The Frequency of Lymphocytes Containing Dumbbell-Shaped Nuclei Depends on Ionizing Radiation Dose and Correlates with Appearance of Chromosomal Aberrations

Meenakshi C., Sivasubramanian K., Venkatraman B.

Radon is a naturally occurring radionuclide in the environment, during decay it emits high linear energy transfer (LET) alpha particles. When radon exposure is accompanied by smoking it has been reported that lung cancer risk is higher. Blood samples were collected after prior consent, 25 smokers and 25 non smokers (only males) exposed in vitro to radon gas with doses ranging between 0.3-12.6mGy Ionizing radiation is a strong clastogenic agent and a potent inducer of MN. Cytokinesis-Blocked Micro Nucleus (CBMN) assay has proven to be a reliable, thoroughly validated and standardised technique in the field of radiation biology. In view of this, an invitro study has been undertaken using CBMN assay to suggest Nucleoplasmic bridges (NPBs) as a potential bio-dosimeter and to investigate the existence of a possible influence of smoking on genetic damage induced by ionizing radiation. The mean percentages frequencies of micronuclei, nucleoplasmic bridges and nuclear buds among the exposed smoker group were 4.82±0.271, 0.6±0.04 and 0.12±0.02 respectively and these values for exposed non-smoker group were 2.78±0.285, 0.4±0.04 and 0.06±0.01 respectively. Spearman rank correlation for the frequency of nucleoplasmic bridge with respect to dose was considered to be significant (P

Hintzsche H., Hemmann U., Poth A., Utesch D., Lott J., Stopper H.

The present review describes available evidence about the fate of micronuclei and micronucleated cells. Micronuclei are small, extranuclear chromatin bodies surrounded by a nuclear envelope. The mechanisms underlying the formation of micronuclei are well understood but not much is known about the potential fate of micronuclei and micronucleated cells. Many studies with different experimental approaches addressed the various aspects of the post-mitotic fate of micronuclei and micronucleated cells. These studies are reviewed here considering four basic possibilities for potential fates of micronuclei: degradation of the micronucleus or the micronucleated cell, reincorporation into the main nucleus, extrusion from the cell, and persistence in the cytoplasm. Two additional fates need to be considered: premature chromosome condensation/chromothripsis and the elimination of micronucleated cells by apoptosis, yielding six potential fates for micronuclei and/or micronucleated cells. The available data is still limited, but it can be concluded that degradation and extrusion of micronuclei might occur in rare cases under specific conditions, reincorporation during the next mitosis occurs more frequently, and the majority of the micronuclei persist without alteration at least until the next mitosis, possibly much longer. Overall, the consequences of micronucleus formation on the cellular level are still far from clear, but they should be investigated further because micronucleus formation may contribute to the initial and later steps of malignant cell transformation, by causing gain or loss of genetic material in the daughter cells and by the possibility of massive chromosome rearrangement in chromosomes entrapped within a micronucleus by the mechanisms of chromothripsis and chromoanagenesis.

Anbumani S., Mohankumar M.N.

Gamma radiation-induced genetic perturbations in aquatic vertebrates is largely unknown at low-dose rate, especially in the wake of a nuclear disaster and/or other environmental outbreaks. Freshwater fish, Oreochromis mossambicus subjected to low-dose rate (2 mGy/min) at 2.5-, 5-, and 10-Gy doses, were analyzed for “exposure signatures” in blood samples drawn on days 3, 6, 12, 18, and 30, respectively. Significant dose-dependent increments in micronuclei frequency and other anomalies such as nucleoplasmic bridges and tailed nuclei were observed and exhibit a strong positive correlation, suggesting that they could be used as prospective signatures of radiation exposure. Similarly increased incidence of apoptosis and DNA repair machinery circuits at high and low doses were noted. This work highlighted “cytogenetic signatures” in fish and the sensitivity of these endpoints toward low-dose rate of radiation exposure.

Sayed A.E., Oda S., Mitani H.

Previous studies have examined the effects of gamma-radiation on Japanese fish, in particular medaka (Oryzias latipes). In the present work, alterations in erythrocytes were recorded as haematological bio-indicators of exposure to gamma-radiation. After exposure of medaka fish to two different doses of radiation (2 Gy and 10 Gy), many malformations in red blood cells were observed in the irradiated fish compared with control fish. These malformations included acanthocytes, crenated cells, amoeboid cells, and sickle cells. More malformations were seen at the higher radiation dose. No micronuclei were seen in any group, but nuclear abnormalities were observed. We conclude that gamma-radiation causes morphological malformations of erythrocytes and is harmful to medaka fish.

Deshuillers P., Raskin R., Messick J.

A 14-year-old, spayed female Domestic Shorthair cat was referred to the Purdue University Veterinary Teaching Hospital (PUVTH) for iodine 131 treatment of hyperthyroidism. Upon arrival, a biochemistry profile and a CBC were performed. Approximately 50% of the neutrophils and all the eosinophils observed were hyposegmented with a mature, condensed chromatin pattern. Nuclei had a band to "dumbbell" shape, and rarely a round shape, suggesting a Pelger-Huët anomaly or a pseudo Pelger-Huët. Based on both a negative FeLV and FIV tests, the absence of any clinical signs to support an inflammatory process, and the persistence of this granulocytic morphology 6 months after its previous admission to the PUVTH, a diagnosis of Pelger-Huët anomaly was established in this cat.

Fenech M., Kirsch-Volders M., Natarajan A.T., Surralles J., Crott J.W., Parry J., Norppa H., Eastmond D.A., Tucker J.D., Thomas P.

Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

Pironon N., Puechberty J., Roizès G.

The mode of evolution of the highly homogeneous Higher-Order-Repeat-containing alpha satellite arrays is still subject to discussion. This is also true of the CENP-A associated repeats where the centromere is formed. In this paper, we show that the molecular mechanisms by which these arrays evolve are identical in multiple chromosomes: i) accumulation of crossovers that homogenise and expand the arrays into different domains and subdomains that are mostly unshared between homologues and ii) sporadic mutations and conversion events that simultaneously differentiate them from one another. Individual arrays are affected by these mechanisms to different extents that presumably increase with time. Repeats associated with CENP-A, where the centromere is formed, are subjected to the same evolutionary mechanisms, but constitute minor subsets that exhibit subtle sequence differences from those of the bulk repeats. While the DNA sequence per se is not essential for centromere localisation along an array, it appears that certain sequences can be selected against. On chromosomes 1 and 19, which are more affected by the above evolutionary mechanisms than are chromosomes 21 and 5, CENP-A associated repeats were also recovered from a second homogeneous array present on each chromosome. This could be a way for chromosomes to sustain mitosis and meiosis when the normal centromere locus is ineluctably undermined by the above mechanisms. We discuss, in light of these observations, possible scenarios for the normal evolutionary fates of human centromeric regions.

Hamza V.Z., Mohankumar M.N.

The effect of radon in inducing DNA damage was investigated in vitro by two well-established cytogenetic assays. Blood samples were irradiated with radon using a novel irradiation assembly. Doses varied between 0 and 127 mGy for chromosome aberration (CA) assay and 0 and 120 mGy for cytokinesis blocked micronucleus (CBMN) assay. Dose-rates varied between 0.000054 and 0.708 mGy/min. After the irradiation period of 3h, excess radon gas was released and cultures were initiated using standard procedures. Chromosome aberrations such as dicentrics, excess acentric fragments, acentric rings, centric rings, chromatid breaks were observed. Micronuclei, nucleoplasmic bridges and nuclear buds were scored by the CBMN assay. A significant increase in the frequency of dicentrics, excess acentric fragments and centric rings was observed with increasing radon dose, whereas total acentric rings plus double minute and chromatid breaks/cell were not significantly elevated. In CBMN assay, the frequency of micronuclei was found to be significantly raised whereas that of nucleoplasmic bridges and nuclear buds were not. Nucleoplasmic bridges and nuclear buds tended to increase with dose but did not achieve statistical significance. There was a strong positive correlation between nucleoplasmic bridges and dicentrics (P

Fenech M.

The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios. Further information regarding mechanisms leading to MNi, NPBs and NBUDs formation is obtained using centromere and/or telomere probes. The assay is being applied successfully for biomonitoring of in vivo genotoxin exposure, in vitro genotoxicity testing and in diverse research fields such as nutrigenomics and pharmacogenomics as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. The procedure can take up to 5 days to complete.

Schueler M.G., Sullivan B.A.

Centromeres are the elements of chromosomes that assemble the proteinaceous kinetochore, maintain sister chromatid cohesion, regulate chromosome attachment to the spindle, and direct chromosome movement during cell division. Although the functions of centromeres and the proteins that contribute to their complex structure and function are conserved in eukaryotes, centromeric DNA diverges rapidly. Human centromeres are particularly complicated. Here, we review studies on the organization of homogeneous arrays of chromosome-specific α-satellite repeats and evolutionary links among eukaryotic centromeric sequences. We also discuss epigenetic mechanisms of centromere identity that confer structural and functional features of the centromere through DNA-protein interactions and post-translational modifications, producing centromere-specific chromatin signatures. The assembly and organization of human centromeres, the contributions of satellite DNA to centromere identity and diversity, and the mechanism whereby centromeres are distinguished from the rest of the genome reflect ongoing puzzles in chromosome biology.

Joseph L.J., Patwardhan U.N., Samuel A.M.

The aim of this study was to evaluate the genotoxic effect of low dose of occupational radiation exposure in Nuclear Medicine Department employees, by using cytokinesis-blocked micronucleus assay in peripheral blood lymphocytes. The study included 46 exposed individuals together with 27 from the same area without occupational exposure to radiation which served as controls. The results obtained were evaluated with respect to age, gender, smoking habits, pathological condition and the occupational exposure to radiation of the individuals. The frequency of micronuclei increased significantly with the age of the subjects (P = 0.007). However there were no significant differences in micronucleus frequency with gender, smoking habits and occupational exposure. The frequency of micronuclei was significantly higher in individuals with presence of pathological condition (P < 0.0001) in comparison to healthy population irrespective of their exposure status.

Norppa H.

As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between these phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of genotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. A specific analysis of the induced type of MN may considerably improve the sensitivity of detecting the exposure effect. MN harbouring chromosomes can be distinguished from those harbouring acentric fragments by the presence of a centromere. The proportion of centromere-positive MN in human lymphocytes increases with age, which primarily reflects an age-dependent micronucleation of the X and Y chromosomes. The X chromosome especially tends to lag behind in female lymphocyte anaphase, being micronucleated more efficiently than autosomes. There is some evidence for an enhanced prevalence of fragments from chromosome 9 in spontaneous human lymphocyte MN and from chromosomes 1, 9 or 16 in MN induced in vitro by some clastogens; the breakage appears to occur in the heterochromatic block of these chromosomes. Although there are indications that centromere identification can improve the detection of clastogenic effects in humans in vivo, smokers have not shown an increase in centromere-negative MN in their cultured lymphocytes, although smoking is known to produce chromosomal aberrations. This may suggest that fragment-containing MN and chromosomal aberrations cover partly different phenomena. Understanding the mechanistic origin and contents of MN is essential for the proper use of this cytogenetic end-point in biomarker studies, genotoxicity testing and risk assessment.

Fenech M., Crott J.W.

We have validated the analysis of nucleoplasmic bridges (NPBs) and nuclear buds as biomarkers of genomic instability within the cytokinesis-block micronucleus assay in long-term lymphocyte cultures. Lymphocytes from 20 subjects were cultured in medium containing 12-120 nM folic acid for 9 days. Binucleate cells were scored for micronuclei (MN), NPBs and nuclear budding on day nine after 24h incubation in the presence of the cytokinesis inhibitor cytochalasin-B. Folic acid concentration was correlated significantly (P

Kravtsov V.Y., Fedortseva R.F., Starkova Y.V., Yartseva N.M., Nikiforov A.M.

A study of peripheral blood smears from irradiated Chernobyl liquidators and other subjects has shown nuclei of some lymphocytes to have a protrusion into the cytoplasm. Such abnormal nuclei are called "tailed" nuclei (TN). Sixteen main morphologic types, observed in human peripheral blood lymphocytes, are described. The frequency of appearance of lymphocytes with TN in a group of Chernobyl liquidators was significantly higher than in control groups (p < 0.001, in all cases). A positive correlation was found between the TN frequency in lymphocytes and dicentric chromosomes in lymphocytes cultured to metaphase (p < 0.001). Elevated frequencies of dicentrics, higher than 0.1%, were found in 17 out of 22 subjects in whom the frequency of lymphocytes with TN was 0.8% and more. Abnormalities of the TN type in lymphocytes are likely to result from breakdown of chromosome bridges formed by dicentrics. The TN can be considered as a possible marker of irradiation and, therefore, their detailed study is important.

Savage J.R.

Kisnanto T., Purnami S., Nurhayati S., Syaifudin M.

The health risk of chronic exposure to elevated natural radiation may include changes in the chromosome that increases genomic instability. There are also other phenomena, which seem to appear in particular frequently in metaphases of lymphocyte of exposed persons. This study aimed to assess a correlation between the incidence of named 8-shaped cells as cytogenetic changes in persons chronically exposed to elevated natural radiation compared to an unexposed control group. Peripheral blood lymphocyte samples obtained from 68 inhabitants of Mamuju and 27 from the control area were assessed. Both samples were then cultured and exposed to cytochalasin-B to arrest cells during the cytokinesis stage, then 8-shaped. Its characteristics were observed in binucleated cells (BNC) with cytochalasin blocked micronuclei (CBMN) assay procedure. The number of 8-shaped cells was evaluated per 1000 BNC for both Mamuju inhabitants as study group and control groups. Results showed that the frequency of 8-shaped in the study area was lower (0.0043 ± 0.0034) compared to the control group (0.0058 ± 0.0024) and statistical difference (p40 years old (y.o) (0.0044 ± 0.0036) than in 0.05). In conclusion, the radio-adaptive response of elevated natural radiation to the cell was observed in Mamuju inhabitants. However, demographic factors, such as age and sex, have often been shown to affect cells. It needs further evaluation of the 8-shaped to be used as optional biomarkers.

Tsakanova G., Babayan N., Karalova E., Hakobyan L., Abroyan L., Avetisyan A., Avagyan H., Hakobyan S., Poghosyan A., Baghdasaryan B., Arakelova E., Ayvazyan V., Matevosyan L., Navasardyan A., Davtyan H., et. al.

The development of new laser-driven electron linear accelerators, providing unique ultrashort pulsed electron beams (UPEBs) with low repetition rates, opens new opportunities for radiotherapy and new fronts for radiobiological research in general. Considering the growing interest in the application of UPEBs in radiation biology and medicine, the aim of this study was to reveal the changes in immune system in response to low-energy laser-driven UPEB whole-body irradiation in rodents. Forty male albino Wistar rats were exposed to laser-driven UPEB irradiation, after which different immunological parameters were studied on the 1st, 3rd, 7th, 14th, and 28th day after irradiation. According to the results, this type of irradiation induces alterations in the rat immune system, particularly by increasing the production of pro- and anti-inflammatory cytokines and elevating the DNA damage rate. Moreover, such an immune response reaches its maximal levels on the third day after laser-driven UPEB whole-body irradiation, showing partial recovery on subsequent days with a total recovery on the 28th day. The results of this study provide valuable insight into the effect of laser-driven UPEB whole-body irradiation on the immune system of the animals and support further animal experiments on the role of this novel type of irradiation.

Zhao H., Cai T., Lu X., Tian M., Liu Q.

Previous studies showed that the yield of cobalt-60 γ-rays-induced nucleoplasmic bridges (NPB) in human peripheral blood lymphocytes is dose dependent. However, the influence of the radiation quality and dose rates on NPB frequencies has not been investigated. The present study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by carbon ions and explore the dose rate effect on cobalt-60 γ-rays-induced NPB. To establish dose-response curves, human peripheral blood samples were irradiated with 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 8.0 Gy of carbon ions at a dose rate of 3.0 Gy/min in vitro. To explore the dose rate effect, human peripheral blood samples were irradiated with 2.0 and 5.0 Gy of cobalt-60 γ-rays at dose rates of 0.2, 0.5, 1.0, 3.0, 5.0 and 10.0 Gy/min in vitro. NPB and micronuclei (MN) in binucleated cells were analyzed with the cytokinesis-block micronucleus cytome assay. Results showed that the dose-response curve of carbon ion-induced NPB frequencies follow a linear-quadratic model (R2 = 0.934). The relative biological effectiveness (RBE) values of carbon ions to cobalt-60 γ-rays decreased with increased NPB frequencies (ranging from 2.47 to 5.86). Compared with group 1.0 Gy/min, the NPB frequencies in groups 10.0 Gy/min (2.0 Gy), 5.0 and 10.0 Gy/min (5.0 Gy) were decreased significantly (P < 0.05). Carbon ion-induced NPB in human peripheral blood lymphocytes have a good dose-response relationship. Cobalt-60 γ-rays-induced NPB frequencies are affected by the specific dose rate.