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Dioxin Alters Gene Expression in Mouse Embryonic Tooth Explants
C. Sahlberg1,*,
E. Peltonen1,
P.-L. Lukinmaa2,3 and
S. Alaluusua1,4
1 Department of Pediatric and Preventive Dentistry, Institute of Dentistry, Biomedicum Helsinki, PL 63 (Haartmaninkatu 8), FIN-00014 UNIVERSITY OF HELSINKI, Helsinki, Finland;
2 Department of Oral Pathology, Institute of Dentistry, University of Helsinki, Helsinki, Finland;
3 Department of Pathology, Helsinki University Central Hospital, Helsinki, Finland; and
4 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland
Correspondence: * corresponding author, carin.sahlberg{at}helsinki.fi
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ABSTRACT
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Dioxins are ubiquitous environmental poisons that cause disturbances in developing organs, including the teeth. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at the cap stage leads to reduced tooth size and deformation of cuspal morphology. Our hypothesis was that TCDD affects the expression of genes specific for tooth development, which leads to these aberrations. Mouse embryonic E14 tooth germs were cultured for 24 hrs with/without 1 µM TCDD. Analysis of total RNA on Affymetrix arrays showed that TCDD altered the expression of 31 known genes by a fold factor of at least 2. Genes implied in tooth development expressed only slight changes. Genes active at the cap stage were selected for quantitative PCR analysis. Of these, the most highly up-regulated were Follistatin and Runx2, while TGFβ1 and p21 were the most down-regulated genes. Incomplete tooth morphogenesis caused by TCDD may thus result from modified expression of developmentally regulated genes.
Key Words: tooth development • TCDD • gene expression • dioxin • tooth morphology
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INTRODUCTION
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Dioxins and related polyhalogenated aromatic hydrocarbons are highly persistent and ubiquitous environmental poisons that are enriched in the food chain. The consequences of dioxin poisoning vary, from a wasting syndrome to chronic effects such as immunosuppression, thymic atrophy, teratogenesis, and carcinogenesis (Pohjanvirta and Tuomisto, 1994; Steenland et al., 2004).
The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been used as a paradigm for research on dioxins, and also recently on its effects on the developmental and reproductive pathways (Matikainen et al., 2001). Developmental defects caused by dioxins in rodents include, e.g., cleft palate, hydronephrosis, retardation of hair growth, accelerated eye opening and incisor eruption, and morphological changes in reproductive organs (Madhukar et al., 1988; Hurst et al., 2002). Even very small amounts are still sufficient to produce adverse developmental effects (Hamm et al., 2000).
In utero and lactational exposure to TCDD affects incisor and molar tooth development in rodents and rhesus monkeys. The effects include quantitative as well as qualitative changes, from missing teeth and shortened roots to defects in matrix secretion and mineralization (Lukinmaa et al., 2001; Yasuda et al., 2005). We have also shown an association between dioxin exposure and tooth abnormalitites in breast-fed children, as well as in children accidentally exposed to dioxins during the tooth-forming period (Alaluusua et al., 1996, 2004).
Our previous studies have shown that exposure of tooth germs to TCDD affects development in a time- and dose-dependent manner. Early exposure until the bud stage will arrest tooth development altogether, while exposure at the ensuing cap stage leads to smaller tooth size and deformation of cuspal morphology . At sequentially later stages, developmental defects will show in the extracellular matrices of both dentin and enamel (Partanen et al., 1998, 2004).
Most if not all of the effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR), a cytosolic protein that binds TCDD and becomes part of a transcription factor complex. This initiates the transcription of xenobiotic-metabolizing enzymes such as CYP1A1, one of the cytochrome P450 compounds that are tightly regulated by the AhR-binding pathway and play important roles in drug, carcinogen, and steroid hormone metabolism (Whitlock, 1999).
Developing teeth express AhR in the epithelium of the early bud stage as well as at the stages of matrix formation and mineralization, indicating special susceptibility to dioxin poisoning (Sahlberg et al., 2002a).
Many AhR ligands stimulate signaling cascades similar to those initiated by growth factors, hormones, and neurotransmitters. Molecular mechanisms occurring downstream of AhR—such as changes in cytosolic signaling proteins, calcium mobilization, growth factors, oncogenes, and cell-cycle proteins—have been characterized (Enan et al., 1998; Carlson and Perdew, 2002). TCDD is thus able to induce a variety of biological responses on both cellular and molecular levels.
The aims of our study were to characterize changes in gene expression in developing mouse E14 tooth germs exposed to TCDD for 24 hrs. In particular, we were interested in identifying candidate genes that may be involved in the effects of TCDD exposure on cultured teeth. For an overall view of induced changes, we first performed microarray hybridization experiments using total RNA isolated from exposed and unexposed mouse teeth. Interesting genes were subsequently selected for testing in quantitative PCR (qPCR) assays.
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MATERIALS & METHODS
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Mice
The age of mouse embryos from the NMRI strain was determined by the day of the vaginal plug (E0). The mice were anesthetized with CO2 and killed by cervical dislocation. The use of animals was approved by the Institutional Animal Care and Use Committee of the Faculty of Science of the University of Helsinki.
Organ Culture
Mandibular molar tooth regions from E14 mouse embryos were dissected and transferred to Trowell-type organ culture as previously described (Sahlberg et al., 2002b). TCDD stock (Department of Chemistry, University of Jyväskylä, Finland) was a 50 µg/mL solution in dimethylsulfoxide. TCDD at the concentration of 1 µM, or dimethylsulfoxide, was added to the culture medium (Partanen et al., 1998). The explants were cultured for 24 hrs, after which they were snap-frozen in liquid nitrogen.
Microarray Analysis
Three separate experiments were combined for each replicate sample. About 60 explants each were pooled, and total RNA was isolated with the RNeasy MiniKit (Qiagen, Hilden, Germany). cDNA was synthesized with Superscript II Choice System (Invitrogen Ltd., Paisley, UK). Biotinylated cRNA was hybridized to a murine U74v2A array (Affymetrix, Santa Clara, CA, USA). Experimental details for the microarrays have been submitted to GEO (http://www.ncbi.nlm.nih.gov/geo/, Series GSE4873).
Each replicate sample was repeated once, and GeneSpring 6.1 software was used to identify and analyze gene changes.
Based on the variability, a two-fold or greater change in gene expression was found to be statistically significant (P < 0.05) for most genes and was used as an indicator of TCDD-induced alteration.
Quantitative PCR
We used the expression data to select genes known to be expressed during tooth development at the cap stage. cDNA synthesis was performed on total RNA by means of a Reverse-iT 1st Strand Synthesis Kit from ABgene. Amplification and analysis of the cDNA was performed with the Absolute qPCR SYBR Green kit (ABGene) and the ABI-PRISM 7000 sequence detection system (Applied Biosystems). Analysis was undertaken as previously described (Livak and Schmittgen, 2001). The endogenous control gene was β-actin. The primers used for the qPCR are listed in Table 1 . Each culture experiment was performed in triplicate and repeated 3 times. The value for β-actin against which the other genes were compared was set at 1.
We ran standard curves to assess the relative efficiencies of PCR amplification of the target and the endogenous control genes. Negative controls with water were included in each run.
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RESULTS
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Microarray
Out of the 12,488 genes on the Affymetrix array, 6060 showed a reliable signal. The number of genes induced or repressed at least two-fold was 31 known genes and 4 expressed sequence tags. Nineteen known genes were up-regulated, and 12 were down-regulated (Fig. 1). We found that genes active in cellular metabolism, intracellular organelles, or membranes showed altered expression levels. Several genes previously implicated in TCDD exposure [CYP1a1 (15.91-fold), CYP1b1 (5.96-fold), Tiparp (3.81-fold), and Nqo1 (2.63-fold)] were highly induced.
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Figure 1. Array gene sets induced or repressed at least two-fold in E14 molars exposed to 1 µM TCDD for 24 hrs in tissue culture. The scale of the X-axis is logarithmic.
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To test genes implicated in tooth development, we ran our results against a list of genes expressed at the cap stage in E14 molars (Table 2). The list was compiled from the Gene Expression in Tooth database maintained by the Tooth and Craniofacial Development Group of the Developmental Biology Programme, Institute of Biotechnology, University of Helsinki (http://bite-it.helsinki.fi/).
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Table 2. TCDD-induced Fold-changes in the Expression of Genes in Molar Teeth during Cap Stage (Affymetrix MG74v2A array)
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The differences in expression profiles of genes implicated in molar tooth development between non-exposed and exposed molars were generally small: All but one were less than twofold up- or down-regulated. The most highly up-regulated genes were Follistatin (Fst) and Transforming growth factor beta 1 (TGFβ1), 1.61- and 1.52-fold, respectively, and the most down-regulated genes were Runx2 (0.70), Retinoid X receptor beta (Rxrb) (0.64), and Cyclin-dependent kinase inhibitor 1a (p21) (0.32).
The Affymetrix MG74v2A array has 5 probe sets associated with Runx2, of which 2 were returned in our study (see Table 1 ). 161378_r_at was the third most down-regulated (0.704) gene, and 92676_at was slightly up-regulated (1.151). Runx2 is a transcription factor essential for regulating tooth development, and, in Runx2 null mutants, development will arrest at bud stage. This stage is most sensitive to TCDD exposure, and Runx2 expression was therefore analyzed in more detail by qPCR.
Quantitative PCR
To verify the results generated by microarray analysis, we selected some essential genes implicated in tooth development for analysis by qPCR.
In general, the results were in line with those of the microarray assay (Fig. 2). Cyp1b1 and Fst were up-regulated 3.22- and 3.35-fold, respectively, and p21 was down-regulated to 0.70-fold of the control expression. However, TGFβ1, which had been slightly up-regulated by microarray data (1.52-fold), was clearly down-regulated in the qPCR experiments (0.58). Runx2, which was slightly down-regulated in the microarray assay (0.74), was up-regulated, as tested by qPCR (1.96-fold). This result correlated more with the Runx2 probe set (92676_at).
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Figure 2. Mean fold changes in qPCR assays. Relative expression ratios showing relative changes in unexposed compared with TCDD-exposed mouse molars after normalization to actin-β (= 1). Up-regulation is seen for CYP1b1 (3.42 ± 1.7), Fst (3.35 ± 2.2), and Runx2 (1.96 ± 1.0), and p21 (0.70 ± 0.1) and TGFβ1 (0.58 ± 0.2) are down-regulated. Each column consists of 9 datapoints for each gene. Results are mean ± SD of triplicate analyses.
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DISCUSSION
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An increasing number of genes is currently being associated with different stages of tooth development. They form a signaling network regulating patterning and morphogenesis of dental tissues and differentiation of the specific cells in the tooth (Jernvall and Thesleff, 2000).
Mouse molars develop well in tissue culture, showing all typical features and gene expression of the developing tooth, such as morphogenesis, cellular differentiation, and early matrix secretion. Our previous studies showed that 1 µM TCDD in the culture medium of E14 molars affected the development of the first and completely arrested the development of the second molar (Partanen et al., 2004), as well as accelerated the apoptotic program in cells destined to undergo programmed cell death. We therefore could anticipate that mainly a subset of genes, involved in the changes in tooth development, would show a marked difference between experiments.
In the current study, we used a general murine genome expression array and qPCR to investigate the transcriptional responses of developing mouse molars following exposure to TCDD in organ culture. We observed altered expression in several genes known to be connected with TCDD exposure, but only slight changes in genes involved in tooth development at the cap stage. We observed differences between microarray and qPCR in the levels of transcription for TGFβ1 and one of the probe sets for the Runx2 gene. Because qPCR is considered more sensitive, especially at low fold changes, the gene expression results discussed below are based on the qPCR results.
Follistatin
Both microarray and qPCR analysis showed that Fst was rapidly up-regulated in the exposed molars. The extracellular glycoprotein follistatin functions as a regulator of activin and BMPs (Balemans and Van Hul, 2002). Antagonistic effects between follistatin and TGFβ-superfamily signals seem to be critical for enamel knot formation and function, as well as for patterning of tooth cusps. In mice overexpressing Fst in the dental epithelium, the first and second molars have misshapen cusps, and all of the third molars are missing (Wang et al., 2004). These features are in line with the results of our in vitro assays, where TCDD exposure resulted in smaller-sized teeth and lower, shallower cusps than in control experiments, and completely arrested development of the second molar (Partanen et al., 2004).
TGFβ-1
TGFβ1 has been implicated in the regulation of odontoblast differentiation and dentin formation. The expression of TGFβ1 varies during tooth development. Expression is first seen during the early bud stage in the dental epithelium, followed by an intense expression in the cervical loop of the dental epithelium at the cap stage. By E18, this expression is almost lost, only to re-appear transiently during ameloblast differentiation (Vaahtokari et al., 1991).
Mice lacking TGFβ1 have teeth with hypomineralized dentin, which results in excessive wear of the teeth and inflammation of the pulpal tissue (D’Souza et al., 1998). Interestingly, we found that molar teeth of rat pups exposed to TCDD during lactation showed delayed dentin mineralization and retention of enamel matrix, indicating retardation of molar mineralization (Lukinmaa et al., 2001; Gao et al., 2004).
TGFβ1 may act in concert with other growth factors to regulate the size and shape of tooth form during morphogenesis. TGFβ1 is known to induce p21 expression. Our qPCR results showed down-regulation of both these genes. Down-regulation of TGFβ1 may also be a result of inhibition by the up-regulated Fst.
p21
The cyclin-dependent kinase inhibitor p21 is associated with the exit from the cell cycle and is expressed in post-mitotic mesenchymal and epithelial cells differentiating into odontoblasts and ameloblasts, respectively (Bloch-Zupan et al., 1998). It is one of the earliest molecular markers of the primary enamel knot, and may be part of the mechanisms that allow the cells not to proliferate while expressing growth-stimulating Fgf-4 (Jernvall et al., 1998). Hence, it may have a role in both enamel knot formation and apoptosis. We found that p21 expression was clearly down-regulated by TCDD. This would imply that the increase in apoptosis by TCDD (Partanen et al., 2004) is not a consequence of an increased activity of p21 in the enamel knots.
Runx2
In contrast to the microarray assay for probe set (161378_r_at), Runx2 expression was up-regulated when analyzed by qPCR. Runx2 regulates epithelial-mesenchymal interactions during tooth development, and may regulate several genes in different signaling pathways. The effect of up-regulated Runx2 induced by TCDD is difficult to interpret. Runx2 expression is, e.g., not affected during arrest of tooth development in Lef1 and Eda mutants, even though teeth fail to develop beyond the bud stage in Runx2 null mutants (Åberg et al., 2004).
Genes with an Expression Change of More Than Two-fold
Enzymes of the cytochrome P450 family play important roles in the initial steps of the metabolic pathways of drugs, toxicants, steroid hormones, and fatty acids. Several members of this family are also involved in the regulation of developmental processes, such as pattern formation, morphogenesis, cell differentiation, and cell growth (Abu-Abed et al., 2001; Stoilov et al., 2004).
Expression of Cyp1a1 and Cyp1b1 was most highly up-regulated in our study. This is in line with results of earlier studies suggesting that these genes are the prime target for TCDD-inducible gene expression (Whitlock, 1999). Cyp1b1 efficiently catalyzes the rate-limiting step in the synthesis of retinoic acid, which is a key determinant of vertebrate embryo patterning and organogenesis (Morriss-Kay and Sokolova, 1996; Chen et al., 2000).
We have earlier shown that Cyp1a1 is expressed at mineralization stages in rat molar ameloblasts and, to a lesser extent, in odontoblasts. This expression is still strong at post-natal day 9 in rat pups exposed to TCDD by dam’s milk, but will be lowered in a dose- and developmental-stage-dependent manner in molars exposed to TCDD (Gao et al., 2004).
To conclude, the change in expression of genes implicated in tooth development was rather small after 24-hour culture. This implies that the effect of TCDD on dental tissues is indirect, as opposed to the fast and high induction of Cyp1a1 and Cyp1b1. Incomplete tooth morphogenesis caused by TCDD may thus result from modified expression of several developmentally regulated genes.
In all, only a handful of genes were induced or repressed more than three-fold, and these were genes well-known as highly inducible by TCDD. This may be a reflection of the rather short exposure time of 24 hrs for tissue culture, as well as the different tissue types developing or already present in the tooth germ at the cap stage. Thus, the responses of complex tissues, as opposed to individual cells, to TCDD were measured. Recent studies have shown that many genes of interest have relatively low levels of array-fold changes, especially in tissue homogenate assays, where only a subpopulation of cells express the genes (Wurmbach et al., 2002).
There may be both temporal and spatial variations in cellular responses to TCDD exposure, which are caused by the different histories of the cells. The identification of disruption in the balance of these regulatory processes may add to the current knowledge of TCDD-induced toxicity.
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ACKNOWLEDGMENTS
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This study was supported by an EU-funded project (QLK4-1999-01446) and by the Research Program for Environmental Health, Academy of Finland (Contract no. 206689). The expert technical assistance of Marjatta Kivekäs is acknowledged.
Received for publication May 23, 2006.
Revision received February 7, 2007.
Accepted for publication March 8, 2007.
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Journal of Dental Research, Vol. 86, No. 7,
600-605 (2007)
DOI: 10.1177/154405910708600704
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ABSTRACT
INTRODUCTION