Mobile phone specific radiation disturbs cytokinesis and causes cell death but not acute chromosomal damage in buccal cells: Results of a controlled human intervention study.

Several human studies indicate that mobile phone specific electromagnetic fields may cause cancer in humans but the underlying molecular mechanisms are currently not known. Studies concerning chromosomal damage (which is causally related to cancer induction) are controversial and those addressing this issue in mobile phone users are based on the use of questionnaires to assess the exposure. We realized the first human intervention trial in which chromosomal damage and acute toxic effects were studied under controlled conditions. The participants were exposed via headsets at one randomly assigned side of the head to low and high doses of a UMTS signal (n = 20, to 0.1 W/kg and n = 21 to 1.6 W/kg Specific Absorption Rate) for 2 h on 5 consecutive days. Before and three weeks after the exposure, buccal cells were collected from both cheeks and micronuclei (MN, which are formed as a consequence of structural and numerical chromosomal aberrations) and other nuclear anomalies reflecting mitotic disturbance and acute cytotoxic effects were scored. We found no evidence for induction of MN and of nuclear buds which are caused by gene amplifications, but a significant increase of binucleated cells which are formed as a consequence of disturbed cell divisions, and of karyolitic cells, which are indicative for cell death. No such effects were seen in cells from the less exposed side. Our findings indicate that mobile phone specific high frequency electromagnetic fields do not cause acute chromosomal damage in oral mucosa cells under the present experimental conditions. However, we found clear evidence for disturbance of the cell cycle and cytotoxicity. These effects may play a causal role in the induction of adverse long term health effects in humans.

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva.

BACKGROUND: The isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages - volume, time, cost, and yield are a few notable ones. OBJECTIVE: We aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. METHODS: Five DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. RESULTS: Out of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. CONCLUSION: In the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Cytotoxicity, oxidative stress, and inflammatory response of smokeless tobacco extracts and cytotoxicity of combustible cigarette whole smoke in a 3D oral organotypic buccal cell model.

Oral disease is frequently associated with viral and environmental exposures and oral hygiene. The use of tobacco is a risk factor in the development of oral disease. Cytotoxicity, inflammatory response, and oxidative stress have been reported to have a role in the development of oral disease. These three endpoints were evaluated in a 3D human oral buccal model, EpiOral™, following exposure to CORESTA reference smokeless tobacco products (CRPs) and cigarette whole smoke. CRPs for Swedish style snus (CRP1), moist snuff (CRP2), and dry snuff (CRP3) were each extracted in complete artificial saliva (CAS) with a ratio of 300 mg CRP to 1 mL of CAS. Each of the CRP extracts (15-300 mg/ml) were applied to the apical side of a 3D organotypic buccal cell model for 24 or 48 h continuously, then cytotoxicity (LDH), oxidative stress (8-isoprostane), and inflammatory response (IP10, IL-1α, and IL-8) were measured. Experiments with 3R4F cigarettes were conducted by exposing the buccal tissues to whole smoke for a maximum of 2.5 h. Cytotoxicity (MTT) was measured 24 h post-exposure. Exposure of buccal tissues to whole smoke from a cigarette induced a dose-dependent cytotoxic response. In contrast, the CRP extracts elicited minimal cytotoxicity (<15%) when compared to CAS (vehicle control), but time- and dose-dependent effects on oxidative stress and inflammatory response were observed. Collectively, these data demonstrate that a 3D organotypic buccal human model may be used to assess biological mechanisms (MOAs) involved in the development of oral disease following exposure to smokeless tobacco products and may be applicable for differentiation between tobacco product categories.

Comparison of Telomere Length between Buccal Cells and Blood Cells.

Telomere length (TL) in blood cells is commonly used as a proxy for TL in other tissue types. The source of DNA of adequate quality and quantity is important for TL analysis. Compared to blood cells, buccal cells easy for genomic DNA preparation would facilitate the rapid and reliable TL analysis. However, the feasibility of buccal cells for TL analysis remains yet unestablished. We characterized TL of buccal cells and blood cells collected from 52 individuals using buccal cell swabs and fingertip sticks. Relative TL (RTL) determined by quantitative PCR showed that there is a strong correlation between buccal RTL and blood RTL (r=0.877, p<0.001), suggesting that buccal cells are adequate sources of DNA for TL analysis. The validity of sampling using buccal cell swabs provides simple operation and good reproducibility for TL analysis, that overcomes the discomfort and risk of infection caused by blood sampling.

Genomic instability in Buccal mucosal cells of children living in abnormal conditions from Santos-Sao Vicente Estuary.

The aim of this study was to evaluate genomic instability and cytotoxicity in buccal mucosa cells of children living in abnormal conditions from Santos Sao Vicente estuary. The study area is located between coordinates 23°58'11.8"S and 46°24'26.3"W, in the southwestern zone of the Sao Paulo State, Brazil. A total of 40 children was distributed into two groups: exposed and non-exposed groups. The frequency of micronuclei increased to buccal mucosa cells of children living in Santos Sao Vicente estuary when compared to the non-exposed group (p < 0.05). No remarkable differences on buccal cells were found inpyknosis, karyorrhexis and karyolysi between groups. Taken together, our results suggest that children living in contaminated areas comprise a high group for genomic instability on buccal mucosa cells. Given that the current investigation is a preliminary study, further analysis with a larger sample of children is interesting as a future perspective.

Effect of Angiotensin-I Converting Enzyme Gene Insertion/Deletion Polymorphism on genome instability in children living in Russian Arctic.

Main risks of arterial hypertension manifest in childhood. Children living in the Far North are especially susceptible to this. There is a need for an inexpensive, non-invasive and simple diagnosis of the risk of childhood pathologies. It was previously found that the genotype DD of the in/del polymorphic marker of the ACE gene is found in people at risk of developing cardiovascular pathologies. Buccal micronucleus cytome assay and genetic analysis were used in the work. In total, 77 schoolchildren from the city of Apatity, aged 15-17 years old, were examined. We have shown that carriers of the D allele have a tendency to an increase in the frequency of cells with micronuclei. In the case of homozygous I/I variant, the frequency of occurrence of cells with karyopycnosis is significantly higher than in carriers of allele D. Polymorphic marker in/del of the ACE gene is associated with apoptotic changes in the cells of the studied children. The in/del polymorphic marker of the ACE gene can be used as a prognostic marker of the processes of genome destabilization at the early stages of development of the human body.

Added value of buccal cell FISH analysis in the diagnosis and management of Turner syndrome.

STUDY QUESTION: Is there an added diagnosis value of buccal cell FISH analysis compared with blood lymphocyte chromosomal investigations in patients with Turner syndrome (TS)? SUMMARY ANSWER: Buccal cell FISH analysis, a non-invasive technique, modified the chromosomal results obtained with the blood karyotype in 17 patients (12%) of our cohort. WHAT IS KNOWN ALREADY: Few studies have evaluated buccal cell FISH analysis and compared them with blood karyotype in patients with TS. STUDY DESIGN, SIZE, DURATION: A prospective, monocentric cohort study was conducted in a rare diseases centre (CMERC) between July 2017 and August 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 142 adult patients with TS, and at least 5% 45,X cells in a previous blood karyotype, were recruited. All the patients' files were included in the CEMARA database. This national database has been declared to the French data protection agency (CNIL approval number 1187326). In compliance with French law, consent regarding non-opposition to collect and use the data was obtained from each patient. A FISH analysis on a buccal smear was performed. MAIN RESULTS AND THE ROLE OF CHANCE: The percentage of 45,X cells was identical between the two tissues in only 32.4% of cases. The discrepancy was higher than 41% for 12% of the cohort. The percentage of 45,X cells was higher in blood in 53 (37.3%) patients, and higher in buccal cells in 43 (30.3%) of cases. In 17 (12%) cases, the blood karyotype had to be reconsidered in regard to the buccal cell analysis. LIMITATIONS, REASONS FOR CAUTION: It would have been interesting to evaluate karyotypes in cells from other tissues such as cells from skin biopsy or from the urinary tract and even from blood vessels or gonads in case of surgery and to compare them with each patient's phenotype. However, most of the time, these tissues are not available. WIDER IMPLICATIONS OF THE FINDINGS: Although blood lymphocyte karyotype remains the gold standard for the diagnosis of TS, buccal cell FISH analysis is an efficient tool to evaluate the global chromosomal constitution in these patients, thus allowing them to have better care and follow-up. For instance, identifying a Y chromosome can prevent the occurrence of a gonadoblastoma, as gonadectomy should be discussed. On the other hand, finding normal XX cells in a patient with a previous diagnosis of homogenous 45,X TS, may be psychologically helpful and relevant for gynaecological care. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was sought for the study. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Age related micronuclei frequency ranges in buccal and nasal cells in a healthy population.

BACKGROUND: Micronuclei (MNs) are extranuclear DNA-containing bodies and determining MN frequencies is a measure of genomic instability. An age-related increase in MN frequencies in lymphocytes has been quantified, but this effect has not yet been measured in nasal and buccal cells. METHODS: We determined the effect of age on the MN frequency distributions in buccal and nasal cells among a sample of a general adult population in Switzerland. To maximize the power to detect an effect of age in our population study, we recruited preferentially younger and older working age adults. We harvested buccal and nasal cells from 32 young (19-36 year) and 33 working age (47-71 years) participants. The collected cells were washed, centrifuged, and stained (Feulgen) before microscopic manual counting in 2000 cells. Based on these results, we developed an age-dependent background MN frequency chart to help interpret an individual's MN frequency score as an early signal for the effect of genotoxic exposure. RESULTS: MN frequencies were respectively 0.53‰ and 0.47‰ for buccal and nasal among the younger and 0.87‰ and 1.03‰ in the older working age group. This corresponded to a multiplicative slope of 14% and 20% per 10 years of age for buccal and nasal cells, respectively. CONCLUSION: Based on our study results, we are able to propose an approach for interpreting an individual's MN screening results.

Buccal Mucosa Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) precipitation in the elderly population increases the need for sensitive biomarkers that can be applied to large population screening. Buccal cells can be obtained easily, noninvasively, and contain many proteins related to cerebral processes. Hence, they offer an ideal candidate for AD biomarker discovery. The purpose of this study is to provide an overview of the current research landscape covering both clinical and methodological issues. A brief summary is given on related laboratory techniques to ascertain protein concentration changes due to AD. At the end, we describe a protocol designed in our laboratory for disease early diagnosis.

Early genotoxic damage through micronucleus test in exfoliated buccal cells and occupational dust exposure in construction workers: a cross-sectional study in L'Aquila, Italy.

AIM: The city of L'Aquila (central Italy) was hit by a strong earthquake in 2009 that caused the collapse of several buildings, deaths and injured people. In the following years, a great number of building sites were activated, building workers resulted intensely exposed and represent a relevant target for research on environmental mutagenesis and epidemiological surveillance. Cells of buccal mucosa are considered an appropriate site for early detecting of cytogenetic damage, since it represents the first barrier in inhalation or ingestion and can metabolize carcinogenic agents into reactive chemicals. Our study is aimed 1) at comparing the early genotoxic damage as measured by the buccal mucosa micronucleus test in two subgroups of workers defined by different occupational exposure and 2) at evaluating possible confounding variables such as lifestyle factors. METHODS AND RESULTS: A cross-sectional study was conducted in L'Aquila, on 24 outdoor workers (OWs) highly exposed on the construction sites and 26 indoor workers (IWs), all subjected to the compulsory occupational surveillance system, in the period 2017-2018. Buccal cells samples were collected and, based on the Micronucleus test, the exfoliated cells were classified in respect of nuclear changes observed. Moreover, a self-report questionnaire composed of 84 items, was administered to the workers. RESULTS: Significant differences were observed between Exp+ (OWs) and Exp- (IWs) in the number of the analyzed cells (expressed as mean value out of 1000 cells): respectively 954.46 vs 990.06 normal cells, (p < 0.001); 19.79 vs 4.95 micronucleated cells, as marker of chromosomal damage (p < 0.001); 13.93 vs 8.96 binucleated cells, as marker of failed cytokinesis (p < 0.001); 2.09 vs 1.18 karyolytic cells, as marker of cell death and damaged DNA (p < 0.05). According with a multivariate regression analysis, in addition to the job exposure (OW vs IW, beta = 12.221, p < 0.001), the only variable independently associated with an increase in Micronuclei (MNs) is the smoking habit (OWs vs IWs, beta = 6.683, p < 0.001) which, even if not associated with dust exposure, worsens cell integrity. Moreover, this worsening effect is weaker in workers not exposed to the site dust (moderation effect). Within social demographic factors, the high educational level only apparently seems to affect MNs number: even if unbalanced in favor of IWs vs OWs, this variable resulted a confounder, since its effect disappears when the interaction between these two factors is considered, because it is a covariate of smoking habit as well as of the job condition. CONCLUSION: Despite some limitation, our findings clearly confirm the role of occupational exposure as a marker of cytogenetic damage associated with MNs number in construction workers. Moreover, smoking status appears as the only other investigated factor independently associated to the outcome. The statistical model, in addition, highlights possible moderation and confounding effects, such as interaction between smoking and occupational exposure and the unbalanced school education level in workers. Micronucleus test in exfoliated buccal cells would be considered a suitable method for studying the early genotoxic damage in the construction occupational setting as well as in evaluating the efficacy of preventive practices.

Hair straightening products and the risk of occupational formaldehyde exposure in hairstylists.

Hair straitening products are widely used by hairstylists. Many keratin-based hair smoothing products contain formaldehyde. This study aimed to investigate occupational formaldehyde exposure among hairstylists dealing with hair straightening products and the relation between genotoxicity biomarkers and the short-term formaldehyde exposure concentrations and the working years. The study was carried out in Cairo, Egypt on 60 hairstylists use hair straightening products divided into two groups according to the working years. All hairstylists were subjected to micronucleus (MN) frequencies in both epithelial buccal cells (EBC) and peripheral blood lymphocytes (PBL). Fifteen-minute (min) formaldehyde exposure concentrations were measured at workplace during hair straightening procedure. Fifteen-minute formaldehyde concentrations in both groups exceeded the National Institute for Occupational Safety and Health and the American Conference of Governmental Industrial Hygienist thresholds levels. The MN frequencies in EBC and PBL showed a significant increase in group II in comparison to control and group I, which in turn showed a significant increase in MN frequency in PBL and a nonsignificant increase in the MN frequency in EBC when compared to control. A positive correlation was found between genotoxicity biomarkers and working years. Occupational exposures to hair straightening products in the studied hairstylist were found to expose them to formaldehyde concentrations that exceeded the standard limits. All selected genotoxicity biomarkers showed a significant increase in exposed workers and were positively correlated to the duration of exposure.

Altered Fluorescence of Buccal Cells as a Candidate Biomarker for Areca Nut Chewing.

Background: Betel nut is used by an estimated 600 million people globally and is the 4th most widely used psychoactive substance in the world. Its use has been shown to cause oral and esophageal cancers. Therefore, cessation programs are needed in which an effective biomarker can be employed. Objectives: Buccal cells are highly exposed to the betel nut during its use and are also easy to collect. However, it is unknown if there are significant changes to these cells upon exposure or how long any changes may last as the turnover of buccal cells is relatively fast. We sought to determine if optical changes could be detected on buccal cells after exposure to betel nut and if detected, how long these changes were sustained. Methods: Flow cytometry was employed to determine whether fluorescence intensities differ between buccal cells exposed to betel nut and naïve cells. We further characterized the optical signature of buccal cells exposed to betel nut and other polyphenol-rich substances using lambda scans performed on a laser scanning confocal microscope. Results: We demonstrate that the fluorescence of betel nut exposed cells is greater than that of cells exposed to other optically active compounds such as polyphenol-rich foods. We also demonstrate that the fluorescence spectra of betel nut quid exposed cells are distinct from that of cells exposed to other polyphenol-rich substances. Conclusions: We conclude that detecting the altered fluorescence of buccal cells following exposure to betel nut quid may serve as a candidate biomarker for betel nut quid use.

Chemical Markers for Short- and Long-Term Areca Nut Exposure.

Background: Areca nut (AN) chewing causes oral cancer. AN cessation programs are the most effective approach to reduce AN chewing induced cancers but require biomarkers to determine program compliance and success. Objectives: To explore chemical markers for short- and long-term AN exposure using non-invasively collected saliva, buccal cells (BCs), and scalp hair of chewers. Methods: Saliva was collected from a male chewer before and up to 2 days after AN chewing. Saliva was separated into supernatant and pellet (BCs) then analyzed by spectrophotometry and liquid chromatography (LC) with UV/VIS detection. Scalp hair was collected from four chewers and analyzed for areca alkaloids using direct analysis in real time-tandem mass spectrometry (DART-MSMS). Results: The red pigmented saliva after chewing showed no valuable signals when either the saliva supernatant or pellet (BCs) were analyzed by spectrophotometry. Saliva analysis by LC-UV/VIS showed diagnostically valuable signals at 488 nm up to 5 and 24 h post chewing in the supernatant and pellet, respectively. DART-MSMS analysis detected two of the four AN specific alkaloids (arecoline and arecaidine) in male but none in female hair. Conclusions/Importance: LC-UV/VIS analysis of the red pigments extracted from saliva and BCs after AN chewing showed distinct signals up to 24 h post chewing while DART-MSMS analysis in BCs and scalp hair showed selective signals of AN alkaloids for several weeks or months after AN exposure. Chemical hair treatment might prevent detection of areca alkaloids in hair. AN cessation trials and other programs now have essential tools for bioverification.

Micronucleus Assay: The State of Art, and Future Directions.

During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.

Interactions Among Odorants, Phenolic Compounds, and Oral Components and Their Effects on Wine Aroma Volatility.

To determine the impact of oral physiology on the volatility of typical wine aroma compounds, mixtures of a synthetic wine with oral components (centrifuged human saliva (HS), artificial saliva with mucin (AS), and buccal epithelial cells (BC)) were prepared. Each wine type was independently spiked with four relevant wine odorants (guaiacol, ß-phenyl ethanol, ethyl hexanoate, and ß-ionone). Additionally, the impact of four types of phenolic compounds (gallic acid, catechin, grape seed extract, and a red wine extract) on aroma volatility in the HS, AS, and BC wines was also assessed. Static headspace was measured at equilibrium by solid phase microextraction-GC/MS analysis. Results showed a significant impact of oral components on the volatility of the four tested odorants. Independently of the type of aroma compound, aroma volatility was in general, higher in wines with BC. Moreover, while guaiacol and ethyl hexanoate volatility was significantly lower in wines with HS compared to wines with AS, ß-ionone showed the opposite behavior, which might be related to metabolism and retention of mucin, respectively. Phenolic compounds also showed a different effect on aroma volatility depending on the type of compound and wine. Gallic acid had little effect on polar compounds but it enhanced the volatility of the most hydrophobic ones (ethyl hexanoate and ß-ionone). In general, flavonoid type polyphenols significantly reduced the volatility of both polar (guaiacol and ß-phenyl ethanol) and hydrophobic compounds (ß-ionone in HS and BC wines), but through different mechanisms (e.g., π-π interactions and hydrophobic binding for polar and apolar odorants respectively). On the contrary, flavonoids enhanced the volatility of ethyl hexanoate, which might be due to the inhibition exerted on some salivary enzymes (e.g., carboxyl esterase) involved in the metabolism of this odorant molecule.

Induction of chromosomal damage in exfoliated buccal and nasal cells of road markers.

Road markers are exposed to various chemicals and particles. The aim of this study was to determine whether road worker exposure induceschromosomal damage which is indicative for increased cancer risks. Micronucleus (MN) cytome assays were thus conducted with exfoliated nasal and buccal cells collected from 42 workers and 42 matched controls. The frequencies of MN (reflecting chromosomal aberrations), nuclear buds (NBuds; reflecting gene amplifications) and binucleated cells (BN; reflecting disturbed mitosis) were scored. Further, the rates of nuclear anomalies indicative of acute cytotoxicity (condensed chromatin, karyorrhexis, karyolysis, pyknosis) were evaluated. Data demonstrated marked induction of MN, NBuds, and BN by 1.34-fold, 1.24-fold and 1.14-fold in buccal cells. In nasal cells, only MN frequencies were elevated, 1.23-fold. These effects were paralleled by increased rates of condensed chromatin, karyorrhexis and karyolysis in both cell types. The effects were more pronounced in individuals who had worked for more than 10 years while smoking did not produce synergistic responses. This is the first investigation concerning the induction of genetic damage in road markers and the results are suggestive for enhanced cancer risks. It is conceivable that exposure to silica dust (known to induce cancer and genetic damage) and/or benzoyl peroxide which forms reactive radicals may be associated with the observed genetic damage in road workers. Further investigations of the cancer risks of these workers are warranted.

In vivo evaluation of fluoride and sodium lauryl sulphate in toothpaste on buccal epithelial cells toxicity.

OBJECTIVES: The present study addresses the effect of fluoride and sodium lauryl sulphate content of toothpaste on oral epithelial cells in vivo conditions. SUBJECTS AND METHOD: Forty volunteers were assigned into two experimental groups, each of them applying the different brand of toothpaste. Every group has been using three different types of toothpaste (non-fluoride and non-SLS, fluoride and non-SLS, and the fluoride and SLS) of the same brand for 6 months, each for 2 months. The buccal epithelial cells were sampled at baseline and 30, 60, 90, 120, 150 and 180 days after the beginning of the research. Effect on DNA damage was analyzed by micronucleus assay Results: After 60 days of use, for both tested kinds of toothpaste with fluoride and without SLS, all studied parameters were not significantly different from the results obtained at the time when the participants used a non-fluoride toothpaste. While, after 60 days of use, for one kind of toothpaste with SLS and fluoride, was observed significantly higher incidence of pyknotic cells (2.20 ± 0.95, 0.00 ± 0.00 vs. 0.05 ± 0.22, respectively; p = .001), cells with karyorrhexis (2.35 ± 1.14, 0.85 ± 0.93 vs. 0.40 ± 0.68, respectively; p = .001), and nuclear buds (1.35 ± 0.68, 0.45 ± 0.51 vs. 0.45 ± 0.60, respectively; p = .001), compared to toothpastes of the same brand with fluoride and without SLS, and without fluoride and without SLS, for the same period. CONCLUSIONS: Based on the results, can be concluded that there is no fluorine-dependent cytotoxic or genotoxic effect, while SLS dentifrice increases the number of nuclear morphological changes in buccal epithelial cells.

[Buccal epitelium cytogenetic status in schoolchildren living in high and middle latitudes.]

The Russian northern regions development is associated with the extraction and processing of natural resources, which leads to environmental pollution and makes the task of sanitary-hygienic monitoring relevant. Buccal micronucleus cytome assay is one of the toxicological methods for human population studies. Studies on the inter-latitudinal comparison of Buccal micronucleus cytome assay are lacking, therefore there are difficulties in comparing the results obtained in the Russian Arctic with data from more southern regions. The aim of this study is to compare cytogenetic abnormalities in the buccal epithelium in two groups of older schoolchildren living in high and middle latitudes. The study was conducted in the city of Apatity (Murmansk region, 67 ° 34'03 ″ N, 33 ° 23'36 ″ E) and the city of Serpukhov (Moscow region, 54 ° 54 '56 ″ N, 37 ° 24 '40 "E). A total of 61 children were examined: 41 children from the Apatity and 20 children from the Serpukhov (16-18 years old). The Buccal micronucleus cytome assay was carried out according to an international protocol. It was shown that the average frequency values of the cells with micronucleus in the comparison groups of schoolchildren living in high and middle latitudes did not significantly differ and did not exceed the values for the average population norm. The frequency of cells with nuclear buds and two nucleus was significantly higher in the group of schoolchildren living in middle latitudes, which, in turn, is compensated by a higher rate of elimination of cells with impaired. Therefore, when comparing Buccal micronucleus cytome assay data, it is quite possible not to take into account the breadth of the studied groups.

Global methylation profiles in buccal cells of long-term smokers and moist snuff consumers.

PURPOSE: Alternations in gene methylation and other epigenetic changes regulate normal development as well as drive disease progression. The aim of this study is to investigate global methylation changes in the buccal cells of smokers and smokeless tobacco users. MATERIALS AND METHODS: Generally healthy adult male subjects were recruited into smoker (SMK), moist snuff consumer (MSC) and non-tobacco consumer (NTC) cohorts (40 subjects/cohort) (ClinicalTrials.gov Identifier: NCT01923402). Global methylation profiling was performed on Illumina 450 K methylation arrays using buccal cell DNAs. RESULTS: The SMK cohort exhibited larger qualitative and quantitative changes relative to MSC. Approximately half of the differentially methylated 1252 gene loci were grouped as combustible tobacco-related (CTR) signatures and a third of the changes, tobacco-related (TR) signatures, were associated with smoking. Very few (41) differentially methylated gene loci were exclusively associated with moist snuff use and were designated as moist snuff-related (MSR) signature. Pathway enrichment analyses revealed that developmental and immune response pathways, among others, were impacted due to tobacco use. CONCLUSIONS: Chronic cigarette smoking causes hyper- and hypo-methylation of genes that could contribute to smoking-related diseases. These results help place combustible and non-combustible tobacco products along a risk continuum and provide additional insights into the effects of tobacco consumption.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

BACKGROUND: ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. METHODS: We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. RESULTS: Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. CONCLUSION: The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.