Binuclear Cells Research Articles

Short‐term “in vivo” study on cellular DNA damage induced by acrylic Andresen activator in oral mucosa cells

To analyse through comet assay and micronucleus test the viability and DNA damage occurred in buccal mucosa epithelial cells after a short-term exposure to Andresen activator resin monomers. Test group consisting of 26 subjects was treated with Andresen activator; 16 subjects who had never undergone orthodontic treatment were enrolled in the control group. Buccal mucosa samples were collected before treatment and after 7, 15, 30, 60 and 90days. The analyses performed on the cells included the following: cellular viability, comet assay and micronucleus test. Mean±SD were calculated for cellular viability, tail moment, tail intensity, tail length, micronuclei, binuclear and bud cells. Significance (P<0.05) was evaluated with Dunnett's test. Cellular viability did not change during observational time, and its trend was similar to the controls. Tail moment and tail intensity significantly increased after 30 and 60days, respectively, whereas tail length remained unchanged over time in the test group; the same parameters did not change in the control group. In the test group, micronuclei, binuclear and bud cells significantly increased after 30, 60 and 90days, respectively. The resin monomers of the Andresen activator cause genotoxic effects detectable through comet assay and micronucleus test, but they do not produce clear cytotoxic effects after a 90days exposure.

Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis.

The in vitro micronucleus test according to OECD Test Guideline 487 (TG 487) is widely used to investigate the genotoxic potential of drugs. Besides the identification of in vitro genotoxicants, the assay can be complemented with kinetochore staining for the differentiation between clastogens and aneugens. This differentiation constitutes a major contribution to risk assessment as especially aneugens show a threshold response. Thus, a novel method for automated MN plus kinetochore (k+) scoring by image analysis was developed based on the OECD TG 487. Compound-induced increases in MN frequency can be detected using the cytokinesis-block (cytochalasin B) method in V79 cells after 24 h in a 96-well format. Nuclei, MN, and kinetochores were labeled with nuclear counterstain and anti-kinetochore antibodies, respectively, to score MN in binuclear or multinuclear cells and to differentiate compound-induced MN by the presence of kinetochores. First, a reference data set was created by manual scoring using two clastogens and aneugens. After developing the automated scoring process, a set of 14 reference genotoxicants were studied. The automated image analysis yielded the expected results: 5/5 clastogens and 6/6 aneugens (sensitivity: 100%) as well as 3/3 non-genotoxicants (specificity: 100%) were correctly identified. Further, a threshold was determined for identifying aneugens. Based on the data for our internally characterized reference compounds, unknown compounds that induce ≥53.8% k+ MN are classified as aneugens. The current data demonstrate excellent specificity and sensitivity and the methodology is superior to manual microscopic analysis in terms of speed and throughput as well as the absence of human bias. Environ. Mol. Mutagen. 60:227-242, 2019. © 2018 Wiley Periodicals, Inc.

Overexpression of Fbxo6 inactivates spindle checkpoint by interacting with Mad2 and BubR1

ABSTRACT The spindle assembly checkpoint prevents chromosome mis-segregation during mitosis by delaying sister chromatid separation. Several F-box protein members play critical roles in maintaining genome stability and regulating cell cycle progress via ubiquitin-mediated protein degradation. Here, we showed that Fbxo6 critically regulated spindle checkpoint and chromosome segregation. Fbxo6 was phosphorylated during mitosis. Overexpression of Fbxo6 lead to faster exit from nocodazole-induced mitosis arrest through premature sister chromatid separation. Moreover, we found substantially more binuclear and multilobed nuclei cells accompanied with impaired cell viability in Fbxo6-overexpressed HeLa cells. Mechanistically, Fbxo6 interacted with spindle checkpoint proteins including Mad2 and BubR1 leading to the premature exit from mitosis. Overall, we revealed a novel role of Fbxo6 in regulating spindle checkpoint, which may shed light on the regulation of genome instability of cancer cells.

Unusual nuclear form hairy cells in hairy cell leukemia discovered using a lymphocyte-binding anti-CD antibody microarray.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder constituting about 2% from all leukemia cases and characterized by typical “hairy” morphology of tumor lymphocytes. We describe an HCL case with atypical nuclear shapes (lymphocytes with clover-leaf-like, horse-shoe-like, ring-shaped nuclei and binuclear cells were present). Morphology and immunophenotype of circulating leukemic cells were studied using a cell-binding microarray - a transparent plastic slide with immobilized monoclonal antibodies against surface antigens of lymphocytes. The cell-binding microarray with immobilized anti-CD11c, anti-CD103 and anti-CD123 permits to study a lymphocyte population enriched with hairy cells. Hairy cells with atypical nuclei constituted 3% of all lymphocytes and 15% of all hairy cells. This unusual hairy cell morphology is the first described in Russia and was found in one out of 85 HCL cases in our practice.

The assessment of radio-adaptive response in graves' hyperthyroidism patients following radioactive iodine uptake.

Low doses of radiation affect the response of cells to higher doses; this phenomenon is called radio-adaptive response, which leads to increased resistance to subsequent higher doses. We have investigated the adaptive response using 0.37 MBq priming dose of I-131 followed by 296–444 MBq challenging dose in peripheral human lymphocyte cells. The study was performed on 42 patients with Graves' disease and 29 healthy adult persons as a control group. The patients were divided into two groups. In the first group, patients were referred for radioactive iodine therapy with a specific dose, and iodine was given to them on the day of referral. In the second group, patients were referred for radioactive iodine uptake and radioactive iodine therapy, and iodine uptake was initially performed, then 24 h later, iodine therapy was done. In both groups, 1 month after treatment, blood samples were taken to cytokinesis-block micronucleus (MN) assay. The number of MN in binuclear lymphocyte cells was counted as an end point test. The mean frequency of MN in first, second, and control groups was 75.86 ± 12.68, 71.45 ± 12.56, and 20.06 ± 7.30, respectively. Our results showed that the frequency of total chromosome aberration in both radiation groups was higher than controls. However, in the first group was higher than another group, but their difference was not statistically significant. According to the results, we cannot approve the hypothesis that 0.37 MBq I-131 administration before iodine therapy could induce a radio-adaptive response in lymphocytes of Graves' patients.

The protein expression disorders of connexins (Cx26, Cx32 and Cx43)and keratin 8 in bovine placenta under the influence of DDT, DDE and PCBs.

Polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and its metabo- lite, dichlorodiphenyldichloroethylene (DDE) can disturb the secretory function of the ovary and both contractions and secretory function of the uterus during the estrus cycle and pregnancy. Additionally, PCBs can pass through the placental barrier into allantoic and amniotic fluid. The presence of PCBs in these fluids is associated with higher frequency of spontaneous abortions and premature births in humans and animals. Therefore, the effect of PCBs, DDT and DDE on the connexins (Cx26, Cx32 and Cx43) and keratin 8 (KRT8) expression in bovine placentomes was investigated. The placentome slices from the second trimester of pregnancy were incubated with PCB153, 126, 77, DDT and DDE (each at doses of 1, 10 or 100 ng/ml) for 48 h. Then, the slices were stained using immunohistochemistry. The density of Cxs staining was measured with Axio- Vision Rel. 4.8 software in fetal-maternal connections and binuclear cells (BNC). None of the tested xenobiotics (XBs) affected the localization of Cxs and KRT8 in the fetal-maternal connec- tion area, but the XBs affected the density of Cxs in fetal-maternal connections and binuclear cells (BNCs). Depend on the doses, in fetal-maternal connections all used PCBs changed the protein expression of different Cxs, while in BNCs, all tested XBs except DDT increased the ex- pression of Cxs. None of investigated XBs affected on KRT8 expression. In summary, used XBs affect the expression of Cxs and change the quantitative relationships between them. Therefore, XBs can unfavorably influence function of the utero-placental barrier in cows.

THE CYTOGENETIC AND PHYTOTOXIC ASSESSMENT OF THE ANTI-ICING MATERIAL ON ONION ASSAY (ALLIUM CEPA)

Allium cepa assay is one of the relevant methods for the cytogenetic analysis. Today modern anti-icing reagents seem to be unstudied in the field of cytogenetics studies. The aim of the experiment is to study the phytotoxic and cytogenetic activity of multi-component anti-icing material using an onion Allium cepa. We researched anti-icing concentrations in the range of from 1 to 100 g/l. The test object was onion «Stuttgarter risen» variety couched in anti-icing solutions during the 72 hours. According to the assessment of phytotoxic effects, there was estimated the number of roots, the length of the longest root and the length of all roots for the every onion plant. The parallel experiment included the phytotoxic research of the same anti-icing using wheat seeds according to method MR 2.1.7.2297-07. Every onion roots were cut away, fixed, painted and pressured for microscopic analysis for the purposes of the cytogenetic research. About 2000 cells were analyzed for each onion. We estimated the number of mitosis per 1000 cells (mitotic index, ‰) and the proportion of cells in each phase of mitosis by the total number of dividing cells (%). For the analysis of cytogenetic damage using micronucleus test that provided the estimation of micronucleus, protrusions, and binuclear cells. An ana-telophase analysis included the estimation of fragments bridges and lagging chromosomes. As a result of analysis of the roots growth, we founded acting concentrations (2; 4; 10 and 100 g/l) and in-acting ones (1.3 and 1). The experiment showed the similar susceptibility of the onion and wheat root response. The mitosis analysis showed that concentrations from 1 to 1.3 g/l increased a mitosis activity and concentration of 10 g/l provided the decline of mitosis. There were no cytogenetic defects in all studied concentrations.

Studies on Oogenesis of the FernLygodium japonicum

Oogenesis of Lygodium japonicum (Thunb.) Sw. is studied using TEM and cytochemical techniques. The early development of the archegonia of L. japonicum is similar to those of the core-leptosporangiate ferns. A mature archegonium always contains an egg, a ventral canal cell (VCC) and a binuclear neck canal cell (NCC). With the development of the egg, a separation cavity forms between the egg and the VCC. However, a pore region, with a diameter of about 3.5 μm, constantly connects the egg and the VCC. The canal cells degenerate gradually accompanied by the accumulation of mucilaginous secretions around the canal cells. PAS reaction reveals that these secretions are polysaccharide in nature. Later development of the archegonia of L. japonicum differs greatly from the core-leptosporangiate ferns. The egg nucleus becomes highly irregular and two types of nuclear evaginations are formed during oogenesis. When the egg matures, no egg envelope or fertilization pore are formed. Only a layer of amorphous material is deposited on the outer surface of the egg. This is the first observation of a fern species that has a pore region but does not result in the formation of a fertilization pore. The pore region appears to determine the formation of the fertilization pore. These observations are consistent with the hypothesized phylogenetic position of L. japonicum relative to the core leptosporangiate ferns.

Distinctive features of hepatocytes in five small mammal species (insectivores and rodents): taxonomic versus ecological specificity

Morphometric parameters of hepatocytes have been studied in five small mammal species with different ecological specificity from two taxonomic groups: a surface-dwelling insectivore (the common shrew, Sorex araneus), a subterranean insectivore (the European mole, Talpa europaea), surface-dwelling rodents (the bank vole, Myodes glareolus and herb field mouse, Apodemus uralensis) and a subterranean rodent (the northern mole vole, Ellobius talpinus). The results show that the hepatocytes of the European mole differ markedly from those of all other species, being characterized by smaller size and nuclear area, low anisocytosis and anisokaryosis, the almost complete absence of binuclear cells and, on the other hand, high nuclear–cytoplasmic ratio and packing density. The common shrew differs from the European mole containing a relatively high proportion of binuclear hepatocytes, and the size of hepatocytes in this species is smaller than in rodents. Differences in the parameters of these cells between rodents are minimal or absent. The differences observed in the characteristics of hepatocytes between the above species cannot be explained solely by either taxonomic or ecological specificity. Comparative research involving a greater number of closely related species (at the levels of families and orders) is needed to reveal and evaluate the taxonomic and ecological specificity of hepatocytes in greater detail.

INFLUENCE OF BASIC VARIABLES ON MICRONUCLEI FREQUENCY AND CHROMOSOMAL ABERRATIONS IN GENERAL POPULATION OF FB&amp;H

The aim of this study was to determine the values of micronuclei (MN) and structural chromosomal aberrations (CA) in peripheral blood lymphocytes from 200 healthy participants of both genders from general population of FB&amp;H, as well as to explore the influence of gender and age on MN and CA frequencies. Standard protocols for MN test, cultivation and micronuclei analysis from peripheral blood binuclear lymphocytes have been applied. MN values ranged from 0 to 8 MN per 1000 binuclear cells. The results suggest that gender and age influence MN frequency, with pronounced effect on 2 MN frequencies. Females on average have higher values of all observed variables of MN test than men. We have also found significant effect of gender – females had increased number of CAs – chromatid type; and of the age in both genders. Frequency distribution of CTAs and CSAs between male and female groups showed predominance of CTAs over CSAs, independently of gender. The results of this study will be incorporated into reference data base for comparative research in future.

The evaluation of the genetic material stability of male patients with paranoid schizophrenia at different stages of treatment using the micronucleus test in the buccal epithelium

The buccal epithelium of male patients with paranoid schizophrenia at different stages of treatment was characterized by a reduced fraction of cells with micronuclei, central circular notches, “broken egg” protrusions, and an increased frequency of cells with karyorrhexis. A modified nonlinear frequency of binuclear cells, cells with karyolysis, “tongue” protrusions and perinuclear vacuoles, was observed. The fraction of cells with perinuclear vacuoles was increased in the spectrum of aberrations during the treatment. Patients with schizophrenia were divided into three groups differing in responses to the treatment of the cellular genetic apparatus. An enhanced stability of the genome of patients with schizophrenia was caused by the expressed antistress action of drugs used for complex therapy.

Single-Cell Analysis Uncovers Clonal Acinar Cell Heterogeneity in the Adult Pancreas

Acinar cells make up the majority of all cells in the pancreas, yet the source of new acinar cells during homeostasis remains unknown. Using multicolor lineage-tracing and organoid-formation assays, we identified the presence of a progenitor-like acinar cell subpopulation. These cells have long-term self-renewal capacity, albeit in a unipotent fashion. We further demonstrate that binuclear acinar cells are terminally differentiated acinar cells. Transcriptome analysis of single acinar cells revealed the existence of a minor population of cells expressing progenitor markers. Interestingly, a gain of the identified markers accompanied by a transient gain of proliferation was observed following chemically induced pancreatitis. Altogether, our study identifies a functionally and molecularly distinct acinar subpopulation and thus transforms our understanding of the acinar cell compartment as a pool of equipotent secretory cells.

1151 Impact of dietary protein levels during late pregnancy on the number of binuclear cells in sheep

1151 Impact of dietary protein levels during late pregnancy on the number of binuclear cells in sheep

Genotoxic and cytotoxic effects of flumetralin in human peripheral blood lymphocytes in vitro.

Flumetralin, a synthetic plant growth regulator with herbicidal activity belonging to the 2,6-dinitroaniline class of chemicals, has been evaluated for its ability to induce genotoxicity in human peripheral blood lymphocytes (PBLs). The potential genotoxic and cytotoxic effects of flumetralin were investigated in vitro by chromosome aberration (CA) and cytokinesis-block micronucleus assays. Human PBLs were treated with 125, 250, 500, and 1000 µg/mL flumetralin for 24 and 48 h. Flumetralin statistically significantly increased the frequency of structural CAs at the three highest concentrations (250, 500, and 1000 µg/mL) for both treatment periods (24 and 48 h) when compared with both the negative and solvent controls. In addition, micronucleus formation was significantly induced at higher concentrations (250, 500, and 1000 µg/mL) for 24 h and at 125 and 500 µg/mL of flumetralin for the 48-h treatment period compared with the controls. Because of the excessive cytostatic effects of flumetralin, binuclear cells could not be detected sufficiently at the highest two concentrations (500 and 1000 µg/mL) for the 48-h treatment period. Furthermore, flumetralin significantly decreased the mitotic index and nuclear division index for all concentrations and treatment times compared with the control groups. The present results indicate that flumetralin was clastogenic and cytotoxic/cytostatic to human PBLs. This study presents the first report of the genotoxic and cytotoxic properties of flumetralin.

Binuclear Cells in the Lactating Mammary Gland: New Insights on an Old Concept?

In a recent paper (Rios et al. Nat Commun. 7:11400, 2016), it was reported that polyploid cells are frequent in lactating mammary tissues. This phenomenon was observed in mammary tissue sampled from five separate mammalian species. According to that report, these binucleated cells occur late in pregnancy and early in lactation. Unfortunately, this paper did not mention a number of earlier observations and findings that remain pertinent to this day (Banerjee et al. Life sciences Pt 2: Biochemistry, general and molecular biology. 10(15):867-77, 1971; Banerjee MR, Wagner JE. Biochem. Biophys. Res. Commun. 49(2):480-7, 1972). In these classical experiments, the authors demonstrated in vivo that DNA synthesis continued without commensurate cell division during late pregnancy and lactation, and that this DNA synthesis was imperative for functional differentiation of the mammary epithelium. Later studies showed that DNA synthesis was indispensable to the induction of milk protein production in explant cultures of mammary tissue from unprimed, nulliparous mice. This dependence on DNA synthesis in mammary explant cultures stimulated by lactogenic hormones was found to be dispensable following a single pregnancy. The absolute requirement for DNA synthesis in nulliparous mouse mammary explants stimulated to synthesize milk protein in vitro has remained unexplained, as has the need for DNA synthesis prior to the onset of lactation. From a historical perspective, it is more likely that binuclear secretory cells in the lactating mammary gland are a consequence of the DNA synthesis requirement for lactation, rather than an essential element.

Protective effect of wheat germ agglutinin on the course of mitosis in the roots of Triticum aestivum seedlings exposed to cadmium

Effect of pretreatment with 28 nM wheat germ agglutinin (WGA) on cell divisions in the root apical meristem of 4-day-old seedlings of wheat (Triticum aestivum L.), distribution of cells among mitotic phases, cadmium-induced disruptions of normal progression through mitosis, and activity of nucleolar organizer regions (NOR) of the chromosomes was studied after 7-h-long exposure to 1 mM cadmium acetate. Pretreatment with WGA has a pronounced protective effect on divisions of root meristem cells exposed to cadmium. Progression of the cells through mitotic phases was normalized, abnormal mitoses became much less numerous, and the share of binuclear cells decreased. Activity of NOR remained at the control level that much depended on the ability of WGA to prevent reduction in cytokinin content under cadmium stress.

COMPARATIVE ASPECTS OF REGENERATIVE POTENCY OF SPHINCTER CERVICAL TISSUES IN RATS WITH CANALIS CERVICIS UTERI TRACTION UNDER EXPERIMENTAL CONDITIONS

Aim - to study the reparative regeneration processes after experimental gradual traction of cervix uteri in rats. Materials and methods. The object of the study is the cervix at the sphincter. We performed gradual traction of cervix uteri in 25 rats under aether anesthesia and gathered the samples.We analyzed the course of reparative regeneration and performed sampling after 1, 3, 5, 7, 10, 15, 21 and 30 days. We carried out complex morphological examination with optical and transmission electron microscopy, and immunohistochemistry. The immunohistochemical examination of cervical tissues was performed using a set of monoclonal antibodies to а-smooth muscle actin. Results. It was determined that cervical traction formed three distinguishable zones: the zone of damage, the periwound zone and the zone of morphological integrity. The zone of damage corresponds to the actual sphincter and the periwound zone - to pre- and postsphincteral compartments. Several myocytes undergo necrosis in the damage zone. There is no necrosis in the periwound area, but we found some abnormality of intercellular contacts due to the change of the morphology of intercellular substance. The experimental traction causes the development of local septic inflammation, affecting all the layers. The synthesis of fibrous connective tissue components starts in the proliferative phase. It leads to restructuring of histoarchitecture and disruption of the functional syncytium structure. Conclusion. Post-traumatic regeneration is performed by changing the phenotype of survived myocytes from mantle to mantle-synthetic myocytes with synthesis of the fiber component of the intercellular substance. Also, there is an incomplete mitosis in several myocytes, which leads to the formation of binuclear or polyploid cells.

Haspin has Multiple Functions in the Plant Cell Division Regulatory Network.

Progression of cell division is controlled by various mitotic kinases. In animal cells, phosphorylation of histone H3 at Thr3 by the kinase Haspin (haploid germ cell-specific nuclear protein kinase) promotes centromeric Aurora B localization to regulate chromosome segregation. However, less is known about the function of Haspin in regulatory networks in plant cells. Here, we show that inhibition of Haspin with 5-iodotubercidin (5-ITu) in Bright Yellow-2 (BY-2) cells delayed chromosome alignment. Haspin inhibition also prevented the centromeric localization of Aurora3 kinase (AUR3) and disrupted its function. This suggested that Haspin plays a role in the specific positioning of AUR3 on chromosomes in plant cells, a function conserved in animals. The results also indicated that Haspin and AUR3 are involved in the same pathway, which regulates chromosome alignment during prometaphase/metaphase. Remarkably, Haspin inhibition by 5-ITu also led to a severe cytokinesis defect, resulting in binuclear cells with a partially formed cell plate. The 5-ITu treatment did not affect microtubules, AUR1/2 or the NACK-PQR pathway; however, it did alter the distribution of actin filaments on the cell plate. Together, these results suggested that Haspin has several functions in regulating cell division in plant cells: in the localization of AUR3 on centromeres and in regulating late cell plate expansion during cytokinesis.

Genotoxicity and cytotoxicity of copper oxychloride in cultured human lymphocytes using cytogenetic and molecular tests.

The genotoxicity of copper oxychloride was investigated in human lymphocytes using chromosome aberration (CA) and micronucleus (MN) tests and the randomly amplified polymorphic DNA-polymerase chain reaction technique. The lymphocytes were treated with 3, 6, and 12µg/mL of copper oxychloride for 24 and 48h. Copper oxychloride increased CA and abnormal cells in a dose-dependent manner. The frequency of MN and micronucleated binuclear cells also increased at all concentrations and treatment periods. However, copper oxychloride cytotoxicity, observed through lower mitotic and nuclear division index, was significantly lower only at the higher concentrations (6 and 12µg/mL). Copper oxychloride increased the polymorphic bands and decreased genomic template stability. In conclusion, in this study it was confirmed that copper oxychloride has genotoxic potential for human lymphocytes in vitro. Additionally, caution is advised for its use as a fungicide, because it may increase the risk of exposure through the food chain.

Influence of Radiotherapy on Frequency of Sister Chromatid Exchange, Micronuclei and Binuclear Cells in Breast Cancer Patients

The aim of this study was to evaluate any cytogenetic change in sister chromatid exchange (SCE), micronuclei (MN) and binuclear cell (BNC) of peripheral blood lymphocytes in breast cancer patients treated with postoperative radiation therapy (RT). Frequency of the SCE, MN and BNC were examined in 22 breast cancer patients received RT and 10 healthy individuals. All parameters were measured before (RT-a), at the completion of (RT-b) and three months after the completion of (RT-c) RT. A significant difference emerged in SCE (p=0.008) and MN (p=0.004) between RT-a and control groups. There was not any statistically significant difference in the SCE frequencies between RT-b and RT-c measurements. The frequencies of MN were also significantly higher in RT-b (p=0.005) and RT-c (p=0.005) than in control group. MN levels decreased to pre-RT levels three months after completion of treatment. Increasing MN and SCE frequencies following radiotherapy is an expected situation. Decrease in MN frequency at 3-month after the completion of RT suggests that expected repair continues. Persistent SCE at the same period suggests that recovery in SCE has not completed yet and a longer period of time is needed.