Binucleate Research Articles

Induction of Genomic Instability in a Primary Human Fibroblast Cell Line Following Low-Dose Alpha-Particle Exposure and the Potential Role of Exosomes.

Simple SummaryExposure to the naturally occurring radioactive gas Radon, and the resulting alpha-particle emitting progeny, dominates human exposure and is the second largest cause of lung cancer after smoking. The work presented shows that not only can very low doses of alpha-particles produce DNA damage in normal HF19 fibroblast cells (detected using comet and micronuclei assays) in the irradiated cell population, but also results in an enhanced yield of DNA damage in the progeny of these cells out to 10 and even 20 population doublings. This persistent elevation of DNA damage is consistent with genomic instability, a well-recognised feature of many tumours with its ability to generate genetic diversity in the dividing population. Environmental exposure is normally associated with a small fraction of cells being irradiated with a single alpha-particle, while the vast majority of cells remain unirradiated. Here, we demonstrate an enhanced yield of DNA damage observed at 10 and 20 population doublings even when less than 1% of the cell population is traversed, with a response similar to that observed when essentially all cells are irradiated. The finding also highlights the potential of exosomes produced by irradiated cells contributing to DNA damage observed in unirradiated bystander cells. Purpose: To study the induction of genomic instability (GI) in the progeny of cell populations irradiated with low doses of alpha-particles and the potential role of exosome-encapsulated bystander signalling. Methods: The induction of GI in HF19 normal fibroblast cells was assessed by determining the formation of micronuclei (MN) in binucleate cells along with using the alkaline comet assay to assess DNA damage. Results: Low dose alpha-particle exposure (0.0001–1 Gy) was observed to produce a significant induction of micronuclei and DNA damage shortly after irradiation (assays performed at 5 and 1 h post exposure, respectively). This damage was not only still evident and statistically significant in all irradiated groups after 10 population doublings, but similar trends were observed after 20 population doublings. Exosomes from irradiated cells were also observed to enhance the level of DNA damage in non-irradiated bystander cells at early times. Conclusion: very low doses of alpha-particles are capable of inducing GI in the progeny of irradiated cells even at doses where <1% of the cells are traversed, where the level of response was similar to that observed at doses where 100% of the cells were traversed. This may have important implications with respect to the evaluation of cancer risk associated with very low-dose alpha-particle exposure and deviation from a linear dose response.

Nuclear abnormalities in umbilical cord blood lymphocytes of newborns from the Ahome and Guasave municipalities in Sinaloa, Mexico.

We measured the frequency of nuclear abnormalities of 210 blood samples from the umbilical cord, since human fetuses are exposed to environmental mixtures of pesticides that induce DNA damage. The determinations were made through the micronucleus assay test in lymphocytes from the umbilical cord blood of newborns whose mothers live in Ahome (n = 105) and Guasave (n = 105), Sinaloa, Mexico. The average frequency of anomalies in 1000 cells were, respectively: micronucleus 0.4 vs. 2.9, pyknotic cells 18.3 vs. 109.2, chromatin condensation 7.7 vs. 150.1, karyolitic cells 1.8 vs. 24.4, and binucleated cells 4.9 vs. 74.6. The calculated Pearson correlation factors of nuclear abnormality frequencies between both municipalities were low and negative, suggesting that they did not correlate between the Ahome and Guasave newborns and indicating a higher number of mothers exposed in Guasave. Our data suggest that monitoring nuclear abnormalities in umbilical cord blood samples could be a useful tool to identify transplacental mutagens perfusion that is being discharged into the local environment.

Evaluation of genotoxic damage in buccal mucosa cytome assays in Iraqi school children exposed to air pollutants emanating from oil fields

Presently, over 135 thousand barrels of crude oil are extracted daily from the Al-ahdeb oil fields (situated at 180 km south east of Baghdad, Iraq). Millions of gallons of untreated toxic wastes, gas and crude oil are released into the environment. Oil mining mediated pollution may cause damage to humans and the environment. To investigate potential health risks for children living in this area, we recruited a sample of 6–8 year old school children residing within a 5 km radius around the oil field, and a control sample of children of the same age from a school 40 km away from the oil-field. Exfoliated buccal mucosa cells obtained from these children were analyzed applying the micronucleus (MN) cytome assay. Possible confounding variables such as x-rays performed during three weeks preceding sampling were obtained by a questionnaire. Nuclear anomalies were significantly elevated in children living near the oil-field. Micronucleated cells 1.3-fold, nuclear buds 3-folds, binucleated cells 3-folds, karyorrhetic cells 4-folds and karyolysis more than 10 folds with a significant difference (P < 0.05). Hence, children living in the proximity of Iraqi oil-fields are at elevated risk of genetic damage, which can cause detrimental health effects in the future.

In vitro and in vivo evaluation of clastogenicity of second-line antitubercular drug loaded PLGA nanoparticles.

Sustained release nanoformulations of second line antitubercular drugs levofloxacin and ethionamide had shown promise in pharmacokinetics and acute and sub-acute toxicity studies. The present study evaluated the clastogenicity potential of the nanoformulations of these antitubercular agents. Clastogenicity was evaluated by (a) in vitro micronucleus assay (b) in vivo micronucleus assay in Swiss albino mice and (c) sister chromatid exchange (SCE) in CHO cell lines. Ethionamide and levofloxacin loaded nanoparticles were 312 ± 64 nm and 245 ± 24 nm in size respectively and drug encapsulation was 35.2 ± 3.1% w/w and 45.6 ± 9.4% w/w, respectively. The frequency of MN-NCE/1000 NCE and MN-PCE/1000 PCE were significantly reduced in mice treated with ethionamide nanoparticle (3.5 ± 0.9, 13.8 ± 16.68) and levofloxacin nanoparticles (5.6 ± 2.7, 16.7 ± 12.7) compared to the mice treated with free ethionamide (11.5 ± 4.1, p = 0.23 and 45.19 ± 19.21, p = 0.38) and free levofloxacin (14.7 ± 1.88, p < 0.0001 and 54.6 ± 18.1, p = 0.0017), respectively. For in vitro, micronucleus assay frequencies of micronuclei per thousand bi-nucleated cells (MN-BN/1000 BN) was 188.3 ± 20.20 and 148 ± 20.42 for ethionamide and levofloxacin nanoparticles as compared to 232.6 ± 16.04 (p = 0.52) and 175 ± 5.56 (p = 0.45) for free ethionamide and levofloxacin, respectively. The average number of SCE per cell for nanoformulation of ethionamide were not different from that of free drug (4.9 ± 0.51 vs 4.1 ± 0.55, p = 0.86). The SCE per cells were not significant difference for nanoformulation of levofloxacin (2.33 ± 1.36 vs 5.46 ± 0.25, p = 0.88). In vitro and in vivo assays have shown relatively less clastogenic potential of equivalent dose of ethionamide nanoparticles as compared to the conventional formulation.

Seasonal variations in the morphology of the seminal vesicles of the African straw-coloured fruit bat (Eidolon helvum).

The study investigated seasonal changes in the morphology of the seminal vesicles of Eidolon helvum in a typical African tropical environment. Seminal vesicles of forty-eight bats were examined during the early rainy, late rainy and peak dry seasons, using gross anatomical, histological and ultrastructural techniques. The interlobular connective tissue septa from the external capsule demarcated the glandular parenchyma into numerous lobules of tubulo-alveolar glands. A simple cuboidal epithelium of mono- or bi-nucleated cuboidal cells lined the glandular mucosa during the rainy season. This was replaced by a bistratified cuboidal epithelium, whose upper layer of cells showed evidence of degeneration, during the dry season. The PAS-positive secretory products appeared to be numerous during the rainy season, but few and clumped together during the dry season. The mean gross weight, interlobular septal thickness, acini diameter and epithelial height of the glands varied significantly with season. Ultrastructural features of the secretory cells during the rainy season showed well-developed cytoplasmic organelles, numerous electron lucid secretory vesicles and electron-dense granules. Secretory epithelial cells of the seminal vesicles are very active during the rainy season, while regeneration of degenerated epithelium in preparation for the next reproductive cycle occurs during the dry season.

Cytogenetic biomonitoring of road tunnel construction workers: buccal micronucleus cytome assay.

Road tunnel construction workers revealed higher frequencies of cells with genotoxic damage (i.e., MN and NBUD). MN and NBUD resulted to be Poisson distributed and counts of these genotoxicity biomarkers were then analysed by Poisson regression. The frequency ratio (FR) for MN was 1.31 (95% CI: 0.84-2.04), with an increase in the exposed subjects; this finding, though indicating a higher genotoxic risk in the exposed subjects, did not reach statistical significance. On the other hand, the FR for NBUD was 3.49 (95% CI: 1.86-6.56), with a statistically significant increased risk of chromosomal damage. Even the frequencies of binucleated cells (a marker of cell proliferation) and pyknotic cells (a cell death biomarker) were significantly higher in tunnel workers. Tunnel construction workers are exposed to complex mixtures of toxic agents, some of which are known to be genotoxic. The aim of this study was to evaluate the genotoxic risk in this occupational setting by comparing tunnel workers with a control group for frequencies of nuclear aberrations in oral exfoliated cells. To evaluate the genotoxic effects of tunnel air pollutants, we conducted a cross-sectional, molecular epidemiological study (35 tunnel workers and 35 healthy controls) using the buccal micronucleus cytome assay. A questionnaire was administered to obtain information about demographic variables, lifestyle, dietary habits, anthropometric data, and occupational history. Buccal mucosa cells were collected by scraping the buccal mucosa with a small-headed toothbrush. Coded slides were examined blind by trained scorers for micronuclei (MN), nuclear buds (NBUD), and other nuclear abnormalities. Our observations provide further knowledge and understanding of the occupational hazards of tunnel workers and confirm the complexity of effects (cytotoxic and genotoxic) probably induced by fumes and dust produced in underground operations.

Micronucleus frequency in buccal mucosa cells of patients with neurodegenerative diseases

Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer’s disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.

Micronucleus Evaluation in Exfoliated Human Buccal Epithelium Cells among E-Waste Workers in Payatas, the Philippines.

Background.The improper recycling of electronic waste (e-waste) by informal recyclers often leads to contamination of the environment. E-waste contains organic and inorganic compounds along with heavy metals and trace elements. These pollutants can have a negative effect on humans. Biomonitoring can provide information on the sources, amount, geographical distribution, and adverse health effects of contaminants.Objectives.The present study aimed to assess risks to the health of informal e-waste recyclers in Payatas, the Philippines due to their exposure to e-waste toxicity by examining the presence of micronuclei in buccal epithelium cells.Methodology.Frequencies of binucleated cells (BNc) and abnormal cells were obtained from the buccal epithelium of the study population composed of e-waste exposed recyclers (n=40) and a control group (n=52). Descriptive statistics and regression analysis were employed for the data analysis.Results.Participants' gender, occupation, smoking status, alcohol consumption, and the number of karyolitic cells of both groups were significantly associated. Only occupation in e-waste recycling and length of e-waste exposure were significantly associated in terms of the number of abnormal cells and micronuclei. Similar trends were found in the linear regression analysis drawn from participants' length of e-waste exposure with a significance of R2= 7346, indicating that as the length of e-waste exposure increased, the number of micronuclei found in the participants' buccal epithelium cells increased as well.Conclusions.Longer exposure to e-waste materials may induce genotoxic damage in human cells which is a serious concern, leading to adverse effects to human health.Competing Interests.The authors declare no competing financial interests.

The Assessment of Radon Emissions as Results of the Soil Technogenic Disturbance.

222Rn is a specific indoor-type pollutant that represents a primary radiological hazard as a main source of ionizing radiation (IR) for humans. Coal mining creates new sources of gas that are formed over mines. This process can significantly increase the density of radon flux. Therefore, the concentration of radon in a room can increase. We investigated the territory of the Leninsk-Kuznetsky district of the Kemerovo region, which is subject to underground mining. Two groups of residential locations and measuring points of radon flux density were selected to identify the higher emanation relationship of radon and mining-affected areas. The first group (Case group) included subjects located within the territory of the underground mine; the other (Control group) included subjects in an area without mining. Radon flux density in coal mining areas was significantly higher than in the rest of the territory; moreover, the percentage of values in the Case group that had a radon flux density above 80 mBq·m−2·s−1 was 64.53%. For the Case group, 20.62% of residential buildings had a radon concentration above 200 Bq/m3. For the studied area, the radon flux density correlates positively (r = 0.79, p = 0.002) with indoor radon. Additional clastogenic/aneugenic effects are also found in dwellings with increased volume activity of radon (VAR) within the territories of underground mines. Ring chromosomes are positively correlated with radon levels in smoker groups but not in non-smokers. An increased frequency of binucleated (BN) cells with micronuclei (MN) is also positively correlated with VAR regardless of smoking status. It has been concluded that reducing the total exposure level of a population to radon can be achieved by monitoring areas with underground mines where radon is emitted heavily.

DNA damage in lens epithelial cells exposed to occupationally-relevant X-ray doses and role in cataract formation

The current framework of radiological protection of occupational exposed medical workers reduced the eye-lens equivalent dose limit from 150 to 20 mSv per year requiring an accurate dosimetric evaluation and an increase understanding of radiation induced effects on Lens cells considering the typical scenario of occupational exposed medical operators. Indeed, it is widely accepted that genomic damage of Lens epithelial cells (LEC) is a key mechanism of cataractogenesis. However, the relationship between apoptosis and cataractogenesis is still controversial. In this study biological and physical data are combined to improve the understanding of radiation induced effects on LEC. To characterize the occupational exposure of medical workers during angiographic procedures an INNOVA 4100 (General Electric Healthcare) equipment was used (scenario A). Additional experiments were conducted using a research tube (scenario B). For both scenarios, the frequencies of binucleated cells, micronuclei, p21-positive cells were assessed with different doses and dose rates. A Monte-Carlo study was conducted using a model for the photon generation with the X-ray tubes and with the Petri dishes considering the two different scenarios (A and B) to reproduce the experimental conditions and validate the irradiation setups to the cells. The simulation results have been tallied using the Monte Carlo code MCNP6. The spectral characteristics of the different X-ray beams have been estimated. All irradiated samples showed frequencies of micronuclei and p21-positive cells higher than the unirradiated controls. Differences in frequencies increased with the delivered dose measured with Gafchromic films XR-RV3. The spectrum incident on eye lens and Petri, as estimated with MCNP6, was in good agreement in the scenario A (confirming the experimental setup), while the mean energy spectrum was higher in the scenario B. Nevertheless, the response of LEC seemed mainly related to the measured absorbed dose. No effects on viability were detected. Our results support the hypothesis that apoptosis is not responsible for cataract induced by low doses of X-ray (i.e. 25 mGy) while the induction of transient p21 may interfere with the disassembly of the nuclear envelop in differentiating LEC, leading to cataract formation. Further studies are needed to better clarify the relationship we suggested between DNA damage, transient p21 induction and the inability of LEC enucleation.

Morphological Analysis of Human Milk Membrane Enclosed Structures Reveals Diverse Cells and Cell-like Milk Fat Globules

Over the past decade, the cellular content of human milk has been a focus in lactation research due to the benefit a potential non-invasive stem cell compartment could provide either to the infant or for therapeutic applications. Despite an increase in the number of studies in this field, fundamental knowledge in regard to milk cell identification and characterisation is still lacking. In this project, we investigated the nature, morphology and content of membrane enclosed structures (MESs) and explored different methods to enrich human milk cells (HMCs) whilst reducing milk fat globule (MFG) content. Using both flow cytometry and immunofluorescence imaging, we confirmed previous reports and showed that nucleated HMCs make up a minority of milk-isolated MESs and are indistinguishable from MFGs without the use of a nuclear stain. HMC heterogeneity was demonstrated by differential uptake of nuclear stains Hoechst 33258 and DRAQ5™ using a novel technique of imaging milk MESs (by embedding them in agar), that enabled examination of both extracellular and intracellular markers. We found that MESs often contain multiple lipid droplets of various sizes and for the first time report that late post-partum human milk contains secretory luminal binucleated cells found across a number of participants. After investigation of different techniques, we found that viably freezing milk cells is an easy and effective method to substantially reduce MFG content of samples. Alternatively, milk MESs can be filtered using a MACS® filter and return a highly viable, though reduced population of milk cells. Using the techniques and findings we’ve developed in this study; future research may focus on further characterising HMCs and the functional secretory mammary epithelium during lactation.

Measurement of transfer of colostral passive immunity in dairy calves

Measurement of transfer of colostral passive immunity in dairy calves

Light and Electron Microscopic Studies of the Seminal Vesicular Glands of the African Straw-coloured Fruit Bat, Eidolon helvum

Background: Secretions from the seminal vesicular gland enhance the survival and reproductive functions of the spermatozoa. The order Chiroptera has evolved great diversity in their reproductive structure influenced by location, abiotic factors and availability of food. Eidolon helvum is a near threatened tropical African fruit bat and little is known of its reproductive biology. The aim of this study was to highlight the structure, histochemistry and ultrastructure of the seminal vesicular glands of Eidolon helvum. Methods: The paired seminal vesicular glands of 16 adult male Eidolon helvum were obtained in May during the early rainy season in tropical Nigeria. The glands were evaluated grossly, histological and ultrastructurally.Result: The spirally coiled tubular organs were divided into numerous lobules of tubular-alveolar glandular acini lined by simple cuboidal epithelium made up of basal cells and mono or bi-nucleated cuboidal secretory cells. PAS positive secretions projected from the apical surfaces. Principal secretory cells contained numerous rough endoplasmic reticulum, mitochondria, Golgi complexes, electron lucid secretory vesicles, electron dense granules and lysosomes. The unique merocrine and apocrine secretions contributed to the formation of vaginal plugs.

Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay

Purpose In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. Materials and methods Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy – background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. Results >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. Conclusion This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90–216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Risk of genotoxic damage in schoolchildren exposed to organochloride pesticides

This study identified and determined organochloride pesticide (OCs) concentrations in hair samples from children at two elementary schools: one exposed to fumigations in agricultural fields, the other unexposed. Three concentrations of OCs levels in the hair were compared (high, medium, low), and total nuclear abnormalities in buccal cells were determined: micronuclei (MNi), condensed chromatin, karyorrhexis, pyknosis, binucleate cells, karyolysis, lobed nuclei, and apoptosis. No significant differences were found for the presence of MNi between the schoolchildren from the exposed and unexposed schools, but the prevalence of OCs in both schools was over 50%, as well as the frequencies of MNi in the children were over 58%. Findings show a significant difference between the frequency of MNi in the total sample of schoolchildren (exposed school + unexposed school) in relation to the concentration of OCs detected in their hair. The children from exposed school that showed the higher concentrations of OCs in hair had higher levels of genotoxic damage in the buccal cells; compared against children with lower concentrations of OCs. The most frequent nuclear abnormalities in the exposed children were lobed nuclei (79.4%), binucleate cells (66.66%), apoptosis (65.07), and MNi (58.7%). We determined the prevalence ratio (PR) and prevalence odds ratio (POR) for the presence of MNi in buccal cells in relation to the OCs concentrations in the hair samples. Both ratios were high for MNi [PR 3.93, 95% confidence interval (CI) 1.97–7.84, p = 0.0003; and POR 7.97, 95% CI 2.62–24.28, p = 0.0003], indicating a 7.97 times greater risk that the exposed children will present > 0.2% of MNi when OCs concentrations exceed 0.447 μg/g. These indicators may be useful biomarkers of genotoxic damage in children exposed to persistent, highly-toxic compounds. Results suggest the potential risk to which those schoolchildren are exposed on a daily basis due to fumigations in nearby agricultural fields.

Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.

THE EFFECT OF APRICOT SEEDS ON MICROSCOPIC STRUCTURE OF RABBIT LIVER

Natural phytosubstances, such as amygdalin, used in alternative medicine has gained popularity. However, some researchers suspect the protective properties of amygdalin due to a lack of clinical studies. The aim of the present in vivo study was to determine the effect of apricot seed administration on microscopic changes in the liver using a rabbit as a biological model. Sixteen male rabbits 45 days old were randomly divided into four groups (control group without any apricot seed administration, and experimental groups fed by crushed apricot seeds at the doses 60, 300 and 420 mg/kg b.w., mixed with commercial feed), which was administered orally a daily during a ten-month period. The liver tissue samples were evaluated by histological analysis. Significant changes were observed in the microscopic structure of rabbit livers after apricot seed ingestion. The morphometric evaluation of rabbit livers after the application of apricot seeds showed an increase of binucleated cells in the vena centralis region (P≤0.001) at the highest dose and in the peripheral zone at all the doses used (P≤0.001, P≤0.01, P≤0.05) compared to control. On the other hand, distinct inhibition in the number of binucleated cells in the region vena centralis at the doses 300 (P≤0.01) and 420 mg/kg b.w. (P≤0.05) and in the peripheral zone at all the doses used (P≤0.001, P≤0.01) was observed. No significant differences between the control and experimental groups in vena centralis after apricot seed treatment were found (P≥0.05). In addition, the effect of apricot seeds on the relative volume of liver structures – vena centralis, stroma and parenchyma after the application of apricot seeds to rabbit males were assessed. No significant differences between control and experimental groups in the relative volume of vena centralis were found (P≥0.05). On the other hand, the relative volume of the stroma was increased at doses 60 (P≤0.05) and 300 (P≤0.01) mg/kg b.w. Interestingly, the relative volume of parenchyma was significantly decreased (P≤0.05) after the application of apricot seeds in two experimental groups 60 and 300 mg/kg b.w. The current study provides experimental evidence that apricot seeds might affect the liver microscopic structure in rabbits in vivo and thus amygdalin present in apricot seeds might present a potential risk for animal health. However, the toxic effect could not be accurately corroborated, as in many cases changes were dose-dependent and not recorded at the highest dose used in the study.

Cytokinesis-Block Micronucleus Cytome Assay Evolution into a More Comprehensive Method to Measure Chromosomal Instability.

This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.

Revisiting cytomorphology, including unusual features and clinical scenarios of 8 cases of alveolar soft part sarcoma with TFE3 immunohistochemical staining in 7 cases.

To present a comprehensive analysis of cytomorphological features, including clinical scenarios, for 8 cases (4 males, 4 females, aged 17-39 years, average=28.5) of, retrospectively diagnosed alveolar soft part sarcoma (ASPS), with TFE3 immunostaining in 7 cases. Conventional Papanicolaou and May Grunwald-Giemsa (MGG) stained smears and corresponding tissue sections were critically reviewed. Fine needle aspiration cytology was performed for primary diagnosis in 6 cases and for metastatic lesions in 2 cases. TFE3 and other immunohistochemical stains were tested using polymer detection technique. Tumour sites were thigh (n=6), shoulder (1) and neck (1). Tumour size (n=6) varied from 5 to 14.5 cm (average=7.2). Seven out of 8 cases were correctly diagnosed on cytosmears. The smears were mostly hypercellular (5), composed of cohesive clusters (8), including cell balls and pseudopapillae (3) and singly scattered cells (8). Tumour cells were round to oval, containing central to eccentric nuclei (8), abundant granular (8) to finely vacuolated (7) cytoplasm that was ill- to well-defined, intracytoplasmic rod-like or needle-shaped crystals (3) and prominent nucleoli (8), Additionally, there were binucleated cells (7), multinucleation (2), intracytoplasmic inclusions (3), intranuclear inclusions (2), intercellular stroma (5) and bare nuclei (8). Immunohistochemically, 7/8 tumours were positive for TFE3. This constitutes the largest series describing cytomorphological spectrum of ASPS with TFE3 immunostaining results. Frequently observed features and rod-like/needle-shaped crystals on MGG smears, can help to differentiate ASPS from its mimics. TFE3 immunostaining aids in substantiating diagnoses, in an appropriate clinicoradiological context.