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.

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.

Analysis of Resin-Based Dental Materials' Composition Depending on Their Clinical Applications.

The objective of this study was to detail the monomer composition of resin-based dental materials sold in the market in 2023 and to evaluate the proportion of bisphenol A (BPA)-derivatives in relation to their applications. A search on manufacturers' websites was performed to reference resin-based dental materials currently on the European market (including the European Union (EU) and United Kingdom (UK). Their monomer composition was determined using material-safety data sheets and was completed by a search on the PubMed database. Among the 543 material compositions exploitable, 382 (70.3%) contained BPA derivatives. Among them, 56.2% contained BisGMA and 28% BisEMA, the most frequently reported. A total of 59 monomers, of which six were BPA derivatives, were found. In total, 309 materials (56.9%) contained UDMA and 292 (53.8%) TEGDMA. Less than one third of materials identified contained no BPA derivatives. These proportions vary a lot depending on their applications, with materials dedicated to the dental care of young populations containing the highest proportions of BPA-derivative monomers. The long-term effects on human health of the different monomers identified including BPA-derivative monomers is a source of concern. For children and pregnant or lactating women arises the question of whether to take a precautionary principle and avoid the use of resin-based dental materials likely to release BPA by opting for alternative materials.

MIH and Dental Caries in Children: A Systematic Review and Meta-Analysis.

(1) Background: Molar-incisor hypomineralization (MIH) is a clinical condition affecting permanent teeth in children, with a documented rising trend in the last two decades. The aim of the present study was to analyze and synthesize the available evidence on caries experience (dmft/DMFT) and MIH in children. (2) Methods: A systematic review and meta-analysis were conducted according to the PRISMA statement. (3) Results: 59 papers published between 2007 and 2022 were included in the qualitative synthesis and 18 in the meta-analysis. The total sample of subjects was 17,717 (mean: 896), of which 2378 (13.4%) had MIH (mean: 119), with a girl/boy ratio of 1:1. The mean age of the enrolled participants was 8.6 (age range 7-10 years). Meta-analysis showed that MIH has a positive correlation with both dmft (effect size of 0.67, 95% CI [0.15, 1.19]) and DMFT (effect size of 0.56, 95% CI [0.41, 0.72]); (4) Conclusions: Children with MIH should be diagnosed correctly and on time. Treatment and management options for moderate and severe forms of MIH should consider prognosis based on known risk factors, and secondary and tertiary prevention policies should also consider the multifactorial nature of caries etiology.

Multi-scale characterization of Developmental Defects of Enamel and their clinical significance for diagnosis and treatment.

Developmental Defects of Enamel (DDE) such as Dental Fluorosis (DF) and Molar Incisor Hypomineralization (MIH) are a major public health problem. Their clinical aspects are extremely variable, challenging their early and specific diagnosis and hindering progresses in restorative treatments. Here, a combination of macro-, micro- and nano-scale structural and chemical methods, including, among others, Atom Probe Tomography recently applied on tooth enamel, were used to study and compare MIH, DF and healthy teeth from 89 patients. Globally, we show that DF is characterized by an homogenous loss of mineral content and crystallinity mainly disrupting outside layer of enamel, whereas MIH is associated with localized defects in the depth of enamel where crystalline mineral particles are embedded in an organic phase. Only minor differences in elemental composition of the mineral phase could be detected at the nanoscale such as increased F and Fe content in both severe DDE. We demonstrate that an improved digital color measurement of clinical relevance can discriminate between DF and MIH lesions, both in mild and severe forms. Such discriminating ability was discussed in the light of enamel composition and structure, especially its microstructure, organics presence and metal content (Fe, Zn). Our results offer additional insights on DDE characterization and pathogenesis, highlight the potentiality of colorimetric measurements in their clinical diagnosis and provide leads to improve the performance of minimally invasive restorative strategies. STATEMENT OF SIGNIFICANCE: Developmental Defects of Enamel (DDE) are associated to caries and tooth loose affecting billions of people worldwide. Their precise characterization for adapted minimally invasive care with optimized materials is highly expected. Here In this study, first we propose the use of color parameters measured by a spectrophotometer as a means of differential clinical diagnosis. Second, we have used state-of-the-art techniques to systematically characterize the structure, chemical composition and mechanical optical properties of dental enamel teeth affected by two major DDE, Dental Fluorosis (DF) or Molar Incisor Hypomineralization (MIH). We evidence specific enamel structural and optical features for DF and MIH while chemical modifications of the mineral nanocrystals were mostly correlated with lesion severity. Our results pave the way of the concept of personalized dentistry. In the light of our results, we propose a new means of clinical diagnosis for an adapted and improved restoration protocol for these patients.

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.

MSX2 in ameloblast cell fate and activity.

While many effectors have been identified in enamel matrix and cells via genetic studies, physiological networks underlying their expression levels and thus the natural spectrum of enamel thickness and degree of mineralization are now just emerging. Several transcription factors are candidates for enamel gene expression regulation and thus the control of enamel quality. Some of these factors, such as MSX2, are mainly confined to the dental epithelium. MSX2 homeoprotein controls several stages of the ameloblast life cycle. This chapter introduces MSX2 and its target genes in the ameloblast and provides an overview of knowledge regarding its effects in vivo in transgenic mouse models. Currently available in vitro data on the role of MSX2 as a transcription factor and its links to other players in ameloblast gene regulation are considered. MSX2 modulations are relevant to the interplay between developmental, hormonal and environmental pathways and in vivo investigations, notably in the rodent incisor, have provided insight into dental physiology. Indeed, in vivo models are particularly promising for investigating enamel formation and MSX2 function in ameloblast cell fate. MSX2 may be central to the temporal-spatial restriction of enamel protein production by the dental epithelium and thus regulation of enamel quality (thickness and mineralization level) under physiological and pathological conditions. Studies on MSX2 show that amelogenesis is not an isolated process but is part of the more general physiology of coordinated dental-bone complex growth.

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.