediatrics, University of Utah, Salt Lake City, UT 84113
1To whom correspondence should be addressed at Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Fax: (414) 456-6517. E-mail: jlough{at}mcw.edu.
Received June 20, 2006; accepted August 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Trichloroethylene (TCE) and its metabolite trichloroacetic acid (TCA) are common drinking water contaminants in the United States. Both chemicals have been implicated in causing congenital heart defects (CHD) in human epidemiological and animal model studies. However, the latter studies have primarily focused on assessment of cardiac morphology at late embryonic stages. Here, we tested whether treating avian embryos with TCE or TCA during an exposure window encompassing cardiac specification (Hamburger-Hamilton [HH] 3+) until the onset of chambering (HH 17) informs the etiology of CHD at later stages of development. Embryos were exposed to TCE or TCA via direct injection into the yolk, over a range of doses that included each compound's maximum contaminant level as established by the U.S. Environmental Protection Agency. A modified TUNEL (Terminal deoxynucleotide transferase mediated dUTP-biotin Nick-End Labeling) assay indicated that neither compound induced apoptotic cell death in ventricular myocytes or endocardiocytes at HH 18. However, mid-range dosages of TCE increased myocyte and endocardiocyte proliferation by this time, as determined by monitoring BrdU incorporation; in contrast, an intermediate dose of TCA inhibited proliferation in endocardiocytes. These cellular changes had no apparent functional consequences because all measured hemodynamic parameters were normal for TCE- and TCA-exposed embryos at HH 18, HH 21, and HH 23. In summary, TCE or TCA exposure during the cardiac specification window has only minimal effects on the developing avian heart. These results sharply contrast with our previously reported observations following administration of equivalent doses during a window of valvuloseptal morphogenesis. Taken together, these findings indicate that, as for other teratogens, sensitivity is dictated by the embryo's stage of development.
Key Words: chick embryo; cell proliferation; congenital heart defects (CHD); trichloroethylene (TCE); trichloroacetic acid (TCA); teratogen.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Trichloroethylene (TCE; C2HCl3) is an industrial chemical that is used heavily in many industries, chiefly in metal degreasing applications (Agency for Toxic Substances and Disease Registry [ATSDR], 1997
). Widespread use of the chemical has led to environmental contamination of toxic waste sites as well as groundwater supplies. For instance, over 60% (852 out of 1416) of Superfund sites on the National Priority List (NPL) prepared by the U.S. Environmental Protection Agency (EPA) and ATSDR are contaminated with TCE (ATSDR, 1997). TCE currently ranks 16th on the list of potentially hazardous substances found at NPL sites (CERCLA, 2005). Environmental TCE release can occur during the chemical's manufacture, use, or disposal. While the vast majority of TCE is released into the atmosphere, the chemical does enter and persist in groundwater. In fact, TCE is the most frequently reported groundwater contaminant in the United States (ATSDR, 1997), and up to 34% of U.S. drinking water has some detectable level of TCE contamination (U.S. EPA, 2001b).
In addition, disinfection of municipal water supplies with chlorine generates several TCE metabolites including trichloroacetic acid (TCA; Miller and Uden, 1983). The level of TCA in U.S. drinking water is regulated at 0.3 mg/l TCA (300 parts per billion [ppb]; U.S. EPA, 2001a). TCA can also be formed during degradation of the industrial chemicals tetrachloroethene and 1,1,1-trichloroethane (Scholer et al., 2003).
A number of studies have associated gestational exposure to TCE or TCA with an increased risk for congenital heart defects (CHD) in offspring. For example, one study reports that children born in locations having groundwater TCE contamination (239 ppb) exhibited a three-fold increased risk for CHD, which was attenuated after contaminated wells were closed (Goldberg et al., 1990). Most malformations were valvuloseptal defects. Similar cardiac malformations were observed during subsequent studies performed on chick and rat embryos exposed to TCE or TCA (Dawson et al., 1990, 1993; Johnson et al., 1998a, 2003; Loeber et al., 1988; Smith et al., 1989); it has also been reported that dichloroacetic acid causes cardiac malformations in rat fetuses (Smith et al., 1992). However, these findings are controversial, as others report no incidence of CHD (Dorfmueller et al., 1979; Fisher et al., 2001; Schwetz et al., 1975).
A limitation of the previous studies is that they evaluated embryos at relatively late stages of development, after completion of the major phases of cardiac morphogenesis. Hence, antecedent effects of these chlorinated hydrocarbons during early heart formation—precardiac specification, terminal differentiation, myocardial tube formation/looping, and cushion formation/chambering—are unknown. To identify a possible cause of the valvuloseptal defects reported in the work cited, we have evaluated the effect of exposing avian embryos to TCE or TCA during two early cardiac developmental windows. One exposure period, termed the "cushion" window, encompassed the sequence of cardiac cushion formation beginning at Hamburger-Hamilton (HH) stage 13 through HH stage 20; results are described in a separate communication (Drake et al., 2006a). The second period is termed the "specification" window, in which embryos were exposed to TCE or TCA during the earliest stages of cardiogenesis, from the initiation of cardiac specification (HH stage 3+) until the onset of chambering (HH stage 17). Results of these latter experiments are reported here.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chicken embryos.
Embryonated White Leghorn chicken eggs (Bovan strain) were obtained from Sunnyside Hatchery (Beaver Dam, WI). For cardiovascular function experiments, White Leghorn chick eggs (Bovan strain) were purchased from Utah State University (Logan, UT). Eggs were stored in a humidified atmosphere at 14°C for no more than 72 h. To resume embryonic development, eggs were placed in a 60% humidified incubator set at 38°C. Embryonic death was determined by the absence of a beating heart; this assessment was made in ovo after carefully removing shell from a
1 cm2 area directly over the embryo. All embryos were staged according to the criteria of Hamburger and Hamilton (1951).
Chemicals.
Stock solutions (1mM) of TCE Sigma-Aldrich (St Louis, MO) #20667 and TCA (Fluka #91228) were prepared fresh daily in 1x phosphate-buffered saline (PBS). Subsequent dilutions were made in 1x PBS to enable administration of each dose in a total volume of 50 µl. Solutions were warmed to 38°C prior to injection.
Embryonic TCE or TCA exposure.
PBS, TCE, or TCA was directly injected into the center of the egg yolk using an established protocol (Drake et al., 2006b). Briefly, eggs were removed from the incubator and wiped with 70% ethanol, followed by introduction of a 1.5-mm-diameter hole in the blunt end using a metal probe. Using a 20-gauge needle, injections were slowly introduced directly into the center of the yolk, after which the injection aperture was sealed with electrical tape, and the egg was rotated 180° and returned to the incubator.
In this manner, TCE or its metabolic product TCA was injected for comparison against vehicle-only controls (1x PBS). Dosages were administered during the "specification window" of embryonic heart development, beginning at Hamburger and Hamilton (1951) stage 3+ when the primary heart field is specified in the primitive streak (Garcia-Martinez and Schoenwolf, 1993) and ending approximately 50 h later at HH stage 17, at the onset of chambering (Fig. 1). Total dosages of 0.2, 2, 4, 20, and 200 nmol were administered via four injections during this window, at stages 3+ (16 h), 6 (24 h), 13 (46 h), and 17 (68 h); hence, each injection contained one fourth of the final total dosage. Embryos were harvested at subsequent developmental stages (HH stage 18 [72 h], 21 [3.5 days], 23 [4 days], or 24 [4.25 days]) for examination of embryonic survival, cardiac function, or cellular parameters.
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Cell death via in situ TUNEL (Terminal deoxynucleotide transferase mediated dUTP-biotin Nick-End Labeling) labeling modified with BrdUTP.
Treated embryos were harvested at HH stage 18, rinsed with 1x PBS, fixed overnight in freshly prepared 4% paraformaldehyde, dehydrated and embedded in paraffin, sectioned in sagittal orientation at 3 µm, and mounted on glass microscope slides (Fisher Scientific, Pittsburgh, PA, #15-188-52). To detect cell death, deparaffinized sections were subjected to a modified TUNEL (Terminal deoxynucleotide transferase mediated dUTP-biotin Nick-End Labeling) protocol (Gavrieli et al., 1992
) in which 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP) incorporates into the 3' hydroxyl ends of nicked DNA, followed by detection with a monoclonal anti-BrdU antibody. Prior to the TUNEL reaction, sections were subjected to limited digestion with proteinase K (20 µg/ml for 1 min at room temperature [RT]) to facilitate access of reagents to DNA repair sites, followed by refixation with 10% formalin/1x PBS for 5 min at RT. After thorough rinsing, TUNEL reaction mixture was applied, consisting of 1x terminal deoxynucleotidyl transferase (TdT) buffer (0.2M cacodylic acid-NaOH [pH = 6.6], 0.25 mg/ml bovine serum albumin [BSA; Fisher #BP1605-100], 1mM CoCl2 [Sigma, St Louis, MO, #C8661], 80 µM BrdUTP [Sigma, St Louis, MO, #B-0631]), 0.1 unit/µl TdT enzyme (Promega, Madison, WI, #M1871) for 90 min at RT. For a negative control, the primary antibody (below) was omitted, in which case only background fluorescence was observed. For an external positive control, sections were treated with 0.01 units/µl deoxyribonuclease I (DNase I; FPLCpure #27-0514-01, GE Healthcare, Chalfont St Giles, UK) for 10 min at 37°C prior to reacting with the TdT reaction mixture. As an internal positive control, sections used for analysis included noncardiac regions that contained concentrations of previously documented cell death. Signal from incorporated BrdU was detected by immunostaining 90 min at RT with monoclonal anti-BrdU antibody (Developmental Studies Hybridoma Bank #G3G4) diluted 1:50 in blocking buffer (TBST [Tris-Buffered Saline with Tween-20] with 10% heat-inactivated goat serum, Sigma, St Louis, MO, #G6767), followed by application of the secondary antibody (Alexa 594 donkey anti-mouse [Invitrogen Corp., Carlsbad, CA, #A-21203]) diluted 1:500 in the same buffer. Nuclei were counterstained with 4'-6-diamidino-2-phenylindole (DAPI; 0.5 µg/ml). Observations were made using an epifluorescence microscope (Nikon TE-DH 100W) and photographs were taken with a Spot II camera. TUNEL-positive cardiac myocytes and endocardiocytes, expressed as a percentage of 400–800 DAPI-stained myocardial cells or 50–100 DAPI-stained endocardial cells, were enumerated in each embryo. Results were tabulated from evaluation of 7-µm sagittal sections removed from the entire heart. Percentages of TUNEL-labeled cells were determined at x400 magnification by evaluating every fourth section; hence, all areas of stage 18 endocardium and myocardium were included in these samples, with the exception of the most laterally situated myocardial cells. Results were compiled from three separate experiments performed in blind. In each experiment, triplicate embryos were treated with PBS vehicle or 0.2, 4, or 200 nmol TCE or TCA.
Cell proliferation via BrdU labeling.
Three hours before embryo harvest, eggs were windowed near the embryo and 50 µl of prewarmed (37°C) 10mM BrdU (Sigma-Aldrich, St Louis, MO, #858811) was slowly injected into the space above the embryo. Upon harvest, embryos were rinsed with ice-cold 1x PBS and transferred to freshly prepared 4% paraformaldehyde/1x PBS for overnight fixation at 4°C. On the following morning, embryos were dehydrated and embedded in paraffin wax, followed by sectioning at 7 µm thickness. After placing sections on slides, BrdU in DNA was unmasked by extraction with 4N HCl for 15 min at RT, followed by thorough washing in PBS. Sections were blocked with 5% BSA/0.05% Triton X-100 for 1 h at RT. Primary mouse anti-BrdU antibody (Becton, Dickinson Co., Franklin Lakes, NJ, #347580) diluted 1:250 in the same blocking solution was applied overnight at 4°C. After thorough washing with blocking solution the secondary antibody, fluorescein-conjugated goat anti-mouse IgG (MP Biomedicals, Irvine, CA, #55496) diluted 1:500, was applied for 90 min at RT. After thorough washing, nuclei were counterstained with propidium iodide (PI; 1 µg/ml). A minimum of three hearts treated with each concentration of TCE or TCA was evaluated in each of three separate experiments. To determine the percentage of BrdU-positive cells in each heart, the strategy described above for TUNEL labeling was used; a minimum of 1000 myocytes and 500 endocardiocytes was enumerated, in blind, and percentages of cycling cells were calculated from the BrdU:PI ratio.
Cardiovascular function assessments.
Cardiovascular effects of TCE or TCA were assessed at HH 18, HH 21, and HH 23 with Doppler ultrasound technology (Hu and Clark, 1989). For each assessment, the egg was placed blunt end-up on a microscope stage and the shell and overlying membranes were removed. Dorsal aortic and atrioventricular blood velocities were measured using a 20-MHz pulsed-Doppler velocity meter (model 545C-3, Department of Bioengineering, University of Iowa, Iowa City, IA) and 0.75-mm piezoelectric crystals. To measure dorsal aortic blood velocity, a crystal was positioned at a 45° angle to the dorsal aorta adjacent to the sinus venosus. To measure atrioventricular blood velocity, a crystal was placed at the apex of the ventricle pointing toward the atrioventricular orifice. Analog waveforms were sampled digitally at 500 Hz via an analog-to-digital board (National Instruments AT-MIO16, Austin, TX) and viewed with custom analysis software (National Instruments Labview).
Data were analyzed over five consecutive cardiac cycles (each cycle is defined as the sequence of events occurring between consecutive diastoles) for each embryo, and heart rate (beats per min) was determined from the interval of cardiac cycles. Dorsal aortic blood flow (mm3/s) was calculated by multiplying the integrated velocity curve by the dorsal aortic cross-sectional area. Stroke volume index (mm3 per beat) was obtained as the quotient of dorsal aortic blood flow against heart rate. Passive atrioventricular blood flow (mm3/s) was calculated using the equation [passive component area/(passive + active areas)] x dorsal aortic blood flow. Active atrioventricular blood flow (mm3/s) was calculated using the equation [active component area/(active + passive areas)] x dorsal aortic blood flow. All data are presented as mean ± standard error (SE).
Statistical analyses.
Data that were binary (e.g., survival data) were analyzed by a logistical regression analysis using the statistical software SAS version 9.1 (SAS Institute, Inc., Cary, NC). SigmaStat statistical software version 3.1 (Systat Software, Point Richmond, CA) was utilized to examine all other data. Specifically, normally distributed data were subjected to an unpaired, two-tailed t-test employing the appropriate variance parameter (equal or unequal variance). Data not normally distributed were examined using the Mann-Whitney U-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We evaluated the consequences of embryonic exposure to a range of TCE or TCA dosages during the cardiac specification window (Fig. 1) of avian heart development. Total dosages ranged from 0.2 to 200 nmol per egg; these exposures correspond to approximately 0.4–400 ppb (Table 1). This range of TCE doses did not affect embryo survival to HH 18 (72 h incubation) or HH 24 (4.25 days incubation; Fig. 2A and 2B). Similarly, all tested TCA dosages did not affect embryonic survival at HH 18 (Fig. 2C) or HH 24 (Fig. 2D); although application of the logistics regression test indicated a significant reduction in survival caused by 0.2 nmol TCA per egg at HH 24 (p < 0.05), this modest effect was not accompanied by a dose response. Gross examination of HH 18 and HH 24 whole embryos and hearts that were exposed to these TCE or TCA dosages revealed no defects in gross morphology or patterning (data not shown). Hematoxylin and eosin (H&E)–stained serial sagittal sections of HH 18 embryos, performed in triplicate for each dosage of TCE and TCA, indicated that the microscopic appearance of hearts exposed to these agents was unremarkable, as evidenced by the absence of extravasated blood within, and normal thickness and cellularity of, the atrial and ventricular myocardial walls.
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We performed in situ TUNEL analysis to assess whether TCE or TCA exposure induced apoptotic cardiac cell death by stage 18. As shown in Figure 3, enumeration of heart sections for myocardial and endocardial cells undergoing apoptosis, normally an infrequent occurrence during this time (Schaefer et al., 2004
), revealed that neither TCE nor TCA at aggregate dosages of 0.2, 4, or 200 nmol per egg significantly affected the incidence of apoptotic cells at HH 18.
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Nuclear incorporation of BrdU was monitored to assess whether exposure to TCE or TCA during the specification window affected the index of cycling myocardial or endocardial cells at HH 18. As shown in Figure 4B, significant increases in endocardiocyte proliferation were observed after exposure to 2 (p = 0.02) or 4 nmol (p = 0.006) TCE. Moreover, 4 nmol TCE significantly increased myocyte proliferation (p = 0.04; Fig. 4A). By contrast, TCA had no effect on myocyte proliferation at any dosage (Fig. 4C), although endocardiocyte proliferation was depressed after exposure to 4 nmol TCA per egg (p = 0.003; Fig. 4D). To assess whether the increased myocyte and endocardiocyte proliferation caused by 4 nmol TCE was apparent at an earlier stage, embryos from eggs injected during the specification window were evaluated at HH 12, a stage just after onset of rhythmic contractions and cardiac looping. Because HH 12 preceded the last two TCE injections in the exposure protocol (Fig. 1), total dosage was held constant by administering two (instead of four) TCE injections at HH stages 3+ and 6. As shown in Figure 5, neither myocyte nor endocardiocyte proliferation was affected by the tested TCE dosages (0.2–200 nmol per egg) in the HH 12 embryo. Examination of these embryos at both gross and light microscopic levels after H&E staining revealed no indications of cardiac dysmorphology (data not shown).
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Considering the TCE- and TCA-induced effects on ventricular cellular proliferation at HH 18, and our previous observations of increased proliferation and reduced cardiac function following TCE and TCA exposure during cushion morphogenesis (Drake et al., 2006a
), it was important to determine whether similar cardiac dysfunctions occurred following specification window exposure. For these determinations, the level of TCE or TCA exposure that maximally affected these parameters (Fig. 4A, 4B, and 4D)—4 nmol per egg—was again administered during the cardiac specification window, and intracardiac blood flow was measured at time points thereafter. Specifically, pulsed-Doppler ultrasound was utilized to assess hemodynamic parameters in HH 18, HH 21, and HH 23 embryonic hearts. In comparison to PBS-treated embryos, all of the functional parameters assessed (cardiac cycle length, heart rate, stroke volume, and dorsal aortic and atrioventricular blood flow) were unaffected in TCE- and TCA-exposed embryos (Tables 2–4). Measurements for control-treated embryos were consistent with previously published stage-specific values (Hu and Clark, 1989; Hu et al., 1991).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We sought to identify developmental anomalies during early cardiogenesis that may precede previously reported findings correlating TCE exposure to CHD (Dawson et al., 1990
, 1993; Drake et al., 2006a; Goldberg et al., 1990; Johnson et al., 1998a,b, 2003; Loeber et al., 1988; Yauck et al., 2004). The chick embryo was selected because the mechanisms of cardiac specification, patterning, and morphogenesis are best understood in this model. Moreover, the chick embryo develops within a relatively constant in ovo environment in contrast to the environment of placental embryos, which is affected by changing maternal metabolism. Avian and human hearts develop in nearly identical fashion with few exceptions (review: Martinsen, 2005), utilizing the same signaling molecules and developmental steps at approximately equivalent embryonic stages.
The TCE dosages tested here, 0.2 to 200 nmol per egg, are relevant to the EPA-established limit for human exposure which is 5 ppb. Specifically, when calculated in the context of average chick egg volume (66.7 ml) and assuming a one-compartment model, each TCE dosage—0.2, 2, 4, 20, 200 nmol—respectively, corresponds to approximately 0.4, 4, 8, 40, and 400 ppb TCE (Table 1). Hence, the 0.2- and 2-nmol dosages are below the 5 ppb EPA limit, while the higher levels are in excess of this limit. Although the 4-nmol dosage (8 ppb) that caused increased cardiac cell proliferation is in modest excess of the 5 ppb limit, it is relevant to consider previous findings (Loeber et al., 1988) indicating that exposures as low as 2.7 ppb (4 µg/kg body weight) are sufficient to induce cardiac teratogenicity at HH stages 29, 34, and 44. Further, the TCE exposures used in our study are environmentally relevant when considering that TCE has contaminated drinking water sources at levels as high as 55 ppb (Bove et al., 1995), 239 ppb (Goldberg et al., 1990), 267 ppb (Lagakos et al., 1986), and 1400 ppb (ATSDR, 1998).
The most important finding of this work is that, with the exception of increased cell proliferation, the consequences of specification window TCE dosing are markedly different from those we recently obtained from a "cushion window" exposure (Drake et al., 2006a). This later period spanned events of endocardial cushion formation, from epithelial induction through mesenchymal migration (HH 13–20), whereas the specification window targeted the earlier events of cardiac specification, patterning and early morphogenesis (HH 3+ to 17). A disturbance in either process could culminate in the septal defects attributed to TCE exposure (Dawson et al., 1990, 1993; Johnson et al., 1998a, 2003), and we sought to distinguish those contributions. In both exposure windows, 4 nmol TCE enhanced proliferative responses within the heart, specifically, the myocytes and endocardiocytes after specification window dosing (Fig. 4A and 4B), and the endocardial cushion mesenchyme after cushion window dosing (Drake et al., 2006a). For the cushion window model, increased cushion cellularity, reduced cardiac output at HH 24, and significant embryo mortality by HH 30 accompanied these changes. By contrast, these defects were not observed after specification dosing (Tables 2–4
; Fig. 2A and 2B). Comparison of these findings has two implications. First, exposure to TCE during the specification window did not alter cardiac morphology or functionality, although, as discussed below, mid-range levels of TCE or TCA did distort the proliferation of cardiac cells, including the endocardial cells (Fig. 4). Second, because TCE exposure during the window of cushion formation not only distorted cardiac cell proliferation but also caused heart dysfunction and embryonic mortality, the cushion window (HH 13–20) is established as a critical window of sensitivity for this teratogen in chick. This result reinforces previous claims that the cardiac cushions may be potential targets for developmental toxicants (Sadler, 2000).
The enhanced cardiocyte proliferation caused by 4 nmol per egg TCE during the specification window (Fig. 4A) also occurred after exposure to 4 nmol TCE during the period of cushion formation (Drake et al., 2006a). Although these two studies addressed different cellular populations (cardiomyocytes and endocardiocytes for the specification window; cushion mesenchyme for the cushion window), the common response of cells within these cardiac compartments to TCE is striking. It may be relevant that, during the TCE exposure period, the heart undergoes significant cellular expansion that is reflected in a remarkable acceleration of the proliferation index, peaking at approximately 40% by HH 22 (Jeter and Cameron, 1971). This is borne out in our own data; the proliferative index of myocardial cells in control embryos at HH stage 12 was only 7% (Fig. 5A), and increased to 28% at HH stage 18 (Fig. 4A and 4C). We speculate that this large increase in proliferative activity reflects a period of unregulated cell cycle control that provides a window of opportunity for exogenous agents, such as TCE, to effect distortions.
Despite the proliferative effects of 4 nmol TCE and TCA after specification dosing, this dosage had no apparent physiologic consequences because all measured cardiac functional parameters (heart rate, dorsal aortic blood flow, atrioventricular blood flow, stroke volume) were normal at HH 18, HH 21, and HH 23 (Tables 2–4). This contrasts with our previously reported observations that an equivalent TCE exposure, when administered during a window of valvuloseptal morphogenesis, causes respective reductions of 23 and 31% in dorsal aortic and atrioventricular blood flow (Drake et al., 2006a). The absence of physiologic effects after TCE treatment during a window encompassing specification and early cardiac morphogenesis may reflect the intimate relationship between cardiac structure and function at that time (Bartman and Hove, 2005), such that the nascent ventricle is able to compensate for toxicant-induced changes in cell proliferation (which normally rapidly increases during this period; Jeter and Cameron, 1971) while maintaining normal cardiac function. The absence of a physiologic defect is further supported by our preliminary findings that the same TCE and TCA levels do not affect ventricular pressure at HH 18 (data not shown).
An intriguing aspect of the effects of TCE on cell proliferation is that the increase occurs near the middle of the dose range. This biphasic dose-dependent response is similar to that reported after exposure of chick embryos to a range of TCE (Loeber et al., 1988), as well as dichloroethylene (Goldberg et al., 1992), concentrations which resulted in dysmorphology of chambering at later stages of development. It was also seen in our investigation of cushion window exposure (Drake et al., 2006a). We speculate that this phenomenon results from induction of TCE-metabolizing enzymes and/or by the utilization of alternative metabolic pathways at the higher TCE levels. There are several metabolic pathways for TCE, involving cytochrome P450 2E1, aldehyde dehydrogenase, alcohol dehydrogenase, glutathione-S-transferase, 
-glutamyltransferase, and dipeptidase. It may be revealing to investigate the contributions of these pathways to TCE's metabolic fate and its cardiac teratogenicity. The levels of these enzymes are likely to vary with developmental stage, adding an additional layer of complexity to the teratogen's effects. Such could explain, for example, our observation that TCE and TCA have opposing effects on proliferation following specification treatment (Fig. 4B and 4D), and parallel effects following cushion exposure (Drake et al., 2006a).
In summary, we conclude that, as for other teratogens including thalidomide (Newman, 1986), retinoic acid (Shenefelt, 1972), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (Lin et al., 2002; Ohsako et al., 2002), the effects of TCE appear to be dependent upon developmental stage at the time of exposure. Our results show that the chick embryo is relatively resistant to TCE when exposure occurs during early cardiogenic stages but is extremely vulnerable when TCE exposure occurs during valvuloseptal morphogenesis. Although these findings may partly explain why some groups observe no developmental cardiac effects after TCE exposure (Dorfmueller et al., 1979; Fisher et al., 2001; Schwetz et al., 1975) while others report positive associations (Dawson et al., 1990, 1993; Johnson et al., 1998a, 2003; Loeber et al., 1988), TCE exposure routes, dosages, and other variables including the animal model must also be considered.
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ACKNOWLEDGMENTS |
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Supported by National Institutes of Health grants ES11738 (J.L. and S.M.S.) and U10HD45944 (N.H.), and American Heart Association Predoctoral Fellowship #0510017Z (V.J.D.).
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