Nuclear Buds Research Articles

DNA damage assessment with buccal micronucleus cytome assay in Turkish coal miners.

The aim of this study was to assess DNA damage in Turkish coal miners with the buccal micronucleus cytome (BMCyt assay as the least invasive and therefore most practical method that may find wider application in coal miner biomonitoring. Buccal epithelial cell samples were taken from 54 coal miners and 42 controls from Zonguldak, Turkey to establish their micronucleus (MN), binucleus (BN), condensed chromatin (CC), karyorrhectic (KHC), karyolytic (KYL), nuclear bud (NBUD), and pyknotic (PYC) frequencies. We also analysed the effects of confounding factors such as age, years of work at the mine, smoking, alcohol drinking, and use of protective equipment on differences in MN frequencies. Two miners had confirmed and three suspect pneumoconiosis, whereas 49 displayed normal chest radiographs. MN, BN, KHC, and NBUD frequencies were significantly higher in coal miners than controls. Years of work at the mine also showed a significant effect on buccal MN frequencies in coal miners, but we found no correlation between MN frequencies and age, smoking, and alcohol consumption. In conclusion, BMCyt assay proved itself an accurate and practical screening method, as it can detect DNA damage much earlier than pneumoconiosis develops.

Localized Toxicity in Patients with Fixed Orthodontic Appliance:A Case Control Study

Fixed orthodontic appliances made of metal alloys are routinely used in dental practise to correct malocclusion. Oral environment favours the biodegradation of metal alloys. The discharged metal ions accumulate cumulatively on the adjacent tissues and results in toxic effects on cellular metabolism and DNA stability. The study was designed to evaluate localized genetic and cytokinetic toxicity due to fixed orthodontic appliance by measuring micronuclei frequency and other nuclear abnormalities in the buccal mucosal cells. Subjects with orthodontic appliance were selected as exposure cohort (n=20) and subjects without orthodontic appliance were selected as comparative cohort (n=20). Buccal smears were collected and DNA specific feulgen stain was used. Micronuclei, nuclear bud and binucleate cells frequency were recorded and compared between the study groups. We found a significant increase in the frequency of cells with micronuclei (p<0.001) and binucleate cells(p=0.002) in the exposure cohort compared to controls. The increase in the indicators of genotoxicity and cytokinetic toxicity in the exposed cohort compared to controls could be attributed to the exposure to the metal ions released from long term orthodontic appliance treatment. Increase in binucleate cells in subjects with fixed orthodontic appliance was demonstrated for the first time in our study. The cellular changes due to toxicity could be the predisposing factor for the occurrence of immune mediated oral mucosal lesions in orthodontic patients.

Fridericia platyphylla (Cham.) L.G. Lohmann root extract exerts cytotoxic and antiproliferative effects on gastric tumor cells and downregulates BCL-XL, BIRC5, and MET genes.

Fridericia platyphylla (Cham.) L.G. Lohmann (FP) has cytotoxic, anti-inflammatory, and analgesic properties. We aimed to characterize the cytotoxic and antiproliferative effects of FP extract on normal (GAS) and tumor-derived (ACP02 and HepG2) cell lines. The effective concentrations (EC50s) by tetrazolium bromide assay (MTT) were 56.16, 43.68, and 42.57 µg mL-1 and 69.38, 41.73, and 52.39 µg mL-1 by neutral red assay for GAS, ACP02, and HepG2 cells, respectively. The extract decreased nuclear division indices, which was not reflected in cell proliferation curves. Flow cytometric analyses showed that even 30 µg mL-1 extract (shown to be noncytotoxic by MTT assay) increased the sub-G1 population, indicating cell death due to apoptosis and necrosis. A cytokinesis-block micronucleus cytome assay showed that 30 µg mL-1 of the extract increased the frequency of nuclear buds in tumor cells. Real-time quantitative polymerase chain reaction showed CCND1 upregulation in doxorubicin-treated GAS cells and BCL-XL, BIRC5, and MET downregulation in 5 or 30 µg mL-1 in FP extract-treated ACP02 cells. In conclusion, FP extract modulated apoptosis- and cell cycle-related genes and presented selective cytotoxicity toward tumor cells that deserves further investigation by testing other cell types. Our results demonstrated that even medicinal plants exert adverse effects depending on the extract concentrations used and tissues investigated.

Evaluation of the genotoxic potential of cypermethrin, chlorpyrifos and their subsequent mixture, on cultured bovine lymphocytes.

Cypermethrin (CYP) and chlorpyrifos (CPF) are insecticides/parasiticides used in the production of fruits, vegetables and beef cattle. These substances or their metabolites are frequently reported as residues in food, whose consumption in a diet implies a genotoxic risk. The potential for chronic toxicity of CYP and CPF is unclear, and only a few genotoxicological evaluations based on their mixture have been performed. The aim of this study was to evaluate the genotoxic potential of CYP, CPF and CYP+CPF in five concentrations, from 5.9 to 175 μg/mL, on bovine lymphocytes. By means of the cytokinesis-block micronucleus cytome assay, a decrease in the cell proliferation index was observed (r=-0.89 p=0.04); and also an increase in the frequencies of binucleated cells (BN) with micronuclei (BNMn) (r=0.93, p=0.02) and BN with nuclear buds (BNBud) (r=0.778 p=0.04), depending on the concentrations of CPF. An increase in BNMn frequencies was observed as a function of CYP concentrations (r=0.89, p=0.04) and also of the CYP+CPF mix (r=0.99, p=0.008). CYP caused greater genotoxic damage (BNMn) than CPF and the mixture on bovine lymphocytes. Cells with simultaneous presentation of micronuclei and nuclear buds were detected, as well as cells with irregular nuclei, something never previously reported, whose origin and significance should be investigated. The genotoxic effect of chlorpyrifos, cypermethrin and their mixture on bovine lymphocytes was observed. We recognized the value of the use of primary bovine cultures, animal species adjacent to man in the food chain, for genotoxicity studies.

Thermal stress causes nuclear and cellular abnormalities of peripheral erythrocytes in Indian major carp, rohu Labeo rohita.

Rise of water temperature as a consequence of global warming is anticipated to affect the physiological activities of fish, especially in tropical regions. In the present experiment, we exposed the Indian major carp, rohu Labeo rohita to three different temperature regimes (30 °C as control and 33 °C and 36 °C) for 60 days and observed the effects of these temperature on: major hemato-biochemical indices (Hemoglobin; Hb, Red blood cell; RBC, White blood cell; WBC and blood glucose levels), erythrocytic nuclear abnormalities (ENA), and erythrocytic cellular abnormalities (ECA) of peripheral erythrocytes along with the formation of differential leucocytes in the blood. Fish were sacrificed at day 7, 15, 30 and 60 after the start of exposure to the temperature regimes. Hb decreased significantly on days 7 and 15 at 36 °C. Throughout the study period, the decrease of RBC and increase of WBC were significant at 36 °C. Blood glucose level increased significantly initially at day 7 but decreased significantly at day 60 at 36 °C. Frequencies of ENA (binucleated, nuclear bud, nuclear bridge, karyopyknosis and notched nuclei) and ECA (twin, fusion, echinocytic, spindle, tear drop and elongated shaped) were significantly increased at the highest temperature (36 °C) at almost all of the sampling days. In the case of differential leucocyte count, high temperature caused a significant increase in the number of neutrophils and a significant decrease in the number of lymphocytes. Overall, these results indicate that chronic exposure to high temperature (36 °C) induces a number of stress responses in rohu and that temperature should be kept below 36 °C in the aquaculture setting to avoid damage to the fish.

Epithelial-mesenchymal transition of tumour buds: A histopathological parameter with potential implication in oncological therapy of colorectal carcinoma

Abstract Background The most recent histopathological guideliness recommend evaluation of tumor buds, as an independent prognostic factor of colorectal carcinoma (CRC). In the present paper, we examined the immunoprofile of tumor buds, using three markers of epithelial mesenchymal transition (EMT): E-cadherin, β-catenin and vimentin, which were evaluated in both tumor core and buds. Methods In 157 CRCs specimens, consecutive cases with curative resection, without preoperative therapy, three molecular subtypes were defined: epithelial (positive for E-cadherin, membrane expression for β-catenin and negative for vimentin, in core and buds), mesenchymal (loss of E-cadherin staining and vimentin positivity and/or nuclear β-catenin, in core and buds) and hybrid cases (epithelial tumor core and mesenchymal tumor buds). Based on the number of buds, which were evaluated at high power field (HPF), cases were divided in two groups: group A or low grade (≤5 buds/HPF) and and group B or high-grade CRCs (>5 buds/HPF). Results It was a similar distribution between the two groups: 74 cases (47.13%) in group A and 83 cases in group B (52.87%). Most of the cases (n = 76; 48.41%) were classified as epithelial-type, followed by hybrid- (n = 63; 40.13%) and mesenchymal-type CRCs (n = 18; 11.46%). On both groups, a similar distribution was seen between epithelial- and hybrid-type cases but 14 out of the 18 mesenchymal-type CRCs belonged to group B (p = 0.01). All of the mesenchymal-type CRCs from group B showed lymph node metastases. Both lymph node status and budding degree proved to be independent prognostic factors. Conclusions In daily practice, the immunoprofile of CRC buds might have a prognostic impact. Further studies are necessary to check the prognostic role of mesenchymal-immunoprofile of tumor buds. Legal entity responsible for the study University of Medicine, Pharmacy, Sciences and Technology, Tirgu-Mures, Romania. Funding University of Medicine, Pharmacy, Sciences and Technology, Tirgu-Mures, Romania. Disclosure All authors have declared no conflicts of interest.

Contamination and genotoxicity biomarker responses in bivalve mussels from the major Lithuanian rivers.

European inland waters are under continuous threat of anthropogenic pollution. Determination of background level of biomarker response and subsequent classification of the impact increases the applicability of results. In the current study, we evaluate the range of chemical contamination by measuring the concentrations of metals, polycyclic aromatic hydrocarbons and polychlorinated biphenyls, and the levels of environmental genotoxicity by using the micronuclei and nuclear bud tests in bivalve mussels of the major Lithuanian rivers. Second, we aimed to evaluate the association between chemical contamination and genotoxicity biomarker responses. Finally, we set to determine the background level of genotoxic effects. Such value (summed frequency of MN and NB) was assessed-6‰. On that basis, we develop a scale of potential genotoxic impact and perform ranging sites into five categories. The results clearly indicate the existence of significant differences in the levels of chemical pollution and genotoxicity in different sites. Increased levels of studied parameters were assessed at the areas affected by municipal and industrial effluents, road runoff, combustion products, and in the area contaminated by accidental spillage. On the other hand, downstream decrease of contamination level, presumably associated with biological degradation and photochemical oxidation, were also observed. Genotoxicity parameters were associated with PAH and metal concentrations measured in mussel tissues as well as in sediments. Results also indicate that in situ genotoxicity assessment performed in the areas affected by long-term contamination of municipal origin might be not sufficiently precise. Study highlights the necessity to combine genotoxicity assessment with chemical analysis. Environ. Mol. Mutagen. 61:338-354, 2020. © 2019 Wiley Periodicals, Inc.

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.

Cytogenotoxic effects of fullerene C60 in the freshwater teleostean fish, Anabas testudineus (Bloch, 1792)

In recent years, carbon nanomaterials, including fullerene C60 is regarded as the building block in nanotechnology because of its widespread use in medicine, industry, cosmetics and commercial products. Despite the special properties, several reports have raised public health concerns due to the unknown and practically unexplored toxic effects of nanomaterials. However, there have been relatively few studies regarding the genotoxic responses of fullerene C60in vivo. Genotoxic effects of DMSO-solublized C60 nanomaterial suspension at sublethal concentrations (5 and 10 mg/L) were investigated on adult freshwater fish, Anabas testudineus using micronucleus and comet assays. An assessment of micronucleus induction showed severe cytoplasmic and nuclear abnormalities in erythrocytes, gill and liver cells. Abnormalities in cytoplasm were identified as formation of sticky cells, vacuolated cytoplasm, cytoplasmic degeneration, echinocyte, acanthocyte, anisochromatic cells and abnormal erythrocyte membrane. The nuclear abnormalities included micronucleus, binucleated cells, nuclear buds, irregular nucleus, vacuolated, notched and serrated nucleus in the erythrocytes compared to the control groups. Similarly, significant increase (P < 0.05) in micronucleus frequencies were observed in gill and liver cells. The high frequency of micronucleus was observed in the gill cells followed by liver and erythrocytes, respectively, at both sublethal concentrations, and the severity was duration and concentration-dependent. In comet assay, significant increase (P < 0.05) in DNA damage was observed using the comet parameter, percent tail DNA. The highest level of comet damage with grade 3 was observed in blood, gill and liver cells on increase in duration and concentration when compared to the respective control groups. Thus the results revealed that fullerene C60 nanomaterials may pose risk to aquatic organisms, especially fish, by the induction of genotoxicity. Further studies are warranted to provide new insights on the mechanisms and consequences of C60 nanomaterials interactions with biological membranes.

In Vitro Genotoxicity of Polystyrene Nanoparticles on the Human Fibroblast Hs27 Cell Line.

Several studies have provided information on environmental nanoplastic particles/debris, but the in vitro cyto-genotoxicity is still insufficiently characterized. The aim of this study is to analyze the effects of polystyrene nanoparticles (PNPs) in the Hs27 cell line. The viability of Hs27 cells was determined following exposure at different time windows and PNP concentrations. The genotoxic effects of the PNPs were evaluated by the cytokinesis-block micronucleus (CBMN) assay after exposure to PNPs. We performed ROS analysis on HS27 cells to detect reactive oxygen species at different times and treatments in the presence of PNPs alone and PNPs added to the Crocus sativus L. extract. The different parameters of the CBMN test showed DNA damage, resulting in the increased formation of micronuclei and nuclear buds. We noted a greater increase in ROS production in the short treatment times, in contrast, PNPs added to Crocus sativus extract showed the ability to reduce ROS production. Finally, the SEM-EDX analysis showed a three-dimensional structure of the PNPs with an elemental composition given by C and O. This work defines PNP toxicity resulting in DNA damage and underlines the emerging problem of polystyrene nanoparticles, which extends transversely from the environment to humans; further studies are needed to clarify the internalization process.

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

ABSTRACTRoad 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.

Oxidative stress, mutagenic effects, and cell death induced by retene

Retene (RET) is the most abundant polycyclic aromatic hydrocarbon (PAH) released upon burning of cellulose, although it is not considered as one of the priority PAHs and is not included for risk assessments by the US Environmental Protection Agency (US-EPA). There are only a few studies concerning the toxic effects of RET. To the best of our knowledge, this study is the first one to examine whether RET, in an environmental concentration, plays a crucial role in the induction of oxidative stress in A549 lung cell line, and its consequence as such as mutagenicity and cell death. Our results revealed that RET was able to significantly decrease cell viability only at 72 h of exposure, increase oxidative stress, mitochondrial membrane potential and mitochondrial contents, leading an increased reactive oxygen species (ROS) production. Mutagenic activity was not detected in Salmonella strains, suggesting that RET does not induce base-pair substitution (TA100), frameshift (TA98 and TA97a) and transition/transversion (TA102) mutations. However, exposure to RET led to a significant increase in micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) frequency, as well as cell death, mainly due to necrosis. Taken together, the results of our study provide new evidence suggesting that RET promotes oxidative stress, contributes to the processes of genomic instability, and favors necrosis. Thus, we highlight the importance of including RET in routine environmental analyses in the future as a potential risk factor involved in complex diseases and carcinogenesis.

Results of buccal micronucleus cytome assay in pesticide-exposed and non-exposed group.

Since many different pesticides have been used occupationally, there have been inconsistent results regarding DNA damages among greenhouse workers. Thus, the aim of the study is to evaluate DNA damages, cell death, and chromosomal instability by using the buccal micronucleus cytome (BMcyt) assay in greenhouse workers and to compare those with a non-exposed group. The BMcyt assay was applied to the exfoliated buccal cell samples collected from 66 pesticide-exposed and 50 non-exposed individuals. We evaluated the frequency of micronucleus (MN), nuclear bud (NBUD), binucleated (BN) cells, and karyolitic (KL), pyknotic (PY), and karyorrhectic (KH) cells. The results showed that the MN, BN, PY, and KH frequencies of the pesticide-exposed group were significantly higher than those of the controls (P˂0.05, P˂0.05, P˂0.01, and P˂0.05, respectively). We observed that the MN, BN, PY, and KH frequencies in the autumn were statistically different compared with those in the control group (P = 0.037 for MN, P = 0.001 for BN, P = 0.016 for PY, and P = 0.033 for KH). The same comparison was done in the spring for the control, and there was a statistically significant difference for MN (P = 0.046) and PY (P= 0.014). We can conclude that pesticide exposure in greenhouse workers was one of the factors that altered DNA damages, cell death, and chromosomal instability in oral mucosa cells.

Investigation of pesticide exposure by genotoxicological, biochemical, genetic polymorphic and in silico analysis

Soybean farmers are exposed to various types of pesticides that contain in their formulations a combination of chemicals with genotoxic and mutagenic potential. Therefore, the objective of this paper was to evaluate the genetic damages caused by this pesticide exposure to soybean producers in the state of Mato Grosso (Brazil), regarding biochemical, genetic polymorphic and in silico analyses. A total of 148 individuals were evaluated, 76 of which were occupationally exposed and 72 were not exposed at all. The buccal micronucleus cytome assay (BMCyt) detected in the exposed group an increase on DNA damage and cell death. No inhibition of butyrylcholinesterase (BchE) was observed within the exposed group. The detection of inorganic elements was made through the particle-induced X-ray emission technique (PIXE), which revealed higher concentrations of Bromine (Br), Rubidium (Rb) and Lead (Pb) in rural workers. A molecular model using in silico analysis suggests how metal ions can cause both DNA damage and apoptosis in the exposed cells. Analysis of the compared effect of X-ray Repair Cross-complement Protein 1 (XRCC1) and Paraoxonase 1 (PON1) genotypes in the groups demonstrated an increase of binucleated cells (exposed group) and nuclear bud (non-exposed group) in individuals with the XRCC1 Trip/- and PON1 Arg/- genes. There was no significant difference in the telomere (TL) mean value in the exposed group in contrast to the non-exposed group. Our results showed that soybean producers showed genotoxic effect and cell death, which may have been induced by exposure to complex mixtures of agrochemicals and fertilizers. In addition, XRCC1 Arg/Arg could, in some respects, provide protection to individuals.

Genotoxic Evaluation in Tadpoles Associated with Agriculture in the Central Cerrado, Brazil.

Many agricultural practices cause environmental degradation that affects the cellular integrity of anurans. In the present study, we provided in situ data of Dendropsophus minutus, Physalaemus cuvieri, and Scinax fuscovarius collected in soybean/corn and conservation units in the Brazilian Cerrado. The in situ data showed no significant variation in the micronucleus frequency between the sites, only the reniform cells had a higher rate for the agricultural environment. A combined analysis of all nuclear erythrocyte abnormalities (ENAs = nuclear buds, reniform nuclei, apoptotic cell, binucleated, and anucleated cells) was recorded higher frequencies in farmland. Overall, Scinax fuscovarius was considered the best potential bioindicator for soybean/corn plantations. Finally, we recommend expanding the micronucleus test for in situ studies to expand our understanding of the sensitivity of native anuran species and provide a more systematic assessment of the adverse effects of environmental pollutants on wildlife.

Methamphetamine (“crystal meth”) causes induction of DNA damage and chromosomal aberrations in human derived cells

Methamphetamine (METH) is a widely consumed psychostimulant drug; its acute toxic effects in brain and liver are well known, furthermore, there is some evidence in regard to its DNA damaging properties in humans. Therefore, we studied the impact of the drug on genomic stability in human derived hepatoma (HepG2) cells, which reflect the activation/detoxification of drugs better than other cell lines. Furthermore, experiments with human buccal derived cells (TR146) were conducted as the drug is consumed orally. Induction of DNA damage in both cell types with doses reflecting the exposure in abusers was found in single cell gel electrophoresis (SCGE) assays (which detect single and double strand breaks as well as apurinic sites). Furthermore, induction of micronuclei (formed as a consequence of structural and numerical chromosomal aberrations) and formation of nuclear buds resulting from gene amplifications was detected. Additional experiments with lesion-specific enzymes showed that the drug causes oxidation of purines and pyrimidines, indicating that its genotoxic effects may be due to oxidation of the DNA. Our findings support the assumption that the drug may cause adverse health effects (such as cancer and infertility) in long-term users which are causally related to DNA damage.

Genotoxic effects induced by beta-myrcene following metabolism by liver HepG2/C3A human cells

ABSTRACTBeta-myrcene [or myrcene (1,6-Octadiene, 7-methyl-3-methylene-)] and the essential oils containing this monoterpene have been widely used in cosmetics, detergents, and soaps, and as flavoring additives for food and beverages. Due to the potentially high level of human exposure to beta-myrcene, and absence of studies involving its genotoxicity in human cells, the aim of this study was to investigate the cytotoxic and genotoxic potential of this terpenoid in non-metabolizing cells (leukocytes) and liver metabolizing cells (HepG2/C3A cells). Prior to the genotoxic assessment by the comet and micronucleus (MN) assays, a range of beta-myrcene concentrations was tested in a preliminary MTT assay. Regarding the MTT assay, the results showed cytotoxic effects for leukocytes at 250 µg/ml and higher concentrations, while for HepG2/C3A cells, absence of cytotoxicity was noted relative to all tested concentrations (after 24 hr exposure). Thus, the concentrations of 2.5, 10, 25, 50, and 100 µg/ml for leukocytes, and 2.5, 100, and 1000 µg/ml for HepG2/C3A cells were selected for subsequent assays. Genotoxicity evaluation demonstrated significant DNA damage in the comet assay and significant chromosomal abnormalities including nucleoplasmic bridges and nuclear buds in HepG2/C3A cells at beta-myrcene concentrations of 100 and 1000 µg/ml. Under our experimental conditions, caution is recommended in the use of beta-myrcene, since this compound produced genotoxic effects especially after metabolic activation using human HepG2/C3A cells, which may be associated with carcinogenic and teratogenic effects previously reported in the literature.

STRUCTURE OF RELATIONS BETWEEN THE FREQUENCY OF MICRONUCLEI IN PERIPHERAL BLOOD LYMPHOCYTES AND AGE, GENDER, SMOKING HABITS AND SOCIO-DEMOGRAPHIC FACTORS IN SOUTH-EAST REGION OF SERBIA

Frequency of micronuclei (MN) in peripheral blood lymphocytes is a measure of genotoxicity and spontaneous chromosomal instability with many modifying factors. The aim of this work was to examine the relation between results of micronucleus test and set of socio-demographic parameters in healthy population in south-east Serbia using structure equation approach. Cytokinesis–block (CB) technique was used for analysis of MN frequency in peripheral blood lymphocytes on 133 healthy volunteers of various ages. Socio-demographic data was collected through a questionnaire. The mean frequency of MN gradually rose with age from 0.56±0.71 in new-borns to 5.48 ±3.65 in the 61-80 years age group (AG), with a decrease in the 81-92 years AG. MN frequency was positively correlated with age, altitude of birth place, altitude of place of residence, nuclear buds and nucleoplasmatic bridges, and negatively with education level and smoking habits. Linear structural model revealed age to be related to all of the examined variables, and indicates probable existence of another factor, independent of age, influencing all of these except nuclear buds. It can be concluded that the frequency of micronuclei is influenced by age and factor/s resembling socioeconomic status or lifestyle and this influence is independent of age.

Nuclear morphological changes in gingival epithelial cells of patients with periodontitis.

This study assessed the frequency of nuclear morphological changes in gingival epithelial cells, as a biomarker for DNA damage, in individuals with periodontitis, before and after implementation of periodontal therapy, and compared the morphology to those with healthy periodontal tissues. Exfoliated gingival cells were taken from 30 participants without periodontal destruction in any teeth and 30 participants with periodontitis before and after 45 and 90days following treatment. Nuclear morphological changes were analyzed using the micronucleus test. Compared with the healthy volunteers, those with periodontitis had a significant increase in the number of cells with nuclear broken eggs (P = 0.048), condensed chromatin (P = 0.015), karyolysis (P < 0.001), or binuclei (P < 0.001). In the periodontitis group, the pretreatment frequencies of cells with micronuclei (P = 0.008), binuclei (P < 0.001), karyolysis (P = 0.038), nuclear buds (P = 0.005), and condensed chromatin (P = 0.015) were significantly higher than 90days after treatment. Our observations suggest that periodontal disease increases the frequency of nuclear morphological changes in gingival epithelial cells and that the implementation of periodontal therapy was associated with a reduction of that number. The micronucleus test could serve as a tool for estimating genotoxic damage in assessing the success of periodontal therapy.

Doramectin induced cytotoxic and genotoxic effects on bovine peripheral lymphocytes and cumulus cells in vitro

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60 ng mL–1) for 24 h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60 ng mL–1, and nucleoplasmic bridges (NPBs) only with 40 ng mL–1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40 µg mL–1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC.