The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation (original) (raw)
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Relative alterations in blood mononuclear cell populations reflect radiation injury in mice
Cytometry, 1998
Damage to the immune and hematopoietic systems following exposure to ionizing radiation, whether accidental or for therapeutic purposes, renders victims susceptible to opportunistic infections and diseases. Elucidating a reliable biological indicator or ''biological dosimeter'' to indicate rapidly the extent of injury sustained by an individual would be desirable. Total leukocyte count has been used historically as an indicator of immune damage, but it is not truly indicative of functional immunity post-irradiation. A flow cytometric (FCM) technique was developed to determine whether a rapid reproducible assay could be developed to assess the extent of radiation damage. To this end, peripheral blood leukocyte populations and subpopulations were monitored. C57BL/6 mice were exposed to 100-, 400-, or 700-cGy whole-body ␥-irradiation (WBI) and blood samples were collected from the retro-orbital sinus 1, 4, and 7 days post-irradiation. The blood samples were prepared for FCM by incubation with monoclonal antibodies (mAb) to various murine leukocyte CD surface markers. The results show that the proportion of CD4؉ T lymphocytes increased approximately 2-fold on day 4 after 700 cGy, the proportion of B lymphocytes declined markedly at all doses relative to unirradiated controls, and natural killer (NK) cells rose dramatically (9-fold) by day 4 after 700 cGy. The patterns of alteration in the relative proportions of peripheral blood mononuclear cells (PBMC) populations observed post-irradiation, particularly in B lymphocytes and natural killer (NK) cells, seem to represent potent and consistent indicators of immune damage, allowing some inference as to the immune competence of the individual. Cytometry 31: 45-52, 1998.
Dose-Response, 2008
ᮀ Previous research has demonstrated that adding a very small gamma-ray dose to a small alpha radiation dose can completely suppress lung cancer induction by alpha radiation (a gamma-ray hormetic effect). Here we investigated the possibility of gamma-ray hormesis during low-dose neutron irradiation, since a small contribution to the total radiation dose from neutrons involves gamma rays. Using binucleated cells with micronuclei (micronucleated cells) among in vitro monoenergetic-neutron-irradiated human lymphocytes as a measure of residual damage, we investigated the influence of the small gammaray contribution to the dose on suppressing residual damage. We used residual damage data from previous experiments that involved neutrons with five different energies (0.22-, 0.44-, 1.5-, 5.9-, and 13.7-million electron volts [MeV]). Corresponding gamma-ray contributions to the dose were approximately 1%, 1%, 2%, 6%, and 6%, respectively. Total absorbed radiation doses were 0, 10, 50, and 100 mGy for each neutron source. We demonstrate for the first time a protective effect (reduced residual damage) of the small gamma-ray contribution to the neutron dose. Using similar data for exposure to gamma rays only, we also demonstrate a protective effect of 10 mGy (but not 50 or 100 mGy) related to reducing the frequency of micronucleated cells to below the spontaneous level.
Dose-Response, 2008
Previous research has demonstrated that adding a very small gamma-ray dose to a small alpha radiation dose can completely suppress lung cancer induction by alpha radiation (a gamma-ray hormetic effect). Here we investigated the possibility of gamma-ray hormesis during low-dose neutron irradiation, since a small contribution to the total radiation dose from neutrons involves gamma rays. Using binucleated cells with micronuclei (micronucleated cells) among in vitro monoenergetic-neutron-irradiated human lymphocytes as a measure of residual damage, we investigated the influence of the small gamma-ray contribution to the dose on suppressing residual damage. We used residual damage data from previous experiments that involved neutrons with five different energies (0.22–, 0.44–, 1.5–, 5.9–, and 13.7-million electron volts [MeV]). Corresponding gamma-ray contributions to the dose were approximately 1%, 1%, 2%, 6%, and 6%, respectively. Total absorbed radiation doses were 0, 10, 50, and 100...
Teratogenesis, Carcinogenesis, and Mutagenesis, 1994
Usingthecytokinesis-blocktechnique, lymphocytesfrom healthy volunteers (n = 9) were evaluated for 1) the radiation dose-response curve for micronuclei (MN) expression; 2) technique variables on the yield of MN; and 3) the shortest lymphocyte incubation time required for the MN assay. We found that the best fitting of relationships between increasing MN production and increasing irradiation dose (U. 0 GJ) was the linear-quadratic model as expressed by the yield equation Y = C + aD + PD (P = 0.0003). When lymphocytes were irradiated in vitro with 2.0 Gy and harvested at various time intervals, MN increased during the entire 84 hr culture time. The radiation caused a division delay in lymphocyte as indicated by an increased frequency of mononucleated cells and a decreased number of mitotic indices. The data showed that a shortened culture time (60 hr) for the MN assay is possible and that binucleated cells with 2 3 MN were found only in cells irradiated at 2 2. 0 Gy. These findings suggest that scoring of MN in lymphocytes may be a practical biological dosimeter for the rapid screening of accidental radiation exposure victims, especially when their clinical manifestations are not obvious.
2011
Aim: Cytokinesis-block micronucleus (CBMN) assay and its comprehensive variant CBMN cytome assay are cytogenetic methods. CBMN is based on assessment of micronuclei in nucleated cells that have completed only one nuclear division. Besides micronuclei, CBMN cytome assay analyzes additional genotoxic (nucleoplasmic bridges and nuclear buds), cytostatic (nuclear division index), and cytotoxic (amount of necrotic and apoptotic cells) parameters. The aim of this study is to evaluate these parameters in human blood lymphocytes after in vitro irradiation and to assess its contribution to biodosimetry. Material and methods: Human blood from 6 donors was in vitro irradiated by 0, 1, 2, 3, 4, or 5 Gy and cultivated for 72 hours. Blood lymphocytes were stimulated with phytohemagglutinin and their cytokinesis was blocked by cytochalasin B. After cultivation, cultures were hypotonically treated, dropped onto glass slides and stained with Giemsa. Slides were evaluated by microscope. Results: We observed significantly increased amount of micronuclei, nucleoplasmic bridges, and nuclear buds measured in binucleated cells, significantly increased amount of micronuclei measured in mononucleated cells and significantly decreased nuclear division index after irradiation by 1, 2, 3, 4, and 5 Gy. Amount of death cells (apoptotic and necrotic) significantly increased after irradiation by 4 and 5 Gy. Conclusion: Although all parameters assessed by CBMN cytome assay have biodosimetric potential, practically feasible is only evaluation of micronuclei in binucleated cells. This parameter was used to construct in vitro linear-quadratic dose-response calibration curve which could be used as a biodosimetric tool for triage of radiation casualties.
The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure
Mutagenesis, 2011
Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus (MN) background frequency, by which only in vivo exposures in excess of 0.2-0.3 Gy X-rays can be detected.
REVIEW The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure
2010
Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally ex-posed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis.
Background: The purpose of this paper is to establish an easy and reliable biodosimeter protocol to evaluate the biological effects of proton beams. Materials and Methods: Human peripheral blood lymphocytes were irradiated using proton beams (LET: 34.6 keV µm −1 ), and the chromosome aberrations induced were analyzed using cytokinesis-blocked (CB) micronucleus (MN) assay. To determine the efficiency of MN assay in estimating the doses received by 50MeV proton beams and to monitor predicted dose of victims in accidental exposure, here we have evaluated the performance of MN analysis in a simulated situation after exposure with proton beams. Peripheral lymphocytes were irradiated by 50MeV proton beams up to 6Gy and analyzed by Giemsa staining of CB MN assay. Results: The detected MN was found to be a significant dose-effect curve in the manner of dose-dependent increase after exposure with proton beams in vitro. When plotting on a linear scale against radiation dose, the line of best fit was Y=0.004+(1.882x10 -2 ±9.701x10 -5 )D+(1.43x10 -3 ±1.571x10 -5 )D 2 . Our results show a trend towards increase of the number of MN with increasing dose. It was linear-quadratic and has a significant relationship between the frequencies of MN and dose (R 2 = 0.9996). The number of MN in lymphocyte that was observed in control group is 5.202±0.04/cell. Conclusion: Hence, this simple protocol will be particularly useful for helping physicians to decide medical therapy for the initial treatment of victims with rapid and precise dose estimation after accidental radiation exposure. Also it has potential for use as a valuable biomarker to evaluate the biological effectiveness for cancer therapy with proton beams. Iran. J. Radiat. Res., 2011; 8(4): 231 236
Iranian Journal of Radiation Research, 2015
Background: Radio-adapve response and bystander effects are known phenomena occurring in cells following exposure to ionizing radiaon (IR). In this study we examined possible radio-adaptaon of lymphocytes following bystander effects induced by CHO-K1 cells. Materials and Methods: Whole blood and CHO-K1 cells were cultured in RPMI-1640 complete medium. Cells were separately irradiated with various doses of gamma rays. A co-culture was set to examine the bystander ef fects induced by CHO-K1 cells on lymphocytes exposed later to a challenge dose of 4 Gy. Treated cells were exposed to cytochalasin-B to arrest cells in cytokinesis stage. Micronucleus (MN) as end point was scored in binucleate cells a2er staining in Giemsa. Results: The frequency of MN increased significantly with increasing dose of radiaon both in lymphocytes and CHO-K1 cells (p 0.05). Co-culture of the non-irradiated lymphocytes with pre-irradiated CHO-K1 cells significantly reduced the mean frequency of MN in lymphocyt...
An in vitro study of the dose responses of human peripheral blood lymphocytes was conducted with the aim of creating calibrated dose-response curves for biodosimetry measuring up to 4 Gy (0.25-4 Gy) of gamma radiation. The cytokinesis-blocked micronucleus (CBMN) assay was employed to obtain the frequencies of micronuclei (MN) per binucleated cell in blood samples from 16 healthy donors (eight males and eight females) in two age ranges of 20-34 and 35-50 years. The data were used to construct the calibration curves for men and women in two age groups, separately. An increase in micronuclei yield with the dose in a linear-quadratic way was observed in all groups. To verify the applicability of the constructed calibration curve, MN yields were measured in peripheral blood lymphocytes of two real overexposed subjects and three irradiated samples with unknown dose, and the results were compared with dose values obtained from measuring dicentric chromosomes. The comparison of the results obtained by the two techniques indicated a good agreement between dose estimates. The average baseline frequency of MN for the 130 healthy non-exposed donors (77 men and 55 women, 20-60 years old divided into four age groups) ranged from 6 to 21 micronuclei per 1000 binucleated cells. Baseline MN frequencies were higher for women and for the older age group. The results presented in this study point out that the CBMN assay is a reliable, easier and valuable alternative method for biological dosimetry.