Mutation Research 699 (2010) 11–16 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres Biodosimetry for high dose accidental exposures by drug induced premature chromosome condensation (PCC) assay Sreedevi Balakrishnan ∗ , kapil Shirsath, Nagesh Bhat, Kshiti Anjaria Radiological Physics & Advisory Division, Bhabha Atomic Research Centre Trombay, Mumbai, India a r t i c l e i n f o Article history: Received 31 August 2009 Received in revised form 8 March 2010 Accepted 16 March 2010 Available online 23 March 2010 Keywords: Ionizing radiation Premature chromosome condensation Biodosimetry Chromosomal aberration Cytogenetic a b s t r a c t The conventional dicentric assay does not provide an accurate dose estimate in the case of accidental exposure to ionizing radiation above 6 Gy due to mitotic delay and poor mitotic index. The present study aims to establish a simple and rapid dose assessment technique based on scoring of rings and fragments in PCC spreads of stimulated lymphocytes. Human peripheral blood lymphocytes were gamma irradiated to different doses (6.2–24.5 Gy), cultured for two days with PHA and were forced to condense prematurely using 500 nM Okadaic acid (OA). The chromosome spreads were prepared, stained with Giemsa and observed under a microscope. The PCC index, PCC rings, and PCC fragments were scored for each dose point to arrive at the dose effect curve for various end points such as induction of rings and fragments and dicentrics. The PCC index varied from 12–18% up to 18 Gy and thereafter dropped to 6–8% at higher doses. The dose dependent increase in rings and fragments was found to be linear with a slope of 0.054 ± 0.001 Gy−1 for rings and 0.45 ± 0.03 Gy−1 for PCC fragments. An experiment was carried out to simulate partial-body exposure by mixing 10 Gy in vitro irradiated blood with un-irradiated blood in different proportions. The ratio of frequency of damaged cells among the total number of cells analyzed was found to be a good index of partial-body exposure. The culture duration was extended to 72 h to overcome the cell cycle delay induced by high doses of radiation. The conventional dicentrics rings and fragments also showed a dose response at high doses. The response can be best fitted to a linear model with a slope of 0.28 ± 0.0007 Gy−1 for the induction of dicentrics. However, long culture duration, technical skill and time required to analyse multi-aberrant cells makes the dicentric assay less suitable for high dose exposures requiring a rapid dose estimate. The PCC assay can be performed in 50 h with biodosimetric information about the irradiated fraction in cases of acute radiation exposures. The automated finding of PCC spreads significantly increased the speed of scoring PCC fragments. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The analysis of chromosomal aberration, particularly dicentrics in cultured peripheral blood lymphocytes (PBL) has been used for biological dosimetry of accidental overexposure cases for over three decades. This technique has been developed into a routine procedure in radiological protection and it supplements physical dosimetry. In the absence of physical dosimetry, this is the most reliable method of quantifying the absorbed radiation dose. The recent IAEA manual [1,2] gives comprehensive account of the radiation induced aberrations, the laboratory procedures and the dose calculation from the chromosomal aberration yields. This method was successfully used for the dose assessment in Chernobyl, Goiania and Tokaimura accidents involving life threatening doses [3–5]. ∗ Corresponding author. Fax: +91 22 25519209. E-mail address: bsdevi@barc.gov.in (S. Balakrishnan). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.03.008 The radiation exposures of >10 Gy to whole body were lethal to human beings. But recently, with the advancement in medical technology by stem cell transplantation and use of several cytokines, it has become possible to save individuals exposed to large doses in the order of 10 Gy [6]. An accurate and quick dose estimate is required for choosing medical treatment for irradiated victims exposed to high doses (8–10 Gy). With high doses, the conventional cytogenetic dosimetry using chromosomal aberrations becomes difficult due to mitotic delay and disappearance of lymphocytes from peripheral blood circulation. Therefore there is a need to establish reliable, easier and quicker methods for biodosimetry. The premature chromosome condensation assay with PBL is being recommended as a rapid method of biodosimetry [7–10]. The PCC assay can also discriminate between whole- and partial-body exposures at low doses. Darroudi et al. [11] has investigated the efficiency of dicentric, PCC and Micronucleus (MN) assay for the assessment of partial-body exposure in Rhesus monkeys exposed to 5 Gy of X-rays and by shielding 6% of marrow cells. The PCC assay could distinguish the in-homogeneity of exposure where 12 S. Balakrishnan et al. / Mutation Research 699 (2010) 11–16 as for dicentrics and MN, shielding of 6% bone marrow cells was found to be too small to estimate the un-irradiated fraction accurately. Recently Lamadrid et al. [12] have shown that using this technique dose effect curve can be set up for 25 Gy of gamma rays and 10 Gy of neutrons. Conventionally, PCC is induced by fusing PBL with mitotic CHO cells using polyethylene glycol (PEG) or Sendai virus [13]. After accomplishing cell fusion, diffusion of mitotic promotion factors (MPF) takes place and it induces PCC. This method is technically demanding, laborious and the PCC index is reported to be too low [8]. Hence a simple procedure has become necessary. Protein phosphatases inhibitors such as Okadaic acid (OA) and Calyculin A are reported to be inducing PCC in lymphocytes stimulated with mitogen for 48 h [14–16]. Simultaneous detection of chromosomal aberrations in G1 and M phases was possible by using painting probes and FISH. OA and Calyculin A are inhibitors of type 1 and 2A protein phosphatases and could induce PCC in any phase of the cell cycle [8]. They also showed that using drug induced PCC and FISH painting, a dose of 40 Gy can be estimated. Kanda et al. [17] proposed an easy method of scoring PCC rings in Giemsa stained PCC spreads induced by OA in PBL cultures. They also estimated the doses in three Tokaimura criticality accident victims using this assay [18]. Gotoh et al. also reported that the number of chromosome pieces increases with radiation dose and it is a good indicator for estimation of radiation doses in emergencies and accidents [19]. As per IAEA guidelines every cytogenetic biodosimetry laboratory should establish dose response curve [1]. The present paper deals with standardization of OA induced PCC assay, dose response studies with cobalt-60 gamma rays in the dose range of 6.2–24.5 Gy for the induction of PCC rings and fragments. The validation studies were also carried out in simulated partial-body exposures. 2. Materials and methods 2.1. Chemicals Phytohaemagglutinin, Colcemid and Okadaic acid were purchased from Sigma chemicals (St. Louis, Mo, USA). RPMI medium and fetal bovine serum was purchased from Gibco chemicals. Rests of the chemicals were analytical grade and purchased from local distributors. 2.2. Blood collection and irradiation The blood donors were two healthy non-smoking males aged 25 and 35 and one female aged 53. Aliquots of heparinised blood from two donors were irradiated to different doses of 0, 6.2, 12.5, 18.4, 24.5 Gy of cobalt-60 gamma rays at a dose rate of 1.34 Gy min−1 using a gamma cell 900 irradiator (BARC Isotope Division, Trombay). The blood sample from the third donor was divided into two parts and one part was irradiated to 10 Gy dose and mixed with un-irradiated blood in different proportions to simulate partial-body exposures. In the mixed blood cultures the percentage of irradiated blood was 0, 15, 50, and 100. 2.3. Culturing and slide preparation 2.3.1. PCC assay The peripheral blood lymphocytes were incubated for 2 h at 37 ◦ C before and after irradiation for repair. Whole blood cultures were set up with RPMI 1640 medium supplemented with 10% FCS and 0.1 ml of PHA (final concentration of 10 ␮g/ml) and incubated 37 ◦ C for 48 h [20,21]. One hour before harvesting, the cultures were centrifuged and the volume was reduced to 2.5 ml and Okadaic acid was added to the culture at a final concentration of 500 nM to induce PCC [9,17]). The cells were harvested with hypotonic treatment (75 mM KCl) for 20 min at 37 ◦ C, washed and fixed with carnoy’s fixative (Methanol: Glacial acetic acid, 3:1, v/v) three times, dropped on a pre cleaned glass slide, air dried, stained with 5% Giemsa solution in phosphate buffer, pH 6.8 and finally mounted with DPX [15]. 2.3.2. Chromosomal aberration and mitotic index Two sets of replicate cultures were set up with the same irradiated blood. One set contained colcemid solution (0.025 ␮g/ml) from the beginning of the culture and terminated at 48 h to compare the mitotic index with PCC. The second set was cultured for 72 h and at 48 h the cells were treated with colcemid to have a final concentration of 0.025 ␮g/ml. The cultures were harvested at 72 h with hypotonic treatment as described earlier. The metaphase preparations made from these cultures were used for chromosomal aberration studies. 2.4. Scoring A minimum of 100 PCC rings were analysed per dose point using a light microscope. A ring with a visible hole with or without centromere and a couple of rings either separate or joined by a centromere in PCC spreads is scored as a ring. The number of chromosome pieces in excess of 46-G2/M PCC or 92-G2/A PCC was considered as fragments. The PCC/mitotic index was obtained by scoring 500 cells as follows: number of metaphases/PCC spreads × 100. number of nucleated cells + number of metaphases/PCC spreads Fifty metaphases were analyzed for the presence of dicentrics, rings, and fragments. Multicentrics were considered as n − 1 dicentrics, n being the no of chromosomes involved in the formation of multicentrics. Analysis of slides was carried out using an automated metaphase finding system (Carl Zeiss Axio-imager and metafer supplied by Metasystems, Germany). This system consists of Carl Zeiss microscope equipped with 8 slide capacity stage, motorized x-, y- and z-axis, computer controlled positioning with the auto focus capabilities and automatic storage of gallery images, relocation, automatic high magnified image for on screen analysis of aberrations and transfer into Ikaros software. 2.5. Statistical analysis The distribution of PCC ring and dicentric was tested for fit to the Poisson expectation by Papworth’s u test [22]. This method makes use of the fact that for a Poisson distribution the variance ( 2 ) equals the mean (y). Thus the two are compared and insignificant differences suggest a Poisson distribution. In this test the quantity u in the following equation approximates to a unit normal deviate: √ u = d − (N − 1)/ Var D where N is the total number of cells scored, d is the coefficient of dispersion defined as (N − 1)  2 /y, where  2 /y is the relative variance and Var(d) is the variance of d given by 2(N − 1) (1 − 1/Ny). For a Poisson distribution, the variance divided by the mean is equal to 1 so that u = 0. If the rings/dicentrics are over dispersed the variance will increase and the relative variance will be >1 resulting in a positive value of u. On the other hand if the rings are under dispersed, the variance will be reduced and the relative variance will be <1, producing a negative value for u. If the absolute value of u is greater than ±1.96, the over – or under dispersion is significant. Only a 5% probability exists for the magnitude of u to be greater than this value when the distribution is Poisson [23,24]. The dose effect relation could be fitted to a best fit linear model. The dose to the irradiated fraction (partial-body dose) was measured by Qpcc method. This method is identical to the Qdr method of Sasaki and Miyata [25] where the unstable dicentric and ring aberration per damaged cells was represented as a function of dose. Here the damaged cells were defined as those with >46 or 92 PCC fragments. The excess PCC fragments (acentrics) per damaged cells were fitted in to a linear model and that data was used for dose calculation of simulated partial-body exposure. 3. Results 3.1. Premature chromosome condensation Fig. 1a shows the Giemsa stained PCC spread from gamma irradiated peripheral blood lymphocytes cultures treated with OA. Centromeres are not visible and hence it is difficult to identify dicentrics. Rings include both acentric and centric rings. Cells that exhibit attached sister chromatid are G2/M PCC and cells with separate sister chromatid are G2/A PCC. Figs. 2 and 3 show the dose response curves for the induction of PCC fragments and rings by gamma radiation. The percentage of PCC spreads induced by 1 h treatment with OA at various doses of gamma radiation varied from 16% to 6% as given in Table 1. The number of excess PCC fragments increased with the dose, from 4% at 0 Gy to 792% at 24.5 Gy. The frequency of PCC rings increased from 0 at 0 Gy to 1.115 at 24.5 Gy. The dose dependent increase in rings and fragments was found to be linear with a slope of 0.054 ± 0.001 Gy−1 for rings and 0.45 ± 0.03 Gy−1 for fragments. The frequency and distribution of rings along with relative variance ( 2 /y) and u values are given in Table 2. The  2 /y values vary from 1.2 to 1.9 and the u values varies from 4.3 to 7.3 showing a significant over-dispersed distribution. The data obtained from simulated partial-body exposure S. Balakrishnan et al. / Mutation Research 699 (2010) 11–16 13 Fig. 2. Dose response for the induction of PCC breaks induced by ␥ radiation. Fig. 1. (a) M-A PCC spread showing two rings and fragments. (b) A multi-aberrant metaphase showing eight dicentrics and fragments. Fig. 3. Dose response for the induction of PCC rings in peripheral blood lymphocytes induced by ␥ radiation. study performed by mixing 10 Gy in vitro irradiated blood with unirradiated blood in different proportions is given in Table 3. The fraction of damaged cells observed in simulated partial-body exposures is given in Table 3. The measured fraction of damaged cells agreed well with the irradiated fraction. The dose to the irradiated fraction was evaluated by the Qpcc method [9] from the yield of excess PCC fragments in damaged cells. The frequency of damaged cells increases with the radiation dose. The dose response of excess PCC fragments in damaged cells for radiation doses up to 24.3 Gy was fitted to a linear relationship with a slope of Table 1 Induction of PCC rings and fragments, PCC index and mitotic index in lymphocytes exposed to various doses of 60 Co gamma rays and cultured for 48 h. Dose in Gy No. of cell analyzed 0 6.2 12.3 18.4 24.5 100 100 100 100 69 No. of rings obtained 0 34 65 100 77 Acentric fragments PCC index (%) Mitotic index in 48 h cultures (%) 4 409 602 665 792 16 16 8 9 6 12 3 1 0 0 Table 2 The yield and distribution of PCC rings induced in gamma irradiated lymphocytes cultured for 48 h. Dose in Gy No. of cells scored 0 6.2 12.5 18.4 24.5 100 200 200 198 69 No. rings obtained in cell 0 1 2 3 4 5 6 7 8 9 – 164 124 101 33 – 19 34 46 15 – 12 35 32 10 – 4 4 10 7 – 1 2 7 3 – – 1 1 – – – – 1 – – – – – – – – – – – – 1 Total no. rings Y = X/N  2 /y u – 59 129 179 77 – 0.295 0.645 0.904 1.115 – 1.7306 1.43 1.544 2.12 – 7.35 4.33 5.43 6.59 14 S. Balakrishnan et al. / Mutation Research 699 (2010) 11–16 Fig. 4. Dose response relationship for excess PCC fragments in damaged cells. 0.46 ± 0.04 Gy−1 (Fig. 4). This data was used for estimation of doses to the irradiated fraction in simulated partial-body exposures. The dose estimate ranged from 5.07 to 6.98 Gy for 15–100% irradiated volume and it is found to be almost 50% less than the actual dose. Fig. 5. Dose response for the induction of dicentrics induced by ␥ radiation. The data points for 2 and 4 Gy are from the low dose calibration curve obtained earlier. 3.2. Chromosomal aberrations The comparative studies with 24 h colcemid treatment also yielded sufficient number of metaphase spreads in 72 h cultures. Even though OA treatment generated PCC cells at 48 h, the mitotic index was found to be only 1–3% in colcemid treated 48 h cultures. Hence the culture time was extended up to 72 h. The data given in Table 4 shows the number of cells analysed, number and frequency of fragments, dicentrics and their distribution, tricentrics, and rings in lymphocyte cultures treated with colcemid at various radiation doses. The dicentric frequency ranged from 2.06 at 6.2 Gy to 6.26 at 24.5 Gy. The dose effect curve can be best fitted to a linear model with a slope of 0.28 ± 0.0007 Gy−1 . The fitted dose response curve is given in Fig. 5. The yield of breaks in metaphase preparations shows saturation at high doses above 18 Gy. The distribution of dicentrics was found to be Poisson with u values ranging from −0.3 to +1.5.The distribution of fragments was found to be over dispersed with respect to Poisson (data not shown). The dose response graph is shown in Fig. 6.There was no increase in the yield of rings from 12 Gy onwards in metaphase preparations where as there was dose dependant increase in PCC preparations. The tricentric yield Fig. 6. Dose response for the induction of acentrics induced by irradiation of PBL by ␥ radiation after culturing for 72 h. Table 3 The yield of damaged cells in simulated non-uniform exposure of 10 Gy 100 PCC spreads are analyzed per simulation. Mixed culture % un-irradiated blood Mixed culture % 10 Gy irradiated blood Damaged cell % Excess PCC breaks per damaged cell Estimated radiation dose (Gy) 100 85 50 0 0 15 50 100 2 10 50 100 2 2.33 2.6 3.21 – 5.07 5.65 6.98 ± ± ± ± 0.05 0.05 0.14 0.15 Table 4 The yield and distribution of dicentrics, tricentrics, rings and fragments in gamma irradiated lymphocytes cultured for 72 h. Dose in Gy 6.2 12.3 18.4 24.5 No. of cells scored 50 50 50 50 No. of normal cells No of dic. 3 0 0 0 91 130 167 223 Distribution of dicentrics 0 1 2 3 4 5 6 7 8 9 9 2 1 0 13 8 5 0 10 8 6 4 8 12 5 5 5 7 7 4 4 6 9 3 1 3 6 10 0 2 3 5 0 1 6 9 0 1 2 5 No of tric No of rings Fragments Dicentrics per cell  2 /y 6 18 31 45 4 15 17 15 79 196 308 316 2.06 3.34 4.58 6.32 1.28 1.37 1.2 1.24 1.23 1.14 0.93 −0.33 u 10 0 10 S. Balakrishnan et al. / Mutation Research 699 (2010) 11–16 also increased in a dose dependant manner from 12% at 12.2 Gy to 19% at 24.5 Gy. 4. Discussion After the introduction of PCC assay by Johnson et al. [13], the applicability of this assay in biodosimetry was proposed by Pantelias and Maillie [7] and thereafter many researchers have examined the reliability of studying chromosomal aberrations in PCC cells using C banding and chromosome painting. The conventional Giemsa staining was introduced by Kanda et al. [17] for studying PCC rings and later it was used by Hayata et al. [5] for the biodosimetry of Tokaimura criticality accident victims. As reported by Kanda et al. [17] we also observed 500 nM OA was ideal for inducing PCC. Longer treatment time was found to be altering the morphology of chromosomes. Durante et al. [16] also reported no significant differences between the Calyculin A induced PCC on cycling lymphocytes and G0 – PCC induced by cell fusion. In the present study the frequency of fragments and rings increased up to 25 Gy suggesting its applicability for evaluation of large whole-body doses. The slope of the dose response curve was found to be slightly lower (0.054 ± 0.001 Gy−1 ) than that reported by Hayata et al. (0.07 ± 0.001 Gy−1 ) for X-rays. In comparison the yield of rings and fragments observed in metaphase preparations was found to be lower than that in PCC preparations up to 25 Gy. Norman and Sasaki reported very little increase in the frequency of centric rings above 5 Gy where as acentric ring frequency continued to increase [26]. This may be partly due to the competition among centric rings and di-/polycentric chromosomes for the finite number of centromeres present in the cell. Also, the frequency of damaged cells as indicated in Table 3 was found to be a good indicator of partial-body exposure than the frequency of fragments. There was poor correlation between the actual dose and estimated partial-body dose in simulated exposures. The in vivo data from therapeutic exposures would be used to validate this assay in partial-body exposures. After high-dose irradiation it is technically simple to use differential white cell counts to assess the exposure levels of victims quickly. But hematology provides less accurate estimate of the doses than cytogenetic assays. This was observed in the cases of Chernobyl accident victims [27]. To estimate the doses of partialbody exposure in accident victims, chromosomes from biopsy samples of locally injured tissues may be a better option than using lymphocytes [28]. The cell fusion induced PCC assay may work in such situation. The comet assay using skin fibroblasts also may be explored for this purpose [29]. The analysis of the PCC slides using metaphase finding software readily detected the PCC spreads and displayed the images in postage stamp mode in the gallery. Following that each spread is observed under 1000× magnifications for detailed analysis. About 100–125 PCC spreads were scanned in 10 min using metapher 2 software. In this paper we have also tried to show the feasibility of classical colcemid block method for the estimation of doses above 10 Gy. Generally mitosis is not observed after high-dose exposures in 48 h cultures, but by extending the culture duration to 72 h, sufficient metaphase spreads can be made available for scoring. The increase in unstable aberration yields can be correlated with the radiation dose even at high-doses. Two authors have reported the frequency of chromosomal aberrations at high doses of 5–12 Gy [5,30]. The frequency of aberrations induced following in vitro exposure was found to be higher than that observed in in vivo exposures. The reason can be the rapid lymphopenia observed in peripheral blood counts after high exposure. This may not be due to cell killing, but may be merely a physiological response 15 due to movement of lymphocytes from circulation to tissues and lymph-nodes which reduces the number of lymphocytes available for cytogenetic studies in peripheral blood. In Tokaimura accident victims the lymphocytes in the circulating and extra vascular pools had reached equilibrium at 9 h, and highly damaged lymphocytes did not selectively move away from the circulatory system during the first rapid depletion of lymphocytes after exposure. The aberration yield remained constant at 9, 23, and 48 h sampling [18]. The PCC assay takes only 48 h of culturing and another 2 h for processing and analysis. The cytogenetic dosimetry report can be made available in 50 h after the collection of the blood sample. In the dose range of 0–6 Gy radiation specific dicentric assay with a low background frequency, may be a better indicator than PCC assay. By using dicentric assay in the triage mode (scoring 20/50 cells) and mutual assistance networking might help in the mass causality event. It has an added advantage of discriminating whole-body and partial-body exposure. However, long culture duration, technical skill and time required to analyse multi-aberrant cells makes the assay less suitable for high dose exposures requiring a rapid dose estimate. So far the cell fusion induced PCC assay was not used in any radiation accident biodosimetry due to highly demanding technical skill and poor yield. The drug induced PCC assay is a simple, rapid method which is amenable for automation and it requires only light microscope. There is no need for specialized technique like FISH and it does not require highly skilled personnel for analysis of the slides. 5. Conclusions We conclude that the frequency of radiation induced rings and fragments in Giemsa stained PCC preparation provide a very useful biodosimetry tool to evaluate acute whole/partial-body exposures in high dose radiation emergencies compared to classical chromosome block method. Automated PCC spread finding significantly enhances the speed of PCC assay. Being easier to implement and cost effective, the OA induced PCC assay is better option for dosimetry of high dose accidental exposures. The assay can be performed in any hospital during accidental emergencies. Conflict of interest None. Acknowledgement Authors gratefully acknowledge the encouragement and support by Dr. Y.S. Mayya, Head, RP&AD. We also thank Mr. Pradosh J. 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