Toxicogenomic Profile of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in the Murine Fetal Heart: Modulation of Cell Cycle and Extracellular Matrix Ge
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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 Biocomputing, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Ambion, Inc. The RNA Company, Austin, Texas 78744
Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville Health Sciences Center, Louisville, Kentucky 40292
Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
1 To whom to address correspondence should be addressed at College of Pharmacy, 2502 Marble NE, Albuquerque, NM 87131. Fax: 505-272-0704. E-mail: mkwalker@unm.edu.
ABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 μg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix–related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.
Key Words: TCDD; aryl hydrocarbon receptor; microarray; heart development; cell cycle.
INTRODUCTION
The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent teratogen in birds, fish, and mammals (Couture et al., 1990; Guiney et al., 2000; Walker and Catron, 2000). TCDD toxicity is believed to be mediated by alterations in gene expression via activation of the aryl hydrocarbon receptor (AhR). The AhR is a cytosolic protein with high affinity for TCDD and other halogenated and polycyclic aromatic hydrocarbons. Upon ligand activation, the AhR translocates into the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT), binds to dioxin responsive elements (DRE) in the upstream regions of regulated genes, and modulates gene expression (Denison et al., 2002).
Although the AhR is highly conserved across vertebrates (Hahn, 1998), its specific physiological function remains enigmatic. It is notable, however, the both the loss of AhR function by genetic deletion and the sustained activation of AhR by environmental pollutants result in a disruption of normal developmental regulation. For example, mice harboring null alleles for the AhR are viable at birth, but they exhibit a small liver as a result of abnormal hepatic vascular development (Fernandez-Salguero et al., 1995; Lahvis et al., 2000; Schmidt et al., 1996). Additionally, AhR null embryos also develop abnormal cardiac hypertrophy and hyperplasia in utero, and this phenotype is dependent, in part, on the maternal AhR genotype (Thackaberry et al., 2003). Likewise, sustained activation of AhR by TCDD also disrupts developmental regulation, resulting in characteristic teratogenicity, including cleft palate and hydronephrosis in rodents and cardiovascular defects in fish and birds (Couture et al., 1990; Guiney et al., 2000; Walker and Catron, 2000). Thus, both loss of AhR function and sustained AhR signaling are detrimental to developmental control.
Importantly, it has been shown that mice lacking AhR expression are significantly less sensitive to the teratogenic effects of TCDD after developmental exposure (Bunger et al., 2003; Mimura et al., 1997; Peters et al., 1999). For example, AhR wild-type fetuses exposed to 25 μg TCDD/kg in utero exhibit a significantly higher incidence of cleft palate and hydronephrosis compared to AhR null fetuses (Peters et al., 1999). Similarly, when fetal palatal shelves are exposed to TCDD in explant culture, mice harboring a mutated AhR with a deleted nuclear localization sequence are resistant to TCDD-induced cleft palate (Bunger et al., 2003). These data support the hypothesis that the teratogenic effects of TCDD are mediated by transcriptional dysregulation of gene expression via the AhR.
One important objective of our laboratory is to characterize the effects of TCDD on cardiac structure, function, and gene expression during murine development, and toward this objective we have applied a non-biased microarray method for identifying those genes associated with the teratogenic effects of TCDD on the developing cardiovascular system. Because inhibition of cardiomyocyte proliferation is one of the most sensitive effects of TCDD in piscine and avian embryos, we targeted a window during murine fetal development when cardiomyocyte proliferation peaks (i.e., GD 14.5–17.5). Here, in the first of two reports, we present the effects of TCDD treatment on global gene expression profiles in the developing murine heart. Our results demonstrate that in utero TCDD exposure significantly alters the expression of cardiac genes involved in cell cycle regulation, extracellular matrix deposition/remodeling, and cardiac homeostasis. Furthermore, these TCDD-induced changes in gene expression are associated with subtle, but potentially significant, cardiac structural alterations and functional deficits, including reduced fetal cardiocyte proliferation and heart size, as well as postnatal bradycardia, which are reported in detail in a companion article (Thackaberry et al., 2005).
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 of the occurrence of a vaginal plug was designated GD 0.5. On GD 14.5 pregnant mice were dosed with corn oil (control) or 1.5, 3.0, or 6.0 μg TCDD/kg in corn oil via oral gavage (5.0 μl/g body weight). Pregnant dams were sacrificed on GD 17.5, fetuses dissected and weighed, and fetal hearts were isolated, pooled within a litter, and frozen for mRNA isolation.
Microarray analysis.
Total mRNA was isolated from pooled frozen fetal hearts (n = 4 litters per treatment, one microarray chip per litter) using Trizol reagent (Invitrogen, Carlsbad, CA), cDNA synthesized with T7-( dT)24 primer, rcRNA synthesized and labeled with biotin, and rcRNA fragmented, after which samples were hybridized to Affymetrix MG_U74Av2 microarray chips (Affymetrix, Santa Clara, CA) for 16 h. After hybridization, chips were washed and stained with streptavidin-phycoerythrin and scanned. Because microarray experiments were conducted on two different dates with four control chips/date, we investigated whether variations existed between the two batches of chips by inspecting the distribution of gene expression values of each control chip and the quantile-quantile plot between control chips. We found that these two data sets could be combined and analyzed as a single experiment with a total of eight control chips. All microarray experiments were carried out using the UNM KUGR core facility, and were MIAME certified. Raw data can be found at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) Web site http://www.ncbi.nlm.nih.gov/projects/geo/; series record #GSE2812). All genes that were absent on 24/24 chips or moderately detected on 6/24 chips were deleted, reducing the total from 12,488 to 7,253 genes. Raw intensity values were normalized using GeneSpring software (Silcon Genetics, Redwood City, CA), and fold differences were calculated from differential interpretational results. Data were analyzed with the GeneSpring software.
Real-time PCR.
Total RNA was isolated from pooled GD 17.5 hearts using Trizol reagent (Invitrogen), and cDNA was generated using reverse transcriptase (Promega, Madison, WI) with oligo dT and an 18s RT primer to amplify the 18s control RNA. The sequence of the 18s RT primer was 5'-AGTTCGACCGTCTTCTCAGC-3'. mRNA expression was then quantified using a BioRad I-Cycler (BioRad, Hercules, CA). The efficiency of each primer set was determined, using a standard curve with known quantities of cDNA, and all primers sets used were 90–100% efficient. Real-time PCR reactions were run in triplicate for the genes of interest and 18s control simultaneously, and the differences between the CT values were calculated. Values were then converted to mean relative expression, using Q-gene software (Simon, 2003), and expressed as a percent of control. The primer sequences are shown in Table 1.
Statistics.
Only genes predicted to be present by the one-sided Wilcoxon's Signed Rank test using the MAS 5.0 default settings (Affymetrix, Santa Clara, CA), and with p < 0.05, compared to control, were reported as "altered." Fold change in expression was determined with GeneSpring software to normalize TCDD-treated values to the control data (set at 1.000). Therefore, a normalized value of 2.0 would represent a 2.0-fold induction, and a normalized value of 0.5 would represent a 2.0-fold repression (expressed here as –2.0). Additionally, we applied multifactor analysis of variance (ANOVA) to identify genes that were differentially expressed across treatments, and we estimated the false discovery rate (FDR) from the rank-ordered ANOVA p-value (Benjamini and Hochberg, 1995). At the FDR of 0.25, we identified 1239 genes that were differentially expressed across treatment groups, but not between control chip sets. GeneSpring software was used for hierarchal clustering, expression correlations, ontological identification, and pathway analysis. To determine correlations between genes, a Pearson correlation around zero was used.
RESULTS
Genes Altered by TCDD and Grouped by Functional Category
To determine the functional classes of genes that were altered by TCDD, we used the simplified gene ontogeny provided by the GeneSpring software. Table 2 shows the number of genes induced or repressed 1.5- and 2.0-fold by TCDD and grouped by functional category. Those categories with relatively high numbers of genes altered in expression included cell growth, defense/immunity, development, ion channels, transcription factors, signal transduction, and transporters.
Differential Dose-Related Responses to TCDD
Next, we used cluster analysis and filtering to determine the primary dose-related response patterns observed with increasing concentration of TCDD (Table 3). In general, genes were more likely to be induced than repressed by TCDD treatment and the greatest number of genes altered in expression occurred at the highest dose (186 induced 1.5-fold; 99 repressed 1.5-fold; p < 0.05). In addition, 41 genes were induced 1.5-fold at all doses, and 11 genes were repressed 1.5-fold at all doses. Six genes were induced by 2-fold at all three TCDD doses (Cyp1a1, Cyp1b1, Reg3g, Fbp1, Scgb1a1, and EST #AA270365), but there were no genes that were repressed 2-fold at all three doses.
Selected examples of genes induced or repressed 2-fold and that clustered into one of the response groups in Table 3 are shown in the Venn diagram in Figure 1. Along with Cyp1a1 and Cyp1b1, an EST homologous to fructose biphosphatase (Fbp1) was upregulated 2-fold at all three doses, whereas the NAD(P)H oxidase p40phox subunit, Ncf4, and the matrix metalloproteinase enzyme, Mmp13, were upregulated only at the two highest doses. Other genes of note included the inwardly rectifying potassium channel (Kcnj5), which was upregulated only at the highest dose, and the thromboxane A2 receptor (Tbxa2r), which was upregulated only at the lowest two doses.
Expression of Genes in AhR Signaling Cascade or Known to be AhR-Responsive
We next investigated the expression of genes known to be involved in the AhR signaling cascade or regulated by the AhR. No change in the expression of any genes involved in the AhR signaling cascade was observed after TCDD treatment, including Ahr, Arnt, Arnt2, or Aip, (Table 4). Of genes known to contain DREs and to be regulated by the AhR, alcohol dehydrogenase 3 (Adh-3), AhR repressor (Ahrr), cytochrome P-450 1A1 (Cyp1a1), cytochrome P-450 1B1 (Cyp1b1), and UDP-glucuronosyl transferase (Ugt1a6), were all upregulated at the highest dose and in some cases in a dose-related manner. Notably, however, the DRE-containing, AhR-regulated, cytochrome P-450 1A2 (Cyp1a2), glutathione-S-transferase-Ya (GSTa1), and NAD(P)H quinone oxidoreductase (Nqo1) were not altered by TCDD treatment in the fetal murine heart, and the expression of all three of these genes was below background in control samples.
Expression of Genes Known to be Involved in Cardiovascular Development
The effects of TCDD on the expression of genes involved in cardiovascular development are summarized in Table 5. The most striking finding was the large number of extracellular matrix genes altered in response to TCDD. The procollagen genes Col4a5, Col6a1, Col9a2, Col13a1, and Col18a1 were significantly altered by TCDD treatment, as were the matrix metalloproteinases Mmp13 and Mmp14. The dose-related expression of these extracellular matrix genes along with Mmp15, mast cell chymase (Mcpt5), tropoelastin (Eln), mannose binding protein (Mbl2), and the vasoactive promitogenic peptide endothelin-1 (Edn), are shown in graphical form in Figure 2. Although a few of these genes, such as Col6a1 and Mcpt5, showed typical dose-related effects on expression, many more of these genes showed significant alterations in expression only at the highest dose or only at the lower doses. In addition to the extracellular matrix genes, the angiogenic cytokines, vascular endothelial growth factor (Vegfa) and angiopoetin (Agpt), were both downregulated, as were the potassium voltage-gated channels Kcna3 and Kcna5.
Genes with Similar Expression Profiles to the Most Highly Induced or Repressed Genes
We used the GeneSpring software correlation function to identify genes whose TCDD dose-related expression profile correlated (>0.95) with the dose-related expression profile of (1) the most highly induced genes, Ahrr, Cyp1a1, and Cyp1b1 (Fig. 3A, Table 6), or (2) the most highly repressed gene, Mcpt5 (Fig. 3B, Table 7). Among the genes correlating with the most highly induced genes was the NAD(P)H oxidase subunit, Ncf4; two immune-response proteins, Ccl1 and Illr2; the matrix metalloproteinase, Mmp13; and two genes with high homology to known carbohydrate metabolism genes, the Na+/glucose cotransporter (EST) and fructose bisphosphatase (Fbp1). Among genes correlating with the most highly repressed gene was a second mast cell–specific gene, mast cell carboxypeptidase A (Cpa3), as well as protocadherin 7 (Pcdh7), bone morphogenetic protein 10 (Bmp10), an inhibitory GTP binding protein (Gnai1), and three transporters (multidrug resistance–associated protein, Abcc3; carnitine transporter, Octn2; and fatty acid transporter, Cd36).
Real-Time PCR Confirms Altered Expression of Selected Genes
To validate the microarray data presented in this study, we used real-time PCR to confirm the expression of a number of key genes at the 6.0 μg TCDD/kg dose (Table 8). Real-time PCR confirmed (1) induction of Cyp1a1, Cyp1b1, Edn, and Mmp13 and (2) the repression of Cd36, Ccne1, Mcpt5, and Vegfa. We also chose five genes whose expression was unchanged at the 6.0 μg TCDD/kg dose by microarray and quantified them by real-time PCR. The results confirmed a lack of alteration of Ccnd1, Cyp1a2, and Cdkn1b; however, real-time PCR was able to identify decreased expression of two cell-cycle–related genes, Ccna2 and Ccne2, which were unaltered on the microarray at the highest TCDD dose, but which were repressed at two lower doses. Of all the genes quantified by real-time PCR, only these two cell cycle genes and mannose-binding protein (Mbl2) differed from the microarray results. Mbl2 was repressed to a similar extent using both methods, but it was not statistically significant by real-time PCR.
DISCUSSION
Although the effects of TCDD on the developing cardiovascular system of piscine and avian species are well documented, relatively little is known about the effects of TCDD on the developing mammalian heart. Given the critical role of AhR-mediated gene dysregulation in the teratogenic responses to TCDD, we used microarray analysis to determine the global changes in gene expression that occur in parallel with TCDD-induced cardiac teratogenicity (reported in our companion article [Thackaberry et al., 2005]).
Comparison of TCDD-Induced Gene Expression Changes with Other Studies
As expected, in utero TCDD exposure significantly induced Cyp1a1, Cyp1b1, AhR repressor (Ahrr), UDP glucuronosyl transferase (Ugt1a6), and alcohol dehydrogenase 3 (Adh-3) in the fetal murine heart, all of which have been reported to be controlled by the AhR (Emi et al., 1996; Lusska et al., 1991; Mimura et al., 1999; Zhang et al., 1998). Interestingly, Cyp1a2, glutathione-S-transferase-Ya (Gsta1) and NAD(P)H quinone oxidoreductase (Nqo1), also previously shown to be AhR-regulated (Favreau and Pickett, 1991; Nemoto and Sakurai, 1993; Paulson et al., 1990), were not altered by TCDD in the fetal murine heart. Furthermore, the expression of these latter genes was at or below background in all groups, suggesting that they are not expressed and are not TCDD-inducible in the fetal murine heart.
Numerous studies have demonstrated that TCDD can induce target gene expression, such as Cyp1a1, to a much greater degree than that observed in this study. This difference likely results from a combination of the tissue type being analyzed and the relatively small amount of TCDD that reaches fetal cardiac tissue. Induction of Cyp1a1 by AhR agonists has been detected primarily in the endothelial cells of the developing heart, and not in the myocardium nor in isolated myocytes (Kanzawa et al., 2004; Stegeman et al., 1991), and thus the degree of induction in the fetal murine heart may appear low because endothelial cells comprise a small component of the total cells in the heart. Additionally, toxicokinetics models predict that 0.04% of the total maternal TCDD dose reaches an individual fetus (Weber and Birnbaum, 1985) and even a smaller percentage of that will distribute to the heart. Finally, it is also notable that of several novel genes with putative DREs that are conserved across human, rat, and mouse, none of them were altered in a dose-dependent manner in our study (Sun et al., 2004).
These results suggest that AhR gene regulation may be tissue-specific. TCDD microarray studies performed using other tissues or cell types show relatively few genes in common with those identified in our study. When comparing changes in gene expression in our study to those from other TCDD microarray studies, we found five induced genes and one repressed gene that matched exactly, including Cyp1a1, Cyp1b1, Adh3, Ugt1a6, Col6a1, and Vegfa (Boverhof et al., 2005; Handley-Goldstone et al., 2005; Martinez et al., 2002; Mizutani et al., 2004; Puga et al., 2000b). This was particularly surprising when we compared our results with those from a microarray study on TCDD-treated embryonic zebrafish with an emphasis on cardiovascular-related genes (Handley-Goldstone et al., 2005). Significant differences in gene expression that were common between the two studies included only Cyp1a1 and Cyp1b1. The lack of greater similarity may be explained, in part, by the difference in timing of exposure. Zebrafish eggs were exposed to TCDD very early in cardiogenesis, whereas murine fetuses were exposed very late in heart development. Finally, if we considered one other microarray study that used the AhR agonist benzo[a]pyrene, we identified two additional repressed genes that matched with our results: cadherin 13 (Cdh13) and procollagen IV alpha 5 (Col4a5) (Karyala et al., 2004).
In addition to finding few genes in common with other microarray studies, we also found that some changes in gene expression were in the opposite direction or not altered when compared to other studies. For example, TCDD induced Cd36 in the liver of adult mice (Boverhof et al., 2005) and Vegfa in human airway epithelial cells (Martinez et al., 2002), but it repressed the expression of both these genes in the fetal murine heart. In addition, it has been shown that inhibition of transforming growth factor- (TGF) signaling may play a role in TCDD-induced cleft palate (Thomae et al., 2005), and TCDD has been shown to dysregulate expression of TGF-related genes in vascular smooth muscle (Guo et al., 2004). Although TGF signaling plays a key role in cardiac morphogenesis, we did not identify any significant changes in expression of TGFs, their receptors, or related genes. Although many of these differences likely result from tissue-specific responses, we also cannot discount that differences in the dose of TCDD and length of exposure may contribute to differences between our results and those reported in other studies.
TCDD Dysregulates Expression of Many Cardiac Genes Related to ECM Homeostasis
The altered expression of extracellular matrix (ECM) genes or of known regulators of ECM deposition/degradation was one of the most striking findings of the current study. Expression of cardiac ECM genes, including tropoelastin (Eln) and five procollagens (Col4a5, Col6a1, Col9a2, Col13a1, and Col18a1), was highly dysregulated by TCDD exposure. In addition, genes that regulate ECM deposition/degradation also were dysregulated in expression, including three matrix metalloproteinases (Mmp13, Mmp14, Mmp15), mast cell chymase (Mcpt5), mast cell carboxypeptidase A (Cpa3), and endothelin-1 (Edn). Cardiac ECM remodeling is highly dependent on the balance between collagen degradation and synthesis, and cardiac dysfunction is frequently associated with induction of MMPs, alterations in ECM structure, and enhanced cardiac fibrosis (Spinale, 2002). Additionally, local cardiac production of angiotensin II (Ang II) and endothelin-1 is associated with increased cardiac fibrosis and cardiac dysfunction (Schwarz et al., 2002; Seccia et al., 2003). Mast cell chymase and carboxypeptidase A are involved in the synthesis and degradation of Ang II, respectively (Lundequist et al., 2004), and the former has been shown to activate MMPs. It also is noteworthy that a number of studies have demonstrated that TCDD can increase the expression of MMPs, including MMP1, the human equivalent of murine MMP13 (Murphy et al., 2004). These results suggest that TCDD may contribute to alterations in ECM remodeling in the fetal murine heart, and these alterations may contribute to altered cardiac structure and function after birth, as described in our companion article (Thackaberry et al., 2005).
TCDD Dysregulates Gene Expression of Cell Cycle Regulators in the Fetal Murine Heart
Alterations in the expression of genes involved in cell cycle regulation were of particular interest, because TCDD is known to inhibit cardiomyocyte proliferation in the developing chick embryo (Ivnitski et al., 2001), zebrafish embryo (Antkiewicz et al., 2005), and, most recently, murine fetus (Thackaberry et al., 2005). Our data show that expression of cyclins A2, B1, C, E, E2, and F was repressed or showed a trend toward repression in at least one dose. Real-time PCR confirmed the downregulation of cyclin E1 and showed downregulation of cyclins A2 and E1 at 6 μg TCDD/kg. Additionally, Bmp10 expression was reduced at all TCDD doses. The A- and E-type cyclins are involved in the progression from G1 to S phase (Woo and Poon, 2003), whereas BMP10 plays an essential role in regulating cardiac growth and its deficiency is associated with cardiac hypoplasia (Chen et al., 2004). Thus, it is tempting to speculate that the reduced cardiomyocyte proliferation described in the companion article results from disrupted regulation of cell cycle regulatory proteins. It is important to note, however, that the microarray analysis was performed using whole-heart homogenates. While cardiomyocytes represent the majority of the volume of the heart, there also is a significant population of cardiac fibroblasts and endothelial cells, in addition to smaller populations of conduction and immune cells. Thus, in situ hybridization and immunohistochemical studies would need to be conducted to determine if the reduced cardiomyocyte proliferation was correlated spatially with altered cell cycle protein expression.
Potential mechanisms by which TCDD may downregulate A- and E-type cyclins, and thus inhibit proliferation, include an AhR-dependent interaction with the retinoblastoma protein (Rb) (Ge and Elferink, 1998; Puga et al., 2000a) and induction of the cdk inhibitor, p27kip1 (Cdkn1b) (Kolluri et al., 1999). It is notable that mice lacking p27kip1 exhibit increased cardiomyocyte proliferation (Poolman et al., 1999), and thus it is possible that TCDD-induced expression of cardiac p27kip1 might inhibit cardiomyocyte proliferation. However, Cdkn1b expression was not induced by TCDD in the fetal murine heart, and this lack of induction was confirmed by real-time PCR. Taken together, these results suggest that TCDD may reduce proliferation of fetal murine cardiomyocytes by altering expression of cell cycle regulators, but that this occurs via a mechanism that is independent of changes in p27kip1 mRNA expression.
Correlation Analysis of Gene Expression with TCDD-Induced or -Repressed Genes
Correlation analysis identified several genes whose dose-related expression correlated with (1) known AhR-responsive genes that were highly induced by TCDD (Cyp1a1, Cyp1b1, and Ahrr) or (2) the one gene whose expression was most highly repressed by TCDD (Mtp5). These highly induced or repressed genes did not all fall into any one common pathway or apparent physiological category, although some appear to be related, such as two carbohydrate metabolism genes (Fbp1 and a putative Na+/glucose transporter), which were highly induced, and two organic transporters (Abcc3 and Octn2), which were highly repressed. While the expression of these genes was similar in a dose-related manner to other TCDD-induced or TCDD-repressed genes, it is not known if these genes are directly regulated by the AhR. Considering that TCDD exposure occurred 3 days prior to RNA isolation, it is possible that alteration of these highly correlated genes occurred as downstream responses to other changes in gene expression. In fact, one of the functional categories with the largest number of TCDD-altered genes was that of transcription factors. Nonetheless, the TCDD-induced, dose-related changes in expression of these correlated genes provides a starting point for the potential identification of novel AhR-regulated cardiac genes and may provide insight into those genes that mediate TCDD-induced cardiac teratogenicity. Thus, in ongoing studies we are determining those TCDD-induced changes in fetal cardiac gene expression that are mediated directly by the AhR by exposing crosses of AhR heterozygote mice.
In conclusion, we have used an unbiased microarray approach to demonstrate significant alterations in gene expression in the fetal murine heart after TCDD exposure in utero. These gene expression changes in particular suggest possible alterations in cell cycle control and extracellular matrix production/remodeling, which may contribute to TCDD-induced cardiac teratogenic and functional alterations in the murine fetus. Future studies will be needed to define those changes in gene expression, as well as post-transcriptional or epigenetic mechanisms not identified here, that mechanistically underlie the developmental cardiovascular teratogenicity of TCDD that is described in our companion article (Thackaberry et al., 2005).
ACKNOWLEDGMENTS
The authors thank Marilee Morgan, Dr. Gavin Pickett, Dr. Scott Ness, and the entire UNM KUGR facility for their valuable assistance. This work was supported by ES012335 to M.K.W., ES012855 to E.A.T., and the P30 National Institute of Environmental Health Sciences (NIEHS) Center ES12072.
REFERENCES
Antkiewicz, D. S., Burns, C. G., Carney, S. A., Peterson, R. E., and Heideman, W. (2005). Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368–377.
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate—A practical and powerful approach to multiple testing. J. R. Stat. Soc B—Methodological 57, 1289–1300.
Boverhof, D. R., Burgoon, L. D., Tashiro, C., Chittim, B., Harkema, J. R., Jump, D. B., and Zacharewski, T. R. (2005). Temporal and dose-dependent hepatic gene expression patterns in mice provide new insights into TCDD-mediated hepatotoxicity. Toxicol. Sci. 85, 1048–1063.
Bunger, M. K., Moran, S. M., Glover, E., Thomae, T. L., Lahvis, G. P., Lin, B. C., and Bradfield, C. A. (2003). Resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and abnormal liver development in mice carrying a mutation in the nuclear localization sequence of the aryl hydrocarbon receptor. J. Biol. Chem. 278, 17767–17774.
Chen, H., Shi, S., Acosta, L., Li, W., Lu, J., Bao, S., Chen, Z., Yang, Z., Schneider, M. D., Chien, K. R., et al. (2004). BMP10 is essential for maintaining cardiac growth during murine cardiogenesis. Development 131, 2219–2231.
Couture, L. A., Abbott, B. D., and Birnbaum, L. S. (1990). A critical review of the developmental toxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Recent advances toward understanding the mechanism. Teratology 42, 619–627.
Denison, M., Rogers, J. M., Rushing, S. R., Jones, C. L., Tetangco, S. C., and Heath-Pagliuso, S. (2002). Analysis of the Ah receptor signal transduction pathway. In Current Protocols in Toxicology (M. Maines, L. G. Costa, D. J. Reed, S. Sassa, and I. G. Sipes, Eds.), p. 4.8.1–4.8.45. John Wiley & Sons, New York.
Emi, Y., Ikushiro, S., and Iyanagi, T. (1996). Xenobiotic response element–mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex. J. Biol. Chem. 271, 3952–3958.
Favreau, L. V., and Pickett, C. B. (1991). Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. J. Biol. Chem. 266, 4556–4561.
Fernandez-Salguero, P., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722–726.
Ge, N. L., and Elferink, C. J. (1998). A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein—Linking dioxin signaling to the cell cycle. J. Biol. Chem. 273, 22708–22713.
Guiney, P. D., Walker, M. K., Spitsbergen, J. M., and Peterson, R. E. (2000). Hemodynamic dysfunction and cytochrome P4501A mRNA expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin during embryonic stages of lake trout development. Toxicol. Appl. Pharmacol. 168, 1–14.
Guo, J., Sartor, M., Karyala, S., Medvedovic, M., Kann, S., Puga, A., Ryan, P., and Tomlinson, C. R. (2004). Expression of genes in the TGF-beta signaling pathway is significantly deregulated in smooth muscle cells from aorta of aryl hydrocarbon receptor knockout mice. Toxicol. Appl. Pharmacol. 194, 79–89.
Hahn, M. E. (1998). The aryl hydrocarbon receptor: A comparative perspective. Comp. Biochem. Physiol. C 121, 23–53.
Handley-Goldstone, H. M., Grow, M. W., and Stegeman, J. J. (2005). Cardiovascular gene expression profiles of dioxin exposure in zebrafish embryos. Toxicol. Sci. 85, 683–693.
Ivnitski, I., Elmaoued, R., and Walker, M. K. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary development is preceded by a decrease in myocyte proliferation and an increase in cardiac apoptosis. Teratology 64, 201–212.
Kanzawa, N., Kondo, M., Okushima, T., Yamaguchi, M., Temmei, Y., Honda, M., and Tsuchiya, T. (2004). Biochemical and molecular biological analysis of different responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo heart and liver. Arch. Biochem. Biophys. 427, 58–67.
Karyala, S., Guo, J., Sartor, M., Medvedovic, M., Kann, S., Puga, A., Ryan, P., and Tomlinson, C. R. (2004). Different global gene expression profiles in benzo[a]pyrene- and dioxin-treated vascular smooth muscle cells of AHR-knockout and wild-type mice. Cardiovasc. Toxicol. 4, 47–73.
Kolluri, S. K., Weiss, C., Koff, A., and Gottlicher, M. (1999). p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev. 13, 1742–1753.
Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 97, 10442–10447.
Lundequist, A., Tchougounova, E., Abrink, M., and Pejler, G. (2004). Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II. J. Biol. Chem. 279, 32339–32344.
Lusska, A. E., Jones, K. W., Elferink, C. J., Wu, L., Shen, E. S., Wen, L. P., and Whitlock, J. P., Jr. (1991). 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces cytochrome P450IA1 enzyme activity by activating transcription of the corresponding gene. Adv. Enzyme Regul. 31, 307–317.
Martinez, J. M., Afshari, C. A., Bushel, P. R., Masuda, A., Takahashi, T., and Walker, N. J. (2002). Differential toxicogenomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in malignant and nonmalignant human airway epithelial cells. Toxicol. Sci. 69, 409–423.
Mimura, J., Ema, M., Sogawa, K., and Fujii-Kuriyama, Y. (1999). Identification of a novel mechanism of regulation of Ah (dioxin) receptor function. Genes Dev. 13, 20–25.
Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., et al. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645–654.
Mizutani, T., Yoshino, M., Satake, T., Nakagawa, M., Ishimura, R., Tohyama, C., Kokame, K., Kangawa, K., and Miyamoto, K. (2004). Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible and -suppressive genes in the rat placenta: induction of interferon-regulated genes with possible inhibitory roles for angiogenesis in the placenta. Endocr. J. 51, 569–577.
Murphy, K. A., Villano, C. M., Dorn, R., and White, L. A. (2004). Interaction between the aryl hydrocarbon receptor and retinoic acid pathways increases matrix metalloproteinase-1 expression in keratinocytes. J. Biol. Chem. 279, 25284–25293.
Nemoto, N., and Sakurai, J. (1993). Activation of Cyp1a1 and Cyp1a2 genes in adult mouse hepatocytes in primary culture. Jpn. J. Cancer Res. 84, 272–278.
Paulson, K. E., Darnell, J. E., Jr., Rushmore, T., and Pickett, C. B. (1990). Analysis of the upstream elements of the xenobiotic compound–inducible and positionally regulated glutathione S-transferase Ya gene. Mol. Cell Biol. 10, 1841–1852.
Peters, J. M., Narotsky, M. G., Elizondo, G., Fernandez-Salguero, P., Gonzalez, F. J., and Abbott, B. D. (1999). Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice. Toxicol. Sci. 47, 86–92.
Poolman, R. A., Li, J. M., Durand, B., and Brooks, G. (1999). Altered expression of cell cycle proteins and prolonged duration of cardiac myocyte hyperplasia in p27KIP1 knockout mice. Circ. Res. 85, 117–127.
Puga, A., Barnes, S. J., Dalton, T. P., Chang, C., Knudsen, E. S., and Maier, M. A. (2000a). Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. J. Biol. Chem. 275, 2943–2950.
Puga, A., Maier, A., and Medvedovic, M. (2000b). The transcriptional signature of dioxin in human hepatoma HepG2 cells. Biochem. Pharmacol. 60, 1129–1142.
Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996). Characterization of a murine Ahr null allele: Involvement of the Ah receptor in hepatic growth and development. Proc. Natl. Acad. Sci. U.S.A. 93, 6731–6736.
Schwarz, A., Godes, M., Thone-Reineke, C., Theuring, F., Bauer, C., Neumayer, H. H., and Hocher, B. (2002). Tissue-dependent expression of matrix proteins in human endothelin-1 transgenic mice. Clin. Sci. (Lond.) 103(Suppl 48), 39–43.
Seccia, T. M., Belloni, A. S., Kreutz, R., Paul, M., Nussdorfer, G. G., Pessina, A. C., and Rossi, G. P. (2003). Cardiac fibrosis occurs early and involves endothelin and AT-1 receptors in hypertension due to endogenous angiotensin II. J. Am. Coll. Cardiol. 41, 666–673.
Simon, P. (2003). Q-gene: Processing quantitative real-time RT-PCR data. Bioinformatics 19, 1439–1440.
Spinale, F. G. (2002). Matrix metalloproteinases: Regulation and dysregulation in the failing heart. Circ. Res. 90, 520–530.
Stegeman, J. J., Smolowitz, R. M., and Hahn, M. E. (1991). Immunohistochemical localization of environmentally induced cytochrome P450IA1 in multiple organs of the marine teleost Stenotomus chrysops (Scup). Toxicol. Appl. Pharmacol. 110, 486–504.
Sun, Y. V., Boverhof, D. R., Burgoon, L. D., Fielden, M. R., and Zacharewski, T. R. (2004). Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucleic Acids Res. 32, 4512–4523.
Thackaberry, E. A., Bedrick, E. J., Goens, M. B., Danielson, L., Lund, A. K., Gabaldon, D., Smith, S. M., and Walker, M. K. (2003). Insulin regulation in AhR null mice: Embryonic cardiac enlargement, neonatal macrosomia, and altered insulin regulation and response in pregnant and aging AhR null females. Toxicol. Sci. 76, 407–417.
Thackaberry, E. A., Nunez, B., Ivnitski-Steele, I. D., Friggens, M. M., and Walker, M. K. (2005). Effects of 2,3,7,8-tetrachlorodibenzo-p-dixoin (TCDD) on the fetal murine heart II: TCDD alters fetal and postnatal cardiac growth, and postnatal cardiac chronotrophy. Toxicol. Sci. 10.1093/toxsci/kfi302.
Thomae, T. L., Stevens, E. A., and Bradfield, C. A. (2005). Transforming growth factor-beta3 restores fusion in palatal shelves exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 280, 12742–12746.
Walker, M. K., and Catron, T. F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167, 210–221.
Weber, H., and Birnbaum, L. S. (1985). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) in pregnant C57BL/6N mice: Distribution to the embryo and excretion. Arch. Toxicol. 57, 159–162.
Woo, R. A., and Poon, R. Y. (2003). Cyclin-dependent kinases and S phase control in mammalian cells. Cell Cycle 2, 316–324.
Zhang, L., Savas, U., Alexander, D. L., and Jefcoate, C. R. (1998). Characterization of the mouse Cyp1B1 gene. Identification of an enhancer region that directs aryl hydrocarbon receptor–mediated constitutive and induced expression. J. Biol. Chem. 273, 5174–5183.(E. A. Thackaberry, Z. Jia)
Department of Biocomputing, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Ambion, Inc. The RNA Company, Austin, Texas 78744
Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville Health Sciences Center, Louisville, Kentucky 40292
Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
1 To whom to address correspondence should be addressed at College of Pharmacy, 2502 Marble NE, Albuquerque, NM 87131. Fax: 505-272-0704. E-mail: mkwalker@unm.edu.
ABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 μg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix–related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.
Key Words: TCDD; aryl hydrocarbon receptor; microarray; heart development; cell cycle.
INTRODUCTION
The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent teratogen in birds, fish, and mammals (Couture et al., 1990; Guiney et al., 2000; Walker and Catron, 2000). TCDD toxicity is believed to be mediated by alterations in gene expression via activation of the aryl hydrocarbon receptor (AhR). The AhR is a cytosolic protein with high affinity for TCDD and other halogenated and polycyclic aromatic hydrocarbons. Upon ligand activation, the AhR translocates into the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT), binds to dioxin responsive elements (DRE) in the upstream regions of regulated genes, and modulates gene expression (Denison et al., 2002).
Although the AhR is highly conserved across vertebrates (Hahn, 1998), its specific physiological function remains enigmatic. It is notable, however, the both the loss of AhR function by genetic deletion and the sustained activation of AhR by environmental pollutants result in a disruption of normal developmental regulation. For example, mice harboring null alleles for the AhR are viable at birth, but they exhibit a small liver as a result of abnormal hepatic vascular development (Fernandez-Salguero et al., 1995; Lahvis et al., 2000; Schmidt et al., 1996). Additionally, AhR null embryos also develop abnormal cardiac hypertrophy and hyperplasia in utero, and this phenotype is dependent, in part, on the maternal AhR genotype (Thackaberry et al., 2003). Likewise, sustained activation of AhR by TCDD also disrupts developmental regulation, resulting in characteristic teratogenicity, including cleft palate and hydronephrosis in rodents and cardiovascular defects in fish and birds (Couture et al., 1990; Guiney et al., 2000; Walker and Catron, 2000). Thus, both loss of AhR function and sustained AhR signaling are detrimental to developmental control.
Importantly, it has been shown that mice lacking AhR expression are significantly less sensitive to the teratogenic effects of TCDD after developmental exposure (Bunger et al., 2003; Mimura et al., 1997; Peters et al., 1999). For example, AhR wild-type fetuses exposed to 25 μg TCDD/kg in utero exhibit a significantly higher incidence of cleft palate and hydronephrosis compared to AhR null fetuses (Peters et al., 1999). Similarly, when fetal palatal shelves are exposed to TCDD in explant culture, mice harboring a mutated AhR with a deleted nuclear localization sequence are resistant to TCDD-induced cleft palate (Bunger et al., 2003). These data support the hypothesis that the teratogenic effects of TCDD are mediated by transcriptional dysregulation of gene expression via the AhR.
One important objective of our laboratory is to characterize the effects of TCDD on cardiac structure, function, and gene expression during murine development, and toward this objective we have applied a non-biased microarray method for identifying those genes associated with the teratogenic effects of TCDD on the developing cardiovascular system. Because inhibition of cardiomyocyte proliferation is one of the most sensitive effects of TCDD in piscine and avian embryos, we targeted a window during murine fetal development when cardiomyocyte proliferation peaks (i.e., GD 14.5–17.5). Here, in the first of two reports, we present the effects of TCDD treatment on global gene expression profiles in the developing murine heart. Our results demonstrate that in utero TCDD exposure significantly alters the expression of cardiac genes involved in cell cycle regulation, extracellular matrix deposition/remodeling, and cardiac homeostasis. Furthermore, these TCDD-induced changes in gene expression are associated with subtle, but potentially significant, cardiac structural alterations and functional deficits, including reduced fetal cardiocyte proliferation and heart size, as well as postnatal bradycardia, which are reported in detail in a companion article (Thackaberry et al., 2005).
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 of the occurrence of a vaginal plug was designated GD 0.5. On GD 14.5 pregnant mice were dosed with corn oil (control) or 1.5, 3.0, or 6.0 μg TCDD/kg in corn oil via oral gavage (5.0 μl/g body weight). Pregnant dams were sacrificed on GD 17.5, fetuses dissected and weighed, and fetal hearts were isolated, pooled within a litter, and frozen for mRNA isolation.
Microarray analysis.
Total mRNA was isolated from pooled frozen fetal hearts (n = 4 litters per treatment, one microarray chip per litter) using Trizol reagent (Invitrogen, Carlsbad, CA), cDNA synthesized with T7-( dT)24 primer, rcRNA synthesized and labeled with biotin, and rcRNA fragmented, after which samples were hybridized to Affymetrix MG_U74Av2 microarray chips (Affymetrix, Santa Clara, CA) for 16 h. After hybridization, chips were washed and stained with streptavidin-phycoerythrin and scanned. Because microarray experiments were conducted on two different dates with four control chips/date, we investigated whether variations existed between the two batches of chips by inspecting the distribution of gene expression values of each control chip and the quantile-quantile plot between control chips. We found that these two data sets could be combined and analyzed as a single experiment with a total of eight control chips. All microarray experiments were carried out using the UNM KUGR core facility, and were MIAME certified. Raw data can be found at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) Web site http://www.ncbi.nlm.nih.gov/projects/geo/; series record #GSE2812). All genes that were absent on 24/24 chips or moderately detected on 6/24 chips were deleted, reducing the total from 12,488 to 7,253 genes. Raw intensity values were normalized using GeneSpring software (Silcon Genetics, Redwood City, CA), and fold differences were calculated from differential interpretational results. Data were analyzed with the GeneSpring software.
Real-time PCR.
Total RNA was isolated from pooled GD 17.5 hearts using Trizol reagent (Invitrogen), and cDNA was generated using reverse transcriptase (Promega, Madison, WI) with oligo dT and an 18s RT primer to amplify the 18s control RNA. The sequence of the 18s RT primer was 5'-AGTTCGACCGTCTTCTCAGC-3'. mRNA expression was then quantified using a BioRad I-Cycler (BioRad, Hercules, CA). The efficiency of each primer set was determined, using a standard curve with known quantities of cDNA, and all primers sets used were 90–100% efficient. Real-time PCR reactions were run in triplicate for the genes of interest and 18s control simultaneously, and the differences between the CT values were calculated. Values were then converted to mean relative expression, using Q-gene software (Simon, 2003), and expressed as a percent of control. The primer sequences are shown in Table 1.
Statistics.
Only genes predicted to be present by the one-sided Wilcoxon's Signed Rank test using the MAS 5.0 default settings (Affymetrix, Santa Clara, CA), and with p < 0.05, compared to control, were reported as "altered." Fold change in expression was determined with GeneSpring software to normalize TCDD-treated values to the control data (set at 1.000). Therefore, a normalized value of 2.0 would represent a 2.0-fold induction, and a normalized value of 0.5 would represent a 2.0-fold repression (expressed here as –2.0). Additionally, we applied multifactor analysis of variance (ANOVA) to identify genes that were differentially expressed across treatments, and we estimated the false discovery rate (FDR) from the rank-ordered ANOVA p-value (Benjamini and Hochberg, 1995). At the FDR of 0.25, we identified 1239 genes that were differentially expressed across treatment groups, but not between control chip sets. GeneSpring software was used for hierarchal clustering, expression correlations, ontological identification, and pathway analysis. To determine correlations between genes, a Pearson correlation around zero was used.
RESULTS
Genes Altered by TCDD and Grouped by Functional Category
To determine the functional classes of genes that were altered by TCDD, we used the simplified gene ontogeny provided by the GeneSpring software. Table 2 shows the number of genes induced or repressed 1.5- and 2.0-fold by TCDD and grouped by functional category. Those categories with relatively high numbers of genes altered in expression included cell growth, defense/immunity, development, ion channels, transcription factors, signal transduction, and transporters.
Differential Dose-Related Responses to TCDD
Next, we used cluster analysis and filtering to determine the primary dose-related response patterns observed with increasing concentration of TCDD (Table 3). In general, genes were more likely to be induced than repressed by TCDD treatment and the greatest number of genes altered in expression occurred at the highest dose (186 induced 1.5-fold; 99 repressed 1.5-fold; p < 0.05). In addition, 41 genes were induced 1.5-fold at all doses, and 11 genes were repressed 1.5-fold at all doses. Six genes were induced by 2-fold at all three TCDD doses (Cyp1a1, Cyp1b1, Reg3g, Fbp1, Scgb1a1, and EST #AA270365), but there were no genes that were repressed 2-fold at all three doses.
Selected examples of genes induced or repressed 2-fold and that clustered into one of the response groups in Table 3 are shown in the Venn diagram in Figure 1. Along with Cyp1a1 and Cyp1b1, an EST homologous to fructose biphosphatase (Fbp1) was upregulated 2-fold at all three doses, whereas the NAD(P)H oxidase p40phox subunit, Ncf4, and the matrix metalloproteinase enzyme, Mmp13, were upregulated only at the two highest doses. Other genes of note included the inwardly rectifying potassium channel (Kcnj5), which was upregulated only at the highest dose, and the thromboxane A2 receptor (Tbxa2r), which was upregulated only at the lowest two doses.
Expression of Genes in AhR Signaling Cascade or Known to be AhR-Responsive
We next investigated the expression of genes known to be involved in the AhR signaling cascade or regulated by the AhR. No change in the expression of any genes involved in the AhR signaling cascade was observed after TCDD treatment, including Ahr, Arnt, Arnt2, or Aip, (Table 4). Of genes known to contain DREs and to be regulated by the AhR, alcohol dehydrogenase 3 (Adh-3), AhR repressor (Ahrr), cytochrome P-450 1A1 (Cyp1a1), cytochrome P-450 1B1 (Cyp1b1), and UDP-glucuronosyl transferase (Ugt1a6), were all upregulated at the highest dose and in some cases in a dose-related manner. Notably, however, the DRE-containing, AhR-regulated, cytochrome P-450 1A2 (Cyp1a2), glutathione-S-transferase-Ya (GSTa1), and NAD(P)H quinone oxidoreductase (Nqo1) were not altered by TCDD treatment in the fetal murine heart, and the expression of all three of these genes was below background in control samples.
Expression of Genes Known to be Involved in Cardiovascular Development
The effects of TCDD on the expression of genes involved in cardiovascular development are summarized in Table 5. The most striking finding was the large number of extracellular matrix genes altered in response to TCDD. The procollagen genes Col4a5, Col6a1, Col9a2, Col13a1, and Col18a1 were significantly altered by TCDD treatment, as were the matrix metalloproteinases Mmp13 and Mmp14. The dose-related expression of these extracellular matrix genes along with Mmp15, mast cell chymase (Mcpt5), tropoelastin (Eln), mannose binding protein (Mbl2), and the vasoactive promitogenic peptide endothelin-1 (Edn), are shown in graphical form in Figure 2. Although a few of these genes, such as Col6a1 and Mcpt5, showed typical dose-related effects on expression, many more of these genes showed significant alterations in expression only at the highest dose or only at the lower doses. In addition to the extracellular matrix genes, the angiogenic cytokines, vascular endothelial growth factor (Vegfa) and angiopoetin (Agpt), were both downregulated, as were the potassium voltage-gated channels Kcna3 and Kcna5.
Genes with Similar Expression Profiles to the Most Highly Induced or Repressed Genes
We used the GeneSpring software correlation function to identify genes whose TCDD dose-related expression profile correlated (>0.95) with the dose-related expression profile of (1) the most highly induced genes, Ahrr, Cyp1a1, and Cyp1b1 (Fig. 3A, Table 6), or (2) the most highly repressed gene, Mcpt5 (Fig. 3B, Table 7). Among the genes correlating with the most highly induced genes was the NAD(P)H oxidase subunit, Ncf4; two immune-response proteins, Ccl1 and Illr2; the matrix metalloproteinase, Mmp13; and two genes with high homology to known carbohydrate metabolism genes, the Na+/glucose cotransporter (EST) and fructose bisphosphatase (Fbp1). Among genes correlating with the most highly repressed gene was a second mast cell–specific gene, mast cell carboxypeptidase A (Cpa3), as well as protocadherin 7 (Pcdh7), bone morphogenetic protein 10 (Bmp10), an inhibitory GTP binding protein (Gnai1), and three transporters (multidrug resistance–associated protein, Abcc3; carnitine transporter, Octn2; and fatty acid transporter, Cd36).
Real-Time PCR Confirms Altered Expression of Selected Genes
To validate the microarray data presented in this study, we used real-time PCR to confirm the expression of a number of key genes at the 6.0 μg TCDD/kg dose (Table 8). Real-time PCR confirmed (1) induction of Cyp1a1, Cyp1b1, Edn, and Mmp13 and (2) the repression of Cd36, Ccne1, Mcpt5, and Vegfa. We also chose five genes whose expression was unchanged at the 6.0 μg TCDD/kg dose by microarray and quantified them by real-time PCR. The results confirmed a lack of alteration of Ccnd1, Cyp1a2, and Cdkn1b; however, real-time PCR was able to identify decreased expression of two cell-cycle–related genes, Ccna2 and Ccne2, which were unaltered on the microarray at the highest TCDD dose, but which were repressed at two lower doses. Of all the genes quantified by real-time PCR, only these two cell cycle genes and mannose-binding protein (Mbl2) differed from the microarray results. Mbl2 was repressed to a similar extent using both methods, but it was not statistically significant by real-time PCR.
DISCUSSION
Although the effects of TCDD on the developing cardiovascular system of piscine and avian species are well documented, relatively little is known about the effects of TCDD on the developing mammalian heart. Given the critical role of AhR-mediated gene dysregulation in the teratogenic responses to TCDD, we used microarray analysis to determine the global changes in gene expression that occur in parallel with TCDD-induced cardiac teratogenicity (reported in our companion article [Thackaberry et al., 2005]).
Comparison of TCDD-Induced Gene Expression Changes with Other Studies
As expected, in utero TCDD exposure significantly induced Cyp1a1, Cyp1b1, AhR repressor (Ahrr), UDP glucuronosyl transferase (Ugt1a6), and alcohol dehydrogenase 3 (Adh-3) in the fetal murine heart, all of which have been reported to be controlled by the AhR (Emi et al., 1996; Lusska et al., 1991; Mimura et al., 1999; Zhang et al., 1998). Interestingly, Cyp1a2, glutathione-S-transferase-Ya (Gsta1) and NAD(P)H quinone oxidoreductase (Nqo1), also previously shown to be AhR-regulated (Favreau and Pickett, 1991; Nemoto and Sakurai, 1993; Paulson et al., 1990), were not altered by TCDD in the fetal murine heart. Furthermore, the expression of these latter genes was at or below background in all groups, suggesting that they are not expressed and are not TCDD-inducible in the fetal murine heart.
Numerous studies have demonstrated that TCDD can induce target gene expression, such as Cyp1a1, to a much greater degree than that observed in this study. This difference likely results from a combination of the tissue type being analyzed and the relatively small amount of TCDD that reaches fetal cardiac tissue. Induction of Cyp1a1 by AhR agonists has been detected primarily in the endothelial cells of the developing heart, and not in the myocardium nor in isolated myocytes (Kanzawa et al., 2004; Stegeman et al., 1991), and thus the degree of induction in the fetal murine heart may appear low because endothelial cells comprise a small component of the total cells in the heart. Additionally, toxicokinetics models predict that 0.04% of the total maternal TCDD dose reaches an individual fetus (Weber and Birnbaum, 1985) and even a smaller percentage of that will distribute to the heart. Finally, it is also notable that of several novel genes with putative DREs that are conserved across human, rat, and mouse, none of them were altered in a dose-dependent manner in our study (Sun et al., 2004).
These results suggest that AhR gene regulation may be tissue-specific. TCDD microarray studies performed using other tissues or cell types show relatively few genes in common with those identified in our study. When comparing changes in gene expression in our study to those from other TCDD microarray studies, we found five induced genes and one repressed gene that matched exactly, including Cyp1a1, Cyp1b1, Adh3, Ugt1a6, Col6a1, and Vegfa (Boverhof et al., 2005; Handley-Goldstone et al., 2005; Martinez et al., 2002; Mizutani et al., 2004; Puga et al., 2000b). This was particularly surprising when we compared our results with those from a microarray study on TCDD-treated embryonic zebrafish with an emphasis on cardiovascular-related genes (Handley-Goldstone et al., 2005). Significant differences in gene expression that were common between the two studies included only Cyp1a1 and Cyp1b1. The lack of greater similarity may be explained, in part, by the difference in timing of exposure. Zebrafish eggs were exposed to TCDD very early in cardiogenesis, whereas murine fetuses were exposed very late in heart development. Finally, if we considered one other microarray study that used the AhR agonist benzo[a]pyrene, we identified two additional repressed genes that matched with our results: cadherin 13 (Cdh13) and procollagen IV alpha 5 (Col4a5) (Karyala et al., 2004).
In addition to finding few genes in common with other microarray studies, we also found that some changes in gene expression were in the opposite direction or not altered when compared to other studies. For example, TCDD induced Cd36 in the liver of adult mice (Boverhof et al., 2005) and Vegfa in human airway epithelial cells (Martinez et al., 2002), but it repressed the expression of both these genes in the fetal murine heart. In addition, it has been shown that inhibition of transforming growth factor- (TGF) signaling may play a role in TCDD-induced cleft palate (Thomae et al., 2005), and TCDD has been shown to dysregulate expression of TGF-related genes in vascular smooth muscle (Guo et al., 2004). Although TGF signaling plays a key role in cardiac morphogenesis, we did not identify any significant changes in expression of TGFs, their receptors, or related genes. Although many of these differences likely result from tissue-specific responses, we also cannot discount that differences in the dose of TCDD and length of exposure may contribute to differences between our results and those reported in other studies.
TCDD Dysregulates Expression of Many Cardiac Genes Related to ECM Homeostasis
The altered expression of extracellular matrix (ECM) genes or of known regulators of ECM deposition/degradation was one of the most striking findings of the current study. Expression of cardiac ECM genes, including tropoelastin (Eln) and five procollagens (Col4a5, Col6a1, Col9a2, Col13a1, and Col18a1), was highly dysregulated by TCDD exposure. In addition, genes that regulate ECM deposition/degradation also were dysregulated in expression, including three matrix metalloproteinases (Mmp13, Mmp14, Mmp15), mast cell chymase (Mcpt5), mast cell carboxypeptidase A (Cpa3), and endothelin-1 (Edn). Cardiac ECM remodeling is highly dependent on the balance between collagen degradation and synthesis, and cardiac dysfunction is frequently associated with induction of MMPs, alterations in ECM structure, and enhanced cardiac fibrosis (Spinale, 2002). Additionally, local cardiac production of angiotensin II (Ang II) and endothelin-1 is associated with increased cardiac fibrosis and cardiac dysfunction (Schwarz et al., 2002; Seccia et al., 2003). Mast cell chymase and carboxypeptidase A are involved in the synthesis and degradation of Ang II, respectively (Lundequist et al., 2004), and the former has been shown to activate MMPs. It also is noteworthy that a number of studies have demonstrated that TCDD can increase the expression of MMPs, including MMP1, the human equivalent of murine MMP13 (Murphy et al., 2004). These results suggest that TCDD may contribute to alterations in ECM remodeling in the fetal murine heart, and these alterations may contribute to altered cardiac structure and function after birth, as described in our companion article (Thackaberry et al., 2005).
TCDD Dysregulates Gene Expression of Cell Cycle Regulators in the Fetal Murine Heart
Alterations in the expression of genes involved in cell cycle regulation were of particular interest, because TCDD is known to inhibit cardiomyocyte proliferation in the developing chick embryo (Ivnitski et al., 2001), zebrafish embryo (Antkiewicz et al., 2005), and, most recently, murine fetus (Thackaberry et al., 2005). Our data show that expression of cyclins A2, B1, C, E, E2, and F was repressed or showed a trend toward repression in at least one dose. Real-time PCR confirmed the downregulation of cyclin E1 and showed downregulation of cyclins A2 and E1 at 6 μg TCDD/kg. Additionally, Bmp10 expression was reduced at all TCDD doses. The A- and E-type cyclins are involved in the progression from G1 to S phase (Woo and Poon, 2003), whereas BMP10 plays an essential role in regulating cardiac growth and its deficiency is associated with cardiac hypoplasia (Chen et al., 2004). Thus, it is tempting to speculate that the reduced cardiomyocyte proliferation described in the companion article results from disrupted regulation of cell cycle regulatory proteins. It is important to note, however, that the microarray analysis was performed using whole-heart homogenates. While cardiomyocytes represent the majority of the volume of the heart, there also is a significant population of cardiac fibroblasts and endothelial cells, in addition to smaller populations of conduction and immune cells. Thus, in situ hybridization and immunohistochemical studies would need to be conducted to determine if the reduced cardiomyocyte proliferation was correlated spatially with altered cell cycle protein expression.
Potential mechanisms by which TCDD may downregulate A- and E-type cyclins, and thus inhibit proliferation, include an AhR-dependent interaction with the retinoblastoma protein (Rb) (Ge and Elferink, 1998; Puga et al., 2000a) and induction of the cdk inhibitor, p27kip1 (Cdkn1b) (Kolluri et al., 1999). It is notable that mice lacking p27kip1 exhibit increased cardiomyocyte proliferation (Poolman et al., 1999), and thus it is possible that TCDD-induced expression of cardiac p27kip1 might inhibit cardiomyocyte proliferation. However, Cdkn1b expression was not induced by TCDD in the fetal murine heart, and this lack of induction was confirmed by real-time PCR. Taken together, these results suggest that TCDD may reduce proliferation of fetal murine cardiomyocytes by altering expression of cell cycle regulators, but that this occurs via a mechanism that is independent of changes in p27kip1 mRNA expression.
Correlation Analysis of Gene Expression with TCDD-Induced or -Repressed Genes
Correlation analysis identified several genes whose dose-related expression correlated with (1) known AhR-responsive genes that were highly induced by TCDD (Cyp1a1, Cyp1b1, and Ahrr) or (2) the one gene whose expression was most highly repressed by TCDD (Mtp5). These highly induced or repressed genes did not all fall into any one common pathway or apparent physiological category, although some appear to be related, such as two carbohydrate metabolism genes (Fbp1 and a putative Na+/glucose transporter), which were highly induced, and two organic transporters (Abcc3 and Octn2), which were highly repressed. While the expression of these genes was similar in a dose-related manner to other TCDD-induced or TCDD-repressed genes, it is not known if these genes are directly regulated by the AhR. Considering that TCDD exposure occurred 3 days prior to RNA isolation, it is possible that alteration of these highly correlated genes occurred as downstream responses to other changes in gene expression. In fact, one of the functional categories with the largest number of TCDD-altered genes was that of transcription factors. Nonetheless, the TCDD-induced, dose-related changes in expression of these correlated genes provides a starting point for the potential identification of novel AhR-regulated cardiac genes and may provide insight into those genes that mediate TCDD-induced cardiac teratogenicity. Thus, in ongoing studies we are determining those TCDD-induced changes in fetal cardiac gene expression that are mediated directly by the AhR by exposing crosses of AhR heterozygote mice.
In conclusion, we have used an unbiased microarray approach to demonstrate significant alterations in gene expression in the fetal murine heart after TCDD exposure in utero. These gene expression changes in particular suggest possible alterations in cell cycle control and extracellular matrix production/remodeling, which may contribute to TCDD-induced cardiac teratogenic and functional alterations in the murine fetus. Future studies will be needed to define those changes in gene expression, as well as post-transcriptional or epigenetic mechanisms not identified here, that mechanistically underlie the developmental cardiovascular teratogenicity of TCDD that is described in our companion article (Thackaberry et al., 2005).
ACKNOWLEDGMENTS
The authors thank Marilee Morgan, Dr. Gavin Pickett, Dr. Scott Ness, and the entire UNM KUGR facility for their valuable assistance. This work was supported by ES012335 to M.K.W., ES012855 to E.A.T., and the P30 National Institute of Environmental Health Sciences (NIEHS) Center ES12072.
REFERENCES
Antkiewicz, D. S., Burns, C. G., Carney, S. A., Peterson, R. E., and Heideman, W. (2005). Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368–377.
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate—A practical and powerful approach to multiple testing. J. R. Stat. Soc B—Methodological 57, 1289–1300.
Boverhof, D. R., Burgoon, L. D., Tashiro, C., Chittim, B., Harkema, J. R., Jump, D. B., and Zacharewski, T. R. (2005). Temporal and dose-dependent hepatic gene expression patterns in mice provide new insights into TCDD-mediated hepatotoxicity. Toxicol. Sci. 85, 1048–1063.
Bunger, M. K., Moran, S. M., Glover, E., Thomae, T. L., Lahvis, G. P., Lin, B. C., and Bradfield, C. A. (2003). Resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and abnormal liver development in mice carrying a mutation in the nuclear localization sequence of the aryl hydrocarbon receptor. J. Biol. Chem. 278, 17767–17774.
Chen, H., Shi, S., Acosta, L., Li, W., Lu, J., Bao, S., Chen, Z., Yang, Z., Schneider, M. D., Chien, K. R., et al. (2004). BMP10 is essential for maintaining cardiac growth during murine cardiogenesis. Development 131, 2219–2231.
Couture, L. A., Abbott, B. D., and Birnbaum, L. S. (1990). A critical review of the developmental toxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Recent advances toward understanding the mechanism. Teratology 42, 619–627.
Denison, M., Rogers, J. M., Rushing, S. R., Jones, C. L., Tetangco, S. C., and Heath-Pagliuso, S. (2002). Analysis of the Ah receptor signal transduction pathway. In Current Protocols in Toxicology (M. Maines, L. G. Costa, D. J. Reed, S. Sassa, and I. G. Sipes, Eds.), p. 4.8.1–4.8.45. John Wiley & Sons, New York.
Emi, Y., Ikushiro, S., and Iyanagi, T. (1996). Xenobiotic response element–mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex. J. Biol. Chem. 271, 3952–3958.
Favreau, L. V., and Pickett, C. B. (1991). Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. J. Biol. Chem. 266, 4556–4561.
Fernandez-Salguero, P., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722–726.
Ge, N. L., and Elferink, C. J. (1998). A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein—Linking dioxin signaling to the cell cycle. J. Biol. Chem. 273, 22708–22713.
Guiney, P. D., Walker, M. K., Spitsbergen, J. M., and Peterson, R. E. (2000). Hemodynamic dysfunction and cytochrome P4501A mRNA expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin during embryonic stages of lake trout development. Toxicol. Appl. Pharmacol. 168, 1–14.
Guo, J., Sartor, M., Karyala, S., Medvedovic, M., Kann, S., Puga, A., Ryan, P., and Tomlinson, C. R. (2004). Expression of genes in the TGF-beta signaling pathway is significantly deregulated in smooth muscle cells from aorta of aryl hydrocarbon receptor knockout mice. Toxicol. Appl. Pharmacol. 194, 79–89.
Hahn, M. E. (1998). The aryl hydrocarbon receptor: A comparative perspective. Comp. Biochem. Physiol. C 121, 23–53.
Handley-Goldstone, H. M., Grow, M. W., and Stegeman, J. J. (2005). Cardiovascular gene expression profiles of dioxin exposure in zebrafish embryos. Toxicol. Sci. 85, 683–693.
Ivnitski, I., Elmaoued, R., and Walker, M. K. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary development is preceded by a decrease in myocyte proliferation and an increase in cardiac apoptosis. Teratology 64, 201–212.
Kanzawa, N., Kondo, M., Okushima, T., Yamaguchi, M., Temmei, Y., Honda, M., and Tsuchiya, T. (2004). Biochemical and molecular biological analysis of different responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo heart and liver. Arch. Biochem. Biophys. 427, 58–67.
Karyala, S., Guo, J., Sartor, M., Medvedovic, M., Kann, S., Puga, A., Ryan, P., and Tomlinson, C. R. (2004). Different global gene expression profiles in benzo[a]pyrene- and dioxin-treated vascular smooth muscle cells of AHR-knockout and wild-type mice. Cardiovasc. Toxicol. 4, 47–73.
Kolluri, S. K., Weiss, C., Koff, A., and Gottlicher, M. (1999). p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev. 13, 1742–1753.
Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 97, 10442–10447.
Lundequist, A., Tchougounova, E., Abrink, M., and Pejler, G. (2004). Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II. J. Biol. Chem. 279, 32339–32344.
Lusska, A. E., Jones, K. W., Elferink, C. J., Wu, L., Shen, E. S., Wen, L. P., and Whitlock, J. P., Jr. (1991). 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces cytochrome P450IA1 enzyme activity by activating transcription of the corresponding gene. Adv. Enzyme Regul. 31, 307–317.
Martinez, J. M., Afshari, C. A., Bushel, P. R., Masuda, A., Takahashi, T., and Walker, N. J. (2002). Differential toxicogenomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in malignant and nonmalignant human airway epithelial cells. Toxicol. Sci. 69, 409–423.
Mimura, J., Ema, M., Sogawa, K., and Fujii-Kuriyama, Y. (1999). Identification of a novel mechanism of regulation of Ah (dioxin) receptor function. Genes Dev. 13, 20–25.
Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., et al. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645–654.
Mizutani, T., Yoshino, M., Satake, T., Nakagawa, M., Ishimura, R., Tohyama, C., Kokame, K., Kangawa, K., and Miyamoto, K. (2004). Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible and -suppressive genes in the rat placenta: induction of interferon-regulated genes with possible inhibitory roles for angiogenesis in the placenta. Endocr. J. 51, 569–577.
Murphy, K. A., Villano, C. M., Dorn, R., and White, L. A. (2004). Interaction between the aryl hydrocarbon receptor and retinoic acid pathways increases matrix metalloproteinase-1 expression in keratinocytes. J. Biol. Chem. 279, 25284–25293.
Nemoto, N., and Sakurai, J. (1993). Activation of Cyp1a1 and Cyp1a2 genes in adult mouse hepatocytes in primary culture. Jpn. J. Cancer Res. 84, 272–278.
Paulson, K. E., Darnell, J. E., Jr., Rushmore, T., and Pickett, C. B. (1990). Analysis of the upstream elements of the xenobiotic compound–inducible and positionally regulated glutathione S-transferase Ya gene. Mol. Cell Biol. 10, 1841–1852.
Peters, J. M., Narotsky, M. G., Elizondo, G., Fernandez-Salguero, P., Gonzalez, F. J., and Abbott, B. D. (1999). Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice. Toxicol. Sci. 47, 86–92.
Poolman, R. A., Li, J. M., Durand, B., and Brooks, G. (1999). Altered expression of cell cycle proteins and prolonged duration of cardiac myocyte hyperplasia in p27KIP1 knockout mice. Circ. Res. 85, 117–127.
Puga, A., Barnes, S. J., Dalton, T. P., Chang, C., Knudsen, E. S., and Maier, M. A. (2000a). Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. J. Biol. Chem. 275, 2943–2950.
Puga, A., Maier, A., and Medvedovic, M. (2000b). The transcriptional signature of dioxin in human hepatoma HepG2 cells. Biochem. Pharmacol. 60, 1129–1142.
Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996). Characterization of a murine Ahr null allele: Involvement of the Ah receptor in hepatic growth and development. Proc. Natl. Acad. Sci. U.S.A. 93, 6731–6736.
Schwarz, A., Godes, M., Thone-Reineke, C., Theuring, F., Bauer, C., Neumayer, H. H., and Hocher, B. (2002). Tissue-dependent expression of matrix proteins in human endothelin-1 transgenic mice. Clin. Sci. (Lond.) 103(Suppl 48), 39–43.
Seccia, T. M., Belloni, A. S., Kreutz, R., Paul, M., Nussdorfer, G. G., Pessina, A. C., and Rossi, G. P. (2003). Cardiac fibrosis occurs early and involves endothelin and AT-1 receptors in hypertension due to endogenous angiotensin II. J. Am. Coll. Cardiol. 41, 666–673.
Simon, P. (2003). Q-gene: Processing quantitative real-time RT-PCR data. Bioinformatics 19, 1439–1440.
Spinale, F. G. (2002). Matrix metalloproteinases: Regulation and dysregulation in the failing heart. Circ. Res. 90, 520–530.
Stegeman, J. J., Smolowitz, R. M., and Hahn, M. E. (1991). Immunohistochemical localization of environmentally induced cytochrome P450IA1 in multiple organs of the marine teleost Stenotomus chrysops (Scup). Toxicol. Appl. Pharmacol. 110, 486–504.
Sun, Y. V., Boverhof, D. R., Burgoon, L. D., Fielden, M. R., and Zacharewski, T. R. (2004). Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucleic Acids Res. 32, 4512–4523.
Thackaberry, E. A., Bedrick, E. J., Goens, M. B., Danielson, L., Lund, A. K., Gabaldon, D., Smith, S. M., and Walker, M. K. (2003). Insulin regulation in AhR null mice: Embryonic cardiac enlargement, neonatal macrosomia, and altered insulin regulation and response in pregnant and aging AhR null females. Toxicol. Sci. 76, 407–417.
Thackaberry, E. A., Nunez, B., Ivnitski-Steele, I. D., Friggens, M. M., and Walker, M. K. (2005). Effects of 2,3,7,8-tetrachlorodibenzo-p-dixoin (TCDD) on the fetal murine heart II: TCDD alters fetal and postnatal cardiac growth, and postnatal cardiac chronotrophy. Toxicol. Sci. 10.1093/toxsci/kfi302.
Thomae, T. L., Stevens, E. A., and Bradfield, C. A. (2005). Transforming growth factor-beta3 restores fusion in palatal shelves exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 280, 12742–12746.
Walker, M. K., and Catron, T. F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167, 210–221.
Weber, H., and Birnbaum, L. S. (1985). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) in pregnant C57BL/6N mice: Distribution to the embryo and excretion. Arch. Toxicol. 57, 159–162.
Woo, R. A., and Poon, R. Y. (2003). Cyclin-dependent kinases and S phase control in mammalian cells. Cell Cycle 2, 316–324.
Zhang, L., Savas, U., Alexander, D. L., and Jefcoate, C. R. (1998). Characterization of the mouse Cyp1B1 gene. Identification of an enhancer region that directs aryl hydrocarbon receptor–mediated constitutive and induced expression. J. Biol. Chem. 273, 5174–5183.(E. A. Thackaberry, Z. Jia)