Mutation Research 699 (2010) 11–16
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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.
Tondlekar and Mr. Shrikant C. Jagtap for analyzing few of the PCC
slides.
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