Mutagenesis pp. 1–6, 2013
Mutagenesis Advance Access published December 16, 2013
doi:10.1093/mutage/get062
Characteristics of nucleoplasmic bridges induced by 60Co γ-rays in human peripheral blood lymphocytes
Hua Zhao, Xue Lu, Shuang Li, De-Qing Chen and Qing-Jie Liu* China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China *To whom correspondence should be addressed. Tel: +86 10 62389629; Fax: +86 10 62012501; E-mail: [email protected]
Few studies have shown that the yields of ionising-radiation-induced nucleoplasmic bridges (NPBs) in human cells are dose dependent. However, a dose–response curve between the NPB frequency and the absorbed dose of ionising radiation has not yet been established. This study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by cobalt-60 (60Co) γ-rays and to establish a dose–response curve. Human peripheral blood samples were collected from three healthy males, and some of these samples were irradiated with 0–6 Gy 60Co γ-rays. A cytokinesis-block micronucleus cytome assay was then carried out to analyse NPBs and micronuclei (MN) in binucleated cells. The remaining blood samples were irradiated with 0, 2 and 5 Gy of γ-rays, and unstable chromosome aberrations (dicentric chromosome, ring chromosome and acentric chromosome fragment) were analysed. The correlation between NPBs and dicentric plus ring chromosome (dic+r) induced by the same γ-ray dose was also analysed. Results showed that the NPB yields among the three subjects at each dose level were not significantly different. NPBs in binucleated cells at all γ-ray doses conformed to Poisson distribution. The dose–response curve of the γ-ray-induced NPB frequencies followed the linear-quadratic model y = (1.39 × 10−3)x2 + (4.94 × 10−3)x. A positive correlation was observed between the frequencies of NPB and dic+r, as well as between the frequencies of MN and acentric fragments. Therefore, NPB is an important biomarker of early chromosome damage event induced by ionising radiation. Introduction Chromosome aberration assay on human peripheral blood lymphocytes, mainly dicentric plus ring chromosomes (dic+r), has been the only available radiation biodosimetre for several decades. This method is the golden standard of radiation biodosimetry and still the most frequently used technique to date (1–3). However, when the victim is exposed to large doses of ionising radiation, most cells die and no sufficient number of metaphases can be collected. Moreover, dic+r assay needs a well-trained scorer and is time consuming to analyse. Chromosome aberration assay is not suitable for mass casualty during large-scale radiation emergencies, such as the Chernobyl and Fukushima nuclear accidents. The cytokinesis-block micronucleus (CBMN) assay based on micronuclei (MN) scoring has been used as an alternative
© The Author 2013. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: [email protected].
Page 1 of 6
Downloaded from http://mutage.oxfordjournals.org/ at University of Lethbridge on October 29, 2015
Received on August 16, 2013; revised on November 15, 2013; accepted on November 22, 2013
method of dic+r assay in the field of radiation emergencies since 1990s. CBMN assay is a simple and high-throughput method for measuring chromosome breakage and chromosome loss (4). The efficacy of automated MN scoring has been recently confirmed (5). Many studies have shown that the yield of radiation-induced MN is strongly correlated with radiation dose (6,7). The applicability of CBMN assay in biological dosimetry has been verified in accidental studies (8). However, the background MN level is affected by several factors, such as age and gender (9,10). Thus, MN is not radiation specific because it also can be induced by various clastogenic and aneugenic agents (11). CBMN cytome assay has been developed by Fenech (12). The biomarker of early chromosome damage such as nucleoplasmic bridge (NPB) in binucleated cells can also be scored. NPB is a continuous DNA-containing structure linking nuclei in a binucleated cell and is an indicator of DNA mis-repair, chromosome rearrangement and telomere end fusions (13). NPB originates from dicentric chromosomes when the centromeres of dicentric chromosomes or chromatids are pulled to opposite poles of the cell at anaphase (14). Another mechanism of NPB is due to telomere end fusion attributed to telomere shortening or telomere sequence damage (15,16). Studies have shown that NPB frequencies are strongly correlated with dic+r chromosome frequencies in the metaphases of the same lymphocyte cultures (13,17). Compared with chromosome aberration assay, NPB can be more efficiently scored because CBMN assay provides a large proportion of binucleated cells. NPB can be easily identified because of its simple morphological criteria that can be mastered even by inexperienced analysts. Moreover, reports have shown that the NPB background frequency is low and unaffected by age, although gender effects on NPB have not yet been elucidated (18,19). Several studies have shown that ionising radiation can induce NPB in human cells (20–22). Irradiation with 2 Gy of cobalt-60 (60Co) γ-ray can significantly increase NPB yields in the B lymphocyte cell line from breast cancer patients and normal humans (20). Another study has shown that the NPB frequency in the lymphocytes of children with pulmonary diseases induced by diagnostic X-rays is significantly higher than that prior to the diagnostic procedure (21). Cheong et al. (22) have shown that NPBs in two normal human lymphoblastoid cell lines (GM15510 and GM15036) increased with 0–2 Gy (0, 0.5, 1, 1.5 and 2 Gy) neutrons, or with 0–4 Gy (0, 0.5, 1, 2, 3 and 4 Gy) 60Co γ-rays. However, NPB frequency in human blood lymphocytes insignificantly increases after exposure to 120-mGy radon irradiation (17). Whether each kind of ionising radiation with different qualities can induce NPBs in human cells needs further investigation. Few studies have shown that the NPB frequency induced by ionising radiation is dose dependent. A study has shown that the NPB yield in isolated human lymphocytes increases with 1, 2 and 4 Gy 137Cs γ-ray doses (13). This result has also been certified by another study with neutron and 60Co γ-ray irradiation (0, 0.5, 1, 2, 3 and 4 Gy) (22). Both studies have indicated that
H. Zhao et al.
NPB is a sensitive biomarker of ionising radiation and NPB frequency was enhanced with increased absorbed dose of ionising radiation. However, the dose–range and dose–response curve of NPB in human peripheral blood lymphocytes induced by ionising radiation have not yet been systematically investigated. In this study, human peripheral blood samples were irradiated with 0, 1, 2, 3, 4, 5 and 6 Gy of 60Co γ-rays in vitro. NPB and MN in binucleated cells were systematically analysed to determine whether NPB frequency was dose dependent and to establish a dose–response curve for the first time. Dic+r and acentric fragments (ace) obtained by chromosome aberration assay were also scored to analyse the correlation between NPB and dic+r, as well as between MN and ace, after exposure to the same 60Co γ-ray dose. Materials and Methods
Blood sample irradiation For NPB analysis, human peripheral blood samples were divided into seven parts and irradiated with 0 (sham irradiation), 1, 2, 3, 4, 5 and 6 Gy, respectively, at a dose rate of 1.00 Gy/min at room temperature using a 60Co γ-ray source provided by Beijing Radiation Centre, Beijing Normal University. The source radioactivity was 130 TBq, and the homogeneous irradiation field was 30 × 30 cm. The exposure setup was calibrated by physical measurement using an ionising chamber. After irradiation, the blood samples were incubated at 37°C for 2 h before starting the cell culture. For chromosome aberration assay, human peripheral blood samples were irradiated with 0, 2 and 5 Gy at a dose rate of 1.00 Gy/min. CBMN cytome assay Approximately 0.4 ml of peripheral blood was added to 2.0 ml of RPMI 1640 culture medium (Life Technologies) containing 20% heat-inactivated fetal bovine serum (Hyclone), 100 U/ml of penicillin (Sigma), 100 μg/ml streptomycin (Sigma) and 0.2% phytohaemagglutinin (PHA; Sigma). After 40 h of culture at 37°C, cytochalasin-B (Sigma) was added to a final concentration 10 μg/ml, and the mixtures were continuously cultured for another 28 h. Cells were collected at 68 h of incubation, the supernatant of each culture was aspirated and the cells were resuspended in a hypotonic solution (0.075 M KCl) for 1 min. The cells were then fixed with methanol/acetic acid (3:1, v:v). This fixation step was repeated twice, and the cells were resuspended in a small volume of fixative solution. Prepared and dried slides were stained with Giemsa
Chromosome aberration assay Approximately 0.7 ml of peripheral blood irradiated with 0, 2 and 5 Gy of 60 Co γ-ray was added to 5.0 ml of culture medium containing 0.2% PHA at a final concentration 0.05 μg/ml of colchicine. Cells were cultured at 37°C and harvested after 56 h of incubation. The supernatant of each culture was aspirated, and the cells were resuspended in a hypotonic solution (0.075 M KCl) for 10 min. The hypotension and fixation steps were the same as those of CBMN assay. The slides were then stained with Giemsa and randomly coded. For each subject, 400 or more cleared and well-scattered metaphases were analysed using an Olympus BH-2 optical microscope under oil immersion. Chromosome aberrations such as dic+r and ace were scored according to the scoring criteria provided by International Atomic Energy Agency (3). Statistical analysis All statistical analyses were performed using SPSS 19.0. The significance of NPB yield in different subjects at same dose level was assessed by Mann– Whitney U test. Distributions of NPBs exposed to 0–6 Gy of 60Co γ-rays were assessed by one-sample Kolmogorov–Smirnov test. The significance of NPB and MN dose–response curves, NPB/MN ratio in different dose levels were assessed by Kruskal–Wallis analysis of variance test. The significance of different doses of NPB, MN, dic+r and ace were assessed by Mann–Whitney U test. Correlations of different biomarkers were assessed by Spearman correlation test. The level of statistical significance was set at P
Mutagenesis Advance Access published December 16, 2013
doi:10.1093/mutage/get062
Characteristics of nucleoplasmic bridges induced by 60Co γ-rays in human peripheral blood lymphocytes
Hua Zhao, Xue Lu, Shuang Li, De-Qing Chen and Qing-Jie Liu* China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China *To whom correspondence should be addressed. Tel: +86 10 62389629; Fax: +86 10 62012501; E-mail: [email protected]
Few studies have shown that the yields of ionising-radiation-induced nucleoplasmic bridges (NPBs) in human cells are dose dependent. However, a dose–response curve between the NPB frequency and the absorbed dose of ionising radiation has not yet been established. This study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by cobalt-60 (60Co) γ-rays and to establish a dose–response curve. Human peripheral blood samples were collected from three healthy males, and some of these samples were irradiated with 0–6 Gy 60Co γ-rays. A cytokinesis-block micronucleus cytome assay was then carried out to analyse NPBs and micronuclei (MN) in binucleated cells. The remaining blood samples were irradiated with 0, 2 and 5 Gy of γ-rays, and unstable chromosome aberrations (dicentric chromosome, ring chromosome and acentric chromosome fragment) were analysed. The correlation between NPBs and dicentric plus ring chromosome (dic+r) induced by the same γ-ray dose was also analysed. Results showed that the NPB yields among the three subjects at each dose level were not significantly different. NPBs in binucleated cells at all γ-ray doses conformed to Poisson distribution. The dose–response curve of the γ-ray-induced NPB frequencies followed the linear-quadratic model y = (1.39 × 10−3)x2 + (4.94 × 10−3)x. A positive correlation was observed between the frequencies of NPB and dic+r, as well as between the frequencies of MN and acentric fragments. Therefore, NPB is an important biomarker of early chromosome damage event induced by ionising radiation. Introduction Chromosome aberration assay on human peripheral blood lymphocytes, mainly dicentric plus ring chromosomes (dic+r), has been the only available radiation biodosimetre for several decades. This method is the golden standard of radiation biodosimetry and still the most frequently used technique to date (1–3). However, when the victim is exposed to large doses of ionising radiation, most cells die and no sufficient number of metaphases can be collected. Moreover, dic+r assay needs a well-trained scorer and is time consuming to analyse. Chromosome aberration assay is not suitable for mass casualty during large-scale radiation emergencies, such as the Chernobyl and Fukushima nuclear accidents. The cytokinesis-block micronucleus (CBMN) assay based on micronuclei (MN) scoring has been used as an alternative
© The Author 2013. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: [email protected].
Page 1 of 6
Downloaded from http://mutage.oxfordjournals.org/ at University of Lethbridge on October 29, 2015
Received on August 16, 2013; revised on November 15, 2013; accepted on November 22, 2013
method of dic+r assay in the field of radiation emergencies since 1990s. CBMN assay is a simple and high-throughput method for measuring chromosome breakage and chromosome loss (4). The efficacy of automated MN scoring has been recently confirmed (5). Many studies have shown that the yield of radiation-induced MN is strongly correlated with radiation dose (6,7). The applicability of CBMN assay in biological dosimetry has been verified in accidental studies (8). However, the background MN level is affected by several factors, such as age and gender (9,10). Thus, MN is not radiation specific because it also can be induced by various clastogenic and aneugenic agents (11). CBMN cytome assay has been developed by Fenech (12). The biomarker of early chromosome damage such as nucleoplasmic bridge (NPB) in binucleated cells can also be scored. NPB is a continuous DNA-containing structure linking nuclei in a binucleated cell and is an indicator of DNA mis-repair, chromosome rearrangement and telomere end fusions (13). NPB originates from dicentric chromosomes when the centromeres of dicentric chromosomes or chromatids are pulled to opposite poles of the cell at anaphase (14). Another mechanism of NPB is due to telomere end fusion attributed to telomere shortening or telomere sequence damage (15,16). Studies have shown that NPB frequencies are strongly correlated with dic+r chromosome frequencies in the metaphases of the same lymphocyte cultures (13,17). Compared with chromosome aberration assay, NPB can be more efficiently scored because CBMN assay provides a large proportion of binucleated cells. NPB can be easily identified because of its simple morphological criteria that can be mastered even by inexperienced analysts. Moreover, reports have shown that the NPB background frequency is low and unaffected by age, although gender effects on NPB have not yet been elucidated (18,19). Several studies have shown that ionising radiation can induce NPB in human cells (20–22). Irradiation with 2 Gy of cobalt-60 (60Co) γ-ray can significantly increase NPB yields in the B lymphocyte cell line from breast cancer patients and normal humans (20). Another study has shown that the NPB frequency in the lymphocytes of children with pulmonary diseases induced by diagnostic X-rays is significantly higher than that prior to the diagnostic procedure (21). Cheong et al. (22) have shown that NPBs in two normal human lymphoblastoid cell lines (GM15510 and GM15036) increased with 0–2 Gy (0, 0.5, 1, 1.5 and 2 Gy) neutrons, or with 0–4 Gy (0, 0.5, 1, 2, 3 and 4 Gy) 60Co γ-rays. However, NPB frequency in human blood lymphocytes insignificantly increases after exposure to 120-mGy radon irradiation (17). Whether each kind of ionising radiation with different qualities can induce NPBs in human cells needs further investigation. Few studies have shown that the NPB frequency induced by ionising radiation is dose dependent. A study has shown that the NPB yield in isolated human lymphocytes increases with 1, 2 and 4 Gy 137Cs γ-ray doses (13). This result has also been certified by another study with neutron and 60Co γ-ray irradiation (0, 0.5, 1, 2, 3 and 4 Gy) (22). Both studies have indicated that
H. Zhao et al.
NPB is a sensitive biomarker of ionising radiation and NPB frequency was enhanced with increased absorbed dose of ionising radiation. However, the dose–range and dose–response curve of NPB in human peripheral blood lymphocytes induced by ionising radiation have not yet been systematically investigated. In this study, human peripheral blood samples were irradiated with 0, 1, 2, 3, 4, 5 and 6 Gy of 60Co γ-rays in vitro. NPB and MN in binucleated cells were systematically analysed to determine whether NPB frequency was dose dependent and to establish a dose–response curve for the first time. Dic+r and acentric fragments (ace) obtained by chromosome aberration assay were also scored to analyse the correlation between NPB and dic+r, as well as between MN and ace, after exposure to the same 60Co γ-ray dose. Materials and Methods
Blood sample irradiation For NPB analysis, human peripheral blood samples were divided into seven parts and irradiated with 0 (sham irradiation), 1, 2, 3, 4, 5 and 6 Gy, respectively, at a dose rate of 1.00 Gy/min at room temperature using a 60Co γ-ray source provided by Beijing Radiation Centre, Beijing Normal University. The source radioactivity was 130 TBq, and the homogeneous irradiation field was 30 × 30 cm. The exposure setup was calibrated by physical measurement using an ionising chamber. After irradiation, the blood samples were incubated at 37°C for 2 h before starting the cell culture. For chromosome aberration assay, human peripheral blood samples were irradiated with 0, 2 and 5 Gy at a dose rate of 1.00 Gy/min. CBMN cytome assay Approximately 0.4 ml of peripheral blood was added to 2.0 ml of RPMI 1640 culture medium (Life Technologies) containing 20% heat-inactivated fetal bovine serum (Hyclone), 100 U/ml of penicillin (Sigma), 100 μg/ml streptomycin (Sigma) and 0.2% phytohaemagglutinin (PHA; Sigma). After 40 h of culture at 37°C, cytochalasin-B (Sigma) was added to a final concentration 10 μg/ml, and the mixtures were continuously cultured for another 28 h. Cells were collected at 68 h of incubation, the supernatant of each culture was aspirated and the cells were resuspended in a hypotonic solution (0.075 M KCl) for 1 min. The cells were then fixed with methanol/acetic acid (3:1, v:v). This fixation step was repeated twice, and the cells were resuspended in a small volume of fixative solution. Prepared and dried slides were stained with Giemsa
Chromosome aberration assay Approximately 0.7 ml of peripheral blood irradiated with 0, 2 and 5 Gy of 60 Co γ-ray was added to 5.0 ml of culture medium containing 0.2% PHA at a final concentration 0.05 μg/ml of colchicine. Cells were cultured at 37°C and harvested after 56 h of incubation. The supernatant of each culture was aspirated, and the cells were resuspended in a hypotonic solution (0.075 M KCl) for 10 min. The hypotension and fixation steps were the same as those of CBMN assay. The slides were then stained with Giemsa and randomly coded. For each subject, 400 or more cleared and well-scattered metaphases were analysed using an Olympus BH-2 optical microscope under oil immersion. Chromosome aberrations such as dic+r and ace were scored according to the scoring criteria provided by International Atomic Energy Agency (3). Statistical analysis All statistical analyses were performed using SPSS 19.0. The significance of NPB yield in different subjects at same dose level was assessed by Mann– Whitney U test. Distributions of NPBs exposed to 0–6 Gy of 60Co γ-rays were assessed by one-sample Kolmogorov–Smirnov test. The significance of NPB and MN dose–response curves, NPB/MN ratio in different dose levels were assessed by Kruskal–Wallis analysis of variance test. The significance of different doses of NPB, MN, dic+r and ace were assessed by Mann–Whitney U test. Correlations of different biomarkers were assessed by Spearman correlation test. The level of statistical significance was set at P
