Use of Dental Defects Associated with Low-Dose di(2-Ethylhexyl)Phthalate as an Early Marker of Exposure to Environmental Toxicants.

BACKGROUND: Markers of exposure to environmental toxicants are urgently needed. Tooth enamel, with its unique properties, is able to record certain environmental conditions during its formation. Enamel formation and quality are dependent on hormonal regulation and environmental conditions, including exposure to endocrine disrupting chemicals (EDCs). Among EDCs, phthalates such as di-(2-ethylhexyl) phthalate (DEHP) raise concerns about their contribution to various pathologies, including those of mineralized tissues. OBJECTIVES: The effects of exposure to low-doses of DEHP on the continually growing incisors were analyzed in mouse males and females. METHODS: Adult male and female C57BL/6J mice were exposed daily to 0.5, 5, and 50µg/kg per day DEHP for 12 wk and their incisors clinically examined. Incisors of males were further analyzed by scanning electron microscopy (SEM), micro X-ray computed tomography (micro-computed tomography; µCT), and nanoindentation for the enamel, histology and real-time quantitative polymerase chain reaction (RT-qPCR) for the dental epithelium. RESULTS: Clinical macroscopic observations of incisors showed various dose-dependent dental lesions such as opacities, scratches, and enamel breakdown in 30.5% of males (10 of 34 total incisors across three independent experiments), and 15.6% of females (7 of 46 incisors) at the highest dose, among which 18.1% (6 of 34 total incisors across three independent experiments) and 8.9% (4 of 46 incisors), respectively, had broken incisors. SEM showed an altered enamel surface and ultrastructure in DEHP-exposed male mice. Further characterization of the enamel defects in males by µCT showed a lower mineral density than controls, and nanoindentation showed a lower enamel hardness during all stages of enamel mineralization, with more pronounced alterations in the external part of the enamel. A delay in enamel mineralization was shown by several approaches (µCT, histology, and RT-qPCR). DISCUSSION: We conclude that DEHP disrupted enamel development in mice by directly acting on dental cells with higher prevalence and severity in males than in females. The time window of DEHP effects on mouse tooth development led to typical alterations of structural, biochemical, and mechanical properties of enamel comparable to other EDCs, such as bisphenol A. The future characterization of dental defects in humans and animals due to environmental toxicants might be helpful in proposing them as early markers of exposure to such molecules. https://doi.org/10.1289/EHP10208.

Protein Kinase D1 (PKD1) Is a New Functional Non-Genomic Target of Bisphenol A in Breast Cancer Cells.

Exposure to bisphenol A (BPA), one of the most widespread endocrine disruptors present in our environment, has been associated with the recent increased prevalence and severity of several diseases such as diabetes, obesity, autism, reproductive and neurological defects, oral diseases, and cancers such as breast tumors. BPA is suspected to act through genomic and non-genomic pathways. However, its precise molecular mechanisms are still largely unknown. Our goal was to identify and characterize a new molecular target of BPA in breast cancer cells in order to better understand how this compound may affect breast tumor growth and development. By using in vitro (MCF-7, T47D, Hs578t, and MDA-MB231 cell lines) and in vivo models, we demonstrated that PKD1 is a functional non-genomic target of BPA. PKD1 specifically mediates BPA-induced cell proliferation, clonogenicity, and anchorage-independent growth of breast tumor cells. Additionally, low-doses of BPA (≤10- 8 M) induced the phosphorylation of PKD1, a key signature of its activation state. Moreover, PKD1 overexpression increased the growth of BPA-exposed breast tumor xenografts in vivo in athymic female Swiss nude (Foxn1nu/nu ) mice. These findings further our understanding of the molecular mechanisms of BPA. By defining PKD1 as a functional target of BPA in breast cancer cell proliferation and tumor development, they provide new insights into the pathogenesis related to the exposure to BPA and other endocrine disruptors acting similarly.

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors.

Endocrine disrupting chemicals (EDCs) play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA), one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH). In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30), of ketosteroid receptors (ERs, AR, PGR, GR, MR), of the retinoid receptor RXRα, and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERß and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation-stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR), whereas the others were 13 to 612-fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step toward understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis.

Androgen Receptor Involvement in Rat Amelogenesis: An Additional Way for Endocrine-Disrupting Chemicals to Affect Enamel Synthesis.

Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERß remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.

Chronic Exposure to Bisphenol A Exacerbates Dental Fluorosis in Growing Rats.

Enamel defects resulting from environmental conditions and way of life are public health concerns because of their high prevalence. Because their etiology is unclear, the aim of this study was to analyze the various forms of enamel hypomineralization, and to characterize the genes involved in this process to determine the mechanisms involved in disruptions of amelogenesis. We used bisphenol A (BPA) and fluoride as models; both are commonly encountered in human populations and utilized in dentistry. Wistar rats were chronically exposed to 5 µg/kg/day BPA from day 1 of gestation to day 65 after birth (P65) and 5 mM fluoride from P21 to P65. Resulting enamel defects were comparable to the human enamel pathologies molar incisor hypomineralization (MIH) and dental fluorosis (DF) respectively, and were more severe in rats exposed to both agents than to each agent alone. Large-scale transcriptomic analysis of dental epithelium showed a small group of genes the expression of which was affected by exposure to BPA or NaF. Among the most modulated, many are directly involved in amelogenesis (Amelx, Enam, Klk4, Mmp12, Slc26a4, and Slc5a8), and can be regrouped as forming the "hypomineralization enameloma." Each of these gene expression perturbations may contribute to enamel defects. Exposure to BPA weakens enamel, making it more prone to generate frequent mineralization defects MIH and DF. Our study identifies hypomineralization genes that may enable the use of dental enamel as an early marker of exposure to environmental toxicants because of its unique ability to retrospectively record ameloblast pathophysiology. © 2016 American Society for Bone and Mineral Research.

Haplosufficiency of PAX3 for melanoma development in Tyr: NRASQ61K; Cdkn2a-/- mice allows identification and sorting of melanoma cells using a Pax3GFP reporter allele.

The role of the Pax3 gene in embryonic development of pigment cells is well characterized. By contrast, the function of Pax3 in melanoma development is controversial. Indeed, data obtained from cultured cells suggest that PAX3 may contribute to melanomagenesis. PAX3 is found to be overexpressed in melanomas and also in nevi compared with normal skin samples. Pax3 homozygous loss of function is embryonic lethal. To assess the role of Pax3 in melanoma development in vivo, we analyzed Pax3 haploinsufficiency in a mouse model of melanoma predisposition. The Pax3(GFP/+) knock-in reporter system was combined with the Tyr::NRAS(Q61K); Cdkn2a(-/-) mouse melanoma model. Melanoma development was followed over 18 months. Histopathological, immunohistochemical, and molecular analyses of lesions at different stages of melanoma progression were carried out. Fluorescence-activated cell sorting on GFP of cells from primary or metastatic melanoma was followed by ex-vivo transformation tests and in-vivo passaging. We report here that Tyr::NRAS(Q61K); Cdkn2a(-/-); Pax3(GFP/+) mice developed metastasizing melanoma as their Tyr::NRAS(Q61K); Cdkn2a(-/-); littermates. Histopathology showed no differences between the two genotypes, although Pax3 mRNA and PAX3 protein levels in Pax3(GFP/+) lesions were reduced by half. The Pax3(GFP) allele proved to be a convenient marker to identify and directly sort heterogeneous populations of melanoma cells within the tumor bulk at each stage of melanoma progression. This new mouse model represents an accurate and reproducible means for identifying melanoma cells in vivo to study the mechanisms of melanoma development.

Enamel hypomineralization due to endocrine disruptors.

There has been increasing concerns over last 20 years about the potential adverse effects of endocrine disruptors (EDs). Bisphenol A (BPA), genistein (G) and vinclozolin (V) are three widely used EDs having similar effects. Tooth enamel has recently been found to be an additional target of BPA that may be a causal agent of molar incisor hypomineralization (MIH). However, populations are exposed to many diverse EDs simultaneously. The purpose of this study was therefore to assess the effects of the combination of G, V and BPA on tooth enamel. Rats were exposed daily in utero and after birth to low doses of EDs mimicking human exposure during the critical fetal and suckling periods when amelogenesis takes place. The proportion of rats presenting opaque areas of enamel hypomineralization was higher when rats were treated with BPA alone than with a combination of EDs. The levels of mRNAs encoding the main enamel proteins varied with BPA treatment alone and did not differ significantly between controls and combined treatment groups. In vitro, rat ameloblastic HAT-7 cells were treated with the three EDs. BPA induced enamelin and reduced klk4 expression, G had no such effects and V reduced enamelin expression. These findings suggest that combinations of EDs may affect enamel less severely than BPA alone, and indicate that enamel hypomineralization may differ according to the characteristics of the ED exposure.

Estrogen and bisphenol A affect male rat enamel formation and promote ameloblast proliferation.

Bisphenol A (BPA) is a widespread endocrine disrupting chemical (EDC) strongly suspected to have adverse health effects. Numerous tissues and cells are affected by BPA, and we showed recently that BPA targets include ameloblasts and enamel. We therefore investigated the effects of BPA on ameloblasts and the possible involvement of the estrogen signaling pathway. Rats were exposed daily to low-dose BPA, and developed enamel hypomineralization similar to human molar incisor hypomineralization (MIH). BPA increased ameloblast proliferation in vivo and in vitro. The proliferation of the rat dental epithelial cell line HAT-7 was also increased by estrogen (E2). Ameloblasts express ERα but not ERß both in vivo and in vitro. The ER antagonist ICI 182,780 was used to inactivate ERα and abolished the effects of E2 on cell proliferation and transcription, but only partially reduced the effects of BPA. In conclusion, we show, for the first time, that: 1) BPA has ER-dependent and ER-independent effects on ameloblast proliferation and gene transcription; 2) the estrogen signaling pathway is involved in tooth development and the enamel mineralization process; and 3) BPA impacts preferentially amelogenesis in male rats. These results are consistent with the steroid hormones having effect on ameloblasts, raising the issues of the hormonal influence on amelogenesis and possible differences in enamel quality between sexes.

Msx1 role in craniofacial bone morphogenesis.

The homeobox gene Msx1 encodes a transcription factor that is highly expressed during embryogenesis and postnatal development in bone. Mutations of the MSX1 gene in humans are associated with cleft palate and (or) tooth agenesis. A similar phenotype is observed in newborn mice invalidated for the Msx1 gene. However, little is known about Msx1 function in osteoblast differentiation and bone mineralization in vivo. In the present study, we aimed to explore the variations of individualized bone shape in a subtle way avoiding the often severe consequences associated with gene mutations. We established transgenic mice that specifically express Msx1 in mineral-matrix-secreting cells under the control of the mouse 2.3kb collagen 1 alpha 1 (Col1α1) promoter, which enabled us to investigate Msx1 function in bone in vivo. Adult transgenic mice (Msx1-Tg) presented altered skull shape and mineralization resulting from increased Msx1 expression during bone development. Serial section analysis of the mandibles showed a high amount of bone matrix in these mice. In addition, osteoblast number, cell proliferation and apoptosis were higher in Msx1-Tg mice than in controls with regional differences that could account for alterations of bone shape. However, Von Kossa staining and µCT analysis showed that bone mineralization was lower in Msx1-Tg mice than in controls due to alteration of osteoblastic differentiation. Msx1 appears to act as a modeling factor for membranous bone; it stimulates trabecular bone metabolism but limits cortical bone growth by promoting apoptosis, and concomitantly controls the collagen-based mineralization process.

Enamel defects reflect perinatal exposure to bisphenol A.

Endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA), are environmental ubiquitous pollutants and associated with a growing health concern. Anecdotally, molar incisor hypomineralization (MIH) is increasing concurrently with EDC-related conditions, which has led us to investigate the effect of BPA on amelogenesis. Rats were exposed daily to BPA from conception until day 30 or 100. At day 30, BPA-affected enamel exhibited hypomineralization similar to human MIH. Scanning electron microscopy and elemental analysis revealed an abnormal accumulation of organic material in erupted enamel. BPA-affected enamel had an abnormal accumulation of exogenous albumin in the maturation stage. Quantitative real-time PCR, Western blotting, and luciferase reporter assays revealed increased expression of enamelin but decreased expression of kallikrein 4 (protease essential for removing enamel proteins) via transcriptional regulation. Data suggest that BPA exerts its effects on amelogenesis by disrupting normal protein removal from the enamel matrix. Interestingly, in 100-day-old rats, erupting incisor enamel was normal, suggesting amelogenesis is only sensitive to MIH-causing agents during a specific time window during development (as reported for human MIH). The present work documents the first experimental model that replicates MIH and presents BPA as a potential causative agent of MIH. Because human enamel defects are irreversible, MIH may provide an easily accessible marker for reporting early EDC exposure in humans.

Regulation of calbindin-D(28k) expression by Msx2 in the dental epithelium.

Amelogenesis involves the coordinated expression of a set of molecules that includes enamel matrix proteins and calcium-binding proteins. Msx2 is a member of the divergent homeobox gene family and is instrumental in dental morphogenesis and biomineralization. This study focused on an EF-hand calcium-binding protein, calbindin-D(28k), which is highly expressed in dental epithelium. In vivo data showed that calbindin-D(28k) levels were higher in ameloblasts from Msx2(+/-) mice than Msx2(+/+) mice. Consistent with this finding, calbindin-D(28k) distribution was affected in transgenic mice with ectopic expression in root epithelium in rests of Malassez in Msx2(+/-) and more clearly in Msx2(-/-) mice. In accordance with these in vivo data, calbindin-D(28k) protein and mRNA levels were decreased in LS8 ameloblast-like cells by exogenous Msx2 overexpression. Furthermore, calbindin-D(28k) promoter activity (nt-1075/+34) was specifically diminished in the presence of Msx2 overexpression, showing that Msx2 behave as a transcriptional repressor for calbindin-D(28k) gene expression. In conclusion, Msx2 may control the spatiotemporally restricted frame of calbindin-D(28k) production in the dental epithelium in relation to enamel mineralization, as previously shown for amelogenin.