Abstract The aim of this study was to investigate the DNA methylation profile in genes encoding catalase (CAT) and superoxide dismutase (SOD3) enzymes, which are involved in oxidative stress mechanisms, and in genes encoding pro-inflammatory cytokines interleukin-6 (IL6) and tumor necrosis factor-alpha (TNF-α) in the oral mucosa of oncopediatric patients treated with methotrexate (MTX®). This was a cross-sectional observational study and the population comprised healthy dental patients (n = 21) and those with hematological malignancies (n = 64) aged between 5 and 19 years. Oral conditions were evaluated using the Oral Assessment Guide and participants were divided into 4 groups: 1- healthy individuals; 2- oncopediatric patients without mucositis; 3- oncopediatric patients with mucositis; 4- oncopediatric patients who had recovered from mucositis. Methylation of DNA from oral mucosal cells was evaluated using the Methylation-Specific PCR technique (MSP). For CAT, the partially methylated profile was the most frequent and for SOD3 and IL6, the hypermethylated profile was the most frequent, with no differences between groups. For TNF-α, the hypomethylated profile was more frequent in the group of patients who had recovered from mucositis. It was concluded that the methylation profiles of CAT, SOD3, and IL6 are common profiles for oral cells of children and adolescents and have no association with oral mucositis or exposure to chemotherapy with MTX®. Hypomethylation of TNF-α is associated with oral mucosal recovery in oncopediatric patients who developed oral mucositis during chemotherapy. CAT (CAT SOD (SOD3 enzymes mechanisms proinflammatory pro inflammatory interleukin6 interleukin 6 interleukin- IL (IL6 factoralpha factor alpha TNFα TNF α (TNF-α MTX MTX® . (MTX®) crosssectional cross sectional n 21 64 1 years groups individuals 2 3 MethylationSpecific Specific MSP. MSP (MSP) TNFα, α, (SOD (IL (MTX® (MSP (MTX

Abstract The association between Periodontitis and Systemic Lupus Erythematosus (SLE) has been primarily based on their similar pathophysiology and both are associated with genetic polymorphisms. Objectives: To investigate an association between the methylation-related gene polymorphisms DNMT3B (rs2424913) and MTHFR (rs1801133) to Systemic Lupus Erythematosus (SLE) and Periodontitis. Methodology: In total, 196 individuals of all genders aged 24 to 60 years old were allocated into four groups based on their systemic and periodontal status, namely: Healthy control (n=60), periodontitis (n=51), SLE (n=47), and SLE + periodontitis (n=38). Individuals with SLE were stratified according to disease activity (SLEDAI) in inactive or active. We performed polymorphism analysis using PCR-RFLP with genomic DNA from mouthwash. We analyzed data using Fisher’s Exact, Chi-square test, and regression models. Results: Periodontal status were similar in subjects with periodontitis alone and combined with SLE. SLE patients with periodontitis had a longer SLE diagnosis than SLE only (p=0.001). For DNMT3 B polymorphism, the periodontitis, SLE, and Inactive SLE + periodontitis groups showed a higher frequency of T allele and TT genotypes compared to healthy controls (p<0.05). Regression analyses showed that the TT genotype is a strong risk factor for periodontitis (OR=4.53; CI95%=1.13–18.05) and also for SLE without periodontitis (OR=11.57; CI95%=3.12–42.84) and SLE with periodontitis (OR=5.27; CI95%=1.25–22.11) when compared to control. Conclusion: SLE patients with periodontitis had a longer length of SLE diagnosis. The DNMT3B (rs2424913) polymorphism was associated with periodontitis and SLE alone or combined with periodontitis. Our study contributes to understanding the genetic mechanisms involved in periodontitis and SLE susceptibility.

Abstract Oral mucositis (OM) is a painful inflammatory oral condition that affects children who undergo chemotherapy. Oxidative stress is a known OM mediator and pro-inflammatory cytokines contribute to the amplification of the immune response. Objective: To investigate the possible associations of rs4880 (superoxide dismutase 2, SOD2 47 C/T), rs7943316 (catalase, CAT −21 A/T), rs1800629 (tumor necrosis factor α, TNF- α −308 G/A), and rs1800795 (interleukin 6, IL-6 −174 G/C) polymorphisms with chemo-induced OM occurrence and severity in oncopediatric patients. Methodology: We conducted a single-center, observational cross-sectional study with sample collection of oral epithelial cells from 95 children and adolescents with hematological cancers who underwent chemotherapy, followed by genomic DNA extraction. Single-nucleotide polymorphisms (SNPs) were assessed with PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Demographic data and information concerning OM occurrence were obtained from dental charts of the multidisciplinary oral care team. Information on OM severity was obtained from appropriately-filled Oral Assessment Guide records. Descriptive and inferential statistics were conducted with Student's T test, chi-squared test, and Fisher's exact test, with p≤0.05. Results: The mean age was 10 years-old and most patients were male individuals (57.89%). Female sex was considered a protective factor for OM occurrence (OR=4.83; CI=[1.14; 16.57]). The AA genotype for CAT was the most frequent amongst individuals with severe OM (p=0.04). The GA genotype for TNF- α was the most frequent amongst individuals without severe OM (p=0.03). For SOD2 and IL-6 , the most frequent genotypes were CT and GG respectively for all groups (p>0.05). Conclusion: The AA genotype for CAT −21 A/T was a tendency among the group with severe OM. Data on TNF- α −308 G/A were inconclusive. No associations were detected for SOD2 47 C/T and IL-6 −174 G/C polymorphisms in oncopediatric patients with chemo-induced oral mucositis.

Abstract Background: the biological activity of vitamin D depends on the activity of its receptor or VDR. On the other hand, the activity of this receptor is influenced by its state of methylation. The objective of this study was to verify if the BsmI polymorphism of the VDR gene influences its methylation profile in adolescents. Secondly, it was to verify if the status of some metabolic factors (oxidative stress, inflammation, lipid profile, and glycemia) in the serum, and gender-adjusted vitamin D levels are independent factors with an influence on the VDR methylation profile. Methods and results: the study included 198 adolescents of both sexes, aged 15-19 years, who underwent testing for VDR gene methylation polymorphisms, serum vitamin D levels, and metabolic, oxidative stress, and systemic inflammation markers. It was observed that the BB genotype was less methylated than the other groups (26.1 % versus 30.3 %, and 29.3 % for Bb and bb, respectively), although without statistical differences between them. The odds ratio indicated a protection of 13 % (partially methylated) for vitamin D status, while alpha glycols increased the risk ratio (of being partially methylated) by 3 %. MDA was protective at a 28 % chance of risk that adolescents with higher levels of lipid peroxidation would be hypomethylated. Conclusion: we conclude that the methylation profile of the VDR gene is not influenced by the different BsmI polymorphism genotypes, and that serum vitamin D and serum markers of oxidative stress and inflammation can modulate this profile.

Resumen Antecedentes: la actividad biológica de la vitamina D depende de la actividad de su receptor, el VDR. Por otro lado, la actividad de este receptor está influenciada por su estado de metilación. El objetivo de este estudio es verificar si el polimorfismo BsmI del gen VDR influye en el perfil de metilación del mismo en los adolescentes. En segundo lugar, verificar si los factores metabólicos (estrés oxidativo, inflamación, perfil lipídico y glucemia) del suero y la vitamina D ajustada por sexo actúan independientemente de los polimorfismos sobre el perfil de metilación del VDR. Métodos y resultados: el estudio incluyó a 198 adolescentes de ambos sexos, de 15 a 19 años de edad, que se sometieron a análisis de polimorfismos de metilación del gen VDR, niveles de vitamina D, marcadores metabólicos, estrés oxidativo e inflamación sistémica. Se observó que el genotipo BB estaba menos metilado que los otros grupos (26,1 % contra 30,3 % y 29,3 % para Bb y bb respectivamente), aunque sin diferencias estadísticas entre ellos. El odds ratio indicó una protección del 13 % (parcialmente metilado) para el estado de la vitamina D, mientras que los alfa glicoles aumentaron el índice de riesgo (de estar parcialmente metilado) en un 3 %. La MDA fue protectora con un 28 % de probabilidad de riesgo de que los adolescentes con niveles más altos de peroxidación lipídica fueran hipometilados. Conclusión: concluimos que el perfil de metilación del gen VDR no está influenciado por los diferentes genotipos del polimorfismo BsmI y que la vitamina D y los marcadores de estrés oxidativo e inflamación en el suero pueden modular este perfil.

Resumo O presente estudo investigou a relação entre cinco polimorfismos genéticos e o desenvolvimento de mucosite oral em pacientes pediátricos recebendo quimioterapia com metrotexato. O estudo longitudinal foi conduzido com 64 pacientes e a mucosite oral avaliada pelo Oral Assessment Guide modificado, que tem como objetivo diagnosticar e classificar a mucosite oral. Células epiteliais bucais foram obtidas por bochecho e o DNA foi extraído. Os polimorfismos MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) e ABCG2 (rs2231142), foram analisados pela técnica de PCR-RFLP. Dados demográficos, hematológicos e bioquímicos foram coletados a partir de registros médicos. Análise estatística foi realizada utilizando o software SPSS adotando um valor de p=0,05. Observou-se que, o sexo masculino foi predominante (56,2%), e a idade média foi de 10,8 anos (± 4.9). A mucosite oral acometeu 65,6% dos pacientes, dos quais, 61,9% desenvolveram a forma grave da doença. Para o gene ABCG2 (rs2231142), o alelo raro A e o genótipo CA foram mais frequentes em indivíduos com mucosite (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). A gravidade da doença foi observada principalmente em pacientes mais jovens (mediana = 9 anos; p=0.02). Além disso, os pacientes com mucosite oral grave apresentaram menor contagem de leucócitos (mediana = 2150 mm3) em comparação aos pacientes com a forma leve/moderada (mediana = 4200 mm3; p=0.03). Pacientes do sexo feminino e aumento a cada 10.000 plaquetas foram fatores de proteção contra o aparecimento de mucosite oral (p=0.02). Concluiu-se que a presença do polimorfismo rs2231142 aumenta o risco de o paciente desenvolver a mucosite oral, bem como pacientes mais jovens e menor contagem de leucócitos contribui com a severidade.

Abstract The study investigated the relationship between genetic polymorphisms and the development of oral mucositis in pediatric patients undergoing chemotherapy involving methotrexate. A longitudinal study was conducted with 64 patients, and oral mucositis was evaluated by the modified Oral Assessment Guide, which aims to diagnose and classify oral mucositis. Epithelial cells were obtained by mouthwash and DNA was extracted. The polymorphisms MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) and ABCG2 (rs2231142) were analyzed by PCR-RFLP method. Demographic, hematological and biochemical data were collected from medical records. Statistical analysis was performed using the SPSS software adopting a p-value of 0.05. Male sex predominated (56.2%), and the mean age was 10.8 years (± 4.9). Oral mucositis affected 65.6% of the patients, of which 61.9% developed the severe form of the disease. For the ABCG2 gene (rs2231142), the rare A allele and CA genotype were more frequent in individuals with mucositis (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). The severity of the disease was mainly observed in younger patients (median = 9 years; p=0.02). Patients with severe oral mucositis presented lower leukocytes count (median = 2.150 mm3) compared to patients with the mild/moderate form (median = 4.200 mm3; p=0.03). Female patients and each 10,000-platelet increase were protective factors against the onset of oral mucositis (p=0.02). It is concluded that rs2231142 polymorphism increases the likelihood of oral mucositis and younger patients and patients with low leukocytes counts are more likely to develop severe form.

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Abstract Background: Defects in DNA methylation have been shown to be associated with metabolic diseases such as obesity, dyslipidemia, and hypercholesterolemia. To analyze the methylation profile of the ADRB3 gene and correlate it with lipid profile, lipid intake, and oxidative stress based on malondialdehyde (MDA) and total antioxidant capacity (TAC), homocysteine and folic acid levels, nutritional status, lifestyle, and socioeconomic variables in an adult population. A cross-sectional epidemiological study representative of the East and West regions of the municipality of João Pessoa, Paraíba state, Brazil, enrolled 265 adults of both genders. Demographic, lifestyle, and socioeconomic questionnaires and a 24-h recall questionnaire were applied by trained interviewers' home. Nutritional and biochemical evaluation (DNA methylation, lipid profile, MDA, TAC, homocysteine and folic acid levels) was performed. Results: DNA hypermethylation of the ADRB3 gene, analyzed in leukocytes, was present in 50% of subjects and was associated with a higher risk of being overweight (OR 3.28; p = 0.008) or obese (OR 3.06; p = 0.017), a higher waist–hip ratio in males (OR 1.17; p = 0.000), greater intake of trans fats (OR 1.94; p = 0.032), higher LDL (OR 2.64; p = 0.003) and triglycerides (OR 1.81; p = 0.031), and higher folic acid levels (OR 1.85; p = 0.022). Conclusions: These results suggest that epigenetic changes in the ADRB3 gene locus may explain the development of obesity and non-communicable diseases associated with trans-fat intake, altered lipid profile, and elevated folic acid. Because of its persistence, DNA methylation may have an impact in adults, in association with the development of non-communicable diseases. This study is the first population-based study of the ADRB3 gene, and the data further support evaluation of ADRB3 DNA methylation as an effective biomarker.

Abstract: Background: epigenomes can be influenced by environmental factors leading to the development of diseases. Objective: To investigate the influence of sun exposure on global DNA methylation and hydroxymethylation status and at specific sites of the miR-9-1, miR-9-3 and MTHFR genes in skin samples of subjects with no history of skin diseases. Methods: Skin samples were obtained by punch on sun-exposed and sun-protected arm areas from 24 corpses of 16-89 years of age. Genomic DNA was extracted from skin samples that were ranked according to Fitzpatrick's criteria as light, moderate, and dark brown. Global DNA methylation and hydroxymethylation and DNA methylation analyses at specific sites were performed using ELISA and MSP, respectively. Results: No significant differences in global DNA methylation and hydroxymethylation levels were found among the skin areas, skin types, or age. However, gender-related differences were detected, where women showed higher methylation levels. Global DNA methylation levels were higher than hydroxymethylation levels, and the levels of these DNA modifications correlated in skin tissue. For specific sites, no differences among the areas were detected. Additional analyses showed no differences in the methylation status when age, gender, and skin type were considered; however, the methylation status of the miR-9-1 gene seems to be gender related. Study limitations: there was no separation of dermis and epidermis and low sample size. Conclusion: sun exposure does not induce changes in the DNA methylation and hydroxymethylation status or in miR-9-1, miR-9-3 and MTHFR genes for the studied skin types.

The purpose of this study was to investigate the effect of aging on the DNA methylation status of two genes involved in tumorigenesis (telomerase gene hTERT and DNA repair gene- MLH1) and one in metabolism (methylenetetrahydrofolate reductase gene- MTHFR) in oral epithelial cells. DNA methylation analysis was performed by Methylation Sensitive Restriction Enzymes (MSRE) of healthy oral epithelial cells of child (6-10 years, n=21), young (20-25 years, n=19) and elderly (over 60 years, n=25). The results for the hTERT gene showed significant variation in the methylation frequency at CpG dinucleotides among the groups (p=0.0001), with the methylated condition more frequently in children and young people. In relation to MLH1 and MTHFR, no differences were observed among the groups and the unmethylated condition were present in most individuals (p>0.05). Thus, it was concluded that aging of oral epithelial cells was associated with hypomethylation of the hTERT gene promoter and this could be a promising marker for screening a set of age-related alterations.

DNA methylation is mediated by DNA methyltransferases (DNMTs) that add a methyl group to the 5'-carbon of cytosine. The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in the rate-limiting step of the cycle involving the methyl donor S-adenosyl-L-methionine (SAM). The MTHFR C677T polymorphism results in a thermolabile enzyme with reduced activity that is predicted to influence the DNA methylation status. In this study, we investigated the impact of the MTHFR C677T polymorphism on the global DNA methylation of oral epithelial cells obtained from 54 healthy subjects. There were no significant differences in global DNA methylation among the MTHFR CC, CT and TT genotypes (p = 0.75; Kruskal-Wallis test).

Os linfócitos são células importantes do sistema imune e têm sido largamente utilizados em estudos morfológicos. Entretanto, a literatura sobre técnicas de preparação dessas células é escassa e antiga, especialmente para linfócitos cultivados in vitro. Portanto, o objetivo desse estudo foi relatar com detalhes as técnicas de isolamento e microscopia de luz de linfócitos mantidos em cultura. Amostras de sangue foram obtidas por punção venosa e centrifugadas em gradiente de densidade de Percoll, para separar os leucócitos. Os linfócitos foram mantidos em frascos de cultura de 25 cm³ a 37ºC. Após a cultura, as células foram fixadas e coradas de acordo com a metodologia utilizada para esfregaços sanguíneos. Nossos resultados mostraram que nem todos os fixadores e corantes utilizados para esfregaços sanguíneos são uma boa escolha para linfócitos cultivados in vitro.

The purpose of this work was to study the isolation and a light microscopy technique for cultured lymphocytes. Blood samples were obtained by venipuncture with an anticoagulant added and centrifuged in a Percoll density gradient to separate the leukocytes. Lymphocytes were placed in 25 cm ³ tissue culture flasks at 37ºC. After culturing, they were fixed and stained with the methods used for blood smears. Results showed that not all fixing solutions and stains were an equally good choice for cultured lymphocytes.