Genotoxicity of Gamma Radiation Against Lymphocytes of Radiation Workers: The Cytokinesis-Block Micronucleus Assay.

<b>Background and Objective:</b> Gamma irradiation induces genotoxicity, characterized by the formation of extra-nuclear bodies and left behind during the anaphase stage of cell division, often referred to as a micronucleus (MN). The present work aims to monitor exposure to ionizing radiation as a genotoxic agent in the lymphocytes of workers at radiation energy centers. <b>Materials and Methods:</b> The lymphocyte cytokinesis block micronucleus assay used and analyzed the correlation between the Nuclear Division Index (NDI), age, blood type and the number of micronuclei (MN). Blood samples were collected from 20 volunteers in heparin tubes, exposed to 2 Gy gamma rays and cultured <i>in vitro</i>. <b>Results:</b> A significant difference in the number of micronuclei between blood group A and blood groups A, B and AB. The Nuclear Division Index (NDI) value for lymphocytes of radiation energy center workers after gamma radiation was significant (1.74±0.1) but still within the normal range. Neither MN frequency nor NDI values correlated with age, but MN frequency showed a correlation with blood type. <b>Conclusion:</b> The gamma irradiation did not induce a cytostatic effect but proved genotoxic to the lymphocytes of radiation energy center workers. Notably, blood type A demonstrated higher sensitivity to gamma radiation.

Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro.

In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett's oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 h (for the micronucleus (Mn) assay and the invasion assay) and 24 h (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-ß in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment. The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett's oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24 h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells. The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma's genotoxicity. The concentration of IL-8 in TK6 cells and IFN-ß in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.

Micronuclei and other nuclear anomalies in exfoliated urothelial cells and urinary 8-hydroxy-deoxyguanosine levels among Turkish hairdressers.

Hairdressers are constantly occupationally exposed to many chemicals have the potential to cause allergies and carcinogenic effects, act as skin and eye irritants and induce oxidative stress and DNA damage. This study aimed to evaluate occupation-induced genotoxicity based on the presence of micronucleus (MN) and other nuclear anomalies in urothelial cells and measure oxidative DNA damage based on the 8-hydroxy-2'-deoxyguanosine level in the urine of Turkish hairdressers. Originality of this study comes from that there was no study on MN and other nuclear anomalies frequencies and oxidative DNA damage in urine samples of hairdressers in the literature. The mean±standard deviation frequency (‰) of micronucleated (MNed) cells was higher in the hairdresser group (n=56) (4.81±7.87, p<0.001) than in the control group (n=56) (0.93±1.85). Nuclear buds were not observed in either group. While the frequency of basal cells was higher in the control group (446.6±106.21) than in the hairdresser group (367.78±101.51, p<0.001), the frequency of binuclear, karyolytic, pycnotic and karyorrhectic cells were higher in the hairdresser group (0.41±0.80, p<0.001; 438.02±118.27, p<0.001; 0.43±0.76, p<0.001; and 47.27±28.40, p<0.001) than in the control group (0.04±0.27, 358.57±95.71, 0.05±0.23 and 24.41±14.50). Condensed chromatins were observed only in the hairdresser group. Specific gravity adjusted 8-hydroxy-2'-deoxyguanosine level was statistically lower in the hairdresser group (908.21±403.25 ng/mL-SG) compared to the control group (1003.09±327.09 ng/mL-SG) (p=0.024). No significant correlation was found between the 8-hydroxy-2'-deoxyguanosine level and the frequency MN. The amount of formaldehyde released during Brazilian keratin treatment was higher than the American Conference of Governmental Industrial Hygienists -Threshold Limit Value (ACGIH-TLV; 0.1 ppm). Similarly, the amount of ethyl acetate released in three salons was above the recommended limit (400 ppm). These findings suggest that hairdressers have an increased risk of genotoxicity and cytotoxicity owing to occupational exposure, regardless of age, working hours, smoking and alcohol consumption.

Telomere length and micronuclei trajectories in APP/PS1 mouse model of Alzheimer's disease: Correlating with cognitive impairment and brain amyloidosis in a sexually dimorphic manner.

Although studies have demonstrated that genome instability is accumulated in patients with Alzheimer's disease (AD), the specific types of genome instability linked to AD pathogenesis remain poorly understood. Here, we report the first characterization of the age- and sex-related trajectories of telomere length (TL) and micronuclei in APP/PS1 mice model and wild-type (WT) controls (C57BL/6). TL was measured in brain (prefrontal cortex, cerebellum, pituitary gland, and hippocampus), colon and skin, and MN was measured in bone marrow in 6- to 14-month-old mice. Variation in TL was attributable to tissue type, age, genotype and, to a lesser extent, sex. Compared to WT, APP/PS1 had a significantly shorter baseline TL across all examined tissues. TL was inversely associated with age in both genotypes and TL shortening was accelerated in brain of APP/PS1. Age-related increase of micronuclei was observed in both genotypes but was accelerated in APP/PS1. We integrated TL and micronuclei data with data on cognition performance and brain amyloidosis. TL and micronuclei were linearly correlated with cognition performance or Aß40 and Aß42 levels in both genotypes but to a greater extent in APP/PS1. These associations in APP/PS1 mice were dominantly driven by females. Together, our findings provide foundational knowledge to infer the TL and micronuclei trajectories in APP/PS1 mice during disease progression, and strongly support that TL attrition and micronucleation are tightly associated with AD pathogenesis in a female-biased manner.

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

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

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.

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.

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.

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.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances.

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Chromosome length and gene density contribute to micronuclear membrane stability.

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.