Micronucleus Assay Of Buccal Mucosal Cells In Smokers And Non–Smokers

The micronuclei assay (MA) in exfoliated buccal cells is an innovative technique which holds promise for the screening of epithelial carcinogens/mutagens. Micronucleus is the small nucleus that forms whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division. The micronucleus is thought to be an indicator of DNA damage consequent to possible carcinogen exposure. The objectives of our study were to evaluate the frequency of micronuclei in exfoliated buccal mucosal cells of smokers and non–smokers and to correlate the mean micronuclei with period of smoking. Buccal smears obtained from 68 age matched male subjects (40 smokers, 28 non–smokers) were included in the study. Papanicoloau stained smears were screened for micronuclei as per Tolbert et al criteria, with 1000 cells being examined in both categories. Results showed a statistically significant difference in the mean micronuclei count in buccal cells of smokers as compared to non smokers. Micronucleus assay can be used as a simple bio–marker for screening of pre–malignant changes in cells.

Micronucleus Formation Paddy Farmers from the East Coast of Malaysia / Jurnal Sains Kesihatan Malaysia

@#Pesticide exposure may cause genotoxic effects by inducing the formation of micronucleus (Mn). Mn are fragments of chromosomes that remains after cells division. The increase in Mn may increase the risk of cancer formation. Our study aimed to determine the effects of lifestyle and pesticide exposure on the formation of Mn in epithelial cells buccal swabs among paddy farmers in Malaysia. About 40 farmers who were exposed to pesticides were chosen as subjects and 30 personnels were possibly not directly exposed to pesticides, were chosen as the control group. Demographic and anthropometric data were obtained from questionnaires developed. Analysis of Mn formation was done using Giemsa staining (10% v/v) and the frequency of Mn formation was scored from 1000 cells per sample. Kruskal-Wallis test done between Mn frequency with age class showed a significant increase (p < 0.05) in Mn frequency as compared to the control in the age group of 30-39 , 40-49 years, and 50-59 years. Significant increased (p < 0.05) were observed between Mn frequency groups of normal BMI, preobese, and grade 1 obese as compared to control. Significant increase of Mn frequency (p < 0.01) was also seen among smokers and farmer’s group (15:39 ± 3.34) as compared to controls (4.76 ± 1.26). The maximum numbers of Mn found in farmers are 7 Mn per cell whereas for control group is only 3 Mn. However, most farmers had only 1 Mn (81.75 ± 6.42%) and 2 Mn (15:28 ± 5.14%). Mn frequency with the duration of exposure to pesticides in a month and the use of PPE revealed no significant difference (p = 0.27). In conclusion, the increased frequency of Mn was influenced by age, gender and BMI of farmers besides commonly repeated duration of exposures and the use of PPE are needed to be studied to analyze the causes of an increased in Mn among farmers.

Cytogenetic Analysis Of Buccal Cells From Farmers In Tanjung Karang And Kelantan Who Were Exposed To Pesticides / Jurnal Sains Kesihatan Malaysia

Pesticides and chemical fertilizers are widely used in agriculture to increase crop productivity among farmers.However, exposure to pesticides will give potential risk to human health. The aim of this study was to analyze thefrequency of micronucleus (MN) and binucleus (BNu) formation in buccal cells from farmers who were exposedto pesticides using the MN assay. Buccal swabs were collected from the farmers in Tanjung Karang (n = 32) andKelantan (n = 43) using wooden tongue depressor. A structured questionnaire was used to obtain demographic dataof the farmers. Cytogenetic analysis was carried out by Acridin Orange (AO) staining 0.0025% (w/v). The frequencyof MN and BNu as the biomarkers for cytogenetic damage was observed by using a fluorescence microscope.Comparison of frequency of MN and BNu is conducted in two areas namely Tanjung Karang, Selangor and Kelantanbecause of the agricultural activity and the type of pesticides used are different. Results showed that the frequencies of bothMN and BNu among farmers in Tanjung Karang were significantly higher (p 0.05) and the practices of PPE (Personal Protective Equipment) (p > 0.05). This may suggeststhat cytogenetic changes were not influenced by these factors. In addition, correlation study shows positive correlationbetween the frequency of MN with the pesticide exposure of farmers in Tanjung Karang (p > 0.05, r = 0.015) and Kelantan(p > 0.05, r = 0.0158). Besides, the frequency of BNu also has a positive correlation with the pesticide exposure amongfarmers in Tanjung Karang (p > 0.05, r = 0.036) and farmers in Kelantan (p > 0.05, r = 0.013). Hence, this present study demonstrated that exposure to pesticides increasedthe formation of MN and BNu among farmers and theprolonged use of pesticides may induce genotoxicity andDNA damage to human.

Micronucleus assay on buccal cells: an indicator of DNA damage due to formaldehyde exposure in anatomy dissection labs.

Background: The occupational exposure to formaldehyde (FA) can lead to various hazards ranging from allergic reactions to genetic damage. Workers of Anatomy lab are at a higher risk of having the hazardous effects of FA. Micronuclei (MN) appear in the cells due to chromosome breakage and dysfunction of the mitotic apparatus which are the indicators for the DNA damage. The present study was carried out to detect the DNA damage in people exposed to FA using buccal cell MN Assay by measuring the MN frequency in buccal cells with respect to the duration of exposure. Methods: Thirty male workers of Anatomy labs of different medial colleges in Bangalore were included in the study. Thirty people with no FA exposure were considered as comparison group. Buccal cells were scraped from the cheek and slides were prepared. A total of 1000 cells were counted for the presence of MN after staining with Geimsa solution. Results: There was a significant increase in the frequency of MN in both buccal cells (p<0.001). A positive correlation was found between the years of exposure and frequency of MN in buccal cells (r=0.5, p=0.03). Conclusion: This study highlights that there is a significant DNA damage in people exposed to formaldehyde which is proportional to the duration of exposure.

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...

Nuclear anomalies in the buccal cells of calcite factory workers

The micronucleus (MN) assay on exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. To determine the genotoxic effects of calcite dust that forms during processing, MN assay was carried out in exfoliated buccal cells of 50 (25 smokers and 25 non-smokers) calcite factory workers and 50 (25 smokers and 25 non-smokers) age- and sex-matched control subjects. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis, karyolysis and 'broken eggs', were also evaluated. Micronuclei and the other aforementioned anomalies were analysed by two way analysis of covariance. The linear correlations between the types of micronucleus and nuclear abnormalities were determined by Spearman's Rho. There was a positive correlation between micronuclei and other types of nuclear abnormalities in accordance with the Spearman's Rho test. Results showed statistically significant difference between calcite fabric workers and control groups. MN and NA frequencies in calcite fabric workers were significantly higher than those in control groups (p < 0.05). The results of this study indicate that calcite fabric workers are under risk of significant cytogenetic damage.

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 and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 µg (median 15 µg, mean 20.71 µg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Células bucais são fontes convenientes de DNA para diagnóstico e estudos epidemiológicos. O objetivo deste trabalho foi desenvolver um método simples e prático para obter células epiteliais, através de bochechos, a fim de serem usadas como fonte de DNA e avaliar a estabilidade do DNA na solução de bochecho no decorrer do tempo. Os procedimentos usados neste estudo evitam o uso de solventes orgânicos permitindo uma pratica laboratorial mais segura. Isto é alcançado pela remoção das proteínas celulares por desidratação e precipitação com uma solução saturada de acetato de amônio. Este protocolo permite a extração de maneira rápida, simples, econômica e garante o processamento de várias amostras ao mesmo tempo, agilizando assim os procedimentos laboratoriais. Nossas análises forneceram evidências consistentes de que o DNA extraído por esta metodologia é suficiente para diversas amplificações por PCR (polymerase chain reaction - reação em cadeia pela polimerase). O produto total de DNA variou de 5 a 93 µg (mediana 15 µg; média 20,71 µg). Além disso, o DNA mostrou-se eficientemente preservado na solução de bochecho, a qual pode ser estocada em temperatura ambiente por até trinta dias.