Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Cytogenetic changes in oral mucosa cells from individuals submitted to oral human immunodeficiency virus pre-exposure prophylaxis use.

OBJECTIVE: The objective of this study was to evaluate cytogenetic changes in individuals submitted to oral human immunodeficiency virus pre-exposure prophylaxis use through the micronucleus test in oral mucosa. METHODS: This study consisted of 37 individuals, of whom 17 comprised the pre-exposure prophylaxis group and 20 comprised the control group. A total of 2,000 cells per slide were analyzed for the determination of micronuclei, binucleation, nuclear buds, and cytotoxicity parameters: pyknosis, karyolysis, and karyorrhexis (KR), in a double-blind manner. The repair index was also evaluated in this setting. RESULTS: In the mutagenicity parameters, the pre-exposure prophylaxis group showed increased frequencies of micronuclei (p=0.0001), binucleation (p=0.001), and nuclear buds (p=0.07). Regarding the cytotoxicity parameters, there was an increase with a statistical difference (p≤0.05) in the karyorrhexis frequency (p=0.001). Additionally, the repair system efficiency decreased in the pre-exposure prophylaxis group. CONCLUSION: These results indicate that individuals undergoing pre-exposure prophylaxis use have geno- and cytotoxicity in oral mucosal cells.

Micronuclei in human peripheral blood and bone marrow as genotoxicity markers: A systematic review and meta-analysis.

Can human peripheral blood cells be used as a surrogate for bone marrow cells, in evaluating the genotoxic effects of stressors? We searched the Pubmed/Medline and PubChem databases to identify publications relevant to this question. Micronucleus formation was the genotoxicity endpoint. Three publications comparing exposed vs. non-exposed individuals are included in this analysis; the exposures were to ethylene oxide or ionising radiation (atomic bomb, thorotrast, or radioiodine therapy). Information was extracted on the types of exposure, the numbers of participants, and the micronucleus frequencies. Relative differences (odds ratios) and absolute differences (risk differences) in the numbers of micronuclei between exposed and non-exposed persons were calculated separately for individual cell types (peripheral blood and bone marrow). Random effects meta-analyses for the relative differences in cell abnormalities were performed. The results showed very small differences in the frequencies of micronuclei between exposed and non-exposed individuals, as measured in either peripheral blood or bone marrow cell populations, on both absolute and relative scales. No definite conclusion concerning the relative sensitivities of bone marrow and peripheral blood cells can be made, based on these publications.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Heritable transcriptional defects from aberrations of nuclear architecture.

Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance1-3. However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer4,5, as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.

Mitotic clustering of pulverized chromosomes from micronuclei.

Complex genome rearrangements can be generated by the catastrophic pulverization of missegregated chromosomes trapped within micronuclei through a process known as chromothripsis1-5. As each chromosome contains a single centromere, it remains unclear how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis6. Here we tracked micronucleated chromosomes with live-cell imaging and show that acentric fragments cluster in close spatial proximity throughout mitosis for asymmetric inheritance by a single daughter cell. Mechanistically, the CIP2A-TOPBP1 complex prematurely associates with DNA lesions within ruptured micronuclei during interphase, which poises pulverized chromosomes for clustering upon mitotic entry. Inactivation of CIP2A-TOPBP1 caused acentric fragments to disperse throughout the mitotic cytoplasm, stochastically partition into the nucleus of both daughter cells and aberrantly misaccumulate as cytoplasmic DNA. Mitotic clustering facilitates the reassembly of acentric fragments into rearranged chromosomes lacking the extensive DNA copy-number losses that are characteristic of canonical chromothripsis. Comprehensive analysis of pan-cancer genomes revealed clusters of DNA copy-number-neutral rearrangements-termed balanced chromothripsis-across diverse tumour types resulting in the acquisition of known cancer driver events. Thus, distinct patterns of chromothripsis can be explained by the spatial clustering of pulverized chromosomes from micronuclei.

Micronuclei in embryos of eight seabird species in northwestern Mexico: A biomarker of exposure to coastal pollution?

The micronucleus (MN) test may be used to evaluate genome instability in birds and the potential of different species to function as biomarkers of genotoxicity. However, little is known regarding genome instability in seabird embryos or the instability present among embryonic development stages. Therefore, the present study aimed to describe the frequencies of micronucleated erythrocytes (MNE) and micronucleated polychromatic erythrocytes (MNPCE) in blood samples collected from the embryos of eight seabird species nesting on the coast of Sinaloa, Mexico. An additional description of blood cell maturation along with embryo development during incubation was conducted based on the proportion of polychromatic erythrocytes (PCE), and the potential relationships between metals (Hg and Cd concentrations in egg content) and the MN frequencies in embryo blood were evaluated. The PCE proportion appears to decline as incubation advances (initial stage > intermediate stage > advanced stage), and the values varied between species (Suliformes/Pelecaniformes < Charadriiformes: Laridae), which may be related to differences among incubation periods and reproductive strategies. Interspecific variation in the MNPCE frequency was found in embryos showing advanced development, which could be related to both variations in life-history traits and ecological factors and not Hg or Cd exposure. The genomic instability values in this study are the first to be reported for embryos of seabird species nesting in a subtropical coastal region.

Resveratrol-Tempeh reduce micronucleus frequencies bone marrow cells and stimulate osteocyte proliferation in aluminum chloride-induced mice.

Aluminum (Al) is widely used for water purification, cooking pots, cosmetic and pharmaceutical preparations, toothpaste tubes, and food processing industries. Although the transport in the digestive tract is very poor but if the load is high, it can be absorbed and accumulated. About 50-70% of Al accumulates in the bones and can have an impact on human health. Resveratrol (RES), isolated from tempeh as an Indonesian food ingredient, can increase cell viability and has promising cytoprotective effects. RES has the capacity to interact with oxidative stress, so it has the potential as a therapy in bone repair. Therefore, this study aimed to evaluate the effect of RES on the number of osteocytes and bone marrow cells in Al-induced mice. Swiss Webster mice were divided into four groups: (1) untreated groups, (2) AlCl3-treated groups, (3) Al+Res5 treated groups, and (4) Al+Res10 treated groups. Al dose 200 mg/kg body weight was administered intraperitoneally. RES was given one hour after administration of Al, with doses of 5 and 10 mg/kg Body Weight. Al and RES administration is carried out for one month. All mice were sacrificed, and mouse bones were isolated for histological preparations and a half for genotoxic assays. Bone marrow cells were collected and stained with My Grunwald. The number of micronuclei polychromatic erythrocytes (MNPCE) was examined in 1,000 PCEs per animal. The number of PCEs is counted by at least 200 erythrocytes (PCE + NCE) per animal. The results showed that the administration of Al significantly increased the number of micronuclei (MN) but after administration of RES at doses of 5 and 10 mg/kg Body Weight significantly reduced the number of MN in bone marrow cells. A dose of RES 10 mg/kg BW stimulates proliferation and increases the number of osteocytes in bone significantly. It can be concluded that Al can cause genotoxicity in bone marrow cells and RES is anti-genotoxic and can stimulate osteocyte proliferation.

Chromosome aberrations, micronucleus frequency, and catalase concentration in a population chronically exposed to high levels of radon.

PURPOSE: To deepen our knowledge on the effects of high levels of indoor radon exposure, we assessed the frequencies of unstable and stable chromosome aberrations and micronucleus (MN), as well as the concentration of an endogenous antioxidant (catalase, CAT), in blood samples of individuals chronically exposed to high indoor radon concentrations in Indonesia (Tande-Tande sub-village, Mamuju, West Sulawesi). Moreover, we also investigated the occurrence of a radio-adaptive response (RAR) in Tande-Tande sub-village inhabitants using the G2 MN assay. MATERIALS AND METHODS: The frequencies of dicentric (DC), acentric (AF), ring (R), and translocation (Tr) chromosomes in Tande-Tande inhabitants were compared to those in people living in a reference area with low levels of indoor radon levels (Topoyo village, Indonesia). The number of MN per 1000 binucleated cells (BNC) and CAT concentration per total protein was quantified and compared between groups. Lastly, we irradiated (2 Gy) phytohemagglutinin-stimulated samples in vitro and measured the frequency of MN to verify the occurrence of a RAR in Tande-Tande sub-village inhabitants. RESULTS AND CONCLUSION: The frequencies of DC, AF, and Tr did not differ between Tande-Tande inhabitants and control subjects (p = 0.350, 0.521, 0.597). The frequency of MN in Tande-Tande inhabitants was significantly lower than that in the control group (p = 0.006). Similarly, CAT concentration in Tande-Tande inhabitants was also significantly lower than that in the control population (p < 0.001). Significant negative correlations were identified for MN number and CAT concentration versus indoor radon concentration, annual effective dose, or cumulative dose both within groups and when all data were analyzed together. Our findings indicate that, despite the high indoor radon levels, Tande-Tande inhabitants are not under oxidative stress, since this group had lower CAT concentration and MN frequency than those in the control group. The negative correlation between MN frequency and indoor radon concentration, annual effective dose, and cumulative dose suggests the occurrence of an RAR phenomenon in Tande-Tande sub-village inhabitants. This interpretation is also supported by the results of the G2 MN assay, which revealed lower MN frequencies after in vitro irradiation of samples from Tande-Tande sub-village inhabitants than those in samples from the control group (p = 0.0069, for cumulative MN frequency; p = 0.0146, for radiation-induced MN only).

Fate of micronuclei and micronucleated cells after treatment of HeLa cells with different genotoxic agents.

Although micronuclei are well-known biomarkers of genotoxic damage, the biological consequences of micronucleus induction are only poorly understood. To further elucidate these consequences, HeLa cells stably expressing histone 2B coupled with green fluorescent protein were used for long-term live cell imaging to investigate the fate of micronuclei and micronucleated cells after treatment of cells with various genotoxic agents (doxorubicin (20, 30 and nM), tert-butyl hydroperoxide (tBHP, 50, 100 and 150 µM), radiation (0.5, 1 and 2 Gy), methyl methanesulfonate (MMS, 20, 25 and 30 µg/ml) and vinblastine (1, 2 and 3 nM)). Most micronuclei persist for multiple cell cycles or reincorporate while micronucleated cells were more prone to cell death, senescence and fatal mitotic errors compared to non-micronucleated cells, which is consistent with previous studies using etoposide. No clear substance-related effects on the fate of micronuclei and micronucleated cells were observed. To further investigate the fate of micronuclei, extrusion of micronuclei was studied with treatments reported as inducing the extrusion of micronuclei. Since extrusion was not observed in HeLa cells, the relevance of extrusion of micronuclei remains unclear. In addition, degradation of micronuclei was analysed via immunostaining of γH2AX, which demonstrated a high level of DNA damage in micronuclei compared to the main nuclei. Furthermore, transduction with two reporter genes (LC3B-dsRed and LaminB1-dsRed) was conducted followed by long-term live cell imaging. While autophagy marker LC3B was not associated with micronuclei, Lamin B1 was found in approximately 50% of all micronuclei. While degradation of micronuclei was not observed to be a frequent fate of micronuclei, the results show impaired stability of DNA and micronuclear envelope indicating rupture of micronuclei as a pre-step to chromothripsis.

Micronucleus assay in the exfoliated cells of buccal mucosa of gasoline station workers in Tehran.

Objectives: One of the most critical landmarks of DNA damage is the micronucleus assay. Enumeration of micronuclei contributes to the early diagnosis of precancerous lesions and cancers; however, there are few studies on the frequency of micronucleus in gasoline station workers. To the best of our knowledge, no study has addressed this issue in Iran. The present study aimed to determine the role of working in the gasoline stations of Tehran city on micronucleus frequency in buccal mucosa. Materials and Methods: In this historical cohort study, buccal mucosa samples were collected from 110 individuals working at gasoline stations and 100 unemployed persons using wet tongue depressors. After Papanicolaou staining, the percentage of cells containing micronucleus as well as the mean number of micronucleus in the micronucleated cells was reported. Student's t-test, Mann-Whitney test, and regression analyses were used to specify the effect of other variables on the frequency and mean number of micronucleus per cell. Results: The mean frequency of micronucleus in the case and control group was 29.8 ± 8.2 and 9.3 ± 3.2, respectively, which was statistically significant (P = 0.0001). Furthermore, the mean number of micronucleus in the micronucleated cells of buccal mucosa was significantly higher in individuals who were exposed to gasoline than the control group (P = 0.0001). Conclusion: The results indicated that exposure to gasoline could increase the frequency of micronucleus. It was also revealed that cigarette and hookah smoking and alcohol consumption, together with working in gasoline stations, increase micronucleus abundance, implying the cumulative carcinogenic effect of these factors.

ESTABLISHMENT OF THE IN VITRO DOSE-RESPONSE CALIBRATION CURVE FOR X-RAY-INDUCED MICRONUCLEI IN HUMAN LYMPHOCYTES.

The cytokinesis-block micronucleus assay has proven to be a reliable technique for biological dosimetry. This study aimed to establish the dose-response curve for X-ray-induced micronucleus. Peripheral blood samples from three healthy donors were irradiated with various doses and scoring criteria by the micronuclei (MN) in binucleated cells. The results showed that the frequency of MN increased with the elevation of radiation dose. CABAS and Dose Estimate software were used to fit the MN and dose into a linear quadratic model, and the results were compared. The linear and quadratic coefficients obtained by the two software were basically the same and were comparable with published curves of similar radiation quality and dose rates by other studies. The dose-response curve established in this study can be used as an alternative method for in vitro dose reconstruction and provides a reliable tool for biological dosimetry in accidental or occupational radiation exposures.

Susceptibility to chromosome instability and occurrence of the regular form of Down syndrome in young couples.

Although the risk of pregnancy with Down syndrome (DS) increases with age, conceptions with trisomy 21 can occur in mothers aged 35 or less. The micronucleus test on peripheral blood lymphocytes is a well-recognized method for studying chromosomal instability. The aim of this study was to evaluate the application of the micronucleus assay and fluorescence in situ hybridization (FISH) for estimation of chromosome instability and occurrence of trisomy 21 in young parents having pregnancy or a child with the regular form of Down syndrome. The study included 54 parents (27 couples) who had previous pregnancy with trisomy 21 at age 35 or less. The control group consisted of 30 couples with two healthy children and no previous spontaneous abortions. Parents with trisomy 21 pregnancy had significantly higher frequencies of micronuclei in binucleated cells. There was no statistically significant difference between the study and control groups in the frequencies of micronuclei in mononuclear cells, nuclear buds, or nucleoplasmic bridges. FISH analysis showed higher percentages of micronuclei containing whole chromosomes as well as statistically significant higher numbers of micronuclei containing chromosome 21 in the peripheral blood of DS parents. There was no statistically significant difference between the two groups in the responses of peripheral blood lymphocytes to treatment with the mutagen mitomycin C. Our results suggest that young parents with a history of the regular form of Down syndrome have a higher susceptibility to chromosome nondisjunction in peripheral blood lymphocytes. The micronucleus assay showed high specificity, but moderate sensitivity, for risk assessment of trisomy 21 pregnancy.

Nuclear chromosome locations dictate segregation error frequencies.

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.

Factors Affecting the Nuclei in Newborn and Children.

It is known that children are more sensitive to the effects of medical treatments and environment than adults. Today there is limited information regarding the differences in genotoxic effects in children. The micronucleus assay is a method that is used to monitor genotoxicity, and it was validated several years before. Today there is international interest for exfoliated buccal cells. Most of the micronuclei studies in children have been performed with the analyses of lymphocytes. However, there is vast interest in using exfoliated cells from the oral cavity. The reason is that other type of cells are acquired non-invasively, this is an important issue in paediatric cohorts. Unfortunately a limitation of measuring micronuclei frequency is that it has been observed to be low in newborns and on the other hand there are a large number of patients and cell sample counts. It has been observed that radiation exposure and environmental pollutants increase the micronuclei frequency in newborn and children. Regarding the medical treatments, there is little data and several studies are needed to optimise the doses. There is the need to observe if there is a relationship between micronuclei in lymphocytes and exfoliated cells and to identify the baseline of the micronuclei levels. Moreover, we evaluate the changes in response to the toxic agents. Prospective cohorts studies will clarify the predictive value of micronuclei for cancer and chronic diseases for both children and adults. Novel molecular technologies will assist in the elucidation of different biological pathways and molecular mechanisms connected with the micronulcei levels in newborn and children.

Rapid and automatic detection of micronuclei in binucleated lymphocytes image.

Cytokinesis block micronucleus (CBMN) assay is a widely used radiation biological dose estimation method. However, the subjectivity and the time-consuming nature of manual detection limits CBMN for rapid standard assay. The CBMN analysis is combined with a convolutional neural network to create a software for rapid standard automated detection of micronuclei in Giemsa stained binucleated lymphocytes images in this study. Cell acquisition, adhesive cell mass segmentation, cell type identification, and micronucleus counting are the four steps of the software's analysis workflow. Even when the cytoplasm is hazy, several micronuclei are joined to each other, or micronuclei are attached to the nucleus, this algorithm can swiftly and efficiently detect binucleated cells and micronuclei in a verification of 2000 images. In a test of 20 slides, the software reached a detection rate of 99.4% of manual detection in terms of binucleated cells, with a false positive rate of 14.7%. In terms of micronuclei detection, the software reached a detection rate of 115.1% of manual detection, with a 26.2% false positive rate. Each image analysis takes roughly 0.3 s, which is an order of magnitude faster than manual detection.

Plant Cytogenetics in the Micronuclei Investigation-The Past, Current Status, and Perspectives.

Cytogenetic approaches play an essential role as a quick evaluation of the first genetic effects after mutagenic treatment. Although labor-intensive and time-consuming, they are essential for the analyses of cytotoxic and genotoxic effects in mutagenesis and environmental monitoring. Over the years, conventional cytogenetic analyses were a part of routine laboratory testing in plant genotoxicity. Among the methods that are used to study genotoxicity in plants, the micronucleus test particularly represents a significant force. Currently, cytogenetic techniques go beyond the simple detection of chromosome aberrations. The intensive development of molecular biology and the significantly improved microscopic visualization and evaluation methods constituted significant support to traditional cytogenetics. Over the past years, distinct approaches have allowed an understanding the mechanisms of formation, structure, and genetic activity of the micronuclei. Although there are many studies on this topic in humans and animals, knowledge in plants is significantly limited. This article provides a comprehensive overview of the current knowledge on micronuclei characteristics in plants. We pay particular attention to how the recent contemporary achievements have influenced the understanding of micronuclei in plant cells. Together with the current progress, we present the latest applications of the micronucleus test in mutagenesis and assess the state of the environment.