Effects of bisphenol A on murine salivary glands and human tumor cell lines.

Bisphenol A (BPA) is an endocrine-disrupting chemical with a potential role in endocrine cancers. However, the effects of BPA on the salivary glands have been barely explored. We investigated the impact of in vivo sub-chronic exposure to BPA and its in vitro effects on human salivary gland mucoepidermoid carcinoma cell lines. Male and female mice were exposed to BPA (30 mg/kg/day). Sublingual and submandibular salivary glands from an estrogen-deficiency model were also analyzed. BPA concentration in salivary glands was evaluated by gas chromatography coupled to ion trap mass spectrometry. Immunohistochemical analysis using anti-p63 and anti-α-SMA antibodies was performed on mouse salivary gland tissues. Gene expression of estrogen receptors alpha and beta, P63 and α-SMA was quantified in mouse salivary gland and/or mucoepidermoid (UM-HMC-1 and UM-HMC-3A) cell lines. Cell viability, p63 and Ki-67 immunostaining were evaluated in vitro. BPA disrupted the tissue architecture of the submandibular and sublingual glands, particularly in female mice, and increased the expression of estrogen receptors and p63, effects that were accompanied by significant BPA accumulation in these tissues. Conversely, ovariectomy slightly impacted BPA-induced morphological changes. In vitro, BPA did not affect the proliferation of neoplastic cells, but augmented the expression of p63 and estrogen receptors. The present data highlight a potential harmful effect of BPA on salivary gland tissues, particularly in female mice, and salivary gland tumor cells. Our findings suggest that estrogen-dependent pathways may orchestrate the effects of BPA in salivary glands.

The Effects of NAM on the Symmetry of the Face and Maxillary Arch in Babies With Unilateral Cleft.

The study aimed to evaluate the effect of nasoalveolar molding (NAM) therapy through reverse engineering, or its absence, to obtain symmetry of the face and maxillary arch. Twenty-six babies with unilateral cleft lip and palate received treatment with NAM, and 12 babies with unilateral cleft lip and palate without presurgical orthopedics (control group). Patients were molded and photographed in 2-stages: the first month of life (T1/pre) and after the use of NAM/before the cheiloplasty (T2/post). In the digital models, the analyses performed were arch perimeter, arch length, and labial frenulum angle. The photographs allowed us to analyze nasal width, mouth width, columella angle, and nostril area. The results demonstrated that there was an increase in arch perimeter and arch length in control and NAM groups in the T2 period in comparison to T1. Labial frenulum angle was reduced in the NAM group compared to the NAM-T1 and control-T2 periods. Treatment with NAM yielded a reduction in nasal width in the period of T2 compared with T1. Columella angle was enhanced after NAM use in T2 and, was different from control group. The nostril area was reduced in the NAM group in T2 compared with control group. Nasoalveolar molding therapy reduced the labial frenulum angle, contributing to a reduction in the extension of the cleft. The NAM protocol improved facial symmetry, mainly through nasal effects, whereas the absence of orthopedic therapy yielded a commitment to the face and maxillary arch symmetry.

Nestin and Neuron-glial antigen 2 transgenes unveil progenitor units in murine salivary glands.

OBJECTIVE: Undifferentiated cells play pivotal roles in sustaining tissue homeostasis during physiological turnovers and after tissue impairment. Nestin and Neuron-glial antigen 2 (NG2) are markers frequently deployed to distinguish progenitor populations. In the salivary gland scenario, these markers remain largely unknown. Particularly for a double-labeled group of progenitor cells (NG2+Nestin+), their phenotype and distribution have never been explored in freshly isolated tissues. Herein, we analyzed a subset of plastic cells that express Nestin and NG2 near the ducts and in the periacinar region of the major salivary glands of murine samples. DESIGN: The major salivary glands tissues of Nestin-GFP/NG2-DsRed mice were analyzed under a fluorescence microscope. The cells marked by GFP and DsRed were counted in the merged image component of random representative images obtained for each gland sample at × 20 magnification. RESULTS: In the parotid, submandibular, and sublingual glands, the population of cells exclusively expressing Nestin was more abundant. There was a predominance of Nestin, NG2, and double-labeled cells in the submandibular gland compared to the parotid gland, mainly near the ductal system. Of note, the sublingual and parotid glands had similar populations of Nestin+ and NG2+, especially in acini, and some positive cells were observed surrounding ducts. CONCLUSIONS: Collectively, our study revealed differential expression patterns of Nestin and NG2, alone or in combination, in the salivary gland subset during homeostasis.

Effects of high-dose bisphenol A on the mouse oral mucosa: A possible link with oral cancers.

Bisphenol A (BPA) is an endocrine disrupting chemical able to promote hormone-responsive tumors. The major route of BPA contamination being oral, the aim of the present study was to investigate BPA effects on oral cells. Here, we evaluated the impact of sub-chronic in vivo exposure to BPA and its in vitro effects on neoplastic and non-neoplastic oral cells. We evaluated the oral mucosa of mice chronically exposed to BPA (200 mg/L). The response of keratinocytes (NOK-SI) and Head and Neck (HN) Squamous Cell Carcinoma (SCC), HN12 and HN13 cell lines to BPA was examined. In vivo, BPA accumulated in oral tissues and caused an increase in epithelial proliferative activity. BPA disrupted the function of keratinocytes by altering pro-survival and proliferative pathways and the secretion of cytokines and growth factors. In tumor cells, BPA induced proliferative, invasive, pro-angiogenic, and epigenetic paths. Our data highlight the harmful effects of BPA on oral mucosa and, tumorigenic and non-tumorigenic cells. Additionally, BPA may be a modifier of oral cancer cell behavior by prompting a functional shift to a more aggressive phenotype.

Saliva biomonitoring using LPME-GC/MS method to assess dentistry exposure to plasticizers.

Bisphenol A and phthalates are endocrine disruptors widely used as chemical additives mainly in plastic products, including materials for dentistry procedures. Besides, many plasticizers have been associated with important diseases requiring performed methods for their quantification. In the present study, an alternative method for the determination of bisphenol A (BPA) and phthalate metabolites in saliva was developed and validated using hollow fiber liquid phase microextraction (HF-LPME) for sample preparation and gas chromatography coupled to ion trap mass spectrometry (GC/MS) for analysis. A mixture of octanol and ethyl octanoate (1:1 v/v) was used as an acceptor phase in hollow fiber to extract the analytes from saliva samples. A Doehlert design was performed to optimize the variable sample agitation and extraction time. The HF-LPME-GC/MS method developed for saliva analysis showed good selectivity, linearity (R2 > 0.900), and precision (CV = 0.86-18.68%). Limits of detection and quantification ranged from 0.03 to 0.53 µg L-1 and 0.09 to 1.78 µg L-1, respectively. A high concentration of BPA in the oral cavity and oropharyngeal space is a warning of the possible association with the main cancer of the mouth. The method developed and validated was applied to patients with oral squamous cell carcinoma (study group, n = 16) and patients who did not present any oral lesion (control group, n = 16). A principal component analysis was performed and showed a tendency for the association between oral squamous cell carcinoma (OSCC) and plasticizers. Graphical abstract.

Avaliação dos efeitos do Bisfenol A em queratinócitos e células de neoplasia de cavidade oral / Evaluation of bisphenol A effects in keratinocytes and oral cavity neoplasia cells

O Bisfenol A (BPA) é um monômero utilizado na produção de garrafas plásticas, embalagens alimentícias, resinas odontológicas e vários outros materiais. Este monômero age como um desregulador do sistema endócrino e seus efeitos estão associados a cânceres em diferentes órgão e tecidos, como mama, próstata e tireóide. O BPA já foi detectado em fluidos humanos, incluindo saliva, mas os seus efeitos na mucosa bucal e em células orais neoplásicas não foram investigados. Os objetivos do presente trabalho foram 1) verificar os efeitos da exposição crônica ao BPA em glândulas salivares e mucosa bucal in vivo e 2) avaliar os efeitos do BPA in vitro em células de neoplasias bucais e queratinócitos; Para atender ao objetivo 1, camundongos machos e fêmeas receberam BPA (200 mg/mL) na água de beber durante 6 semanas. As mucosas orais (palato, língua e mucosa jugal) e as glândulas submandibulares foram avaliadas. Para atender ao objetivo 2, a resposta ao BPA foi examinada nas linhagens NOK-SI (queratinócito), HN12, HN13 (CCE de cavidade oral), UM HMC1 e UM HMC3a (neoplasia de glândula salivar). Os seguintes parâmetros foram avaliados: viabilidade, proliferação, invasão, angiogênese, produção de citocinas e fatores de crescimento e possíveis mecanismos de ação do BPA. Resultados. A exposição de camundongos ao BPA resultou em alterações microscópicas caracterizadas pelo aumento da espessura do epitélio da mucosa oral (palato, língua e mucosa jugal) e uma redução no número de ácinos das glândulas submandibulares. Foi observado também um acúmulo de BPA nos tecidos orais. In vitro, nas linhagens de CCE, o BPA aumentou a proliferação, invasão celular e os níveis da proteína vimentina, e ainda induziu a secreção de citocinas e fatores de crescimento, e a acetilação de histonas H3. Em queratinócitos orais, o BPA aumentou a proliferação celular e induziu a secreção de fatores de crescimento e a expressão de receptores de estrógeno (ER) α e ß. Os efeitos do BPA foram revertidos na presença do antagonista puro do ER. Nas linhagens de neoplasias de glândula o BPA não alterou a proliferação e induziu a expressão de p63. Em conclusão, o BPA induz alterações morfológicas nos tecidos bucais e alterações moleculares nos queratinócitos e nas células de CCE de cavidade oral. Os mecanismos pelos quais o BPA induz estas alterações são dependentes da interação BPA-ER e da acetilação de histonas.

Bisphenol A (BPA) is a monomer used to product plastic bottles, food packaging, inner coating of food cans, thermal papers, medical devices, dental resins and various other materials. Due to its chemical structure, this monomer acts as a deregulator of the endocrine system and its effects are associated with cancers in different organs and tissues such as breast, endometrium, ovary, prostate, testis and thyroid. BPA has been detected in several human fluids, including saliva, however its effects on the normal oral mucosa and neoplastic oral cells have not been investigated yet. Thus, the objectives of the present study were 1) to verify the effects of chronic exposure to BPA in salivary glands and oral mucosa in vivo and 2) to evaluate the effects of BPA in vitro on oral tumor cells and keratinocytes. To meet objective 1, male and female mice received BPA (200 mg / mL) in drinking water for 6 weeks. The oral mucosa (palate, tongue and buccal mucosa) and submandibular salivary glands were evaluated microscopically. To meet objective 2, the response to BPA was examined in immortalized cell lines NOK SI (keratinocyte); HN12, HN13 (OSCC), UM HMC1 and UM HMC3a (salivary gland tumor). The following parameters were evaluated: viability, proliferation, invasion, angiogenesis, cytokine and growth factors production. Results. Exposure of mice to BPA resulted in microscopic changes characterized by increased thickness of the oral mucosa epithelium (palate, tongue and buccal mucosa) and a reduction in the number of submandibular salivary glands acini. There was also an accumulation of BPA in the oral tissues. In vitro, in OSCC cells, BPA increased cell proliferation and invasion, vimentin expression, induced secretion of cytokines and growth factors, and induced histone H3 acetylation. In oral keratinocytes, BPA increased cell proliferation and induced secretion of growth factors and estrogen receptor (ER) α and ß expression. The effects of BPA were reversed in the presence of the pure ER antagonist. In salivary gland tumor cell lines, BPA did not alter the proliferation and induced the expression of p63. BPA mechanism of action involves its interaction with ER, since the effects were reverted in the presence of pure receptor antagonist. In conclusion, BPA induces morphological changes in oral tissues and molecular changes in keratinocytes and OSCC cells. The mechanisms which BPA induces these changes are dependent to the BPA-ER interaction and histone acetylation.

Intraosseous xanthoma of the mandible: A multi-institutional case series with a literature review.

BACKGROUND: The aim of the present study was to report the clinicopathologic, radiographic and immunohistochemical features of five South American cases of intraosseous xanthomas of the mandible and to compare them to those detected in a literature review. METHODS: Clinical data were collected from the records of three Oral and Maxillofacial Pathology services in South America and compared with those compiled from a literature review based on a search of three electronic databases (PubMed, Web of Science and Scopus). All cases were evaluated by haematoxylin and eosin staining and immunohistochemistry for CD68 and S-100. RESULTS: The series comprised four females (80%) and one male (20%) with a mean age of 23.3 ± 10.9 years (range: 13-45 years). In four cases, there was involvement of the posterior region of the mandible (80%). The lesions presented radiographically as unilocular (60%) radiolucencies with punched-out margin (80%). All cases predominantly consisted of CD68-positive and S-100-negative xanthomatous cells. No recurrences were observed after curettage, with a median follow-up of 27 months. CONCLUSION: Intraosseous xanthoma of the jaws is a rare benign disorder. We report here five additional cases affecting the mandible, for a total of 36 cases of the jaws reported in the literature. Overall, this lesion has predilection for posterior sites of the mandible of asymptomatic young adults.

Multiple Desmoplastic Cellular Neurothekeomas in Child: Report of the First Oral Case and Review of the Literature.

Cellular neurothekeomas (CNs) are distinctive benign tumors of uncertain histogenesis, with predilection for the skin of the head and neck region. We describe the first case of multiple desmoplastic CNs (DCNs) affecting the oral cavity in a 9-year-old girl. Histopathologic evaluation showed a proliferation of spindle and epithelioid cells, forming nests and bundles, supported by exuberant fibrous stroma, as well as scattered multinucleated floret-like giant cells. The tumor cells were immunopositive for vimentin, CD63, CD56, whereas AE1/AE3, S100, CD34, α-SMA, GFAP, EMA, CD57 and NSE were negative. Ki-67 was <2%. Multiple DCNs should be considered in the differential diagnosis of oral nodular lesions.

Exclusive Primary Lesion of Oral Leishmaniasis with Immunohistochemical Diagnosis.

A case of oral leishmaniasis without cutaneous involvement affecting the upper alveolar ridge mucosa/gingiva and the hard palate is reported in a 41-year-old Brazilian man. Microscopic examination disclosed scarce amastigotes and the definitive diagnosis was facilitated by immunohistochemical analysis. The clinical presentation of this lesion is unusual and underlies the importance of considering leishmaniasis in the differential diagnosis of oral lesions, especially in endemic areas. A literature review of the cases of mucosal leishmaniasis with exclusive primary lesions of the oral mucosa was also performed.

The in vitro exposure to cypermethrin does not inhibit the proliferative response of peripheral blood mononuclear cells.

This study evaluated the effect of in vitro exposure to cypermethrin on peripheral blood mononuclear cells proliferative response, considering reduced peripheral blood mononuclear cells proliferative response observed in individuals occupationally exposed to pyrethroids. Peripheral blood mononuclear cells were obtained from 21 healthy subjects (28.0 ± 9.0 years old). The effect of cypermethrin (at 0.5, 1.0 and 5.0 mg/ml) on cell viability was evaluated by flow cytometry using an apoptosis detection kit. Cell proliferation (PI) was evaluated by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence decay using flow cytometry. Cells labeled with CFSE were exposed, in vitro, to cypermethrin (0.5, 1.0, 2.0, 2.5 and 4 µg/ml) and stimulated with phytohemagglutinin (PHA 1.0 or 5.0 µg/ml) for 5 d (37 °C, 5% CO2). The in vitro treatment of peripheral blood mononuclear cells with cypermethrin did not induce apoptosis or necrosis after 5 d in culture. Stimulation by PHA induced cell proliferation (PI = 1.29 ± 1.09 and 2.01 ± 0.62, PHA at 1.0 and 5.0 µg/ml, respectively, mean ± SD) and in vitro exposure to cypermethrin did not alter cellular proliferative response to PHA (PI = 1.80 ± 0.50, 2.60 ± 0.05 and 2.10 ± 1.20 for cypermethrin at 1.0, 2.0 and 4.0 µg/ml, respectively, and PHA at 5.0 µg/ml). In vitro treatment of peripheral blood mononuclear cells with cypermethrin, at the doses tested, does not affect cell viability or proliferation. These findings suggest that the reduction of proliferation observed on lymphocytes derived from individuals occupationally exposed to pesticides may be related to other mechanisms than direct action of cypermethrin on lymphocytes.