Katia Jedeon, Sophia Loiodice, Clémence Marciano, Alexia Vinel, Marie-Chantal Canivenc Lavier, Ariane Berdal,and Sylvie Babajko

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1 ENDOCRINE-DISRUPTING CHEMICALS Estrogen and Bisphenol A Affect Male Rat Enamel Formation and Promote Ameloblast Proliferation Katia Jedeon, Sophia Loiodice, Clémence Marciano, Alexia Vinel, Marie-Chantal Canivenc Lavier, Ariane Berdal,and Sylvie Babajko Centre de Recherche des Cordeliers (K.J., S.L., C.M., A.B., S.B), Institut National de la Santé et de la Recherche Médicale UMRS 1138, Laboratory of Molecular Oral Pathophysiology; Université Paris- Descartes (K.J., S.L.C.M.,A.B., S.B.); Université Pierre et Marie Curie-Paris (K.J., S.L., C.M., A.B., S.B); and Université Paris-Diderot (K.J., A.B., S.B.), UFR d Odontologie, F-75006, Paris, France; I2MC (A.V.), Institut National de la Santé et de la Recherche Médicale U1048, équipe 9 and Université Paul Sabatier (A.V.), Toulouse, France; Institut National de la Recherche Agronomique UMR 1324 (M.-C.C.L.), Centre des sciences du gout et de l alimentation - BP ; CNRS UMR 6265 (M.-C.C.L.), Centre des sciences du gout et de l alimentation; and Université de Bourgogne (M.-C.C.L.), Centre des sciences du gout et de l alimentation, Dijon, France; and Centre de Référence des maladies rares de la face et de la cavité buccale MAFACE hôpital Rothschild (A.B.), AP-HP, Paris, France 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 ER but not ER both in vivo and in vitro. The ER antagonist ICI 182,780 was used to inactivate ER 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. (Endocrinology 155: , 2014) Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) first synthesized at the end of the 19th century and is now one of the most massively produced chemicals in the world, with annual production at over 2.7 billion kilograms (1). It is commonly used in the plastics industry as the base compound for the manufacture of polycarbonates and epoxy resins and may be leached in certain situations (2, 3). Its widespread use in food packaging and its extensive environmental distribution are now issues of major concern. BPA monomers are also present in dental ISSN Print ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received December 20, Accepted June 28, First Published Online July 8, 2014 sealants and resins used in conservative and restorative dentistry, constituting an additional source of BPA contamination (4). Recent studies linking the number of dental resin composites to adverse health effects have reported higher levels of anxiety, depression, social stress and interpersonal relationship problems in children with more than two composites (5). These findings are consistent with epidemiological and experimental data linking BPA concentrations and exposure to behavioral problems (6, 7). Despite health agency warnings and safety policies, Abbreviations: BPA, Bisphenol A; DMSO, dimethyl sulfoxide; EDC, endocrine disrupting chemical; ER, estrogen receptor; ERE, estrogen receptor element; MIH, molar incisor hypomineralization; TDI, tolerable daily intake. doi: /en Endocrinology, September 2014, 155(9): endo.endojournals.org 3365

2 3366 Jedeon et al E2 and BPA Modulate Amelogenesis Endocrinology, September 2014, 155(9): more than 95% of the population is contaminated by BPA (8). Many recent data report its adverse health effects affecting diverse organs including mammary gland, prostate, and testis, but also pancreas and brain. Exposure to BPA seems to contribute to carcinogenesis, diabetes, and obesity, and to reproductive and immune response dysregulations (9 11). Recently, we discovered that low-dose exposure to BPA during perinatal period affects dental enamel mineralization (12). The structural and biochemical abnormalities of rat incisors affected by BPA are very similar to those of human teeth affected by molar incisor hypomineralization (MIH), a recently described pathology affecting 15% to 18% of children (13). Enamel is produced by ameloblasts that secrete enamel matrix proteins (amelogenin, enamelin, ameloblastin, amelotin and tuftelin) and proteases (klk4 and mmp20) that degrade the enamel matrix allowing subsequent mineral crystal growth. We identified klk4 and enamelin as the two main target genes of BPA and found that modulations of their expression lead to enamel hypomineralization (12). Several studies have reported that BPA can promote cell proliferation especially in estrogen-dependent cancer cells, such as breast cancer cells and ovarian cancer cells, but also in prostate cancer cells, testicular seminoma cells, neural progenitor cells, dendritic cells (14, 15, 16). However, the mechanism of action of BPA remains unclear. BPA specifically binds to the estrogen receptors (ER), ER and ER with an equilibrium dissociation constant (K d )of about 100 nm (17, 18, 19). Estrogens regulate many physiological processes, including normal proliferation, development, and tissue-specific gene regulation in the reproductive tract, the central nervous system, and skeletal system. Estrogens also influence pathological processes underlying hormone-dependent diseases, such as breast, endometrial, and ovarian cancers. The biological actions of estrogens are essentially mediated by their binding to the ERs (ER and ER ). BPA was reported to have estrogen-like activity in the 1930 s (20), but the signaling pathway involved, especially at low doses, has not been identified and may even depend on the cell type considered. Note that ameloblasts express ER, but, little is known about the involvement of estrogen in amelogenesis (21). The aim of this study was to pursue the characterization of the effects of BPA on enamel hypomineralization and to compare them with those of estrogen, if any. We investigated BPA involvement in ameloblast proliferation both in vivo and in vitro. Rats were exposed continuously from the conception to the adult stage (day 30) to a low dose of BPA (5 g/kg/d corresponding to one-tenth the tolerable daily intake (TDI)/10) to mimic human exposure occurring during the critical fetal and suckling periods when the teeth are developing. In parallel, the rat dental epithelium HAT-7 cell line and the estrogen responsive MCF-7 cells as controls, were treated with BPA or estrogen. ER signaling was assessed by specific inactivation using the inhibitor ICI 182,780 to determine the involvement of the estrogen pathway in the effects of BPA on ameloblasts. Materials and Methods Animals and biological samples Animals used in this study were bred as described elsewhere (12). Briefly, Wistar Han rats were purchased from Harlan France Sarl. All animals were maintained in accordance with the French Ministry of Agriculture guidelines for care and use of laboratory animals (B EA). Cages and bottles made of polypropylene were used to avoid any contamination by BPA or phthalates. Purified diet was used to avoid phytoestrogens and drinking water was filtered through charcoal to eliminate pesticide residues. On gestational day 1 (identified by daily inspection to detect the presence of spermatozoa in vaginal smears or the vaginal plug), each dam was identified by a numbered ear tag, caged individually, and assigned randomly to either the control or the BPA group (15 16 animals/group). From gestational day 1 (E1) until weaning on postnatal day 21 (P21), 5 g/kg/d BPA (Sigma- Aldrich) in 0.5 ml of corn oil was daily administered orally with a micropipette, to each of the rats in one group of pregnant females; the rats of the second group (controls) were treated in a similar manner, but with corn oil alone (22). After weaning, young rats were exposed daily to BPA as described above until they were killed on P30. Sixteen control rats (n 16 for each group) and sixteen treated rats (n 16 for each group) randomly chosen in independent litters were used in this study. They were anesthetized by isofurane inhalation, killed, and their mandibles were immediately dissected out. The surrounding soft tissues were removed, and the mandibular bone encasing the lower incisors was carefully taken off, under a stereomicroscope (Leica M125, France), to expose the entire labial surface. The incisors were then extracted. Left incisors were microdissected and used for RNA extractions. Right incisors were fixed in 4% paraformaldehyde and embedded in paraffin as described previously (12) for cervical loop analysis. Assessment of ameloblast secretion stage and cervical loop lengths After left incisor extraction and epithelial cell removal, the total incisor lengths were measured accurately with a graduated thread. The labial surfaces of incisors were gently cleaned with a wet paper. Enamel was dried in air for 1 minute, revealing the location of the early maturation stage as a transient white opaque area (23). The secretory stage determined as the distance between the root apex and the start of the white opaque zone was measured immediately (illustrated in Figure 1). Cervical loop length was measured on right incisors by counting the frontal histological sections (each 8 m thick) from the beginning of the cervical loop at the apical level to the beginning of the secretory phase identified according to ameloblast differentiation by microscopy.

3 doi: /en endo.endojournals.org 3367 for 24 or 48 hours. The day of measurement, 80 L of MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (Sigma, 2.5 mg/ ml) was added to 400 L of culture medium in each well, and the plates were incubated at 37 C for 2 hours. The medium was removed and 200 L of DMSO (Sigma-Aldrich) was added to each well to dissolve the violet tetrazolium salt crystals. Plates were shaken for 5 minutes and read in the spectrophotometer (Biochrom WPA Biowave DNA Life Science Spectrophotometer) at 540 nm. Three samples were used for each experimental point and the experiment was repeated six times independently. Figure 1. Analysis of mandibular incisors from rats exposed to BPA. A, Mandibular incisors from BPA-treated rats exhibited white opacities reflecting enamel hypomineralization. B, Comparison of the prevalence of affected incisors in males (75%) and females (31%). C, In males, the ameloblast secretion stage was significantly longer in BPA-treated rats (8.8 mm 0.1) than in controls (8.1 mm 0.1). In females, the length of the secretion stage was not significantly affected by BPA treatment. D, In males and in females, the total incisor length was not significantly different between control and BPA-treated rats (P.6). E, In males, the cervical loop was significantly longer in BPA-treated rats (365 3 m) than in control rats (240 5 m). In females, the cervical loop length was similar in control and BPA-treated groups. F, Diagram showing the position of the cervical loop (CL), and the secretion (S), the transition (T), and the maturation (M) stages of the rat mandible. All values reported are means SD (n 16), ns indicates non significant. **, P.01; ***, P.001 by Mann-Whitney test. BrdU cell proliferation assay Cells were seeded in 96-well plates at a density of cells per well. The labeling solution of 5-bromo-20-deoxyuridine (BrdU) was added simultaneously with the start of treatment. Ten hours later, incorporated BrdU was measured according to the manufacturer s protocol (Roche). Briefly, cells were fixed and DNA was denatured with FixDenat. Cells were incubated with peroxidase-conjugated anti-brdu antibody (diluted 1:100) for 90 minutes at room temperature. Finally, 100 L of substrate (luminol/4-idophénol) was added and chemiluminescence was measured with a Berthold luminometer (Tristar LB 941) after 3 minutes (relative luminescence unit RLU/4). The experiment was repeated six times independently. Cell cultures and treatments Rat HAT-7 ameloblast cells (24) were grown in DMEM/F-12 without phenol red (Gibco) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin (50 IU/mL). Human MCF-7 breast cancer cells were cultured in DMEM without phenol red (Gibco) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin (50 IU/mL). All culture media were filtered through charcoal to remove any steroids. Cells were cultured without serum for the 24 hours before treatments and during experiments. Cells were treated with either BPA or estrogen (E2) (Sigma-Aldrich) alone or with a mixture of both solubilized in dimethyl sulfoxide (DMSO; Sigma-Aldrich); controls were exposed to culture medium containing the same amount of DMSO. For dedicated experiments, ICI 182,780 (Tocris-Bioscience), an estrogen antagonist (25, 26), was mixed with either BPA or E2 and applied to the cells 1 hour before the beginning of the treatments. MTT cell viability assay Cells were seeded in 24-well plates at various densities (5 10 3, , and cells per well) and treated Cell transfections The luciferase reporter plasmids, ERE-luc and MMTV-luc (27), were used to transfect HAT-7 cells according to the manufacturer s instructions (Qiagen). After 48 hours of treatment and transfection, cells were lysed for protein content determination and promoter activity assays. Protein content was determined with a BCA (bicinchoninic acid) protein assay kit (Pierce) according to the manufacturer s instructions. Luciferase activity was assayed by mixing 25 L of protein extract with 75 L of Luciferase Assay Substrate (Promega Corp), and red in a B941 TriStar microplate reader (Berthold France). The levels of luciferase activity in transfected cell extracts are directly dependent on the activity of the promoter considered. The experiment was repeated six times independently. RNA extraction and RT-qPCR analysis Total RNAs were extracted with Trireagent (Euromedex- France) according to the manufacturer s procedure. RNA concentration and purity were determined by spectrophotometry at 260 nm (NanoDrop 1000). Reverse transcription was carried

4 3368 Jedeon et al E2 and BPA Modulate Amelogenesis Endocrinology, September 2014, 155(9): Table 1. Primer Sequences Used for Real-Time Polymerase Chain Reaction Analyses Gene Amplicon size Primer sequences ER rat 112 bp 5 -CCAGCTACAAACCAATGCACCATC-3 Estrogen receptor 5 -GGTCTTTTCGTATCCCGCCTTTC-3 ER mouse 211 bp 5 -AAGAGAGTGCCAGGCTTTGG-3 Estrogen receptor 5 -ACGTAGCCAGCAACATGTCA-3 ER 92 bp 5 -CCATGATCCTCCTCAACTCCAGTATGT-3 Estrogen receptor 5 -CGCGTTCAGTAGGTGTGTCAGCTT-3 PCNA 240 bp 5 -AGGACGGGGTGAAGTTTTCT-3 Proliferating cell nuclear antigen 5 -CGATCTTGGGAGCCAAATAA-3 RS bp 5 -GGCTTGTAGGTGATGGAGAA-3 Ribosomal protein S CTTCCGCAAGTTCACCTACC-3 TBP1 101 bp 5 -CACGAACAACTGCGTTGATC-3 TATA box-binding protein 1 5 -TTTTCTTGCTGCTAGTCTGGAT-3 out with 1 g total RNAs for 45 minutes at 42 C, using a random primer oligodt primer mix, according to the manufacturer s instructions (Superscript II-Invitrogen). Real-time quantitative PCR was performed using Opticon Monitor device (Bio-Rad Laboratories). Each PCR was repeated in triplicate independently and the results were normalized against those for RS15 and TBP1, two selected reference genes which expression didn t vary under our experimental conditions. Details of the primers and the corresponding amplicon sizes are presented in (Table 1). The standard curve method was used to calculate the values corresponding to the relative amounts of test and standard (reference) RNAs. Mean ratios of test RNA/standard RNA were calculated for each sample. Similar data were obtained with the [/delta/] Ct method. Statistical analysis Data resulting from at least three independent experiments are presented as means SD and were analyzed with GraphPad Prism4 software using the nonparametric Mann- Whitney test, two-tailed. When P.05 (*), P.01 (**), P.001 (***), the values compared were considered to be significantly different. Results Figure 2. Modulation of ameloblast proliferation activity by BPA. A, MTT cell viability assay showed a significant increase of the cell number after both 24 and 48 hours of treatment with 10 9 M BPA (n 6). B, Proliferation of BPA-treated cells, determined by BrdU proliferation assay, was 2.3-fold greater than that of control cells (n 6). C, PCNA mrna level in the cervical loop in vivo was significantly increased by BPA (n 4). All values reported are means SD. *, P.05; **, P.01; ***, P.001 by Mann-Whitney test. BPA indicates bisphenol A, OD indicates optical density. BPA impacts amelogenesis stages, preferentially in male rats The control mandibular incisor enamel of Wistar rats was homogeneously yellow to orange whereas incisors from BPA-treated rats showed enamel white spots either symmetrically or asymmetrically affecting the incisors to varying degrees (Figure 1A) (12). The lower incisors of 12 of 16 male rats (75%) and only 5 of 16 females (31%) presented white opaque spots after the administration of BPA whereas none of the control rats were affected (Figure 1B). Rat mandibular incisors were extracted, and the ameloblast secretion stage and total incisor lengths were measured carefully. The ameloblast secretion stage of male BPA-treated

5 doi: /en endo.endojournals.org 3369 rats was longer (8.8 mm 0.1) than that of control rats (8.1 mm 0.1) (Figure 1, C and F). However, there was no significant difference in the total mandibular incisor length between control and BPA-treated male rats (Figure 1D). Unlike male BPA-treated rats, treated female rats didn t differ significantly from control rats either for total incisor or secretion lengths (Figure 1, C and D). The length of the cervical loop was evaluated on in vivo dental microdissections and the cervical loop was significantly larger in BPA-treated (365 3 m) than control rats (240 6 m) (Figure 1, E and F). BPA induces amelolast proliferation in vitro and in vivo MTT assays after both 24 and 48 hours of BPA treatment, showed a significant increase in the number of viable HAT-7 cells from baseline, whatever the cell density (Figure 2A). BrdU proliferation assays were used to determine whether the increased number of viable cells was due to cell proliferation. The proliferative activity of BPA-treated ameloblasts was 2.3-fold greater than that of the control cells (Figure 2B). The cervical loop that constitutes a proliferative niche of dental cells was isolated by microdissection in vivo. PCNA mrna levels in the cervical loop were determined by RT-qPCR and were significantly higher (1.75-fold) in BPA-treated rats than controls (Figure 2C). The effects of BPA on ameloblast proliferation is partially mediated by the estrogen receptor ER Because BPA effects may involve ERs, the expression of ERs in ameloblasts was investigated. Rat and mouse dental epithelia expressed ER but not ER (Figure 3, A and B, Supplemental Figure 1). Similar levels of ER were measured in males and in females. Uterus and ovary tissues were used as a positive control for ER expression confirming the absence of detectable ER mrna in dental epithelium (Figure 3E, Supplemental Figure 1). ER expression was higher in the cervical loop where there are proliferative cells than in the postmitotic differentiated ameloblasts of the secretion and maturation stages (Figure 3A). The protein was detected by immunohistochemistry with a specific polyclonal antibody directed against ER (Supplemental Figure 2). HAT-7 cells, cultured in vitro, Figure 3. Expression of ERs in rat dental epithelium. A, ER mrna levels standardized to RS15 and TBP1 in the cervical loop (CL), the secretion (S) maturation (M) stages, and HAT-7 cells. B, ER mrna was not detected in rat dental epithelium. C, The levels of ER mrna in rat dental epithelium were not significantly modulated by BPA. D, The levels of ER mrna in rat dental epithelium were not significantly modulated by BPA despite the detection of a weak band. E, The PCR products were separated by a 2% agarose gel electrophoresis, stained by ethidium bromide, and photographed under UV light. The sizes of the amplified products are indicated in bp on the left. PCR was performed with non reverse-transcribed RNAs (negative control, lane 1), and cdnas from the uterus (positive control, lane 2), ovary (positive control, lane 3), male control dental epithelium (lane 4), male BPA-treated dental epithelium (lane 5), female control epithelium (lane 6), and female BPA-treated epithelium (lane 7).

6 3370 Jedeon et al E2 and BPA Modulate Amelogenesis Endocrinology, September 2014, 155(9): also contained the mrna for ER but not that for ER, consistent with the findings for ameloblasts in vivo (Figure 3A). In addition, HAT-7 cells didn t express neither ERR nor GPR30, two putative BPA receptors related to estrogen pathway (data not shown). ER expression was not affected by BPA treatment in either males or females (Figure 3C). Despite the absence of quantitative differences and detections, a weak band corresponding to the ER amplicon was observed in the dental epithelia of BPAtreated rats (Figure 3E). To test whether BPA acts through the ER pathway to affect ameloblastic proliferation, we used ICI 182,780, a specific antagonist of ERs. We first established dose-response curves to determine the optimal doses of each molecule (Figure 4, A and D). Smaller cell numbers were measured in the presence of 10 6 M BPA or ICI 182,780 (or 10 7 M to a lesser extend) than in controls suggesting toxic effects at these high doses (Figure 4, A and D) to 10 8 M BPA increased HAT-7 cell proliferation and lower concentrations had no mitogenic effects. Concerning ICI 182,780, the selected concentration for further studies was M because lower concentrations did not inhibit effects of 10 9 M BPA (Figure 4, A and D). MTT assays showed that the number of viable cells was lower in samples treated with both M ICI and 10 9 M BPA (selected as optimal doses) than those treated with 10 9 M BPA alone; but in both cases, the numbers of viable cells were higher than in control samples (Figure 4B). BrdU proliferation assays confirmed these findings: the proliferation activity after combined 10 9 M BPA and M ICI treatment was lower than that after 10 9 M BPA treatment but significantly higher than the control (Figure 4C). MCF-7 breast cancer cells expressing ERs were used as a positive control (14); cell numbers increased after 10 9 M BPA treatment (Figure 4, D and E) and M ICI addition completely abolished the proliferative effects of BPA (Figure 4F). Figure 4. Involvement of ERs in the mediation of BPA effects on proliferation. A, Concentration response analysis for BPA, ICI182,780, and ICI182, M BPA on HAT-7 cell number. BPA concentrations (round points) 10 7 M were toxic for HAT-7 cells whereas 10 9 Mto 10 8 M induced cell proliferation. ICI182,780 concentrations (squares) 10 7 M were toxic for HAT-7 cells whereas concentrations 10 8 M were inactive when used in combination with 10 9 M BPA (triangles) M ICI was the dose selected for subsequent experiments. B, Forty-eight hours after treatments, the number of viable HAT-7 cells was lower in samples treated with 10 9 M BPA M ICI than in those treated with 10 9 M BPA alone but was still significantly higher than that in the control or with ICI alone (n 6). C, BrdU proliferation assays gave results similar to those of MTT cell viability assays for BPA-treated HAT-7 cells (n 6). D, Concentration response analysis for BPA, ICI, and ICI 10 9 M BPA on MCF-7 cell number. E, After 48 hours of treatment with 10 9 M BPA, the number of viable MCF-7 cells was greater than that of the control or ICI alone, and ICI completely inhibited this effect (n 6). F, The proliferation activity in BPA-treated MCF-7 cells was greater (3.2-fold) than that in controls and ICI inhibited the effect of BPA (n 6). All values reported are means SD. *, P.05 by Mann-Whitney test. Estrogen induces ameloblast proliferation via the ER pathway With the aim of assessing the influence of estrogen on ameloblast proliferation activity, cells were pretreated (or not) with M ICI and then treated for 48 hours with various concentrations of E2. Viable cell counts were significantly higher after treatment with 10 7 to M E M ICI pretreatment resulted in a cell number significantly lower than with 10 9 ME2 alone and similar to control values (Figure 5A, B). HAT-7 cell proliferation as evaluated by the BrdU assay was significantly greater (3.7-fold) for E2-treated samples than controls; for samples treated with both M ICI and 10 9 M E2, proliferation was similar to that of controls and significantly lower than for samples treated with E2 alone (Figure 5C). MCF-7 cells were used as positive controls and as expected, cell proliferation was induced by

7 doi: /en endo.endojournals.org 3371 Figure 5. Effects of estrogen (E2) on ameloblast proliferation and ERE promoter activity. A, Concentration response analysis for E2, ICI182,780, and ICI182, M E2 on HAT-7 cell number. E2 (round points) was mitogenic whatever the concentrations tested (from 10 7 Mto10 11 M). ICI182,780 (squares) concentrations 10 7 M were toxic for HAT-7 cells whereas concentrations 10 8 M didn t inhibit the induction of proliferation by E M ICI was the dose selected for subsequent experiments (B) MTT cell viability assays evidenced an increase (1.6-fold) of ameloblast proliferation activity after 48 hours of treatment with 10 9 M E2, with the addition of ICI inhibiting this effect (n 6). C, BrdU proliferation assays revealed that the proliferation activity was significantly increased (3.7-fold) by treatment with 10 9 M E2 and that M ICI inhibited the effects of 10 9 ME2(n 6). D, Concentration response analysis for E2, ICI, and ICI 10 9 M E2 for the modulation of EREpromoter activity in HAT-7 cells. E, E2-induced ERE promoter activity was abolished by treatment with M ICI treatment. F, ERE promoter activity was not significantly affected by 48 hours of treatment with 10 9 M BPA or BPA/ICI combination. G, Concentration response analysis for E2, ICI, and ICI 10 9 M E2 on MCF-7 cell number. H, Estrogen at a concentration of 10 9 M increased the number of viable MCF-7 cells and M ICI neutralized this effect (n 6). I, Estrogen increased MCF-7 cell proliferation (to 4.2-fold that of the control) and ICI inhibited this

8 3372 Jedeon et al E2 and BPA Modulate Amelogenesis Endocrinology, September 2014, 155(9): M E2, and this induction was completely abolished by M ICI (Figure 5, G I). Ameloblasts transfected with estrogen receptor element (ERE) promoter and treated for 48 hours with E2 exhibited an increase of ERE activity (1.8-fold). The activity of MMTV promoter [which does not contain ERE (27)] wasn t induced by E2 (data not shown). The addition of ICI completely abolished the induction by E2 (Figure 5, D and E). Similar experiments carried out with BPA revealed no variation of ERE promoter activity in either HAT-7 or MCF-7 cells (Figure 5, F and L). The combination of BPA and estrogen substantially increases ameloblast proliferation HAT-7 cells were treated with BPA or E2 alone, or in combination. Ameloblast proliferation was greatest in samples treated with the BPA and E2 combination (7.2- fold higher than control) and lower in samples treated with BPA (2.4-fold) or E2 (3.7-fold) alone (Figure 6). Pretreatment with ICI decreased this effect but did not completely abolish it; proliferation of ICI-treated samples was higher than for controls (Figure 6). Discussion BPA is an EDC with many adverse effects on different cells, tissues, and organs. We recently reported that BPA modulates the expression of key enamel genes thereby affecting amelogenesis and enamel mineralization. Rats exposed to BPA present enamel defects with characteristics similar to those of MIH-affected teeth (12). The effects and mechanism of action of BPA in dental cells are still unknown, and need to be documented. Several studies in animal models have demonstrated that adverse effects are observed upon exposure to low doses of BPA, similar to the doses humans are currently exposed to (2, 28, 29). We therefore used low doses of BPA in both in vivo and in vitro experiments. We found that the secretion stage of ameloblasts was longer in male BPA-treated rats than controls and this difference was not due to differences in total size (Figure 1). This difference was only evidenced in male rats and not in females and is reliable to the preferential BPA impact on enamel of male rats. The rodent incisor cervical loop constitutes a niche of proliferative progenitor cells (30), so we measured both cervical loop length and PCNA expression to follow cell proliferation. Both measures were increased Figure 6. Effects of a combination of BPA and E2 on ameloblast proliferation. BrdU assays revealed that a combination of 10 9 M BPA with 10 9 M E2 induced ameloblast proliferation (7.2-fold) more strongly than 10 9 M BPA (2.4-fold) or 10 9 M E2 (3.7-fold) alone M ICI decreased this effect but did not completely abolish it (n 6). All values reported are means SD. *, P.05; **, P.01 by Mann-Whitney test. by BPA indicating that BPA presented a mitogenic activity on preameloblasts in vivo. In vitro, BPA also clearly increased ameloblast proliferation, similarly to its effect on MCF-7 breast cancer cells used as positive controls (14). The stimulation of ameloblast proliferation is likely to modify their differentiation program during amelogenesis. In the normal physiological case, the ameloblast maturation stage is the longest stage during amelogenesis (31). In the presence of BPA, the length of the secretion stage of ameloblasts is increased, thus leading to an acceleration of the differentiation process and consequently leaving less time for the maturation stage to complete mineralization of the enamel matrix. The resulting hypomineralized enamel is manifest as white opacities on teeth, as we described previously (12). A prolonged secretory phase has been observed in other pathological situations and has been found to be associated with an accelerated eruption rate (32, 33). And conversely, an accelerated eruption rate (Figure 5 continued). effect, such that the values for ICI- and estrogen-treated samples were not different from those for controls (n 6). J, Concentration response analysis for E2, ICI, and ICI 10 9 M E2 for the modulation of ERE-promoter activity in HAT-7 cells. K, E2-induced ERE promoter activity in MCF-7 cells. This activity was significantly repressed after ICI treatment. L, ERE promoter activity was not induced by treatment with 10 9 M BPA. All values reported are means SD. ns indicates non significant, OD indicates optical density. *, P.05; **, P.01; ***, P.001 by Mann-Whitney test.

9 doi: /en endo.endojournals.org 3373 has been linked to enamel hypomineralization characteristics (34). Interestingly, dental development is also accelerated in MIH (35) in accordance with our proposition of BPA as a causal factor of MIH. The fragile and often decayed teeth of patients with MIH lead dentists to use filling materials that may contain BPA based monomers thus maintaining the pre-existing BPA contamination. The estrogenic activity of BPA is probably involved in many of its effects. Several developmental and experimental studies have demonstrated the expression of ERs in odontoblasts and the dental pulp tissue (36 40). ER expression has also been reported in rat ameloblasts, and especially in preameloblast proliferative cells (21). The mechanisms of hormonal regulation in human toothforming tissues have not been characterized in detail, especially the responsiveness of ameloblasts that is unknown (21). Here, we show that HAT-7 rat ameloblast proliferation is rapidly induced by E2 probably through a nongenomic pathway. This is the first time, as far as we are aware, that the functional significance of ER in ameloblasts has been demonstrated. ER (but not ER ) isexpressed in the rat HAT-7 cell line and in the cervical loop containing preameloblasts, implicating estrogen/er in dental cell proliferation during enamel formation. Accordingly, ER expression was weaker in ameloblasts of the maturation stage that no longer proliferate. BPA binds both ER and ER, and is considered to be an environmental xeno-estrogen (11, 20). Here, ICI 182,780 totally abolished the mitogenic effects of E2 in the HAT-7 cell line, as expected, but only partially inhibited the effects of BPA. This suggests that BPA affects ameloblast proliferation through other signaling pathways in addition to ER. The results obtained with the combination of BPA and E2 were consistent with this interpretation. Exposure to such mixtures may be similar to conditions of development in vivo, because E2 may be essential for dental cell proliferation and BPA that pass through the placental barrier may modulate E2 effects. Thus, cells are presumably exposed to low doses of BPA, inducing proliferation and thereby altering enamel formation. ERE promoter activity is not responsive to BPA, indicating that the ER signaling pathway is not involved in the long-term genomic effects of BPA on ERE activation and thus on the modulation of E2 target gene transcription. It has become increasingly evident that BPA can activate different signal transduction pathways. There is now evidence that BPA triggers nonclassical estrogen-activated pathways by binding to membranous ERs and GPR30 (16, 41 43). In addition, the actions of BPA may also involve other signaling systems such as androgen (44), ERR (45, 46), and ROR pathways (47) that need to be investigated in the dental epithelium. Consequently, any interpretation of the effects of BPA on ameloblasts based solely on estrogenmimicking actions would undoubtedly be too simplistic. A recent epidemiological study indicated that the sexual dimorphism of tooth size might be controlled, at least in part, by intrauterine levels of testosterone (48), providing new data of a role for steroid hormones in tooth development. The data presented here are consistent with this hypothesis. Interestingly, they show a preferential impact of BPA on males in accordance with the findings of other studies focusing on metabolism or the brain (6, 42), but the reason still remains unclear. A possible hypothesis is that proliferation of cervical loop cells is induced by BPA through an increase in estrogenic activity. Because E2 levels are lower in males, the variation in estrogenic activity due to BPA is higher in males than in females resulting in a stronger impact. Consequently, an increase in estrogenic activity inducing dental cell proliferation as observed after exposure to BPA leads to an irreversible impact on dental enamel. This characteristic could thus be used as an early marker of exposure to EDCs acting in the same way as BPA (12). In conclusion, we report that BPA stimulates ameloblast proliferation. Its mechanism of action is not entirely exerted through ER in these cells especially on gene expression modulations, indicating that there are other additional pathways affected by BPA. We also demonstrate, for the first time, that dental epithelial cells are estrogen targets and that an increase in proestrogenic activity has a greater effect on the enamel in male than in female rats. We thus provide evidence of hormonal influence on amelogenesis and probably on sexual differences of enamel quality. Acknowledgments We are grateful to Raymond Berges, Sofiane Boudalia, Laurence Decoq, Bruno Pasquis, and Elise Lalarme (UMR 1324 INRA, Dijon) for animal breeding. We thank Pr Hidemitsu Harada (Department of anatomy, Iwate Medical University, Japan), Dr Patricia Forgez (Inserm UPMC U938, Hôpital Saint-Antoine, Paris) for the cells, as well as Hong Sun and Xinru Wang (Jiangsu Provincial Centre for Disease Prevention and Control, Nanjing , China) for ERE containing plasmids. We acknowledge Pr Pierre Chambon and Dr Andrée Krust (IGBMC, Strasbourg, France) for providing the ER / mice. Address all correspondence and requests for reprints to: Sylvie Babajko, Centre de Recherche des Cordeliers, INSERM, UMRS 1138, Laboratory of Molecular Oral Pathophysiopathology, rue de l Ecole de Médecine, Paris cedex 06, France. sylvie.babajko@crc.jussieu.fr.

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