Toxicological Sciences 53, 109-117 (2000)
Copyright © 2000 by the Society of Toxicology
Trichloroethylene Inhibits Development of Embryonic Heart Valve Precursors in Vitro
Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724
Received January 15, 1999; accepted May 11, 1999
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous epidemiological studies with humans and laboratory studies with chickens and rats linked trichloroethylene (TCE) exposure to cardiac defects. Although the cardiac defects in humans and laboratory animals produced by TCE are diverse, a majority of them involves valvular and septal structures. Progenitors of the valves and septa are formed by an epithelial-mesenchymal cell transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract areas of the heart. Based on these studies, we hypothesized that TCE might cause cardiac valve and septa defects by specifically perturbing epithelial-mesenchymal cell transformation. We tested this hypothesis using an in vitro chick-AV canal culture model. This study shows that TCE affected several elements of epithelial-mesenchymal cell transformation. In particular, TCE blocked the endothelial cell–cell separation process that is associated with endothelial activation. Moreover, TCE inhibited mesenchymal cell formation throughout the concentration range tested (50–250 ppm). In contrast, TCE had no effect on the cell migration rate of the fully formed mesenchymal cells. Finally, the expression of 3 proteins (selected as molecular markers of epithelial-mesenchymal cell transformation) was analyzed in untreated and TCE-treated cultures. TCE inhibited the expression of the transcription factor Mox-1 and extracellular matrix (ECM) protein fibrillin 2. In contrast, TCE had no effect on the expression of
-smooth muscle actin. These data suggest that TCE may cause cardiac valvular and septal malformations by inhibiting endothelial separation and early events of mesenchymal cell formation in the heart. Key Words: cardiogenesis; epithelial-mesenchymal cell transformation; cardiac valve formation; TCE; Mox-1.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Halogenated hydrocarbons such as trichloroethylene (TCE), dichloroethylene (DCE) and closely related compounds are among the most common water supply contaminants in the United States and abroad (World Health Organization, 1995
). Although considerable information is available regarding the short- and long-term toxicity of these agents and their general teratogenicity, little was known about their specific cardiac teratogenesis until recently. Two epidemiologic studies found an association between these halogenated hydrocarbons and an increased incidence of major cardiac malformations in children born to mothers who lived in areas of water contamination (Goldberg et al., 1990; Swan et al., 1985). The epidemiologic study by Goldberg et al. (1990) revealed that the relative odds ratio for congenital heart disease for offspring of those who lived or worked in an area of contaminated groundwater in Tucson was 3. Though there were other, lesser, contaminants, drinking water concentrations of TCE in the area were measured at 270 ppb. There was a variety of defects observed in the study, but the most common defects included atrial septal (13%) and ventricular septal (53%), plus pulmonary or aortic valvular stenosis (21%). Moreover, the increased incidence of cardiac defects disappeared after the contaminated wells were closed (Goldberg et al., 1990). Though these studies suggested an association between TCE and congenital heart defects, they did could not prove cause and effect.
Cardiac teratogenicity of TCE was subsequently studied in both chicken and rat (Dawson et al., 1990, 1993; Loeber et al., 1988). In chicken, TCE (doses calculated to be 15–150 ppm) were injected in ovo at various stages of development and 7.3% of TCE-treated hearts had defects (Loeber et al., 1988). Cardiac defects observed in the chick study included both inflow and outflow anomalies. Rats were exposed to TCE by either intrauterine osmotic minipumps (Dawson et al., 1990) or through maternal drinking water (Dawson et al., 1993). These studies demonstrated TCE cardiac teratogenicity in rats down to a dose of 1.5 ppm (Dawson et al., 1993; Johnson et al., 1998).
Other metabolites of TCE, including dichloroethylene, trichloroacetic acid and dichloroacetic acid, were also shown to produce heart defects (Epstein et al., 1992; Johnson et al., 1998). Interestingly, exposure to dichloroacetic acid produced a specific cardiac defect in the rat, a high ventricular (membranous) septal defect (Epstein et al., 1992). In both species, the effective level of TCE or metabolites in these experiments remained at least 2 orders of magnitude higher than seen in the Tucson groundwater.
Our understanding of valve and septal formation in the heart provides an avenue for further cellular and molecular analysis of the teratologic effects of TCE. The basic events of cardiac valve formation can be summarized as follows. Early in development, the heart is a hollow tube-like structure with 2 cell layers. The outer surface is a myocardial cell layer and the inner luminal surface is an endothelium. Between the two cell layers lies an expanse of extracellular matrix (ECM). At a specific time in development, a subpopulation of endothelial cells lining the atrioventricular (AV) canal detaches from adjacent cells and invades the underlying ECM (Markwald et al., 1984). This event is termed an epithelial-mesenchymal cell transformation (Fig. 1A). These endothelial-derived mesenchymal cells migrate towards the surrounding myocardium and begin to proliferate in order to populate the entire AV canal ECM. Cardiac mesenchyme provides the cellular constituents of the septum intermedium and the valvular leaflets of the mitral and tricuspid valves. The septum intermedium subsequently contributes to the lower portion of the atrial septum and the membranous portion of the ventricular septum (Markwald et al., 1984; Wessels et al., 1996) (Fig. 1B).
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At least 3 distinct events occur during cardiac epithelial-mesenchymal cell transformation: endothelial cell activation (Stage 14 in chicken), mesenchymal cell formation (Stage 16) and mesenchymal cell invasion (migration) into the ECM (Stages 17 and 18) (Boyer et al., 1999a
). The initial signal for cell transformation comes from the AV canal myocardium and mediates an inductive tissue interaction between the myocardium and the AV canal endothelium (Krug et al., 1987; Krug et al., 1985; Markwald et al., 1984; Runyan and Markwald, 1983). Activation of the endothelium is characterized by a loss of cell–cell contacts, cellular hypertrophy and polarization, and an increased expression of ECM molecules (Bolender et al., 1980; Crossin and Hoffman, 1991; Krug et al., 1985). Activated endothelial cells subsequently undergo morphological changes to become mesenchymal cells surrounded by ECM (Krug et al., 1985; Runyan et al., 1990; Wunsch et al., 1994).
Progress in understanding epithelial-mesenchymal cell transformation in the AV canal of the heart is mainly due to the development of an in vitro culture system (Bernanke and Markwald, 1982; Runyan and Markwald, 1983). The in vitro AV canal culture mimics the in situ temporal and regional specificity of cardiac epithelial-mesenchymal cell transformation. Components of cardiac epithelial-mesenchymal cell transformation, including activities of cardiac cushion cells, endothelial cell activation, and mesenchymal cell transformation and migration have been extensively studied in the chick system (Boyer et al., 1999a,b; Brown et al., 1996, 1999; Krug et al., 1987, 1985; Loeber and Runyan, 1990; Mjaatvedt et al., 1987; Potts et al., 1991, 1992; Potts and Runyan, 1989; Ramsdell and Markwald, 1997; Runyan et al., 1992).
In order to examine the molecular mechanisms of TCE effects on cardiac development, we used the in vitro chick AV canal explants model to study the process of epithelial-mesenchymal cell transformation in the presence of TCE. Although other metabolites may be more potent, application of this toxicant to the target tissue permitted a direct evaluation of TCE as a potential cardiac teratogen. In this study, we show that TCE perturbs endothelial cell separation and mesenchymal cell formation processes during epithelial-mesenchymal cell transformation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagen gel assay.
Fertilized White Leghorn chicken eggs (Rosemary Farm, San Metro, CA) were incubated at 37.5°C and 60% relative humidity for 64 h and dissected in 4°C Tyrode's solution (Gibco, Bethesda, MA). AV canal explants from Stage 16 chick embryo (Hamburger and Hamilton, 1951
) were isolated as described (Potts et al., 1991). Three-dimensional hydrated collagen gels were prepared in a 16-mm Nunclon, 4-well culture dish (Roskilde, Denmark), according to previously published procedures, 24 h prior to use (Bernanke and Markwald, 1982; Potts et al., 1991). The collagen gels were incubated overnight with medium 199+ containing 0 or 50–250 ppm TCE. Doses were selected empirically to extend an initial effective dose of 250 ppm at 50 ppm intervals. (Doses less than 50 ppm were not tested as the observed inhibition of mesenchymal cell formation at 50 ppm suggested that the assay would be insensitive to lower doses.) Excess medium was decanted from the gel-containing well to permit attachment of the explant to the gel surface. AV explants were then placed onto collagen gels. After 6 h of incubation, additional medium 199+ and 50–250 ppm TCE were added to the explants. Explants were incubated for an additional 48 h (with replacement of medium containing TCE at 24 h) and fixed in 4% paraformaldehyde (PFA) for 30 min.
Measurement of epithelial-mesenchymal cell transformation.
Epithelial-mesenchymal cell transformation was measured by counting the number of mesenchymal cells inside the collagen gel matrix (Fig. 1D
) for each explant on an Olympus IMT-2 inverted microscope equipped with Hoffman Modulation Optics (Hoffman Optics, Brooklyn, NY). A total of 15 explants were counted for each dose of TCE or control.
Endothelial cell density measurement.
Endothelial cells from 250 ppm TCE-treated and control AV explant cultures were visualized with Hoffman Modulation Optics. Micrographs of endothelial cells were taken with a Dage CCD camera and a Scion frame grabber on a Macintosh 7500 computer using NIH Image software. A frame of 220 µm x 220 µm was placed over each micrograph, with the center in between the edge of the endothelial cells and the edge of myocardium. The number of endothelial cells within the frame was counted. Micrographs from 7 explants were counted for each treatment. Statistical analysis was performed using the Student's t-test.
Cell migration assay.
In order to produce a population of mesenchymal cells, AV-canal explants from Stage 17 chick embryos were placed onto collagen gels and incubated for 18 h. Cultures then were treated with medium 199+ or TCE (250 ppm). Thirty min after the addition of medium 199+ or TCE, the lateral migration of a population of mesenchymal cells in each culture was videotaped for 2 h. Videomicrography was performed, using an inverted Olympus IMT microscope equipped with an incubator stage, maintained at 37°C and infused with CO2 to maintain the pH of the medium. Cellular migration distance was obtained by measuring the change in location of the centroid of each cell at 5-min intervals using a computer equipped with a Bioquant Image Analysis System (R & M Biometrics, Nashville, TN).
Immunohistochemistry.
AV canal explants from Stage 16 chick embryos were placed onto medium 199+ (control for TCE) or TCE-treated (250 ppm) collagen gels as described above. The explants were treated with medium 199+ or TCE after 6 h of incubation. Explants then were incubated for an additional 48-h and fixed in 1% PFA for 30 min. Collagen gels were rinsed extensively in phosphate-buffered saline (PBS) before immunostaining. Primary antibodies used were anti--smooth muscle actin (mouse monoclonal, 1:400 dilution, Sigma, St. Louis, MO), Mox-1 (mouse monoclonal, 1:200 dilution, gift of Dr. C. Wright), and fibrillin 2 (mouse monoclonal, 1:200 dilution, gift of Dr. C. Little). Primary antibody incubation was carried out overnight at 4°C. Secondary antibody (Cy-5 conjugated anti-mouse antibody at 1:200 dilution) incubation was overnight at 4°C. Immunostained collagen gels were placed on slides, covered with glycerol, and sealed with nail polish. Immunostained AV explants then were viewed on a Leica confocal microscope. For each experimental group (control vs. TCE), the immunostaining procedures, and the settings on the confocal microscope were identical.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TCE Inhibition of Epithelial-Mesenchymal Cell Transformation Is Dose Dependent
A hydrated collagen gel assay was developed to examine the epithelial-mesenchymal cell transformation during heart valve formation (Bernanke and Markwald, 1982
; Runyan and Markwald, 1983). This in vitro assay represents the in vivo specificity of epithelial-mesenchymal cell transformation both temporally and spatially (Runyan and Markwald, 1983). The collagen gel assay is depicted in Figure 1. Stage 16 chicken AV canal explant has 3 cell types: endothelium, mesenchyme, and myocardium (Fig. 1A). The endothelium-derived mesenchymal cells are the progenitors of valve and septa in the adult heart (Fig. 1B). In the in vitro assay, the Stage 16 AV explant is placed on the surface of a collagen gel. The endothelium forms a single layer on top of the collagen gel surface surrounding the myocardium (Fig. 1C). Upon receiving activation signals such as TGFß-3 from the myocardium, the endothelial cells will separate from each other and transform into mesenchymal cells, which in turn invade into the collagen gel (Fig. 1D) (Runyan et al., 1992).
In order to test the effects of TCE on epithelial-mesenchymal cell transformation, AV-canal explants from Stage 16 chick embryos were placed on the surface of collagen gels containing 0–250 ppm TCE, and cultured as described in Materials and Methods. After 6 h of culture, additional medium containing TCE at the desired concentration was added, and the explants were cultured for an additional 48 h before fixation in 4% PFA. The effect of TCE on epithelial-mesenchymal cell transformation is shown in Figure 2. Endothelial cells form a monolayer on the surface of untreated control cultures, displaying a polygonal organization and the cell–cell separation indicative of activation (Fig. 2A) (Bolender et al., 1980; Crossin and Hoffman, 1991; Krug et al., 1985). In TCE-treated cultures, endothelial cells formed a monolayer with a lesser degree of cell–cell separation (Fig. 2B). Figure 2C shows mesenchymal cells from control explant invading inside the collagen gel. TCE-treated cultures (250 ppm) had fewer mesenchymal cells compared to control (compare Figs. 2C and 2D). Therefore, TCE treatment inhibited both endothelial cell activation and normal mesenchymal cell formation in AV cushion cultures.
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The number of mesenchymal cells visible in the collagen gel was counted in both control and TCE-treated cultures as a measure of completed epithelial-mesenchymal cell transformation. Quantitative analysis of mesenchymal cell formation in explant cultures indicated that TCE inhibition of mesenchymal cell formation is dose dependent (Fig. 3
). There was a statistically significant reduction of the average number of mesenchymal cells formed for each dosage of TCE-treated culture (50 ppm–250 ppm). Furthermore, the degree of inhibition increased with increasing TCE concentration. At 250 ppm, TCE inhibited epithelial-mesenchymal cell transformation by 50%. To examine whether TCE perturbs earlier events of endothelial cell activation during chick cardiac epithelial-mesenchymal cell transformation, Stage 14 AV explants also were prepared and analyzed. We found that TCE inhibition of Stage 14 AV explants was indistinguishable from that observed with Stage 16 explants (data not shown). These data suggest that TCE inhibition of epithelial-mesenchymal cell transformation occurs after initial endothelial cell receipt of the signal from myocardium (i.e., Stage 14) and at a later stage of endothelial activation.
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TCE Inhibits Endothelial Cell Separation
The reduction in mesenchymal cell numbers observed in TCE-treated cultures could be due to an inhibition of endothelial cell–cell separation and a corresponding reduced ability of mesenchymal cells to invade collagen gel. Comparative morphological examination of endothelial cells suggested a potentially greater density of endothelial cells in TCE-treated cultures (compare Figs. 2A and 2B
). To confirm this observation, endothelial cell layers from 7 pairs of 250-ppm TCE-treated and control explants were photographed and compared. Endothelial cell densities were analyzed within the frame of 220 µm x 220 µm, and the result is shown in Figure 4. The average endothelial cell density was 31% higher in TCE-treated cultures than in control cultures. These data suggest that the normal endothelial cell–cell separation process associated with epithelial-mesenchymal cell transformation is inhibited by TCE.
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TCE Does Not Inhibit Mesenchymal Cell Migration
We previously documented that cardiac mesenchymal cell migration can be perturbed by TGFß Type II and Type III receptor antibodies and pertussis toxin (Boyer et al., 1999a
; Brown et al., 1996, 1999). These reagents inhibit signal transduction processes that contribute to the initiation and maintenance of mesenchymal cell migration. In this study, we examined whether TCE also inhibits the migration of mesenchymal cells. To produce a population of mesenchymal cells within the gels, Stage 17 AV cushion explants were placed on collagen gels and incubated overnight (as described in Materials and Methods). The rate of lateral migration of a sample of mesenchymal cells was measured in the presence and absence of 250 ppm TCE. TCE did not alter the mesenchymal cell migration rate (Table 1). Therefore, effects of TCE on epithelial-mesenchymal cell transformation are not due to an inhibition of mesenchymal cell migration.
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TCE Inhibits Protein Marker Expression
Even though the molecular mechanisms mediating epithelial-mesenchymal cell transformation are largely unknown, a number of proteins expressed during epithelial-mesenchymal cell transformation have been identified as markers of the transformation process. These protein markers include
-smooth muscle actin, Mox-1 and fibrillin 2. The pattern of expression of each of these proteins has been described in the developing AV canal. -Smooth-muscle actin is first expressed in activated endothelium and then in the mesenchyme and is associated with the early stage of the endothelial-cell activation process and cardiac mesenchymal cell migration (Nakajima et al., 1997). Mox-1 is a homeobox transcription factor expressed in endothelial and mesenchymal cells (Boyer et al., 1999a; Candia et al., 1992). Fibrillin 2 is a secreted ECM protein expressed in activated endothelium and mesenchyme and is a marker of mesenchymal cells (Rongish et al., 1998; Wunsch et al., 1994).
Immunofluorescent staining revealed that expression of Mox-1 and fibrillin 2 was affected by TCE (Fig. 5). Mox-1 and fibrillin-2 immunostaining intensity in mesenchymal cells was strongly inhibited by TCE (compare Figs. 5A and 5B, 5C and 5D). In contrast, -smooth muscle actin expression was not altered by TCE treatment (Figs. 5 E and 5F). These data suggest that TCE has no effect on early stages of endothelial cell activation prior to cell–cell separation. However, TCE does have a specific effect on a subset of mesenchymal cell markers expressed during endothelial cell–cell separation and mesenchymal cell formation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies demonstrated that many infants born with congenital heart defects in TCE contaminated regions have defects involving the valves and septa formation (Goldberg et al., 1990
; Swan et al., 1985). The heart valves and septa progenitor cells are formed via a process known as epithelial-mesenchymal cell transformation (Eisenberg and Markwald, 1995; Markwald et al., 1996; Runyan et al., 1992). Numerous studies on the molecular mechanisms of cardiac epithelial-mesenchymal cell transformation showed that both G protein-mediated and TGFß-mediated signal transduction pathways are involved (Boyer et al., 1999a,b; Brown et al., 1996, 1999; Potts et al., 1991, 1992; Ramsdell and Markwald, 1997). Epithelial-mesenchymal cell transformation in the heart is also mediated by other components of the ECM such as SBA lectin-binding proteins (Sinning et al., 1995), HLAMP-1 (Sinning, 1997), activin (Moore et al., 1998) and ES/130 (Mjaatvedt et al., 1991; Rezaee et al., 1993).
Based on these studies, we hypothesize that TCE mediates cardiac valve and septa malformation by perturbing one or more specific events of epithelial-mesenchymal cell transformation. The objectives of our studies were to investigate the cellular and molecular mechanism of the cardiac teratogenicity of TCE. First, we sought to determine whether TCE perturbs epithelial-mesenchymal cell transformation. Second, using specific proteins as molecular markers of transformation, the effects of TCE on cardiac epithelial-mesenchymal cell transformation were confirmed at the molecular level. We exposed Stage 16 chicken AV canal cultures to 0–250 ppm TCE in vitro and determined that the epithelial-mesenchymal cell transformation is sensitive to TCE exposure. In particular, we observed that mesenchymal cell numbers, as a measurement of epithelial-mesenchymal cell transformation, are inhibited by TCE at all doses tested. In order to identify TCE-sensitive events during valve morphogenesis, we treated Stage 14 AV canal cultures with TCE and found that there is no difference in the effect of TCE on epithelial-mesenchymal cell transformation between younger and older stages. This observation suggests that TCE is perturbing processes immediately proximal to the actual change in endothelial cell phenotype and that it has little effect on the preparatory and inductive events during epithelial-mesenchymal cell transformation that took place at earlier stages (Boyer et al. 1999a; Ramsdell and Markwald, 1997). The decrease of mesenchymal cell numbers in TCE-treated cultures is probably due to a perturbation of cell–cell adhesion that occurs within the endothelial layer.
An inhibition of endothelial cell–cell separation similar to that seen with TCE was also observed in cultures treated with pertussis toxin (an inhibitor of Gi proteins), or blocking antibodies toward both TGFß-2 and the TGFß Type III receptor (Boyer et al, 1999a,b). Since all of these reagents also inhibited mesenchymal cell migration, the observation that TCE has no effect on cell migration suggests that TCE may perturb epithelial-mesenchymal cell transformation through a separate mechanism from TGFß and G protein signal transduction processes.
To determine the TCE effect on epithelial-mesenchymal cell transformation at the molecular level, the expression of molecular markers that are associated with epithelial-mesenchymal cell transformation was examined. Though a variety of proteins is known, the 3 molecules chosen here provide a representative sample of markers. Mox-1 is a mediator of mesenchymal cell formation and Mox-1 protein and mRNA are expressed in both endothelial and mesenchymal cells in the AV canal at the time of transformation (Boyer et al., 1999a; Wendler and Runyan, in preparation). We found that expression of Mox-1 protein was dramatically inhibited by exposure to TCE. The expression of an ECM protein fibrillin 2, as a marker of mesenchymal cell formation, was also greatly inhibited. In comparison, the early endothelial cell activation marker, -smooth muscle actin, was unaffected by TCE. In addition, a mesenchymal cell migration marker, Type I collagen (Sinning et al., 1988) was not affected by TCE (data not shown).
The contrasting effects of TCE on fibrillin 2 and -smooth muscle actin and the normal rate of migration seen in the presence of TCE suggest that this toxicant perturbs specific developmental processes. The expression of cell migration marker Type I collagen and the rate of mesenchymal cell migration are unperturbed by the TCE, indicating that transformed mesenchymal cells are insensitive to TCE. TCE-sensitive events during epithelial-mesenchymal cell transformation are restricted to the period of visible cell–cell separation and cell shape change. The observation that Mox-1 expression is reduced could account for the loss of mesenchymal cell formation and the reduced expression of fibrillin 2. Antisense oligonucleotide experiments demonstrate that Mox-1 is required for epithelial-mesenchymal cell transformation in cardiac explant cultures (Wendler and Runyan, unpublished). However, since several transcription factors are specifically expressed in the AV canal of the heart, including brachyury (Huang et al., 1995), slug (Romano and Runyan, submitted), GATA 4/5/6 (Jiang et al., 1998), Id (Evans and O'Brien, 1993), and NF-ATc (de la Pompa et al., 1998; Ranger et al., 1998), we were unable to distinguish whether the effect of TCE on Mox-1 is direct or indirect. Since our current model of epithelial-mesenchymal cell transformation in the heart suggests that mesenchymal cell formation is the product of multiple, independent signal transduction pathways into and within the target cell (Boyer et al., 1999a), TCE inhibition of Mox-1 may be sufficient to reduce mesenchymal cell numbers.
Although the present study points to TCE inhibition of elements of epithelial-mesenchymal cell transformation as the basis of cardiac valvular and septal defects, not all cardiac defects seen in treated animals are due to defects in early valve formation. Other potential sources of cardiac defects include a loss of neural crest cells (Kirby and Waldo, 1990), altered blood flow patterns (Hogers et al., 1997), and myocardial cell deficits (Vikstrom et al., 1996). It is likely that several of these elements could be affected by TCE. In a concurrent study, TCE-treated rat embryo hearts are being examined for changes in gene expression compared to untreated controls. To date, we have identified more than 40 molecules whose expressions are either up- or down-regulated in response to 110 ppm TCE exposure in maternal drinking water. These molecules include a variety of stress-response genes, housekeeping genes, cytoskeletal components, and developmentally expressed genes (Collier et al., in preparation). Ongoing studies will explore the localization of gene expression in the developing heart and the functional significance of changes in several of these candidates.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Christopher Wright for the Mox-1 antibody and Drs. Charles Little and Brenda Rongish for the fibrillin 2 antibody. We also thank Drs. Clark Lantz, Ornella Selmin, and Glenn Sipes for critically reviewing the manuscript. These studies were funded by NIH ES04940 and HL58696 and supported through the resources of the Southwest Environmental Health Sciences Center (ES-06694). ASB was supported by a fellowship from the Arizona Affiliate of the American Heart Association while WTF was supported by the Howard Hughes Medical Foundation (71195–521303).
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NOTES |
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1 These authors contributed equally.
2 To whom correspondence should be addressed at Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Life Sciences North, Rm. 421, Tucson, AZ 85724. Fax: (520) 626-2097. E-mail: rrunyan{at}u.arizona.edu.
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