The correlation between micronuclei count and smoking behavior: a hospital-based study / Correlación entre recuento de micronúcleos y hábito tabáquico: un estudio hospitalario

Background: Micronucleus is a microscopically visible cyto-plasmic chromatin mass in the extranuclear vicinity, originating from aberrant mitosis, which consists of eccentric chromosomes that have failed to reach spindle poles during mitosis. The present study was designed to evaluate and compare cytogenetic changes in the buccal mucosa of smokers and non-smokers based on the occurrence of micronuclei. The study aimed to determine the correlation between the micronuclei count and the frequency and duration of smoking habit. Materials and Methods: Two groups (smokers and non-smokers) of 34 individuals each were examined. Cytological buccal smears were taken from participants using a moistened wooden spatula and stained with standard Papanicolaou stain. Presence of micronuclei was assessed at 40X magnification using a light microscope and a count per 500 cells was determined. The results of the study were analyzed statistically using Mann-Whitney U test, Spearman's rank correlation coefficient and Student t-test. Result: Smears from smokers showed a significant increase in the total number of micronuclei per 500 cell count compared to non-smokers. There was a strong positive correlation between the occurrence of micronuclei and the frequency and duration of smoking. A age-related increase in older age groups was also observed. Conclusion: The study reveals a strong positive correlation between the occurrence of micronuclei and the frequency and duration of smoking. This observation is vital in the utilization of the micronuclei detection in smears as a prognostic, educational and interventional tool in the management of patients with smoking habits.

Antecedentes: El micronúcleo es una masa de cromatina citoplasmática microscópicamente visible en el área extranuclear, que se origina a partir de la mitosis aberrante, y que consiste en cromosomas excéntricos que no han podido alcanzar los polos del huso durante la mitosis. El presente estudio fue diseñado para evaluar y comparar los cambios citogenéticos en la mucosa bucal de fumadores y no fumadores en función de la aparición de micronúcleos. El estudio tuvo como objetivo determinar la correlación entre el recuento de micronúcleos y la frecuencia y duración del hábito de fumar. Materiales and Métodos: Se examinaron dos grupos (fumadores y no fumadores) de 34 individuos cada uno. Se tomaron frotis bucales citológicos de todos los participantes con una espátula de madera humedecida y se tiñeron con la tinción estándar de Papanicolaou. La presencia de micronúcleos se evaluó al microscopio óptico con un aumento de 40X y se determinó un recuento por 500 células. Los resultados del estudio se analizaron estadísticamente utilizando la prueba U de Mann-Whitney, el coeficiente de correlación de rango de Spearman y la prueba t de Student. Resultados: Los frotis de fumadores mostraron un aumento significativo en el número total de micronúcleos por 500 células en comparación con los no fumadores. Hubo una fuerte correlación positiva entre la aparición de micronúcleos y la frecuencia y duración del tabaquismo. También se observó un aumento relacionado con la edad en los grupos de mayor edad. Conclusión: el estudio revela una fuerte correlación positiva entre la aparición de micronúcleos y la frecuencia y duración del tabaquismo. Esta observación es vital en la utilización de la detección de micronúcleos en frotis como una herramienta pronostica, educativa e intervencionista en el manejo de pacientes con hábitos de fumar.

Phenotype changes of oral epithelial stem cells after in vitro culture

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Genotoxicidade e citotoxicidade dos raios x em crianças submetidas à radiografia panorâmica / Genotoxicity and cytotoxicity of x-rays in children exposed to panoramic radiography

RESUMO Objetivo: Analisar a genotoxicidade e a citotoxicidade produzidas por raios X no epitélio da mucosa oral de crianças durante a obtenção da radiografia panorâmica. Métodos: A amostra foi constituída por 30 crianças saudáveis, sendo 19 do sexo feminino e 11 do masculino, com faixa etária de 4 a 10 anos (média de 7 anos de idade). As células epiteliais da mucosa oral foram coletadas por meio de citologia esfoliativa em base líquida imediatamente antes e após sete dias da obtenção da radiografia panorâmica. Os esfregaços foram processados e corados utilizando a técnica de Feulgen Rossenbeck modificada. Foram analisadas e quantificadas projeções nucleares dos tipos bud e broken egg, alterações genotóxicas na forma de micronúcleo e alterações citotóxicas dos tipos picnose, cariólise e cariorrexe. Resultados: A frequência de picnose, bud e broken egg foi significativamente maior após a exposição aos raios X (p<0,05), porém não houve diferença estatisticamente significante em relação ao sexo, bem como nas demais alterações estudadas. Conclusões: A exposição aos raios X emitidos durante a obtenção da radiografia panorâmica pode induzir à morte celular no epitélio da mucosa oral de crianças. Não foi encontrado indício significativo de efeito genotóxico.

ABSTRACT Objective: To assess the genotoxicity and cytotoxicity produced by X-rays in the epithelium of the oral mucosa of infants exposed to panoramic radiography. Methods: The sample consisted of 30 healthy children, 19 females and 11 males, ranging in age from 4 to 10 years (average of 7 years of age). Oral mucosa cells were collected by liquid-based cytology immediately before and after seven days following the exposure to panoramic radiography. Smears were processed and stained using the modified Feulgen Rossenbeck technique. Bud and broken egg nuclear projections, changes in the form of micronuclei, and genotoxic and cytotoxic changes of pyknosis, karyorrhexis and karyolysis were analyzed and quantified. Results: The frequency of pyknosis, buds and broken eggs was significantly higher after exposure to X-rays (p<0.05), but there was no statistically significant difference regarding gender, as well as in the other changes studied. Conclusions: Exposure to X-rays emitted during submission to panoramic radiography may induce cell death in the epithelium of children's oral mucosa. No evidence was found for a significant genotoxic effect.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

Aloinjertos análogos de mucosa bucal en ratas no consanguíneas / Oral mucosa analog allografts in non-consanguineous rats

Resumen Introducción: A pesar de que existen opciones terapéuticas para el tratamiento de defectos de la mucosa bucal, persiste la necesidad de encontrar sustitutos funcionales, anatómicos y estéticamente similares al tejido que se va a reemplazar, así como soluciones que reduzcan la morbilidad de los injertos autólogos. Objetivo: Determinar la compatibilidad clínica e histológica de aloinjertos equivalentes de mucosa bucal elaborados mediante ingeniería tisular en ratas no consanguíneas. Materiales y métodos: Se utilizó una muestra de mucosa bucal de ratas Sprague Dawley para la obtención de un cultivo de fibroblastos y otro de queratinocitos y fibroblastos. En ambos casos, se usó una membrana de colágeno comercial como soporte. Después de diez semanas de cultivo, las membranas resultantes se injertaron en cuatro ratas Wistar. La primera fase del estudio consistió en la elaboración de los tejidos análogos de mucosa bucal mediante ingeniería tisular, los cuales se implantaron en ratas Wistar inmunocompetentes; posteriormente, se evaluaron las características clínicas e histológicas del aloinjerto. Resultados: La evaluación in vivo de los tejidos análogos demostró que se habían integrado correctamente en los huéspedes inmunocompetentes, y se había logrado el aumento del biotipo periodontal y la creación de una zona con mayor queratinización. Desde el punto de vista histológico, el tejido adquirió características similares a las de la muestra de mucosa bucal de control, sin ningún tipo de reacción inflamatoria ni signos clínicos o histológicos de rechazo. Conclusión: Hubo compatibilidad clínica e histológica de los aloinjertos equivalentes de mucosa bucal obtenidos mediante ingeniería tisular.

Abstract Introduction: Although there are therapeutic options for the treatment of oral mucosa defects, the need for functional, anatomical and aesthetically similar substitutes persists, as well as for solutions to reduce autologous grafts morbidity. Objective: To determine clinical and histological compatibility of equivalent oral mucosa allografts generated through tissue engineering in non-consanguineous rats. Materials and methods: We used a sample of oral mucosa from Sprague Dawley rats to obtain a fibroblast culture and a keratinocytes and fibroblasts co-culture. In both cases, we used a commercial collagen membrane as "scaffold". After ten weeks of culture, we grafted the resulting membranes into four Wistar rats. The first phase of the study was the development of the oral mucosa equivalents generated by tissue engineering. Then, we implanted them in immunocompetent Wistar rats, and finally we evaluated the clinical and histological features of the allografts. Results: In vivo evaluation of mucosal substitutes showed a correct integration of artificial oral mucosa in immunocompetent hosts, with an increase in periodontal biotype and the creation of a zone with increased keratinization. Histologically, the tissue was similar to the control oral mucosa sample with no inflammatory reaction nor clinical or histological rejection signs. Conclusion: The equivalent oral mucosa allografts generated by tissue engineering showed clinical and histological compatibility.

Effect of different presentations of resveratrol on cell proliferation and epitelial thickness of the oral mucosa of wistar rats

Introduction: Grape is one of the most important fruit crops across the world and can be consumed in different ways. There has been a growing interest in the role of antioxidants such as resveratrol, which can be found in grape skin, in oral and dental tissues. Thus, the objective of this study was to analyze the effect of different presentations of resveratrol on cell proliferation and epithelial thickness of the oral mucosa of Wistar rats. Methods: Fifty male Wistar rats were randomly divided into five groups: water/control, red wine, grape juice, 12% alcoholic solution/ethanol and aqueous solution of resveratrol. Samples of palatal and tongue mucosa were collected for a histomorphometric analysis using hematoxylin-eosin staining and the argyrophilic nucleolar organizer region (AgNOR) technique for quantification of cell proliferation. Results: As to epithelial thickness, both the tongue and the palate showed a statistically significant difference between the control group and the other groups, with greater decrease in the resveratrol and the wine groups. In the suprabasal layer of both the tongue and the palate epithelium, red wine reduced the rate of cell proliferation, while ethanol increased it. In the basal layer of the tongue epithelium, there was a statistically significant difference between the control, the grape juice and the resveratrol groups and the ethanol group, with increased cell proliferation in the ethanol group. Conclusions: Wine does not interfere in the physiological renewal of the basal layer of the buccal epithelium and exerts a protective action by reducing the cell proliferation rate of the suprabasal layer (AU)

Cell phone radiation effects on cytogenetic abnormalities of oral mucosal cells

The aim of this study was to evaluate the effects of exposure to cell phone electromagnetic radiation on the frequency of micronuclei, broken eggs cells, binucleated cells, and karyorrhexis in epithelial cells of the oral mucosa. The sample was composed of 60 cell phone users, who were non-smokers and non-drinkers, and had no clinically visible oral lesions. Cells were obtained from anatomical sites with the highest incidence of oral cancer: lower lip, border of the tongue, and floor of the mouth. The Feulgen reaction was used for quantification of nuclear anomalies in 1,000 cells/slide. A slightly increase in the number of micronucleated cells in the lower lip and in binucleated cells on the floor of the mouth was observed in individuals who used their phones > 60 minutes/week. The analysis also revealed an increased number of broken eggs in the tongue of individuals owning a cell phone for over eight years. Results suggest that exposure to electromagnetic waves emitted by cell phones can increase nuclear abnormalities in individuals who use a cell phone for more than 60 minutes per week and for over eight years. Based on the present findings, we suggest that exposure to electromagnetic radiation emitted by cell phones may interfere with the development of metanuclear anomalies. Therefore, it is demonstrated that, despite a significant increase in these anomalies, the radiation emitted by cell phones among frequent users is within acceptable physiological limits.

Utilidad de la prueba de micronúcleos y anormalidades nucleares en células exfoliadas de mucosa oral en la evaluación de daño genotóxico y citotóxico / Utility micronucleus test and nuclear abnormalities in exfoliated cells of oral mucosa in the evaluation of genotoxic and cytotoxic damage

Los micronúcleos son fragmentos o cromosomas completos que quedan fuera del núcleo durante la mitosis; mediante su estudio se pueden evaluar los efectos de genotóxicos ambientales y ocupacionales. Esta prueba es ampliamente utilizada y es una alternativa eficaz, sencilla y económica para detectar la perdida de material genético. Por otra parte, la cavidad oral puede reflejar el estado de salud de los individuos, debido a que la mucosa que la recubre, puede presentar evidencias a nivel microscópico como macroscópico de cambios indicativos de enfermedad local sistémica o por exposición a sustancias tóxicas así como efectos secundarios por tratamientos. Dichas ventajas, favorecen su utilización en pruebas para evaluar genotóxicos o citotóxicos. La mucosa es una barrera protectora del resto del organismo, es un punto de contacto de agentes potencialmente peligrosos; por tanto, se torna susceptible de sufrir daños. El epitelio de revestimiento oral (60 por ciento) es estratificado no queratinizado formado por células con abundante citoplasma, permite la penetración de colorantes y facilita la observación e identificación de características morfológicas del núcleo y membrana celular. Además la mucosa tiene elevada capacidad proliferativa y aunque esta particularidad mantiene la población celular constante, por otro lado, se vuelve más vulnerable a daño al ADN. Esto cobra relevancia ya que el 90 por ciento del cáncer tienen origen epitelial, así que la mucosa oral es usada para monitorear eventos genotóxicos tempranos causados por cancerígenos inhalados o ingeridos. Este epitelio es de fácil acceso, poco invasivo, por lo que al tomar la muestra a los individuos, se les genera mínimo estrés. Por todo lo anterior, el epitelio oral es un tejido ideal para aplicar la técnica de micronúcleos y detección de anormalidades nucleares sin necesidad de cultivos celulares, lo que representa una oportunidad para realizar estudios epidemiológicos en poblaciones de alto riesgo.

Micronucleus are fragments or whole chromosomes that are outside the nucleus during mitosis. Through this study we can evaluate the environmental and occupational the genotoxic effects. This test is widely used because it is a very effective alternative, it is a simple, fast and inexpensive way to detect the loss of genetic material. Meanwhile a healthy oral cavity is evidenced because in the overlying mucosa changes indicative of local or systemic disease, toxic exposure and side effects of treatments can be observed. This favors their use in tests to assess the presence of genotoxins or cytotoxins. Although protective barrier from the rest of the body is the point of contact of potentially dangerous agents thus becoming susceptible to damage. Coating and oral epithelium (60 percent) are formed by stratified non-keratinized cells with abundant cytoplasm, allowing the absorption of dyes and facilitating microscopic observation and identification of nucleus and membrane morphological characteristics. It has a particularly proliferative capacity, and even though this particularity maintains constant cell population, on the other hand, becomes more vulnerable to DNA damage. This information is relevant as 90 percent of all cancers are of epithelial origin. Therefore, the oral mucosa is used to monitor early events caused by inhaled or ingested genotoxic carcinogens. Epithelium is easily accessible and minimally invasive, thereby generating less stress when samples are obtained from study participants. In view of the above, oral epithelium tissue is ideal for implementing micronucleus assay and for the detection of nuclear abnormalities without the need for cell cultures, which presents a unique opportunity for epidemiological studies in high-risk populations.

Genetics analysis with down syndrome and histopathological examination of buccal epithelial cells / Análisis genético y examen histopatológico de las células epiteliales bucales en pacientes con síndrome de down

Down syndrome is primarily caused by trisomy of chromosome 21. We reviewed cytogenetic studies performed on 1048 patients who were referred to the Cytogenetics Unit at Dicle University Hospital, Diyarbakir, Southeast Turkey, between 2000 and 2009. The cases were grouped according to the reason of referral for cytogenetic analysis. The highest frequencies of abnormal karyotypes were found among cases that were referred due to suspicion of Down syndrome (84.8 percent). For histologic examination to persons with Down syndrome and normal, buccal mucosa smear was prepared by rubbing. Down syndrome are disabled and control groups were compared statistically buccal epithelial cells and nuclei (p<0.05). Periphery of the nucleus in some patients with Down's syndrome, while the bud structures in the form of micronuclei was observed in the karyolytic cells.

El síndrome de Down es causado principalmente por la trisomía del cromosoma 21. Se revisaron los estudios citogenéticos realizados en 1.048 pacientes que fueron remitidos a la Unidad de Citogenética del Dicle University Hospital, Diyarbakir, sudeste de Turquía, entre los años 2000 y 2009. Los casos se agruparon de acuerdo a la razón de referencia para el análisis citogenético. Las frecuencias más altas de cariotipos anormales se encontraron ent los casos que fueron remitidos por sospecha de síndrome de Down (84,8 por ciento). Para el estudio histológico de las personas con y sin síndrome de Down, se realizó el frotis de mucosa oral por hisopado. Los grupos con síndrome de Down y de control (sin síndrome) se compararon estadísticamente en relación a las células epiteliales orales y los núcleos (p <0,05). Se observaron núcleos periféricos en algunos pacientes con síndrome de Down, mientras que estructuras de tipo brotes en la forma de micronúcleos se observaron en las células cariolíticas.

Isolamento, caracterização e análise da resposta à injúria de células tronco epiteliais da mucosa bucal humana / Isolation, characterization and response to damage of humam epithelial stem cells from human oral mucosa

Avanços na identificação e caracterização de diferentes populações de células tronco tem melhorado as perspectivas do seu uso clínico. A maioria dos estudos obtêm as células-tronco com base na expressão de marcadores de superfície celular e testa as suas propriedades por meio de ensaios funcionais in vitro e in vivo. Na presente pesquisa foram isoladas diferentes populações de células epiteliais com base na expressão de dois marcadores de células-tronco epiteliais descritos pela literatura, o receptor de neurotrofina p75 (p75NTR) e o receptor de transferrina 1 (CD71). Uma vez isso feito, foi avaliado a co-expressão de outros marcadores de células-tronco epiteliais, como as integrinas 6 e 1, e realizado ensaios de eficiência de formação de colônias, potencial de crescimento celular, capacidade de reconstrução epitelial in vitro, análise da resposta ao trauma e avaliação da capacidade de auto renovação. Os resultados mostram que as células p75NTR+ tem um desempenho funcional melhor do que as p75NTR- na maioria dos ensaios funcionais realizados, exceto pela capacidade de responder ao trauma e de se autorenovar. Ademais, comprovou-se que células p75NTR+ expressam em maior proporção as integrinas 6 e 1 e que o isolamento de células por dupla marcação (p75NTR+CD71-) possibilita a obtenção de uma população de células epiteliais ainda mais enriquecida com estes marcadores. No entanto, foi observado também que independentemente da população celular estudada e do tempo em cultivo, as populações de células epiteliais alteraram drasticamente o seu fenótipo quando de tal forma que as populações celulares inicialmente positivas se tornaram majoritariamente negativas e que as populações celulares inicialmente negativas passaram a ter células positivas em seu meio.

Recent advances in cell sorting and characterization technics of stem cells have provided new insights and perspectives for clinical applications of this particular cell population. Most of the studies isolat stem cells based on the expression of cell surface markers and evaluate their stem cells-like proprieties by performing in vitro and in vivo assays. Here we isolated different populations of keratinocytes based on the expression of the epithelial stem cell markers neurotrophin receptor p75 (p75NTR) and transferrin receptor 1 (CD71). The co-expression of other epithelial stem markers such as integrins 6 and 1 was also assessed and in vitro functional assays such as colony-forming efficiency; long-term growth potential, in vitro epithelial reconstruction-capacity and response to damage and self-renew capacity. Our results showed that p75NTR+ve cells have better functional proprieties in most of the assays however they did not respond to damage nether presented self-renew capacity. Additionally, p75NTR-ve cells express higher percentage of other epithelial stem cell markers such as integrins 6 and 1 when compared to p75NTR-ve cells and the double staining (p75NTR+veCD71-ve) contributes to isolate a more enriched cell population based on the expression of the integrins. Nevertheless, independently of the cell population or of the amount of time in culture, cells have changed dramatically their phenotype in a way that the cell population initially p75NTR+ve lost the expression of the p75NTR and the negative population have gained it.

Frequência de micronúcleos em células esfoliadas da mucosa bucal de pacientes obesos / Micronuclei frequency in exfoliated buccal cells of obese patients

A crescente incidência da obesidade e suas comorbidades constituem-se em um grande desafio para a saúde no mundo. Além da doença cardiovascular e do diabetes, dados epidemiológicos demonstraram ligação entre a obesidade e diversos tipos de câncer. As alterações citogenéticas tem sido utilizadas como biomarcadores para identificação de danos celulares. Este estudo teve como objetivos: comparar a frequência dos tipos celulares (células basais e diferenciadas), anormalidades nucleares (células binucleadas, picnóticas, cariorréticas, cariolíticas e com cromatina condensada) e de danos celulares (micronúcleos e brotos nucleares) de células bucais esfoliadas em um grupo de obesos (teste) e em um grupo de indivíduos com peso normal (controle). A amostra foi composta por 30 indivíduos, sendo o grupo teste constituído por indivíduos obesos mórbidos (n=15) e o grupo controle por indivíduos com peso normal (n=15). A classificação do peso corporal foi feita de acordo com o Índice de Massa Corporal (IMC); um questionário forneceu informações sobre exposições ocupacionais e não ocupacionais; hábitos e dieta. As células da mucosa bucal foram coletadas da mucosa jugal, de ambos os lados, processadas e analisadas microscopicamente. Para cada indivíduo foram avaliadas 1000 células para a caracterização dos tipos celulares (basal e diferenciada) e alterações nucleares (células binucleadas, picnose, cariólise, cariorrexe, cromatina condensada) e 2000 células diferenciadas para verificar a presença de danos celulares (micronúcleos e brotos nucleares). Os dados foram processados e analisados estatisticamente, por meio do teste de Mann Whitney. Considerou-se o nível de significância de 5% (p<0,05). Observaram-se diferenças nas frequências de tipos celulares e anomalias nucleares para os dois grupos, porém estas diferenças não foram significativas (p>0,05). Quanto ao tipo de dano celular, notou-se a mesma frequência de brotos nucleares para ambos os grupos, entretanto...

The increasing incidence of obesity and its co-morbid conditions poses a great challenge to global health. In addition to cardiovascular disease and diabetes, epidemiological data demonstrate a link between obesity and multiple types of cancer. The buccal micronucleus cytome assay has been used as a biomarker for identification of cell damage. This study aimed to compare the frequency of cell types (basal and differentiated), nuclear anomalies (binucleated, pyknotic, karyorrhectic, karyolitic and condensed chromatin cells), and cell damage (micronuclei and nuclear buds) in exfoliated buccal cells in a group of obese (test) and a group of normal weight (control).The sample consisted of 30 subjects, the test group comprised of individuals with morbid obesity (n = 15) and a control group of normal weight (n = 15). The classification of body weight was made according to Body Mass Index (BMI), a questionnaire provided information on occupational exposures and non-occupational, lifestyle and diet. The oral mucosa cells were collected from the buccal mucosa, on both sides, processed and analyzed microscopically. For each individual 1000 cells were evaluated for the characterization of cell types (basal and differentiated) and nuclear abnormalities (binucleated cells, pyknosis, karyolysis, karyorrhexis, condensed chromatin) and 2000 differentiated cells for the presence of cellular damage (micronuclei and nuclear buds). The data were processed and statistically analyzed using the Mann-Whitney test. We considered the significance level of 5% (p <0.05). Differences were observed in the frequencies of cell types and nuclear abnormalities in both groups, but these differences were not significant (p> 0.05). Regarding the type of cell damage, we noticed the same frequency of nuclear buds for both groups, however, the frequency of micronuclei was higher in the obese group (p <0.001). In this study, there was a higher frequency of micronuclei in...

Frequência de micronúcleos em células esfoliadas da mucosa bucal de pacientes obesos / Micronuclei frequency in exfoliated buccal cells of obese patients

A crescente incidência da obesidade e suas comorbidades constituem-se em um grande desafio para a saúde no mundo. Além da doença cardiovascular e do diabetes, dados epidemiológicos demonstraram ligação entre a obesidade e diversos tipos de câncer. As alterações citogenéticas tem sido utilizadas como biomarcadores para identificação de danos celulares. Este estudo teve como objetivos: comparar a frequência dos tipos celulares (células basais e diferenciadas), anormalidades nucleares (células binucleadas, picnóticas, cariorréticas, cariolíticas e com cromatina condensada) e de danos celulares (micronúcleos e brotos nucleares) de células bucais esfoliadas em um grupo de obesos (teste) e em um grupo de indivíduos com peso normal (controle). A amostra foi composta por 30 indivíduos, sendo o grupo teste constituído por indivíduos obesos mórbidos (n=15) e o grupo controle por indivíduos com peso normal (n=15). A classificação do peso corporal foi feita de acordo com o Índice de Massa Corporal (IMC); um questionário forneceu informações sobre exposições ocupacionais e não ocupacionais; hábitos e dieta. As células da mucosa bucal foram coletadas da mucosa jugal, de ambos os lados, processadas e analisadas microscopicamente. Para cada indivíduo foram avaliadas 1000 células para a caracterização dos tipos celulares (basal e diferenciada) e alterações nucleares (células binucleadas, picnose, cariólise, cariorrexe, cromatina condensada) e 2000 células diferenciadas para verificar a presença de danos celulares (micronúcleos e brotos nucleares). Os dados foram processados e analisados estatisticamente, por meio do teste de Mann Whitney. Considerou-se o nível de significância de 5% (p<0,05). Observaram-se diferenças nas frequências de tipos celulares e anomalias nucleares para os dois grupos, porém estas diferenças não foram significativas (p>0,05). Quanto ao tipo de dano celular, notou-se a mesma frequência de brotos nucleares para ambos os grupos, entretanto...

The increasing incidence of obesity and its co-morbid conditions poses a great challenge to global health. In addition to cardiovascular disease and diabetes, epidemiological data demonstrate a link between obesity and multiple types of cancer. The buccal micronucleus cytome assay has been used as a biomarker for identification of cell damage. This study aimed to compare the frequency of cell types (basal and differentiated), nuclear anomalies (binucleated, pyknotic, karyorrhectic, karyolitic and condensed chromatin cells), and cell damage (micronuclei and nuclear buds) in exfoliated buccal cells in a group of obese (test) and a group of normal weight (control).The sample consisted of 30 subjects, the test group comprised of individuals with morbid obesity (n = 15) and a control group of normal weight (n = 15). The classification of body weight was made according to Body Mass Index (BMI), a questionnaire provided information on occupational exposures and non-occupational, lifestyle and diet. The oral mucosa cells were collected from the buccal mucosa, on both sides, processed and analyzed microscopically. For each individual 1000 cells were evaluated for the characterization of cell types (basal and differentiated) and nuclear abnormalities (binucleated cells, pyknosis, karyolysis, karyorrhexis, condensed chromatin) and 2000 differentiated cells for the presence of cellular damage (micronuclei and nuclear buds). The data were processed and statistically analyzed using the Mann-Whitney test. We considered the significance level of 5% (p <0.05). Differences were observed in the frequencies of cell types and nuclear abnormalities in both groups, but these differences were not significant (p> 0.05). Regarding the type of cell damage, we noticed the same frequency of nuclear buds for both groups, however, the frequency of micronuclei was higher in the obese group (p <0.001). In this study, there was a higher frequency of micronuclei in...

Estudian las características histopatológicas y moleculares de las células de Langerhans en la mucosa bucal / Langerhans cells in oral mucosa: hystopatological and molecular characterization

Actualmente, las células dendríticas en tejidos periféricos, piel y mucosas son centro de numerosas publicaciones. De acuerdo con su localización se clasifican en subtipos de diferente denominación. Su posición apropiada les permite ejercer un papel crucial en la detección y la captación de antígenos y su presentación a los linfocitos T en centros linfoides, iniciando la inmunidad innata y la inmunidad adaptativa. Debe destacarse además su actuación en la tolerancia inmunitaria y su participación en diversas enfermedades autoinmunitarias como la enfermedad periodontal e incluso el cáncer de células escamosas. Presentamos una actualización resumida de los últimos estudios realizados y destacamos el conocimiento de los marcadores inmunológicos y las características fundamentales de los subtipos, células de Langerhans y células plasmocitoides, especialmente en la mucosa oral. La disminución de los distintos fenotipos de células dendríticas en lesiones cancerizables y en la vejez, como su ausencia en neoplasias malignas son aspectos destacables. Comunicamos a la vez nuestro primer acercamiento a la microscopia electrónica de barrido para observarlas en tejido mucoso sano y de tres lesiones: mucosa hiperplásica, gingivitis descamativa y úlceras aftosas recidivantes con vasculitis.

Single exposure of human oral mucosa fibroblasts to ultraviolet B radiation reduces proliferation and induces COX-2 expression and activation

The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV) sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX)-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM), primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001). HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05). The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

Indicadores de riesgo en pacientes etilistas - fumadores: estudio preliminar / Risk indicators in drinker - smoker patients: preliminary study

Lesiones precancerosas y cáncer bucal están frecuentemente asociados en su etiología al tabaco y al alcohol. En este trabajo evalúanse las alteraciones citológicas en 19 pacientes con adicción tabaco alcohol que no presentan lesiones orales clínicas manifiestas en relación con la intensidad de dichos factores de riesgo, incluyéndose un grupo de control que no desarrollaba ninguno de los hábitos mencionados precedentemente. Se realizaron exámenes estomatológicos que incluían un interrogatorio sobre hábitos. En todos los pacientes se tomaron muestras por técnica de raspado de mucosa yugal, dorso de lengua y piso de boca. Los extendidos se fijaron en alcohol 95º y coloreados mediante técnica estándar de Papanicolaou, fueron evaluados según presentasen alteraciones o modificaciones -citoplasmáticas y nucleares - en escamas, hipercromasia y cariomegalia, que la relacionaron con indicadores de riesgo paquete/año; alcohol/año y un índice combinado tabaco-alcohol/año. Los resultados obtenidos revelaron que del grupo etilista fumadores 7 (37 por ciento) pacientes presentaban alteraciones citoplasmáticas y nucleares, de ellos 3 (16 por ciento) con cariomegalia, presentaban indicadores tabaco-alcohol/año superiores al valor promedio para dicho grupo, no observándose modificaciones de ningún tipo en el grupo testigo. En un significativo porcentaje de pacientes alcoholistas-fumadores, se observaron alteraciones nucleares mínimas que podrían considerarse como estadíos previos a lesiones precancerosas, que en relación a los indicadores propuestos servirían para prevenir y detectar precozmente la aparición de dichas lesiones, si bien un número mayor de casos deberían ser estudiados para confirmar esta observación incial.

Precancerous and cancerous lesions of oral mucosa are frequently by associated in their etiology to tobacco and alcohol intake, In this presentation the cytological alterations of 19 patients addicted to tobacco and alcohol without clinical evidence of disease related to the intensity of these habits were evaluated. A control group without any of these habits was included. The clinical examinations included all pertinent clinical data and samples from yugal mucosa, dorsal aspect of tongue and floor of the mouth were taken in each patients. The smears were fixed in 95 per cent alcohol, stained according the Papanicolaou technique and evaluated according the presence of squames, hiperchromasia and cariomegaly and their relationship to risk indicators such as tobacco pack / year; alcohol /year and combined index of tobacco-alcohol /year. The obtained results revealed that 7 (37 per cent) patients of the alcohol tobacco group presented cytoplasmic and nuclear changes and 3 (16 per cent) of them with cariomegaly presented indicators of tobacco-alcohol / year higher to the average for that group while no changes of any type were observed in the control group. In a significant percentage of alcoholic-smoker patients minimal nuclear changes that could be considered as previous stages to precancerous lesions were observed. These observations related to the proposed indicators could be useful to prevent and detect increment changes, although a larger number of cases should be evaluated to confirm this initial observation.

ApoE polymorphisms and diarrheal outcomes in Brazilian shanty town children

A series of studies have shown that the heavy burdens of diarrheal diseases in the first 2 formative years of life in children living in urban shanty towns have negative effects on physical and cognitive development lasting into later childhood. We have shown that APOE4 is relatively common in shanty town children living in Brazil (13.4 percent) and suggest that APOE4 has a protective role in cognitive development as well as weight-for-height in children with heavy burdens of diarrhea in early childhood (64/123; 52 percent), despite being a marker for cognitive decline with Alzheimer’s and cardiovascular diseases later in life. APOE2 frequency was higher among children with heaviest diarrhea burdens during the first 2 years of life, as detected by PCR using the restriction fragment length polymorphism method, raising the possibility that ApoE-cholesterol balance might be critical for growth and cognitive development under the stress of heavy diarrhea burdens and when an enriched fat diet is insufficient. These findings provide a potential explanation for the survival advantage in evolution of genes, which might raise cholesterol levels during heavy stress of diarrhea burdens and malnutrition early in life.

Cytotoxicity of latex and non-latex orthodontic elastomeric ligatures on L929 mouse fibroblasts

This study investigated the cytotoxicity exists between latex and non-latex Orthodontic elastomeric ligatures. Six elastomeric ligatures (1 latex, 2 latex-free and 3 polyurethane) from different manufacturers were divided into 6 groups of 15 elastics each: A (Latex-free, American Orthodontics), M (Polyurethane, Morelli), G (Polyurethane,GAC International), Te (Polyurethane, Tecnident), TP (Natural latex,TP Orthodontics) and U (Latex-free,3M Unitek). The cytotoxicity assay was performed using cell cultures (L929 mouse fibroblast cell line), which were subjected to the cell viability test with neutral red ("dye-uptake") at 1, 2, 3, 7 and 28 days. Data were analyzed statistically by ANOVA and Tukey's test (α=0.05). No statistically significant differences (p>0.05) were observed between Groups M and Te in all experimental periods, except at 2 days. No significant differences (p>0.05) in cell viability were found either among Groups A, G, TP and U or between Groups M and Te at 24 h or among Groups CC, A, G, TP and U at 2 and 28 days. It may be concluded that latex-free elastomeric ligatures from American Orthodontics and Unitek trademarks induced less cell lysis compared to latex and polyurethane ligatures.

Este estudo investigou a citotoxicidade entre ligaduras elásticas ortodônticas de látex e não-látex. Seis ligaduras elásticas de diferentes fabricantes (1 látex, 2 não-látex e 3 poliuretano) foram divididos em 6 grupos de 15 elásticos cada: Grupo A (látex-free, American Orthodontics), M (Poliuretano, Morelli), G (Poliuretano, GAC International), Te (Poliuretano, Tecnident), TP (látex natural, TP Orthodontics) e U (Látex-free, 3M Unitek). O ensaio de citotoxicidade foi realizado utilizando culturas de células (células da linhagem L929, fibroblastos de camundongo) que foram submetidos ao teste de viabilidade celular com vermelho neutro ("dye-uptake") em 1, 2, 3, 7 e 28 dias. A análise de variância (ANOVA), com comparações múltiplas e teste de Tukey foram empregados (α=0,05). Os resultados mostraram que não houve diferença estatisticamente significante entre os Grupos M e Te em todos os tempos experimentais (p>0,05), exceto em 2 dias. Não houve diferença estatisticamente (p>0,05) entre a viabilidade das células nos grupos A, G, TP e U ou entre os grupos M e Te em 24 h, ou entre os grupos CC, A, G, TP e U em 2 e 28 dias. Concluiu-se que as ligaduras elásticas látex-free das marcas American Orthodontics e Unitek induziram menor quantidade de lise celular comparado às ligaduras de látex ou poliuretano.

Buccal cells submitted to three different storage conditions before DNA extraction

This study evaluated quantitatively and qualitatively the effect of the storage time of samples before the application of the cell lysis solution (CLS) for extracting DNA from buccal cells (BC). BC from the upper and lower gutter region were collected from 5 volunteers using special cytobrushes (Gentra), totaling 3 collections for each individual. In the control group (n=10), CLS was applied soon after BC collection. In the other two groups, samples were stored at room temperature (n=10) or at 4°C (n=10). After CLS application, DNA was extracted according to the manufacturer's instructions (Puregene DNA Buccal Cell Kit; Gentra Systems, Inc.). The DNA obtained was evaluated by two calibrated blind examiners using spectrophotometry and analysis of DNA bands (0.8 percent agarose gel electrophoresis). The obtained data were submitted to one-way ANOVA. The means and standard deviations for DNA extracted under immediate, room temperature and cooling temperature conditions were 3.5 ± 0.7, 3.0 ± 0.6 and 4.1 ± 1.8 µg, respectively (p=0.385). No significant differences were found in relation to the amount of DNA for the different storage conditions. However, in the visual analysis of the DNA bands, no trace of DNA degradation was detected when CSL was applied soon after DNA collection, while DNA bands with degradation could be observed in the other groups. Within the limitations of the study, it may be concluded that CLS should be applied soon after DNA collection in order to obtain high-quality DNA from BC.

Comparison between DNA obtained from buccal cells of the upper and lower gutter area

This study compared quantitatively and qualitatively the DNA extracted from buccal cells collected from the upper or lower gutter areas. Buccal cells were collected from the upper (n=15) and lower gutter (n=15) region from 15 volunteers using a special cytobrush (Gentra), totaling 2 collections from each individual. DNA was extracted from the samples according to the manufacturer's instructions. The DNA obtained was qualitatively and quantitatively evaluated by 2 calibrated blind examiners using spectrophotometry and analysis of DNA bands (0.8 percent agarose gel electrophoresis). Data was statistically analyzed by one-way ANOVA (?=0.05). Means and standard derivation (SD) for total DNA yield from the upper and lower gutter area were 12.2 ?g (12.0) and 9.4 ?g (8.5), respectively (p=0.821). There was higher (p<0.05) DNA purity for the upper gutter (1.79; 0.05) when compared to lower gutter area (1.66; 0.10). Regarding to the DNA quality, no differences were observed between the 2 location sites, but all samples showed similar degree of degradation. In conclusion, it would be recommendable that buccal cells for DNA extraction be collected from the upper gutter area in the attempt to increase DNA purity.

O objetivo do presente estudo foi comparar quantitativamente e qualitativamente o DNA extraído de células epiteliais bucais coletadas do fundo de sulco superior e inferior. Foram coletadas células bucais do fundo de sulco superior (n=15) e inferior (n=15) de 15 voluntários utilizando escovas citológicas especiais (Gentra), totalizando 2 coletas por voluntário. Após a coleta o DNA foi extraído conforme o protocolo indicado pelo fabricante (Puregene DNA Buccal Cell Kit; Gentra Systems, Inc.). O DNA obtido foi avaliado quantitativamente e qualitativamente por dois examinadores calibrados cegos utilizando espectrofotometria e análise das bandas de DNA (gel de agarose 0,8 por cento, por eletroforese). Os dados foram submetidos a ANOVA a um critério, com p<0,05. As médias e desvio padrão (DP) para o rendimento total de DNA do fundo de sulco superior e inferior foram respectivamente 12,2 ?g (12,0) e 9,4 ?g (8,5) (p=0,821). Houve maior (p<0,05) pureza de DNA no fundo de sulco superior (1,79; 0,05) quando comparado com o fundo de sulco inferior (1,66; 0,10). Quanto à qualidade do DNA, não foi observado diferenças entre os dois locais testados, no entanto todas as amostras mostraram níveis de degradação semelhantes. Em conclusão seria recomendável coletar células bucais, para extração de DNA, do fundo de sulco superior na tentativa de aumentar a pureza do DNA.

A simple method to measure cell viability in proliferation and cytotoxicity assays

Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 çm and resorufin at 570 çm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 çm) and green (500 to 600 çm) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01) and with the cellular concentrations (r² = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.