Protocol for chromatin immunoprecipitation of histone modifications in frozen adipose tissue.

Chromatin immunoprecipitation (ChIP) combined with sequencing has revolutionized our understanding of gene regulation; however, its application to frozen adipose tissue presents unique challenges due to the high levels of lipid content. Here, we present a protocol for ChIP of histone modifications in human frozen adipose tissue. We describe steps for tissue preparation, chromatin isolation, sonication, pre-clearing of chromatin, and immunoprecipitation. We then detail procedures for elution, crosslink reversal, chromatin purification, quality control, and library synthesis.

Occupational exposure to radiation among health workers: Genome integrity and predictors of exposure.

The current study aimed to investigate genomic instabilities in healthcare workers who may experience varying levels of radiation exposure through various radiological procedures. It also sought to determine if factors related to the work environment and dosimeter reading could effectively explain the observed genomic instabilities. Utilizing the cytokinesis-block micronucleus assay (CBMN) on peripheral blood lymphocytes, we assessed a spectrum of genomic aberrations, including nucleoplasmic bridge (NPB), nuclear budding (NBUD), micronucleus (MN) formation, and total DNA damage (TDD). The study uncovered a statistically significant increase in the occurrence of distinct DNA anomalies among radiology workers (with a significance level of P < 0.0001 for all measurements). Notably, parameters such as total working hours, average work duration, and time spent in projection radiography exhibited significant correlations with MN and TDD levels in these workers. The dosimeter readings demonstrated a positive correlation with the frequency of NPB and NBUD, indicating a substantial association between radiation exposure and these two genomic anomalies. Our multivariable models identified the time spent in projection radiography as a promising parameter for explaining the overall genomic instability observed in these professionals. Thus, while dosimeters alone may not fully explain elevated total DNA damage, intrinsic work environment factors hold potential in indicating exposure levels for these individuals, providing a complementary approach to monitoring.

The emergent role of mitochondrial RNA modifications in metabolic alterations.

Mitochondrial epitranscriptomics refers to the modifications occurring in all the different RNA types of mitochondria. Although the number of mitochondrial RNA modifications is less than those in cytoplasm, substantial evidence indicates that they play a critical role in accurate protein synthesis. Recent evidence supported those modifications in mitochondrial RNAs also have crucial implications in mitochondrial-related diseases. In the light of current knowledge about the involvement, the association between mitochondrial RNA modifications and diseases arises from studies focusing on mutations in both mitochondrial and nuclear DNA genes encoding enzymes involved in such modifications. Here, we review the current evidence available for mitochondrial RNA modifications and their role in metabolic disorders, and we also explore the possibility of using them as promising targets for prevention and early detection. Finally, we discuss future directions of mitochondrial epitranscriptomics in these metabolic alterations, and how these RNA modifications may offer a new diagnostic and theragnostic avenue for preventive purposes. This article is categorized under: RNA Processing > RNA Editing and Modification.

Evaluation of Mitochondrial Copy Number in Thyroid Disorders.

OBJECTIVE: The purpose of this study was to determine whether the mitochondrial DNA (mitDNA) copy number in blood samples of patients with thyroiditis, benign nodules or malignant nodules is different from that in healthy individuals, and to examine whether mtDNAcn has the ability to distinguish between different thyroid diseases. MATERIALS AND METHOD: This study consists of principal groups as thyroid patients and control group. The thyroid patient group comprised 30 patients with malignant nodules, 33 with benign nodules and 31 with thyroiditis, whereas the control group was composed of 21 healthy individuals. Blood samples were collected from the patients before treatment. Results were evaluated between groups. RESULTS: We could not find an adequate number of participants for inclusion to match the groups. Similarly, since there is a gender difference in terms of disease prevalence, it was not possible to pair the populations in terms of gender. Instead, the results were analyzed with an adjusted model, including man characteristics as cofounders. We found that the mtDNAcn of the thyroid patients was significantly lower than that measured for the control group (p = 0.01). Furthermore the mtDNAcn of the benign group was significantly lower than that measured in the control group (p = 0.0001). A similar significant difference was found between the thyroiditis group and the control group (p = 0.005). CONCLUSION: It was observed that mtDNAcn in the malignant group was significantly higher than that measured in the benign group (p = 0.004), which would indicate that it may be used as a diagnostic and therapeutic marker in thyroid diseases.

Evaluation of circulating cell-free nucleic acids in health workers occupationally exposed to ionizing radiation.

Radiology workers might constantly be exposed to low-dose ionizing radiation due to their profession. Low doses of radiation in a short exposure time have the potential to alter the genome, which might potentially lead to diseases. The main objective of this study was to determine whether the amount of cell-free nucleic acids in plasma samples of radiation-exposed workers was different from the general public, in other words, non-exposed individuals. In this context, we investigated the association between radiation exposure and cell-free nucleic acids concentration by using radiation exposure parameters. The study consisted of 40 radiology workers and 40 individuals who were not exposed to ionizing radiation. The plasma concentrations of cell-free DNA, RNA, and miRNA were measured fluorometrically. We found that the ccfRNA concentration of the radiation-exposed group was significantly different from that of the non-exposed group (p = 0.0001). However, there are no differences between both groups in terms of ccfDNA and ccfmiRNA concentration. The concentration of ccfDNA is significantly correlated with working time in the fluoroscopy field (p < 0.05). We found that the concentration of ccfmiRNA was significantly correlated with working time in plain radiography (p < 0.01) and computed tomography (p < 0.05) and with total working time (p < 0.01). Similarly, the concentrations of ccfRNA were significantly correlated with working time in computed tomography (p < 0.01) and with the total working time (p < 0.05) of the workers. We found that imaging number in computed tomography significantly altered the level of ccfRNA (p = 0.006) and that working time in the computed tomography field significantly affected the ccfRNA concentration (p = 0.03, R2 = 0.36 for model). Finally, we determined that total working time was significantly associated with total ccfRNA concentration (p < 0.05, R2 = 0.25 for model). In conclusion, total RNA measured in radiation-exposed workers has the potential to predict the radiation exposure risk. Furthermore, total working time and working time in the tomography field significantly alter the level of free nucleic acids.

RNA modifications as emerging therapeutic targets.

The field of epitranscriptome, posttranscriptional modifications to RNAs, is still growing up and has presented substantial evidences for the role of RNA modifications in diseases. In terms of new drug development, RNA modifications have a great promise for therapy. For example, more than 170 type of modifications exist in various types of RNAs. Regulatory genes and their roles in critical biological process have been identified and they are associated with several diseases. Current data, for example, identification of inhibitors targeting RNA modifications regulatory genes, strongly support the idea that RNA modifications have potential as emerging therapeutic targets. Therefore, in this review, RNA modifications and regulatory genes were comprehensively documented in terms of drug development by summarizing the findings from previous studies. It was discussed how RNA modifications or regulatory genes can be targeted by altering molecular mechanisms. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > RNA Editing and Modification.

m6A Regulators in Human Adipose Tissue - Depot-Specificity and Correlation With Obesity.

Background: N6-methyladenosine (m6A) is one of the most abundant post-transcriptional modifications on mRNA influencing mRNA metabolism. There is emerging evidence for its implication in metabolic disease. No comprehensive analyses on gene expression of m6A regulators in human adipose tissue, especially in paired adipose tissue depots, and its correlation with clinical variables were reported so far. We hypothesized that inter-depot specific gene expression of m6A regulators may differentially correlate with clinical variables related to obesity and fat distribution. Methods: We extracted intra-individually paired gene expression data (omental visceral adipose tissue (OVAT) N=48; subcutaneous adipose tissue (SAT) N=56) of m6A regulators from an existing microarray dataset. We also measured gene expression in another sample set of paired OVAT and SAT (N=46) using RT-qPCR. Finally, we extracted existing gene expression data from peripheral mononuclear blood cells (PBMCs) and single nucleotide polymorphisms (SNPs) in METTL3 and YTHDF3 from genome wide data from the Sorbs population (N=1049). The data were analysed for differential gene expression between OVAT and SAT; and for association with obesity and clinical variables. We further tested for association of SNP markers with gene expression and clinical traits. Results: In adipose tissue we observed that several m6A regulators (WTAP, VIRMA, YTHDC1 and ALKBH5) correlate with obesity and clinical variables. Moreover, we found adipose tissue depot specific gene expression for METTL3, WTAP, VIRMA, FTO and YTHDC1. In PBMCs, we identified ALKBH5 and YTHDF3 correlated with obesity. Genetic markers in METTL3 associate with BMI whilst SNPs in YTHDF3 are associated with its gene expression. Conclusions: Our data show that expression of m6A regulators correlates with obesity, is adipose tissue depot-specific and related to clinical traits. Genetic variation in m6A regulators adds an additional layer of variability to the functional consequences.

Up-to-date on the evidence linking miRNA-related epitranscriptomic modifications and disease settings. Can these modifications affect cross-kingdom regulation?

The field of epitranscriptomics is rapidly developing. Several modifications (e.g. methylations) have been identified for different RNA types. Current evidence shows that chemical RNA modifications can influence the whole molecule's secondary structure, translatability, functionality, stability, and degradation, and some are dynamically and reversibly modulated. miRNAs, in particular, are not only post-transcriptional modulators of gene expression but are themselves submitted to regulatory mechanisms. Understanding how these modifications are regulated and the resulting pathological consequences when dysregulation occurs is essential for the development of new therapeutic targets. In humans and other mammals, dietary components have been shown to affect miRNA expression and may also induce chemical modifications in miRNAs. The identification of chemical modifications in miRNAs (endogenous and exogenous) that can impact host gene expression opens up an alternative way to select new specific therapeutic targets.Hence, the aim of this review is to briefly address how RNA epitranscriptomic modifications can affect miRNA biogenesis and to summarize the existing evidence showing the connection between the (de)regulation of these processes and disease settings. In addition, we hypothesize on the potential effect certain chemical modifications could have on the potential cross-kingdom journey of dietary plant miRNAs.

Exposure to glyphosate and tetrachlorvinphos induces cytotoxicity and global DNA methylation in human cells.

Two organophosphate pesticides-glyphosate and tetrachlorvinphos-have been announced as carcinogens to humans by various authorities, including the European Chemical Agency and the Environmental Protection Agency. We aimed to investigate molecular mechanisms associated with carcinogenicity and to examine changes in global m5C DNA methylation and cytotoxic potential in A549 lung epithelial cells in response to glyphosate and tetrachlorvinphos, and differential gene expression of m5C DNA methyltransferase genes in Sprague Dawley rats to Roundup (commercial formulation of glyphosate). Global m5C level significantly increased after 1500 µM glyphosate exposure for 24 h. We determined that exposure to tetrachlorvinphos did not significantly increase the m5C level in A549 cells for 24 h. Additionally, we did not observe significant DNA methylation alteration for both pesticides after 12 h exposure. In the animal study, we observed that DNA methyltransferase genes (DNMT3b and DNMT3a) showed significantly higher expression in Roundup-exposed rats than the control group in the liver and kidney. We also observed that a significant cytotoxic effect was determined after the treatment of the cells with higher concentrations of glyphosate and tetrachlorvinphos. Our results revealed that DNA methylation could be modified by exposure to glyphosate and that exposure to Roundup was associated with the differential expression level of m5C DNA methylation methyltransferase. Finally, exposure to both pesticides increased cytotoxicity.

RNA A-to-I editing, environmental exposure, and human diseases.

Epigenetic modifications have gained attention since they can be potentially changed with environmental stimuli and can be associated with adverse health outcomes. Epitranscriptome field has begun to attract attention with several aspects since RNA modifications have been linked with critical biological processes and implicated in diseases. Several RNA modifications have been identified as reversible indicating the dynamic features of modification which can be altered by environmental cues. Currently, we know more than 150 RNA modifications in different organisms and on different bases which are modified by various chemical groups. RNA editing, which is one of the RNA modifications, occurs after transcription, which results in RNA sequence different from its corresponding DNA sequence. Emerging evidence reveals the functions of RNA editing as well as the association between RNA editing and diseases. However, the RNA editing field is beginning to grow up and needs more empirical evidence in regard to disease and toxicology. Thus, this review aims to provide the current evidence-based studies on RNA editing modifying genes for genotoxicity and cancer. The review presented the association between environmental xenobiotics exposure and RNA editing modifying genes and focused on the association between the expression of RNA editing modifying genes and cancer. Furthermore, we discussed the future directions of scientific studies in the area of RNA modifications, especially in the RNA editing field, and provided a knowledge-based framework for further studies.

Assessment of the genotoxic potential of tetrachlorvinphos insecticide by cytokinesis-block micronucleus and sister chromatid exchange assays.

Tetrachlorvinphos is an organophosphate that is classified as a carcinogen in humans by several authorities. Due to very limited data regarding the genotoxic potential, we aimed to comprehensively investigate in vitro genotoxic potential of tetrachlorvinphos. We performed our study by applying the cytokinesis-block micronucleus cytome and sister chromatid exchange (SCE) assays to human peripheral blood lymphocytes. We evaluated micronucleus (MN) and SCE frequencies and cytokinesis-block proliferation index in both exposed and non-exposed lymphocytes. We also calculated the chromosomal instability level in response to exposure by combining the results of MN and SCE. We found that MN frequency did not increase with exposure to tetrachlorvinphos (0-50 µg/ml). In contrast, we observed that SCE frequencies significantly increased with exposure to ≥5 µg/ml tetrachlorvinphos. Furthermore, exposure to tetrachlorvinphos at concentrations of 50 µg/ml induced a significant increase in chromosomal instability level (p < 0.05). Cytokinesis-block proliferation index level did not significantly decrease in response to tetrachlorvinphos exposure. Our findings reveal that tetrachlorvinphos resulted in different DNA damages that were measured by two assays. Furthermore, our findings suggested that exposure to tetrachlorvinphos increased chromosomal instability that is a hallmark of many malignancies. We conclude that although tetrachlorvinphos does not significantly increase the MN level, the significant increase of both SCE and CIN frequencies indicates the genotoxic potential of tetrachlorvinphos in human peripheral lymphocytes. Additionally, tetrachlorvinphos is not cytotoxic in the range of tested concentrations.

Evaluation of DNA damages in congenital hearing loss patients.

In the current study, we aimed to compare the level of genetic damages measured as micronucleus (MN), nucleoplasmic bridge (NPB), and nuclear bud formation (NBUD) in congenital hearing loss patients (n = 17) and control group (n = 24). The cytokinesis-blocked micronucleus assay (CBMN) was applied to the blood samples to measure the frequency of the markers in both groups. The frequencies of MN of hearing loss patients were found to be consistently significantly higher than those obtained for the control group (p < 0.0001). Similarly, we found significantly higher frequency of NPB in patients was obtained for the patient group (p < 0.0001). Finally, the frequencies of NBUD in patients is significantly higher than the level measured in the control group (p < 0.0001). Furthermore, the age-adjusted MNL, BNMN, NPB, and NBUD frequencies in the patients were significantly higher than those obtained in the control group. We observed that the frequency of MN in patients was positively correlated with NBUD frequency which may indicate a common mechanism for these biomarkers in the patient group. We found, for the first time, that there were statistically significant higher levels of MN, NPB, and NBUD in sensorineural hearing loss patients. Since the markers we evaluated were linked with crucial diseases, our findings might suggest that sensorineural hearing loss patients are susceptible to several crucial diseases, especially cancer. Furthermore, the results demonstrated the significance of the MN, NPB, and NBUD level and thus provides a potential marker for the diagnosis of congenital hearing loss patients.

Genotoxic and cytotoxic effects of polyethylene microplastics on human peripheral blood lymphocytes.

Currently, we need emerging initial data regarding how plastic exposures affect cellular and molecular components and how such interactions will be crucial for human health. We aimed to determine the genotoxic and cytotoxic effects of microplastic (MPs,10-45 µm, polyethylene) on human peripheral lymphocytes by using the cytokinesis-block micronucleus cytome (CBMN) assay, which is a comprehensive method to reveal a range of mechanisms, not only diseases but also response to environmental exposures. We measured micronucleation (MN), nucleoplasmic bridge formation (NPB), and nuclear bud formation (NBUD) in human peripheral blood lymphocytes. We also measured the cytokinesis-block proliferation index (CBPI) to calculate cytostasis, which indicates cytotoxicity in lymphocytes treated with five different MPs concentrations for 48 h. Even lower concentrations of MPs increased the level of genomic instability. We found that the in vitro MP exposure significantly increased MN, NPB, and NBUD frequencies. Since we investigated the effect of larger particles relative to the lymphocytes, mechanic interaction of MPs with cells, the release of monomer and additives from MPs could be suggested as possible mechanisms accounting for increasing genomic instabilities. We did not observe a decrease in the cell proliferation index, indicating a lack of MPs' cytotoxic potential. To the best of our knowledge, our study is the first to identify MPs' genotoxic potential in human peripheral blood lymphocytes. We suggested further studies to investigate the genotoxic and cytotoxic potential of smaller plastics and the chronic effect of MP on the human population.

Impacts of elevated pCO2 on Mediterranean mussel (Mytilus galloprovincialis): Metal bioaccumulation, physiological and cellular parameters.

Ocean acidification alters physiology, acid-base balance and metabolic activity in marine animals. Near future elevated pCO2 conditions could be expected to influence the bioaccumulation of metals, feeding rate and immune parameters in marine mussels. To better understand such impairments, a series of laboratory-controlled experiment was conducted by using a model marine mussel, Mytilus galloprovincialis. The mussels were exposed to three pH conditions according to the projected CO2 emissions in the near future (one ambient: 8.10 and two reduced: 7.80 and 7.50). At first, the bioconcentration of Ag and Cd was studied in both juvenile (2.5 cm) and adult (5.1 cm) mussels by using a highly sensitive radiotracer method (110mAg and 109Cd). The uptake and depuration kinetics were followed 21 and 30 days, respectively. The biokinetic experiments demonstrated that the effect of ocean acidification on bioconcentration was metal-specific and size-specific. The uptake, depuration and tissue distribution of 110mAg were not affected by elevated pCO2 in both juvenile and adult mussels, whereas 109Cd uptake significantly increased with decreasing pH in juveniles but not in adults. Regardless of pH, 110mAg accumulated more efficiently in juvenile mussels than adult mussels. After executing the biokinetic experiment, the perturbation was sustained by using the same mussels and the same experimental set-up, which enabled us to determine filtration rate, haemocyte viability, lysosomal membrane stability, circulating cell-free nucleic acids (ccf-NAs) and protein (ccf-protein) levels. The filtration rate and haemocyte viability gradually decreased by increasing pCO2 level, whereas the lysosomal membrane stability, ccf-NAs, and ccf-protein levels remained unchanged in the mussels exposed to elevated pCO2 for eighty-two days. This study suggests that acidified seawater partially shift metal bioaccumulation, physiological and cellular parameters in the mussel Mytilus galloprovincialis.

Environmental epitranscriptomics.

Chemical modifications of RNA molecules have gained increasing attention since evidence emerged for their substantive roles in a range of biological processes, such as the stability and translation of mRNA transcripts. More than 150 modifications have been identified in different organisms to date, collectively known as the 'epitranscriptome', with 6-methyladenosine (m6A), 5-methylcytidine (m5C), pseudouridine and N1-methyladenosine (m1A) the most extensively investigated. Although we are just beginning to elucidate the roles of these modifications in cellular functions, there is already evidence for their dysregulation in diseases such as cancer and neurodevelopmental disorders. There is currently more limited knowledge regarding how environmental exposures affect the epitranscriptome and how this may mediate disease risk, but evidence is beginning to emerge. Here, we review the current evidence for the impact of environmental exposures such as benzo[a]pyrene, bisphenol A, pesticides, metals and nanoparticles upon RNA modifications and the expression of their 'writers' (methyl transferases), 'erasers' (demethylases) and 'readers'. We discuss future directions of the field and identify areas of particular promise and consider the technical challenges that are faced.

Environmental exposures and RNA N6-Methyladenosine modified long Non-Coding RNAs.

Recent advances in the field of RNA modifications and long non-coding RNAs (lncRNAs) have provided substantial evidence on important biological functions. LncRNAs are defined as longer than 200 nucleotides which are not translated into proteins. The term "epitranscriptome" refers to all modifications in RNA types. Adenine-6 methylation (m6A) is the most common, dynamic and prominent modifications in coding and non-coding RNAs and has critical and previously unappreciated functional roles. Accumulation evidence indicated the association between RNA m6A modification and cancer and nonmalignant diseases. Recent studies reported that several lncRNAs including MALAT1, MEG3, XIST, GAS5, and KCNK15-AS1 are subject to m6A modification. It can be suggested that lncRNAs modified by m6A modification have substantive roles in diseases. Currently limited data are available regarding how environmental exposure affects m6A-modified lncRNAs. Furthermore, we do not know the interaction of environmental exposure and m6A-modified lncRNAs in development of adverse human health outcomes. Thus, in this systematic review, we aimed to present the data of the studies that reported a significant association between environmental exposure and expression/DNA methylation of m6A-modified long non-coding RNAs.

Age and mitochondrial DNA copy number influence the association between outdoor temperature and cognitive function: Insights from the VA Normative Aging Study.

BACKGROUND: General cognitive function deteriorates with aging, a change that has been linked to outdoor temperature. Older individuals have reduced ability to adapt to changes in outdoor temperature than younger people. However, to what extent short-term changes in outdoor temperature interact with mitochondria to affect cognition in older people has not yet been determined. METHODS: Our study included 591 participants of the Normative Aging Study who underwent multiple examinations between 2000 and 2013. Cognitive function was evaluated via the Mini-Mental State Examination. Outdoor temperature was estimated at residential addresses 1 day before the examination using on a validated spatiotemporal temperature model. Mitochondrial DNA copy number (mtDNAcn) was determined using buffy coat samples. RESULTS: We found an interaction between temperature, age, mtDNAcn, and cognition. In individuals 84 years of age or older, cooler temperature was associated with low cognition (odds ratio = 1.2; 95% confidence interval = 1.05, 1.35 for a 1°C decrease in temperature; P = 0.007). We found higher odds ratio per 1°C decrease in temperature among individuals with lower mtDNAcn (ß3 = 0.12; 95% confidence interval = 0.01, 0.22; P interaction = 0.02). CONCLUSIONS: Our findings, albeit potentially underpowered, suggest that older individuals may be more susceptible to the influence of short-term temperature exposure on cognition. Moreover, the level of mtDNAcn may also modify the association between temperature and cognitive function, indicating a possible role of these cellular elements in this relationship.

Different working conditions shift the genetic damage levels of pesticide-exposed agriculture workers.

In the current study, we had two main purposes. Firstly, we aimed to compare genetic damages in the agricultural workers of two different types of environmental conditions including the greenhouse and open fields. Secondly, we aimed to compare genetic damages in the total agricultural workers as the exposed group (greenhouse and open field workers) (n = 114) and the non-exposed control group (n = 98) living in the same area in Canakkale, Turkey. For these purposes, we investigated the incidence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in peripheral blood lymphocytes. We observed that the frequencies of MN, NPB, and NBUD obtained for the greenhouse workers were statistically significantly higher than those obtained for the open field workers. When the results of the control group were compared with those of the total workers, there were statistically significant differences in terms of MN and NBUD frequencies. We found that age and MN were correlated at a significant level in both the agricultural workers and the control group. The MN frequency of the female workers was 1.5 times greater than that of the male workers, and it was a significant level in the agricultural workers.

Evaluation of circulating cell free DNA in plasma as a biomarker of different thyroid diseases / Avaliação do DNA circulante livre de células no plasma como um biomarcador de diferentes doenças da tireoide

Abstract Introduction: Many studies have been done on proteomics, genomics, epigenetic, immunogenetics in many body fluids. Among these, circulating cell-free DNA (ccfDNA) entered the literature in 1948, but it has not been studied for many years due to technological deficiencies. Following recent advances, geno-metastasis has been mentioned and new research is needed in this area. ccfDNA is known to be an important biomolecule in this regard. Objective: The presence of cell-free DNA in the circulatory system may offer a tremendous opportunity to provide novel biomarkers for thyroid diseases. This experimental study was conducted to determine the amount of ccfDNA in different thyroid diseases, then to evaluate whether the ccfDNA concentration varied between the disease groups and control group. Methods: In total, we included 121 individuals in the present study. We collected blood samples and then determined the ccfDNA concentration in plasma of collected blood samples from three groups: thyroiditis (n = 33), benign (n = 37), and malignant (n = 30) and from a control group (n = 21). Results: The median values of the ccfDNA groups were found as 1610, 1665, 1685 and 576 ng/mL for the thyroiditis, benign, malign, and control groups, respectively. Findings showed that the ccfDNA of the three groups was significantly higher than the control (p < 0.0001). Each group was compared in terms of ccfDNA and the p-values of benign-thyroiditis, benign-malign, and thyroiditis-malign were 0.09, 0.65, and 0.29, respectively. Conclusions: The clear differences between thyroid diseases and controls suggest that ccfDNA is worthy of attention as a biomarker for further evaluation of different thyroid diseases. Likewise, it might indicate a clear tendency that ccfDNA can also be used to distinguish different thyroid diseases.

Resumo Introdução: Muitos estudos foram realizados em proteômica, genômica, epigenética e imunogenética em vários fluidos corporais. Entre esses, o DNA circulante livre de células (cfDNA) despontou na literatura em 1948, mas não foi estudado por muitos anos devido a deficiências tecnológicas. Após recentes avanços, a genometástase é mencionada e novas pesquisas tornam-se necessárias nessa área. Nesse sentido, o cfDNA é conhecido por ser uma importante biomolécula. Objetivo: A presença de DNA livre de células no sistema circulatório pode oferecer uma excelente oportunidade para fornecer novos biomarcadores para doenças da tireoide. Este estudo experimental foi conduzido para determinar a quantidade de cfDNA em diferentes doenças da tireoide e então avaliar se a concentração de cfDNA variou entre os grupos com doença e o grupo controle. Método: No total, 121 indivíduos foram incluídos no estudo. Coletamos amostras de sangue e, em então, determinamos a concentração de cfDNA no plasma de amostras de sangue de três grupos: tireoidite (n = 33), benigno (n = 37) e maligno (n = 30) e de um grupo controle (n = 21). Resultados: As medianas dos valores dos grupos de cfDNA foram de 1.610, 1.665, 1.685 e 576 ng/mL para os grupos tireoidite, benigno, maligno e controle, respectivamente. Os achados mostraram que o cfDNA dos três grupos com doença era significativamente maior do que o do grupo controle (p < 0,0001). Cada grupo foi comparado em termos de cfDNA e os p-valores de benigno-tireoidite, benigno-maligno e tireoidite-maligno foram de 0,09, 0,65 e 0,29, respectivamente. Conclusões: Como resultado, as óbvias diferenças entre as doenças da tireoide e os controles sugerem que o cfDNA é digno de atenção como um biomarcador para avaliação adicional das diferentes doenças da tireoide. Da mesma forma, isso pode indicar uma clara tendência de que o cfDNA também pode ser utilizado para distinção das diferentes doenças da tireoide.

Assessment of the genotoxic potential of a migraine-specific drug by comet and cytokinesis-block micronucleus assays.

Background: Eletriptan is a migraine-specific drug-containing the triptan group. In terms of drug safety, the present study aimed to investigate the genotoxic potential of eletriptan.Research design & methods: We conducted our study by using the cytokinesis-block micronucleus cytome (CBMN) assay, a comprehensive method for measuring micronucleus formation, and a sensitive method for detecting DNA-strand breaks. In the assay, cytokinesis-block proliferation index and the frequency of micronuclei were evaluated in lymphocytes treated with three different concentrations (1, 10 and 25 µg/ml) of eletriptan for 48 hours. In comet assays, DNA damage was evaluated in leucocytes treated with three different concentrations (1, 10 and 25 µg/ml) of eletriptan for an hour.Results: Eletriptan did not induce cytotoxicity nor any increased micronuclei frequencies. While the comet parameters % DNA in tail, tail moment, and the olive moment was found to be significantly increased at 10 and 25 µg/ml, the cytokinesis-block proliferation index values were not.Conclusion: These findings suggest that eletriptan is non-cytotoxic but potentially weakly genotoxic at higher concentrations (10 and 25 µg/ml).