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• W1991486688 abstract "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-timePCR, 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. 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-timePCR, 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. CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The environment has become increasingly contaminated by various pollutants. This contamination has led to an increase in the incidence and gravity of known conditions and/or the emergence of new conditions. Recently, the appearance of a distinct enamel condition was identified and called molar incisor hypomineralization (MIH) in recognition that it is most likely to be found affecting permanent first molars with frequent involvement of the permanent incisors.1Jälevik B. Klingberg G. Barregård L. Norén J.G. The prevalence of demarcated opacities in permanent first molars in a group of Swedish children.Acta Odontol Scand. 2001; 59: 255-260Crossref PubMed Scopus (135) Google Scholar, 2Weerheijm K.L. Merjare I. Molar incisor hypomineralisation: a questionnaire inventory on its occurrence in member countries of the European Academy of Paediatric Dentistry (EAPD).Int J Paediatr Dent. 2003; 13: 411-416Crossref PubMed Scopus (76) Google Scholar MIH is diagnosed in children at approximately 6 to 8 years of age and presents as random white opacities on the enamel of affected teeth. MIH prevalence is highly variable, with 2.4% to 40.2% (mean of approximately 18%) of children affected.3Jälevik B. Prevalence and diagnosis of molar-incisor-hypomineralisation (MIH): a systematic review.Eur Arch Paediatr Dent. 2010; 11: 59-64Crossref PubMed Scopus (205) Google Scholar To date, the cause of MIH remains unclear. However, given that MIH affects those teeth that are undergoing mineralization around the time of birth, it is clear that the enamel-forming ameloblasts are only sensitive to the causative agent(s) responsible for MIH in a specific time window. MIH is indicative of some adverse event(s) occurring during early childhood that effect enamel development.1Jälevik B. Klingberg G. Barregård L. Norén J.G. The prevalence of demarcated opacities in permanent first molars in a group of Swedish children.Acta Odontol Scand. 2001; 59: 255-260Crossref PubMed Scopus (135) Google Scholar, 2Weerheijm K.L. Merjare I. Molar incisor hypomineralisation: a questionnaire inventory on its occurrence in member countries of the European Academy of Paediatric Dentistry (EAPD).Int J Paediatr Dent. 2003; 13: 411-416Crossref PubMed Scopus (76) Google Scholar Diverse environmental conditions, such as medication (amoxicillin), hypoxia, hypocalcaemia, dioxins, polychlorinated biphenyls, and prolonged breastfeeding, have been associated with MIH.4Alaluusua S. Aetiology of Molar-Incisor Hypomineralisation: a systematic review.Eur Arch Paediatr Dent. 2010; 11: 53-58Crossref PubMed Scopus (211) Google Scholar The vast number of putative causative agents and the difficulty in retrospectively linking a specific exposure during the developmental window when enamel is susceptible to MIH make epidemiologic studies inconclusive. An increasing prevalence of numerous adverse health effects, such as diabetes, obesity, infertility, cancers, and autism, has been linked to endocrine-disrupting chemicals (EDCs).5Alonso-Magdalena P. Quesada I. Nadal A. Endocrine disruptors in the etiology of type 2 diabetes mellitus.Nat Rev Endocrinol. 2011; 7: 346-353Crossref PubMed Scopus (313) Google Scholar, 6Vom Saal F.S. Nagel S.C. Coe B.L. Angle B.M. Taylor J.A. The estrogenic endocrine disrupting chemical bisphenol A (BPA) and obesity.Mol Cell Endocrinol. 2012; 354: 74-84Crossref PubMed Scopus (347) Google Scholar Bisphenol A (BPA) is a typical EDC widely used in the production of polycarbonate plastics and epoxy resins. Its widespread use in food packaging and environment is controversial and hotly debated. Despite health agency concerns and safety policies, >95% of the population is contaminated by BPA.7Pirard C. Sagot C. Deville M. Dubois N. Charlier C. Urinary levels of bisphenol A, triclosan and 4-nonylphenol in a general Belgian population.Environ Int. 2012; 48: 78-83Crossref PubMed Scopus (105) Google Scholar BPA affects different organs and physiologic key functions, such as reproduction8Hunt P.A. Lawson C. Gieske M. Murdoch B. Smith H. Marre A. Hassold T. Vandevoort C.A. Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey.Proc Natl Acad Sci U S A. 2012; 109: 17525-17530Crossref PubMed Scopus (171) Google Scholar and sex determinism, brain development, and behavior.6Vom Saal F.S. Nagel S.C. Coe B.L. Angle B.M. Taylor J.A. The estrogenic endocrine disrupting chemical bisphenol A (BPA) and obesity.Mol Cell Endocrinol. 2012; 354: 74-84Crossref PubMed Scopus (347) Google Scholar, 9Poimenova A. Markaki E. Rahiotis C. Kitraki E. Corticosterone-regulated actions in the rat brain are affected by perinatal exposure to low dose of bisphenol A.Neuroscience. 2010; 167: 741-749Crossref PubMed Scopus (141) Google Scholar It may also increase breast cancer risk10Tharp A.P. Maffini M.V. Hunt P.A. VandeVoort C.A. Sonnenschein C. Soto A.M. Bisphenol A alters the development of the rhesus monkey mammary gland.Proc Natl Acad Sci U S A. 2012; 109: 8190-8195Crossref PubMed Scopus (127) Google Scholar and lead to obesity.6Vom Saal F.S. Nagel S.C. Coe B.L. Angle B.M. Taylor J.A. The estrogenic endocrine disrupting chemical bisphenol A (BPA) and obesity.Mol Cell Endocrinol. 2012; 354: 74-84Crossref PubMed Scopus (347) Google Scholar, 11Nadal A. Obesity: fat from plastics?.linking bisphenol A exposure and obesity Nat Rev Endocrinol. 2012; 9: 9-10Crossref PubMed Scopus (31) Google Scholar, 12Rubin B.S. Soto A.M. Bisphenol A: perinatal exposure and body weight.Mol Cell Endocrinol. 2009; 304: 55-62Crossref PubMed Scopus (222) Google Scholar Although molecular mechanisms of action are still being researched, specific BPA-target genes associated with specific disease states have been identified in differentiated cell types in epidemiologic surveys.8Hunt P.A. Lawson C. Gieske M. Murdoch B. Smith H. Marre A. Hassold T. Vandevoort C.A. Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey.Proc Natl Acad Sci U S A. 2012; 109: 17525-17530Crossref PubMed Scopus (171) Google Scholar, 13Soriano S. Alonso-Magdalena P. García-Arévalo M. Novials A. Muhammed S.J. Salehi A. Gustafsson J.A. Quesada I. Nadal A. Rapid insulinotropic action of low doses of bisphenol-A on mouse and human islets of Langerhans: role of estrogen receptor β.PLoS One. 2012; 7: e31109Crossref PubMed Scopus (160) Google Scholar The effects of BPA on dental cells are unknown. Interestingly, sensitivity to BPA in humans is highest during the perinatal period.8Hunt P.A. Lawson C. Gieske M. Murdoch B. Smith H. Marre A. Hassold T. Vandevoort C.A. Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey.Proc Natl Acad Sci U S A. 2012; 109: 17525-17530Crossref PubMed Scopus (171) Google Scholar, 14Varayoud J. Ramos J.G. Bosquiazzo V.L. Lower M. Muñoz-de-Toro M. Luque E.H. Neonatal exposure to bisphenol A alters rat uterine implantation-associated gene expression and reduces the number of implantation sites.Endocrinology. 2011; 152: 1101-1111Crossref PubMed Scopus (93) Google Scholar This period corresponds to the temporal window when the enamel of the permanent incisors and first molars is being formed. Thus, our hypothesis is that EDCs such as BPA may be involved in MIH by having an adverse effect on amelogenesis. Amelogenesis begins with a secretory stage during which a partially mineralized enamel matrix is elaborated. The enamel matrix is composed of amelogenins, enamelin, ameloblastin, and amelotin, which are subject to extracellular processing by matrix metalloprotease 20. The matrix proteins are essential for the correct enamel formation as evidenced by the fact that mutations in any of the proteins involved leads to amelogenesis imperfecta.15Wright J.T. The molecular etiologies and associated phenotypes of amelogenesis imperfecta.Am J Med Genet A. 2006; 140: 2547-2555Crossref PubMed Scopus (111) Google Scholar Once the full thickness of the enamel has been deposited, amelogenesis enters the maturation phase during which the serine protease kallikrein 4 (KLK4) degrades the enamel matrix proteins. Abnormal retention of proteins or indeed any extraneous proteins, such as serum albumin, lead to the eruption of hypomineralized enamel.16Robinson C. Kirkham J. Brookes S.J. Bonass W.A. The chemistry of enamel development.Int J Dev Biol. 1995; 39: 145-152PubMed Google Scholar The rodent incisor provides a running record of how an effect on amelogenesis affects future development as the incisor continues to erupt.17Berdal A. Hotton D. Pike J.W. Mathieu H. Dupret J.M. Cell- and stage-specific expression of vitamin D receptor and calbindin genes in rat incisor: regulation by 1,25-dihydroxyvitamin D3.Dev Biol. 1993; 155: 172-179Crossref PubMed Scopus (75) Google Scholar, 18Lacruz R.S. Smith C.E. Chen Y.B. Hubbard M.J. Hacia J.G. Paine M.L. Gene-expression analysis of early- and late-maturation-stage rat enamel organ.Eur J Oral Sci. 2011; 119: 149-157Crossref PubMed Scopus (35) Google Scholar Once amelogenesis is complete, the ameloblasts and the overlying outer enamel epithelium cells degenerate and are ultimately lost through abrasion after tooth eruption. As a consequence, enamel defects are irreversible and provide a permanent record of any disturbances that occur during enamel development. This record of previous disturbances allows retrospective studies to be performed and provides a means of temporally fixing a pathologic event at some point during development.17Berdal A. Hotton D. Pike J.W. Mathieu H. Dupret J.M. Cell- and stage-specific expression of vitamin D receptor and calbindin genes in rat incisor: regulation by 1,25-dihydroxyvitamin D3.Dev Biol. 1993; 155: 172-179Crossref PubMed Scopus (75) Google Scholar The objective of the present study was to assess the possible effect of BPA on enamel development and elucidate any underlying mechanism of action. Rodents were exposed daily in utero and after birth to a low dose of BPA to mimic human exposure occurring during the critical fetal and suckling periods when the teeth are developing. Bona fide human MIH enamel was also compared with enamel from BPA-treated rat to investigate whether any structural features of MIH were replicated in the BPA-treated rat teeth. Eight-week-old Wistar Han rats were purchased (Harlan France Sarl, Gannat, France). All animals were maintained in accordance with the French Ministry of Agriculture guidelines for care and use of laboratory animals (B2 231010EA). Cages and bottles were made of polypropylene to avoid any contamination by BPA or phthalates, and drinking water was filtered through charcoal to eliminate pesticides. Animals were fed a purified phytoestrogen-free diet consisting of 18% casein, 40% corn starch, 20% maltodextrin, 6% sucrose, 5% corn oil, 5% cellulose, 5% mineral mixture, and 1% vitamin mixture (INRA, Jouy en Josas, France) and provided with water ad libitum. At gestational day 1, determined by the presence of an intravaginal sperm plug, the dams were randomly divided into two groups. From gestational day 1 until weaning day 21, one group of pregnant females was orally administered 5 μg/kg of BPA daily (Sigma-Aldrich, St. Louis, MO) in 0.5 mL of corn oil, whereas the control group was administered corn oil alone. After weaning, young rats were exposed daily to BPA as described until sacrifice at day 30 or day 100. At each stage, 16 male control rats and 16 male treated rats were used in the present study. Half (n = 8) of each group were randomly selected, anesthetized by isoflurane inhalation, and perfused with 4% paraformaldehyde (Sigma-Aldrich) in PBS (1×, pH 7.4). Perfused right hemimandibles were analyzed by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), whereas perfused left hemimandibles were prepared for histologic analysis. The remaining rats (n = 8) in each group were sacrificed, and their mandibles were immediately dissected. The surrounding soft tissues were removed and the mandibular bone encasing the lower incisors was carefully taken off under a stereomicroscope (Leica M125; Leica, Paris, France) to expose the entire labial surface. The incisors were then extracted, and the cervical loops were removed. For right hemimandibles, rat enamel organ, now easily accessible, was carefully harvested in its entirety and put in the Tri-Reagent (Euromedex, Paris, France) for RNA extraction. For left hemimandibles, epithelial cells from the secretion stage and the maturation stage were separately dissected using the molar reference line for isolation18Lacruz R.S. Smith C.E. Chen Y.B. Hubbard M.J. Hacia J.G. Paine M.L. Gene-expression analysis of early- and late-maturation-stage rat enamel organ.Eur J Oral Sci. 2011; 119: 149-157Crossref PubMed Scopus (35) Google Scholar, 19Smith C.E. Nanci A. A method for sampling stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection.Anat Rec. 1989; 225: 257-266Crossref PubMed Scopus (90) Google Scholar and placed in Tri-Reagent (Euromedex) for RNA extraction. Secretory-stage enamel was then harvested from all mandibles using a scalpel20Hiller C.R. Robinson C. Weatherell J.A. Variations in the composition of developing rat incisor enamel.Calcif Tissue Res. 1975; 18: 1-12Crossref PubMed Scopus (120) Google Scholar for the sequential extraction of matrix proteins. Patients were recruited in the Centre de Référence des Maladies Rares de la Face et de la Cavité Buccale MAFACE (Hôpital Rothschild, Paris, France). Inclusion and exclusion criteria were based on those established by the international consensus on MIH diagnosis.21Weerheijm K.L. Duggal M. Mejàre I. Papagiannoulis L. Koch G. Martens L.C. Hallonsten A.L. Judgement criteria for molar incisor hypomineralisation (MIH) in epidemiologic studies: a summary of the European meeting on MIH held in Athens.Eur J Paediatr Dent. 2003; 4: 110-113PubMed Google Scholar Positive diagnosis was based on the clinical singularity of enamel defects: random discolored patches, hypomineralized status assessed by probing, tooth-type selectivity, increased nociceptivity, and increased susceptibility to caries. Differential diagnosis with amelogenesis imperfecta, enamel fluorosis, and punctual inflammation–induced hypomineralization of permanent teeth was established by their distinct clinical features and familial and medical antecedent analysis. MIH and control teeth, obtained after elective extraction, were collected (n = 10) and immediately immersed in 4% paraformaldehyde in PBS buffer (pH 7.4) for 48 hours. Samples were then dehydrated using increasing concentrations of ethanol for 48 hours each and prepared for resin embedding as described previously.22Molla M. Descroix V. Aïoub M. Simon S. Castañeda B. Hotton D. Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.Am J Pathol. 2010; 177: 2516-2526Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Human molars were sectioned parallel to the longitudinal axis of the tooth using cutting equipment (Exakt, Norderstedt, Germany). Sections were then polished with graded sandpaper to a thickness of 40 μm. Each section was bonded onto an aluminum pin stub using adhesive carbon disks. In the case of rat hemimandibles, the incisor cervical margin and the first molar furcation point were used as anatomical reference points so that specimens could be prepared consistently. Mandibles were cut transversely close to these anatomical reference points using a rotating diamond wheel and then ground back to the selected anatomical reference point with graded sandpaper. Samples were dehydrated in ethanol for 48 hours each at 4°C, stuck on a calibrated aluminum pin stub using clear polyester resin, and ultrasonicated for 15 minutes. Human and rat enamel surfaces were etched with 37% orthophosphoric acid for 30 and 15 seconds, respectively, to remove any smear layers. Each sample was coated with platinum 6 nm thick in a vacuum evaporator, and the enamel microstructure was observed with SEM (Carl Zeiss Supra 40; Carl Zeiss AG, Oberkochen, Germany) at 10 kV. After the initial observations, the samples were polished, etched, rinsed with 2.5% sodium hypochlorite for 2 minutes, and observed a second time after coating with platinum. After SEM, the samples were analyzed by EDX using an X-ray detector system attached to an SEM (JSM-6100; Jeol, Tokyo, Japan) at 15 kV. For each specimen, 15 distinct points distributed within the enamel were analyzed to measure Ca and C content. Semiquantitative data were submitted to the ZAF correction method [atomic number effect correction (Z), absorption effect correction (A), and fluorescent excitation effect correction (F)]. Left perfused hemimandibles were postfixed by immersion in 4% paraformaldehyde solution for 24 hours. After rinsing in PBS, hemimandibles were decalcified at 4°C in pH 7.4 PBS solution containing 4.13% EDTA (Sigma-Aldrich) and 0.2% paraformaldehyde for 2 months. The decalcification solution was changed twice weekly. After washing in PBS for 4 hours at 4°C, the samples were dehydrated in ethanol, rinsed in Safesolv (Labonord SASA, Templemars, France), and finally paraffin embedded (Paraplast Plus; Sigma-Aldrich). Serial frontal sections (8 μm thick) were cut using a microtome (RM 2145; Leica). Sections were deparaffinized and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidases were blocked by incubation for 20 minutes in a freshly made solution of 3% H2O2 in PBS. Sections were then washed in PBS and blocked with 5% milk in PBS for 20 minutes at 4°C. Primary anti-amelogenin antibody (Kamya Biomedical Company, Seattle, WA) (1:300), anti-enamelin (1:300),23Brookes S.J. Kingswell N.J. Barron M.J. Dixon M.J. Kirkham J. Is the 32-kDa fragment the functional enamelin unit in all species?.Eur J Oral Sci. 2011; 119: 345-350Crossref PubMed Scopus (11) Google Scholar anti-ameloblastin (M-300 sc-50534; Santa Cruz Biotechnology, Santa Cruz, CA) (1:300), or anti-albumin (M-140 sc-50536; Santa Cruz) (1:400) were applied for 1 hour at room temperature. Sections were incubated with Alexa Fluor 594 secondary antibody (A-11072; Life Technologies, Carlsbad, CA) (1:500) for 1 hour in the dark. After rinsing with PBS, section were immersed in DAPI (010M4003; Sigma-Aldrich) (1:100,000) for 1 minute. They were finally mounted with aquamount (13,800; Lerner Laboratories, Pittsburgh, PA). For albumin staining, sections were incubated for 30 minutes with Impress reagent (TM reagent ImmPRESS kit; Vector Laboratories, Burlingame, CA) containing secondary peroxidase-conjugated antibodies (1:5000) and immunocross-reactivity was visualized by adding peroxidase substrate (K3468; Dako, Carpinteria, CA). They were finally rinsed with water, dehydrated, and mounted with DePeX resin (BDH Laboratory, Poole, England). Secretory-stage enamel matrix was sequentially extracted with 30 μL of 50 mmol/L Tris (pH 7.4) (to extract freely soluble proteins), then with 30 μL of 100 mmol/L PBS (pH 7.4) (to extract mineral bound proteins), and finally with 60 μL of 1% SDS (to extract remaining aggregated proteins). The use of phosphate buffer is akin to the elution buffer used in chromatography columns for protein adsorbed to hydroxyapatite. Phosphate buffer at 100 mmol/L is able to extract all mineral bound proteins from developing enamel.24Brookes S.J. Lyngstadaas S.P. Robinson C. Shore R.C. Wood S.R. Kirkham J. Enamelin compartmentalization in developing porcine enamel.Connect Tissue Res. 2002; 43: 477-481PubMed Google Scholar, 25Brookes S.J. Kirkham J. Shore R.C. Wood S.R. Slaby I. Robinson C. Amelin extracellular processing and aggregation during rat incisor amelogenesis.Arch Oral Biol. 2001; 46: 201-208Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Twenty micrograms of the first (Tris) and third (SDS) fractions and 10 μg of the second fraction (PBS) extracted proteins were subjected to 12% SDS-PAGE. Gels were electrotransferred onto nitrocellulose membranes, which were subsequently blocked with 5% milk for 2 hours. Blocked membranes were incubated at 37°C for 90 minutes with anti-enamelin or anti-amelogenin polyclonal antibodies (1:500). The nitrocellulose membranes were washed and incubated for 45 minutes with a goat polyclonal anti–rabbit IgG antibody coupled to horseradish peroxidase (Sigma-Aldrich) diluted 1:2000. Immunoreactivity was visualized by chemiluminescence (ECL Western blotting detection system; Amersham Pharmacia Biotech, GE Healthcare Life Sciences, Velizy-Villacoublay, France) using a bioimager (ImageQuant LAS 4000; Uppsala, Sweden). Loading and electrotransfer efficiency were checked by staining membranes for total proteins with Ponceau red. Rat HAT-7 ameloblastic cells26Kawano S. Morotomi T. Toyono T. Nakamura N. Uchida T. Ohishi M. Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway.Connect Tissue Res. 2002; 43: 409-412Crossref PubMed Google Scholar were grown in Dulbecco’s modified Eagle’s medium/F-12 without phenol red supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 50 U/mL of penicillin-streptomycin. HAT-7 cells were transfected with plasmids that contained the promoter regions of interest or the plasmids alone according to the manufacturer instructions (Qiagen, Courtaboeuf, France). Forty-eight hours after BPA treatments (Sigma-Aldrich), 5 × 106 cells were either collected for RNA extraction or lyzed for protein content determination and promoter activity assays. Briefly, protein content was determined using a bicinchoninic acid protein assay kit (Pierce, Paris, France) according to the manufacturer’s instructions, and luciferase activity was measured by mixing 25 μL of protein extract with 75 μL of luciferase assay substrate (Promega Corp., Madison, WI). Luciferase activity was measured with the B941 TriStar microplate reader (Berthold, Bruyères, France). The experiment was repeated four times independently. Rat enamelin (−1468/+1 nt) and Klk4 (−1397/+1 nt) promoter regions were amplified using the Phusion high-fidelity Taq polymerase (ThermoScientific, Villebon-sur-Yvette, France) and the following primers: 5′-CCGGGTACCGGCTCACAGACTGAACCACC-3′ and 5′-TACACAGAACGAGGAACCGAGGAGCTCGCC-3′ for rat enamelin promoter and 5′-CCGGGTACCCTGAACTCCAGGGTCTCCCACTGG-3′ and 5′-CAGAAGTAAAGGTCCTCGGTTAGAGCTCGCC-3′ for rat Klk4 promoter. The purified fragments were cloned into PGL4.17 plasmid in front of luciferase reporter gene (Promega) by insertion in KpnI/XhoI (italicized) sites. Total RNA extraction was performed using Tri-Reagent (Euromedex) according to the manufacturer procedure. RNA concentration and purity were determined by a spectrophotometry at 260 nm (NanoDrop 1000; ThermoScientific). Reverse transcription was performed on 1 μg of total RNA for 45 minutes at 42°C, using a primer mix oligodT and random primers according to the manufacturer instructions (Superscript II; Invitrogen). Quantitative real-time PCR (qPCR) was performed using Opticon Monitor device (Bio-Rad Laboratories, Hercules, CA). Each PCR was repeated in triplicate independently, and the results were normalized against Gapdh and Rs15. Details of the primers and the corresponding amplicon sizes are presented in Table 1. Results were calculated by the method of standard curves. Similar data were obtained when ΔΔCt method was applied.Table 1Primer Sequences Used for Real-Time PCR AnalysiscDNAAmplicon size (pb)Primer sequencesRs153155′-GGCTTGTAGGTGATGGAGAA-3′5′-CTTCCGCAAGTTCACCTACC-3′Gapdh2605′-GACCCCTTCATTGACCTCAACTAC-3′5′-AAGTTGTCATGGATGACCTTGGCC-3′Amelogenin2715′-ACACCCTTCAGCCTCATCAC-3′5′-GAGAACAGTGGAGGCAGAGG-3′Enamelin4395′-CATGTGGCCTCCGCCAGTCC-3′5′-GTCATCTGGGGGCGGGTCCT-3′Ameloblastin2585′-TGCAGCCTCACCAGCCAGGA-3′5′-CCCGAGACAGCGAATGGGCG-3′Tuftelin2025′-CTCCCCTGTCCGCAGCAAGC-3′5′-GGCGTCCATGTGCTGCTGGT-3′Amelotin3795′-GCAACAAAACCGACTCCAG-3′5′-CTCCATTCTGCACATCTGG-3′Mmp203205′-CTGGGCCTGGGCCATTCCAC-3′5′-CTGGTGATGGTGCTGGGCCG-3′Klk43205′-GCATCCGCAGTGGGTGCTGT-3′5′-CACACTGCAGGAGGCTGGGC-3′ Open table in a new tab Data resulted from at least three independent experiments are presented as means ± SEM and were analyzed on GraphPad Prism software version 4.0 using the two-tailed nonparametric U-test. Values were considered significantly different at P < 0.05. Extracted human MIH teeth were compared with BPA-treated rat incisors (Figure 1), and both presented asymmetrical white spots that affected the enamel. At day 30, the lower incisors of 12 of 16 rats (75%) were affected after administration of BPA, whereas 100% of control rats were unaffected. Control mandibular incisor enamel was homogeneously yellow to orange, whereas incisors from BPA-treated rats had enamel white spots either symmetrically or asymmetrically affecting the incisors to varying degrees (Figure 1 and Table 2). The severity of the phenotype was scored using criteria based on various ind" @default.
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• W1991486688 date "2013-07-01" @default.
• W1991486688 modified "2023-09-30" @default.
• W1991486688 title "Enamel Defects Reflect Perinatal Exposure to Bisphenol A" @default.
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