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Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
Painting by Cytogenetic and Molecular techniques.
xiv
Chapter 1
Introduction
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
Painting by Cytogenetic and Molecular techniques.
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1.1. Introduction:
Radiation is a ubiquitous energy. Radioactive materials and radiation have always
existed as a part of our environment. The increased use of radioactive materials,
which is a direct outgrowth of the current military and energy policies of the
developed world, poses a need for studying the health effects of radiation (Figure 1.1).
As of 2004, nuclear power provided 6.5% of the world's energy and 15.7% of the
world's electricity (1). As of 2007, the IAEA reported there are 439 nuclear power
reactors in operation in the world, operating in 31 countries (2).
Figure 1.1: World nuclear power consumption showing a rising trend (3)
These statistics show that, of late, artificial radiation sources have become of immense
significance by contributing to human welfare in agriculture, medicine, industry and
research. Figure 1.2 shows the worldwide statistics for radiation exposure due to
different sources (3). The widespread use of ionizing radiation (IR) for medical,
industrial, military and research purposes has increased the risk of accidental
occupational exposures. This is particularly the case for a large number of individuals
exposed to various levels of IR caused by nuclear accidents such as Chernobyl,
atmospheric nuclear testing prior to the early 1960s, the atomic bombing at Hiroshima
and Nagasaki, various medical radiological procedures, and occupational exposures
for which dosimetric information may be poor or absent (4-5). Apart from these,
several other potential radiation exposure scenarios can occur, which include
detonation of nuclear weapons; terrorist attacks on nuclear reactors, and dispersal of
radioactive substances with the use of conventional explosives, resulting in mass
Asia Pacific
Europe and Eurasia
North America
Rest of the world
1981 to 2006
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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casualties. These disasters can result in different radiation exposure types: whole
body, localized or partial body, internal contamination, external contamination, and
contaminated burns and wounds (6).
Figure 1.2: World radiation exposure statistics due to various sources
There is a need for reliable methods to assess past exposure to clastogens and the
related risk. A systematic investigation of radiation-induced damage is of great
importance for the population of radiation workers at risk in the nuclear industry and
medical sector. Radiation accidents in foreign countries have pointed to the need for a
reliable methodology, allowing quick and accurate measurement of the radiation
burden received, not only for a restricted number of individuals but also for a sizeable
fraction of the population. At lower doses, exposure assessment is useful in evaluating
risk to late health effects. Furthermore, dose assessment may be used to reassure non
exposed persons that they have not received any significant exposure.
The onset, nature, severity, and duration of clinical symptoms following radiation
exposure are determined primarily by the casualty’s absorbed dose. But, these factors
are also influenced by the radiation field and quality, the dose rate, the individual’s
inherent radiosensitivity and general medical health status (7).
Dosimetry is hailed as the cornerstone of radiological protection. But, non-availability
or erroneous initial dose estimates, within hours to weeks after exposure, could result
18.9% exposure is due to artificial radiation sources used in the technological sector
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
Painting by Cytogenetic and Molecular techniques.
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in sub-optimal medical intervention. In all potential radiation exposure scenarios, it is
unlikely that physical dosimeters will be available for dose assessment to aid clinical
management of mass casualties. For early treatment of radiation victims, it is
recommended that medical personnel rely heavily on clinical signs and biological
dose assessments.
Since IR induces cellular and molecular changes, the most important cellular target of
radiation damage is the deoxyribonucleic acid (DNA). It is this characteristic that is
employed in biological dosimetry. The damage that is incurred by any living cell can
be observed by diverse techniques tailored for specific end points referred to as
biomarkers. The visualization and analysis of radiation associated damage forms the
basis and origin of these biodosimeters. The advances in cell and molecular biology
make it possible to screen human population for changes in biomarkers of exposure.
To be useful, a biomarker for exposure and risk assessment should employ an end
point that is highly quantitative, stable over time, and relevant to human risk. For any
biomarker to be considered as a biodosimeter, it needs to possess some basic features.
First, sampling should be easy, and the taken sample should represent the whole body.
Second, biomarkers must change as a function of exposure or dose enabling
establishment of in vitro dose response curves. Third, it is important for the assay to
be rapid and ultimately capable of automation if biomarkers are to be useful following
low-level exposures (8). Also, the biomarker needs to be present at a very low
background level and variability between individuals should be small (9). The
important characteristics that must be considered in the design of a radiation
biodosimeter assay system for field use are: hardenability, low maintenance,
portability, including low weight and footprint, operation by non-specialist personnel
and high-throughput.
One of the earliest and most direct methods of dose determination following radiation
exposure involves charting daily counts of different cell types circulating in the
peripheral blood. Total leukocyte counts decline rapidly in the first week following
radiation exposures in excess of about 1 Gy and the extent and duration of the decline
and subsequent recovery have been shown to correlate well with dose (10). Total
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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body irradiation doses of 1 Gy and higher can also be well estimated from peripheral
blood neutrophil counts. The dose estimates derived from these two methods agree
closely with each other and were widely used and confirmed following the Chernobyl
accident as well as other well documented accidents at research facilities in Russia
(11).
The hematopoietic system contains some of the most radiation sensitive and easily
sampled cells in the human body. This has been exploited by many of the
biodosimetry methods developed to date, including those based on cytogenetics,
somatic mutation and gene expression. The use of these biomarkers for biodosimetry
purpose has been discussed.
1.2. Cytogenetic Biodosimetry:
1.2.1. Chromosome aberrations – Dicentric chromosome (DC):
Double strand breaks induced by radiation in individual chromosomes can give rise to
exchanges resulting in an abnormal chromosome with two centromeres referred to as
the DC. Centric Rings, which are formed due to breaks and rejoining within the same
chromosome, are also considered as equivalents of DC chromosomes. Because of the
ease of identification and the low baseline frequency, the use of DC and ring
chromosomes became the standard for detection of past radiation exposure (12, 13).
Even today scoring of DC is considered as most specific for IR damage. Very few
clastogens can be confounding factors for this assay. Biodosimetry, based on the
analysis of DC chromosomes in circulating lymphocytes, is considered the “gold
standard” for estimating radiation injury, and is used to make informed decisions
regarding the medical management of irradiated persons. Figure 1.3 shows dicentrics
induced by gamma radiation as observed by Giemsa staining.
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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Figure 1.3: Dicentric chromosomes seen in cells exposed to 2 Gy gamma
radiation
Peripheral blood lymphocytes from exposed individuals are mitogen-stimulated and
then blocked at the metaphase stage with the spindle inhibitor, namely, Colchicine
(14). Cells are scored for the presence of DC chromosomes. Radiation dose is then
estimated from comparison to a standardized calibration curve obtained from human
lymphocytes irradiated in vitro. Some individual differences in the response for the
induction of this biomarker may arise due to variation in genetic composition and
DNA repair capacity. Hence, DC can also serve as a useful indicator of biological
response of individuals. Significant increase in DC frequencies has been documented
following in vitro doses above 0.02 Gy (15). The lower limit for detection is 0.05 Gy
for X-rays, 0.10 Gy for gamma radiation and 0.10 Gy for fast fission neutrons (16).
Doses over a wide range of 0.1 - 6 Gy can be detected by this technique.
As centromeres are the site of chromosome attachment to the mitotic spindle,
chromosomes with two centromeres will be unable to segregate properly into
daughter cells at mitosis. This means that DC are unstable aberrations and the
lymphocytes bearing these informative chromosomes in peripheral blood decline over
time with kinetics that is not yet fully understood (17). According to Ramalho et al
(18), the rate of decline of DC frequency may depend on the initial dose, with higher
doses declining most rapidly and lower doses producing more stable DC frequencies.
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Despite uncertainties in interpretation of DC frequencies obtained much later after
radiation exposure, this assay is still considered the most practical one shortly after
exposure. Measurement of DC yield has provided very reliable dose estimate during
several accidental scenarios (19, 20).
As with every technique, this assay also has some limitations. The need of a well-
experienced scorer is one of them. The scoring of DC is not only time consuming, but
also, not amenable to automation. Recent advances such as metaphase scanners and
centromeric painting by antikinetocore antibodies can enhance the scoring speed and
reduce errors in the identification of DC. Further, using the DC assay, the nature of
radiation exposure can be assessed by the distribution pattern of DC. In accidents
involving whole body exposures in the lethal range (3-6 Gy), scoring of just 25 - 50
metaphases is adequate for providing the preliminary information required for
medical management of victims. However, at higher doses most of the severely
damaged cells fail to go through cell division and it may be very difficult to find even
a few metaphases. At very low doses (0.10 to 0.20 Gy), where only a few DC are
involved, the error associated with dose estimates can be very large (16)
1.2.2. Chromosome aberrations: Translocations (TL)
Radiation induced unstable chromosomal exchanges like DC, rings and deletions are
eliminated from the body within 1-3 years depending on the exposure condition. As a
result, there is considerable uncertainty in this dosimetry for past exposures (21-22).
Scoring stable chromosomal exchanges such as TL, is a possible approach to
overcome this problem.
Studies of the Japanese A-bomb survivors and patients receiving radiotherapy have
shown TL to persist in peripheral blood lymphocytes many years after exposure and
repeated cytogenetic analyses have also indicated that the frequencies of cells with TL
remain unchanged (23-24). Thus, they are potentially a better indicator of cumulative
dose. This persistence reflects the induction of aberrations in stem cells with
subsequent constant replenishment of the mature lymphocyte pool (17).
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Many laboratories initially explored the use of G-banding to identify stable
chromosomal aberrations – TL for biodosimetry purpose (25-28). Even in this modern
era of the fluorescent technology, some laboratories still prefer G-banding to FISH,
due to the cost viability of this technique (29).
Detection of TL by conventional block staining techniques will only identify those
with obvious length changes and is therefore inefficient and probably subject to scorer
bias (30). Analysis using G-banding, allows the identification of rearrangements
involving any chromosome in the genome and also the precise location of breakpoints
within each chromosome (Figure 1.4). But, it is more time consuming and tedious.
Automation of the same demands good quality of banded preparations.
Figure 1.4: Translocation between chromosomes 7 and 14 observed in peripheral
blood lymphocytes exposed to gamma radiation
Since the beginning of the 1990s, Fluorescent In Situ Hybridisation (FISH) has been
used as a cytogenetic tool for the detection of genome damage. The availability of
whole chromosome specific libraries has enabled painting of individual chromosomes
by fluorescence in situ hybridization technique (31). FISH using whole chromosome
paints enables the detection of TL involving selected chromosomes. Since this
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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technique does not necessarily require well-spread metaphases for analysis, it is
possible to increase the number of analyzable metaphases compared with the banding
technique.
Chromosome painting (FISH) is a simpler, more objective and practical method for
detecting chromosome rearrangements than conventional banding analyses. A number
of studies, using different combinations of painted chromosomes, have adopted the
approach of Lucas et al (32). The results point out that FISH for TL in as few as three
chromosomes, when combined with screening of numerous metaphases, provides
sensitivity comparable with that provided by G-banding, which covers the whole
genome (33-35). Figure 1.5 shows a reciprocal translocation between chromosomes 1
(WCP1 SpectrumGreen) and 3 (WCP3 SpectrumOrange) observed in a peripheral
blood lymphocyte metaphase exposed to gamma radiation.
Figure 1.5: Translocation observed in peripheral blood lymphocytes exposed to
gamma radiation by FISH painting of chromosomes 1 and 3
The translocation frequency obtained for the painted chromosomes is extrapolated to
the whole genome on the assumption that, radiation-induced exchanges are produced
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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randomly (36). Therefore, the data can be also examined for excesses and deficits in
the involvement of specific chromosomes or chromosome regions in rearrangements.
In principle, chromosomes from any cell can be subjected to FISH. However, the
method usually uses peripheral blood lymphocytes obtained from the individual to be
evaluated. The lymphocytes are cultured and metaphase spreads are deposited on
glass slides using standard cytogenetic methods. A cocktail of composite
chromosome-specific DNA probes can be used in conjunction with pancentromeric
probes to discriminate between TL and DC (37-39). TL involving exchange of parts
between the painted chromosomes and counterstained (unpainted) chromosomes are
visualized as bicoloured structures.
FISH assay not only makes the identification of TL very easy, but also increases the
sensitivity by its ability to score events, which the conventional banding may fail to
detect. In recent years many laboratories have explored the potential of FISH assay of
TL as a biological dosimeter (31, 36, 40-46). Since only a part of the genome is
painted (10-20%), the information for the whole genome is derived by extrapolation
of the response obtained for the painted fraction. As, it is likely that individual
chromosomes may differ in their radiosensitivity, there is a need to obtain calibration
curves with different cocktails of painted chromosomes. Some of the previous reports
suggest the usefulness of FISH assay for retrospective biological dosimetry of
radiation (34, 36, 47-51).
1.2.3. Whole genome painting: multicolour-FISH and Spectral Karyotyping
Until rather recently, it was usually assumed that virtually all chromosome exchanges
are simple, i.e. involve only two chromosome breaks. However, chromosome
"painting" techniques have now shown that complex aberrations, involving more than
two breaks in a single configuration, are common. Many whole-chromosome painting
techniques are based on FISH (31). More recent and sophisticated painting
techniques, such as mFISH or spectral karyotyping, employ combinatorial
hybridization schemes, allowing recognition of most exchanges between heterologous
chromosomes (52-56). Still further extensions of this approach allow better
recognition of exchanges between homologous chromosomes, better localization of
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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exchange breakpoints within a chromosome, and better recognition of inversions (57-
60). The intricate aberration spectra uncovered by mFISH/SKY give extra
information about the mechanisms and geometric aspects of radiation damage.
Whole genome analysis by SKY / mFISH is useful especially in the high dose range
to correct aberration data for complex exchange aberrations (61-62). m-FISH excites
and detects each of the five employed fluorochromes with narrow band pass filters
while SKY simultaneously excites multiple fluorophores separately with narrow
band-pass excitation/emission. These multicolor karyotyping technologies are being
used to detect subtle interchromosomal rearrangements that are otherwise below the
resolution of conventional banding methods (54). Pouzoulet et al (63) demonstrated
through their study that more TL were detected with m-FISH than with conventional
three colour FISH, and so m-FISH is expected to improve the accuracy of
chromosome aberration analyses in some situations. Figure 1.6 is a colourful picture
of chromosomal aberrations observed in peripheral blood lymphocytes exposed to
gamma radiation by mFISH whole genome painting of chromosomes.
Figure 1.6: Chromosomal aberrations observed in peripheral blood lymphocytes
exposed to gamma radiation by mFISH whole genome painting of chromosomes.
Despite their wide horizon of detection, their limitations are ill-defined. Multicolor
karyotyping results need to be interpreted with care. Structural rearrangements, which
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juxtapose non-homologous chromosome material, frequently resulting in overlapping
fluorescence at the interface of translocated segments lead to “flaring.” Flaring
obscures or distorts the fluorescence pattern of adjacent chromatin, leading to
misinterpretation.
Szeles et al anticipate that radiation induced chromosomal aberrations may be more
complex than expected from conventional and single chromosome painting analyses
(64). While conventional Giemsa staining is generally accepted as the method of
choice for a triage situation, it is expected that extended mFISH / SKY analysis will
add to the knowledge of underlying mechanisms for irradiation associated
chromosomal aberrations. Some studies suggest that a 24-color FISH approach gives
a more complete picture of radiation-induced aberrations and aids in the detection and
quantification of genetically determined intrinsic radiosensitivity (65). The usefulness
of these whole genome scanning techniques can be understood while reviewing the
number of such studies being conducted these days and the explosive increase in
cytogenetic data, which, together with computer-assisted modeling, allows new
insights into the formation of radiation-induced chromosome aberrations. (53, 66-72).
1.2.4. Premature chromosome condensation (PCC)
Radiation damage can also be detected in interphase cells by the premature
chromosome condensation (PCC) assay (73). This method classically uses fusion of
the test cells with mitotic cells, which transmit a signal for dissolution of the nuclear
membrane and condensation of the interphase chromosomes as if in preparation for
mitosis. The 46 chromosomes have a single chromatid appearance (Figure 1.7). At
this stage, damage induced by radiation appears as excess breaks. Excess PCC
fragments have been shown to increase with increasing radiation exposure (74).
An important advantage of this method is that, information on the exposure can be
derived within 48 hours of obtaining the blood sample. Further, since the technique
does not involve cell division, technical factors associated with post irradiation
stimulation and progression through the cell cycle, do not interfere with the analysis.
Even in accidents involving exposure to doses in excess of 5 Gy, the cells can easily
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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undergo condensation (although they may fail to reach metaphase). PCC technique
would be very informative in such situations.
Figure 1.7: Chromosomal aberrations observed in peripheral blood lymphocytes
exposed to gamma radiation by the PCC assay.
The applications of this technique with respect to biodosimetry are diverse. Since
PCC is one of the techniques that allows visualization of initial DNA damage
incurred, studies pertaining to DNA break induction kinetics can be performed,
yielding useful information (75-76). The method is also suitable for studying radio-
sensitivity (77-78), partial body exposures (79), high dose exposures (80) and
improves detection of even low dose exposures (81-82). The use of PCC FISH can
simplify the analysis further by increasing the speed and accuracy of the assay (83-
85). In spite of the speed of analysis, this technique is in use in very few centres.
1.2.5. Micronucleus Assay (MN assay)
Much more than a century ago, MN have been described by many scientists. MN refer
to small nuclei formed from chromatin material, which fail to get incorporated in
either of the daughter nuclei formed during cell division. It is now known that
sometimes, even entire chromosomes can lag behind during cytokinesis and form MN
(86-87). The in vitro cytochalasin-B block methodology to score MN in cells, which
divided once in culture, became an important and useful tool to distinguish
clastogenic and aneugenic from each other. Exposure to clastogenic agents like
radiation results in a dose dependent increase in the frequency of MN.
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Cells are cultured in the presence of a cytokinesis inhibitor - Cytochalasin-B. The
nucleus goes through mitosis, but the daughter cells fail to separate, leading to a bi-
nucleated cell (Figure 1.8). This ensures scoring of first division cells, which are a
must for biodosimetry (88). In the recent years the in vitro MN test has become an
attractive tool for genotoxicity testing because of its simplicity of scoring (89) and
wide applicability in different cell types.
Figure 1.8: Micronucleus observed in binucleated cells exposed to gamma
radiation.
A few laboratories have investigated the use of MN as a biological dosimeter (90-94).
Scoring does not require skilled or experienced personnel. Around 1000 cells can be
easily scored in an hour to determine the MN frequency. The sensitivity of this assay
is 0.25Gy. However due to high interindividual variability in the spontaneous
frequency of MN, its sensitivity in the low dose region is poor. In this aspect, the use
of chromosome-specific centromeric probes in cytokinesis-blocked binucleated cells
permits an accurate analysis of non-disjunction (95) allowing rapid scoring of only
acentric MN. This has enhanced the specificity and lowered the dose detection limit
of this assay to between 0.1–0.2 Gy (17).
An adaptation of this assay to flow cytometry has rendered it by far the most sensitive
test for induced chromosome damage (96-97). However, MN are not radiation
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specific and the background frequency seems to be much larger and variable. Hence
care needs to be taken in interpretation of observed frequency in terms of radiation
dose. Overall, the simplicity, accuracy, multi-potentiality and large tissue applicability
of the MN technology make it an attractive assay of choice.
1.2.6. Comet Assay
The “Comet Assay” (single cell gel electrophoresis) is a sensitive, rapid and relatively
inexpensive method for measuring DNA damage in individual cells (98). Single cells
are embedded in agarose on microscope slides, lysed to remove majority of the
proteins, electrophoresed and stained with a fluorescent dye in order to visualize the
DNA. When visualized using a fluorescent microscope, DNA of undamaged cells
appears as a spherical mass occupying the cavity formed by the lysed cell. Following
radiation damage, the smaller the fragment size and the greater the number of
fragments of DNA, the greater the percentage of DNA that it is able to migrate in an
electric field, forming a comet image (Figure 1.9).
Figure 1.9: DNA damage observed in peripheral blood lymphocytes exposed to
gamma radiation by the Comet assay.
The assay can be performed under alkaline conditions to examine DNA single strand
breaks (SSB), or in non-denaturing (neutral) conditions to measure double strand
breaks (DSB) in individual cells. The advantages of the technique include: collection
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of data at the level of individual cells, making it possible to identify different
populations of cells within the same sample; the need for small numbers of cells per
sample; sensitivity for detecting DNA damage and that virtually any eukaryotic cell
population is amenable to analysis (99).
Many variant versions of this assay exist. One such evolved version is the halo-comet
assay. The halo-comet assay uses whole blood and does not require post sampling
incubation and is characterized as having a short processing time requirement,
yielding a potentially good slide processing profile. The persistence of the halo-comet
assay can accommodate its use in biodosimetry. (85)
As contradistinct from the normal comet assay, the halo-comet uses nuclear
suspensions. Samples are analyzed after a sufficient interval of time such that initial
strand break repairs have been substantially completed. The halo-comet analysis
exhibits persistent DNA conformational effects.
In the halo assay permeabilised cells have their nuclear protein extracted with high
salt. The DNA remains within a residual nucleus-like structure called a nucleoid. If
the nucleoid DNA contains breaks, a halo of DNA extends around the original form
of the nucleus. The presence of breaks in DNA relaxes supercoiling and loops. The
loops are free to extend outside the nuclear matrix. Measurements of the radius of the
halo give an indication of the size of the loops. In the halo-comet assay, sufficient
time is given, prior to scoring, to eliminate the transient damage repair (single strand-
double strand breaks) component and arrive at a persistent radiation dose effect
(approximately 24 hours). The benefit of cytogenetic halo-comet assay complemented
by Flow Cytometry lies in its ability to estimate partial body exposure effects.
The comet assay is suitable for in vivo human biomonitoring, especially in cases of
incidental exposure to IR (100), as a predictor of radiosensitivity (101) and a
biomarker in assessment of DNA damage during occupational exposures (99) and
predictor of radiotherapy (102-103).
1.3. Somatic Mutation Assays
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Somatic cell mutations are a major cause of cancer formation and hence their
evaluation can be an important tool in predicting cancer risk for radiation-exposed
populations. Repair of DNA damage caused by radiation in hematopoietic stem cells
can result in somatic mutations in certain loci that can be monitored as biological
indicators of dose. Mutations in several different loci have been exploited for
detection of radiation exposure, including expression of glycophorin A (GPA)
variants in erythrocytes and mutations at the T-cell receptor (TCR) or hypoxanthine
guanine phosphoribosyltransferase (HPRT) loci in T-lymphocytes. These assays may
be used for individual assessment of long-term health consequences after the
irradiation, because persons with elevated frequencies of mutant cells may represent a
group at high risk in respect to oncological diseases. A drawback common to these
somatic mutation end-points is their relative lack of specificity for radiation exposure
as other environmental exposures or physiological states can also increase the
observed mutant frequencies in vivo.
1.3.1. GPA variants
The Glycophorin-A (GPA) assay in erythrocytes has been widely used for
biodosimetry. Glycophorin A is a sialoglycoprotein found exclusively and abundantly
in the red-cell membrane. The gene for glycophorin A in humans has two equally
prevalent alleles, M and N, whose gene products differ by two amino acids and are
the basis for the M and N blood types. The Glycophorin A somatic-mutation test is
limited to the 50% of humans who are MN by blood type and, hence, normally
express the M and N gene products on the surface of every red cell.
The test uses flow cytometry and differently labelled fluorescent monoclonal
antibodies to the M and N products to measure their content on the surface of each of
several million red cells. Three mutant types are scored: the M0 and N0, which are
those cells respectively lacking expression of either the N or M product but with
normal amounts of the counterproduct; and the MM, which are those cells lacking the
N product but with double amounts of the M product. M0 and N0 are interpreted as
simple loss-of-function mutations in which the Glycophorin A gene product from one
chromosome 4 has been modified (lost or changed) to the point of being
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unrecognizable by the antibody, whereas the gene product from the other
chromosome 4 is expressed normally. The M0 and N0 mutational events are
statistically independent but otherwise identically likely events and can be averaged
into a common estimate of the gene-loss endpoint. The MM appears to have two
functioning and identical genes and probably represents reduction to homozygosity
through some type of recombinational event. The background mutant frequencies are
in the range of 5–20 per million cells for each of the three endpoints, depending on
measurement method and age of the subject. Figure 1.10 shows a radiation dose
response correlation with GPA frequency in cancer patients receiving radiotherapy.
Figure 1.10: Peripheral blood lymphocytes with elevated GPA frequency due to
radiation exposure as detected by Flow Cytometry (104).
The atom-bomb survivors were one of the original populations to be investigated with
the glycophorin A method, and from the beginning, they showed significant radiation
dose-responses (105). By now, well over 1,000 survivors have been studied, with
results that are remarkably consistent (106). Other studies performed showed the
reliability of the GPA assay in giving dose estimations similar to those obtained by
other biodosimetric assays (107). Some studies undertaken for biodosimetry, debate
on the usefulness of this assay (108-111).
G PA (Vf) Dose Re spo nse Cu rv e O bta ine d fro m Ce rv ix Cance r
pa tie nts e xop se d to Gamma Rad iatio n
y = 0 .02 02x + 0.0 269
R2 = 0.8 082
0
0.0 2
0.0 4
0.0 6
0.0 8
0. 1
0.1 2
0.1 4
0.1 6
0.1 8
0. 2
0 1 2 3 4 5 6 7
Eq .Do se (Gy)
VF
x 1
0-4
.4
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As any other biodosimeter, the GPA assay has its limitations. First there is the
problem of individual variability with the glycophorin A assay. The assay is
reasonably stable for repeat measurements on an individual but its standard deviation
is only slightly larger than expected for counting errors. A target population of
approximately 105 cells is needed to score an average of one mutation, and well over
106 cells to estimate a reasonable frequency of mutation. A human, particularly one
who has just been irradiated, has barely enough hematopoietic stem cells to measure a
continuous response with this test. In short, the glycophorin A test, useful as it is for
large, ordered populations, cannot function as an individual dosimeter. The only other
potentially helpful strategy is to combine the test with other mutational endpoints into
a composite estimate per person. However, this possibility is yet to be explored (112).
1.3.2. T-Cell Receptor
The T-cell receptor (TCR) mutation assay for in vivo somatic mutations is a sensitive
indicator of exposure to IR. In this light, the T-cell receptor mutation assay is known
to be a sensitive indicator of IR. This assay requires only a small volume of blood and
can be performed in a few hours.
For the TCR to be expressed on the T cell surface, the complete TCR/CD3 complex is
required. Any defect in any of the components of the TCR results in the loss of CD3
expression on the T cell surface. Therefore cell surface expression of CD3 can be
used as a marker of TCR mutation rate. Figure 1.11 shows loss of CD3 expression in
peripheral lymphocytes exposed to gamma radiation.
Flow cytometric detection of TCR mutations has made this assay easily amenable to
automation and hence numerous studies involving the TCR have been carried out for
biodosimetry purposes. In one report, the TCR mutation assay appeared to be a useful
biological dosimeter even after a period of 40 years since radiation exposure, where
internal radiation exposure was suspected (113). Smirnova et al suggest the usefulness
of the TCR assay in revealing the nature of radiation exposure (to be internally
localized) (114). Another study undertaken to determine TCR and GPA mutation
frequencies in persons exposed professionally to IR or a result of accidents at nuclear
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power plants showed that the TCR method is more sensitive and informative for
biological dosimetry of recent radiation, than the GPA test (115).
Figure 1.11: Loss of CD3 expression (TCR) in peripheral lymphocytes exposed to
gamma radiation.
CD3 staining CD4 staining CD3 / CD4 staining
One disadvantage of this assay was that the half life of the majority of mutated cells
was around 2-3 years, with the number of mutant cells declining gradually to near
base line after 10 years of exposure (116). Also, this assay cannot be immediately
applied after radiation exposure because expression of a mutant phenotype may
require as long as several months. However, Ishioka et al have eliminated this time
lag and improved the TCR mutation assay making it a useful biological dosimeter for
recent radiation exposure (117).
1.3.3. HPRT and mutant spectra
Functional inactivation of the HPRT gene has probably been the most extensively
used of the T-cell biodosimetry assays. In contrast to the erythrocyte assays, the T-cell
assays monitor mutations occurring directly in the circulating peripheral cells. The
HPRT gene codes for a salvage pathway enzyme that allows the phosphoribosylation
of hypoxanthine and guanine as precursors for DNA synthesis. It can also utilize
purine analogs, such as 6-thioguanine, which can then incorporate into DNA and kill
the cells. Mutant cells that have lost this enzyme can grow in concentrations of
6-thioguanine that are toxic to wild type cells, thus allowing mutant selection.
Furthermore, the location of the hprt gene on the human X-chromosome means it is
functionally hemizygous, allowing detection of the loss of a single allele.
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An assay using T-cell cloning and hprt mutant fraction determination has been used to
show a strong relationship between dose and induced mutations in atomic bomb
survivors and patients receiving high doses of radiation therapy (118). An increase in
hprt mutant fractions may also be detectable following lower dose exposures but these
results seem more variable depending on the time of sampling (119). Albertini et al
show that radiation quality affects both the efficiency of induction and the molecular
spectrum of HPRT mutations in human T lymphocytes both in vitro and in vivo. The
mutational spectrum may be relatively specific for radiations of different quality and
thus allow a more precise measurement of the induction of somatic gene mutations
resulting from individual exposures to radiation, thereby providing more sensitive
assessments of health risks (120). The results of the study conducted by Thomas et al
illustrate the sensitivity of HPRT somatic mutation as a biomarker for populations
with low dose radiation exposure, and the dependence of this sensitivity on time
elapsed since radiation exposure (121). Moreover, the HPRT mutational assay has
also been shown to reveal dose rate differences and hence serves as a useful parameter
for risk estimation in radiation protection (122).
1.4. Molecular biodosimetry
1.4.1. Molecular profiling by gene expression
Mutation induction in cells directly exposed to radiation is currently regarded as the
main component of the genetic risk of IR for humans. Recent technological advances
may allow an additional exploitation of the molecular responses of cells to IR.
Radiation dose, dose rate, radiation quality, and elapsed time since exposure result in
variations in the response of stress genes suggesting that gene expression signatures
may be informative markers of radiation exposure. Irradiation initiates a plethora of
signal transduction cascades responsible for maintaining cellular homeostasis and
promoting interactions with neighboring cells. Large-scale changes in gene expression
have also been found after irradiation, and microarrays have helped discern these
subsequent transcriptional alterations.
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Exposure of cells to DNA-damaging agents elicits a highly complex molecular
response, much of which is mediated through changes in gene expression. A
transcriptional response to genotoxic stress, estimated to involve 1% or more of the
genome, was initially identified in yeast and similar complex transcriptional responses
were soon confirmed in mammalian cells (123-124). The stress response pathways
responding to different environmental and physiological stresses have many
overlapping components, including growth factors, cytokines, oncogenes and genes
involved in cell cycle, apoptosis, signaling pathways and DNA repair. The recent
development of functional genomic approaches to simultaneously quantify expression
of thousands of genes in a single experiment may allow the determination of
expression signatures indicative of exposure to IR or other environmental toxins.
Although earlier it was in the speculative realm, this approach is highly attractive as it
is amenable to rapid, even automated, non-invasive analysis and may additionally
have the potential to discern competing effects from incidents involving different
quality radiations or mixed chemical and physical exposure components.
A number of high-throughput gene expression measurement methods are currently
available, including serial analysis of gene expression (SAGE), oligonucleotide arrays
and cDNA arrays (125-126). The premise is developed that stress gene responses can
be employed as molecular markers for radiation exposure using a combination of
informatics and functional genomics approaches (17). A generalised post-exposure
profile may identify exposed individuals within a population, while more specific
fingerprints may reveal details of a radiation exposure. Changes in gene expression in
human cell lines occur after as little as 0.02 Gy rays, and in peripheral blood
lymphocytes alter as little as 0.2 Gy (127).
Accumulated evidence also implies that the biological effects of low-dose and high-
dose IR are not linearly distributed. According to the study done by Ding et al the
predominant functional groups responding to low-dose radiation are those involved in
cell-cell signaling, signal transduction, development and DNA damage response
(128). At high dose, the responding genes are involved in apoptosis and cell
proliferation. Interestingly, several genes, such as cytoskeleton components ANLN
and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond
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to low-dose radiation but not to high-dose radiation. Pathways that are specifically
activated by low-dose radiation are also evident. These quantitative and qualitative
differences in gene expression changes may help explain the non-linear correlation of
biological effects of IR from low dose to high dose.
While some studies have focused on low dose-rate experiments, others have analyzed
the gene expression response of IR compared to other DNA damaging agents. Very
few genes have been found to be consistently up-regulated by IR, but that set includes
GADD45, CDKN1A, connexin-43 and genes associated with the nucleotide excision
repair pathway (129-130)
Low doses of radiation have an identifiable biosignature in human tissue, irradiated in
vivo with normal intact three-dimensional architecture, vascular supply, and
innervation. The ability to detect a distinct radiation response pattern following low
dose IR exposure has important implications for risk assessment in both therapeutic
and national defense contexts (131).
1.5. Protein biodosimetry
1.5.1. Gamma h2ax assay
The need to detect DNA damage by radiation requires specific markers that can be
easily seen and quantified, and gamma-h2ax foci formation is one such event that can
be used in this scenario. An early event after introduction of DSB is the
phosphorylation of a special form of histone 2A, denoted h2ax that is part of 10% of
all nucleosomes in the cell (88, 132-133). Histone 2AX contains a distinct C-terminal
extension, with a consensus phosphorylation at serine 139. It is known that h2ax
phosphorylation is specific to sites of DNA damage and is also indicative of amount
of DNA damage (134). However, in order to use gamma-h2ax as a quick screening
tool, it must be optimized for sensitivity and rapidity.
Gamma-h2ax can be tagged with a fluorescently labeled antibody, and can then be
detected with excellent sensitivity using in situ image analysis (Figure 1.12). IR is an
efficient inducer of DSB and most of the early research on gamma-h2ax has been
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done with IR (135-136). Both DSB and thus gamma-h2ax are formed linearly with
dose from very low to extremely high (> 10 Gy) doses (135). It has also been shown
that exposure to 10-3Gy of X-rays, induces a significant elevation in h2ax
phosphorylation in human fibroblasts (137), making it more sensitive to low doses.
The gamma-h2ax system well complements the MN system as a radiation
biodosimeter (138), requiring much shorter processing times as the cells do not have
to be cultured. Furthermore, the gamma-h2ax foci reach their maximum value within
about 30 minutes of irradiation (138), decaying over 24-36 hours post-exposure (139).
This is contrasted with MN that appear about 24 hours post-exposure and decay over
months or years (140).
Figure 1.12: Fluorescent antibody tagged detection of gamma h2ax: each signal
represents a double strand break.
The local formation of gamma-h2ax allows microscopical detection of distinct foci by
fluorescent gamma-h2ax-specific antibodies that most likely represent a single DSB
(137-138, 141). The potential to detect a single focus within the nucleus makes this
the most sensitive method currently available for detecting DSB in cells. This method
is, however, labor-intensive and will be difficult to adapt in clinical practice. In
contrast, flow cytometry allows simple detection of gamma-h2ax in a large number of
cells (142). Several reports show that the level of gamma-h2ax as detected by flow
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cytometry correlates well with the number of DNA strand breaks, to the level of cell
death and radiosensitivity (135, 143-144).
1.6. Electron paramagnetic resonance
Electron paramagnetic resonance (EPR) is a physical measurement of absorbed dose
that can be applied to biological material. Although samples of fingernails and
clothing have been used in ESR determinations of dose shortly after exposure, dental
enamel is the most widely used material.
1.6.1. Electron paramagnetic resonance of dental enamel
Tooth enamel as a detector for in vivo dosimetry has been known for more than three
decades (145). The usefulness of enamel for dosimetry results from its high content of
hydroxyapatite (146). Carbonate impurities, which are incorporated into or attached to
the surface of hydroxyapatite crystals during formation, are converted to CO2 radicals
through absorption of IR (147). The concentration of radicals increases with absorbed
dose. The intensity of the resultant EPR absorption is a measure for the absorbed
dose. Examples of the use of EPR in dose reconstruction include the dose evaluation
of survivors of the atomic bomb explosions in Hiroshima and Nagasaki (148-149),
nuclear workers in the South Urals (150), residents of the Techa river basin (151), the
populations of contaminated areas in the Urals (152), the population living near the
Chernobyl nuclear reactor (153) and workers in the Chernobyl Sarcophagus (154).
Finally, EPR dosimetry was applied to a population from an uncontaminated area in
Russia (155) demonstrating the potential to estimate the absorbed dose from natural
background radiation.
International comparisons on EPR tooth dosimetry were carried out (156-160). These
comparisons were designed to check the consistency and reliability of EPR dose
reconstruction among different laboratories. These comparisons led to critical
revisions and improvements to the different variations of the EPR dosimetry method
applied by the participants. Moreover, the capability of EPR dosimetry to measure
low doses in the range of 100 mGy was demonstrated. Today, EPR dosimetry with
tooth enamel is a leading method for retrospective dosimetry of individual radiation
exposures.
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Teeth that are extracted for health reasons are readily available for dose reconstruction
and can be archived for a prolonged period before examination. EPR dosimetry has
the capability to measure the volume of samples required for epidemiological studies
(159). Dose reconstruction can be applied to the distinctive tissues that comprise a
tooth, namely enamel, and dentine. Tooth enamel is preferred in retrospective
dosimetry because this tissue is completely formed in childhood and once formed, is
never remodelled, even after abrasion. Therefore, the accumulated concentration of
radiation-induced radicals in the exposed enamel is preserved. At 25°C, a lifetime of
107 years was determined for the CO2 radicals in fossil tooth enamel (161). Hence,
EPR dosimetry with tooth enamel is suitable for dose reconstruction after long periods
of exposure and for many years after the exposure. The complementary measurement
of the absorbed dose in dentine offers the possibility to measure the dose resulting
from ingested radionuclides deposited in dentine, in addition to the dose from external
sources. This technique has been applied to the measurement of the accumulated dose
resulting from the intake of the bone-seeking radionuclide strontium (151).
There are several strong indications that EPR dosimetry gives correct and accurate
dose assessment even long after the exposure event. Among them are the results of
two international comparisons (162-163), several blind comparisons of the results of
EPR dose reconstruction, and data of operational personal monitoring for nuclear
workers (150-152, 164).
However, there are also certain shortcomings of EPR biodosimetry (165). It is not
always possible to obtain extracted teeth from all individuals in the study group. For
bone-seeking radionuclides (e.g. Sr-90) the reconstruction of the individual dose is
complicated and in certain cases impossible (166). EPR dose reconstruction
procedures are also considered to be time-and labour-consuming. For these reasons, at
present, EPR biodosimetry will likely not be used as the sole method applied to large
cohorts, but will remain invaluable for validation purposes.
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1.7. Biodosimetry - Which assay is reliable and future trends?
In response to radiation exposure, rapid and reliable dose estimates are crucial for risk
assessments during investigation of real or suspected exposed victims. The
employment of biodosimetry can represent more than a complementary methodology
to physical dosimetry in individual monitoring. Quantification of the biologically
relevant dose is required for the establishment of cause-and-effect between radiation
dose and important biological outcomes. Most epidemiological studies of
unanticipated radiation exposure fail to establish cause and effect because of an
inability to construct a valid quantification of dose for the exposed population.
Knowledge about the quantity of absorbed dose, a number together with its unit, is
certainly not sufficient to evaluate the risks associated with radiation exposure. In
addition, the comparison between the changes in biological indicators as a result of an
irradiation, with the same alterations caused by other physicochemical agents may be
important for better understanding of radiation hazards and the risks associated with
them. This will aid professionals, as well as lay people, in a better observance of
radioprotection practices.
With a host of assays available, it is difficult to say which is the best one. No single
biodosimetric technique (biophysical or biological) meets all the requirements of an
ideal dosimeter and thus qualifies as a "gold standard." An approach of using
combined dosimetry from the most appropriate methods in a given situation has been
advocated. However cytogenetic methods of dose determination, have dominated the
biodosimetry arena due to their precise and increasingly informative nature, in the
time immediately following exposure. It should be noted, however, that some of these
more complex analyses may require a high degree of expertise and so may be more
useful for research purposes than in the field (17).
Several attempts have already been made to automate biodosimetric assays (167).
During the last decade many engineers and scientists have teamed-up in the quest for
designing automated systems for medical applications featuring higher and higher
throughputs. Many innovations and new developments with motorized microscopes,
Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome
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automated slidescanning workstations, digital imaging, and the associated computer
software programs have made the cytogenetic biodosimetry process more efficient
and less time consuming. For example, automated systems can quickly scan a
microscope slide and locate the scoreable cells in minutes compared to the hours
required using manual methods. Several prototypes have been designed (168).
The search for biomarkers of effective dose and the early effects of ionizing radiation
exposure in both humans and experimental animals has a history spanning several
decades and is still an ongoing process. Blood cells and serum have proven to be
abundant sources of human radiation biomarkers, including those of DNA damage
and repair, chromosomal aberrations, serum proteomic profiles, and gene expression
profiles determined by both microarrays and RT-PCR. The use of global profiling
technologies has contributed substantially to the understanding of the radiation
cellular stress response and has contributed to the elucidation of many of the complex
biological networks associated with gene expression and signal transduction. Now,
identification of radiation-induced metabolic changes is under study for development
of reliable metabolomic markers to assess radiation exposure and extent of injury.
However, it is unlikely that the more established, tested and well proven
biodosimetric assays, such as cytogenetic biodosimetry will become outdated.
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1.8. Aims and objective of the study:
As nuclear activity in India is on the rise due to the immense potential of nuclear
energy, radiation monitoring and safety bodies like the Atomic Energy Regulatory
Board (AERB), India, are preparing various centers all across the country for
biodosimetry purpose. Hence, the current study served as a good attempt in
establishing biodosimetry through the construction and validation of the in vitro dose
response curves. As many biodosimetry assays are available, there is a need to
validate the usefulness of these assays. Cytogenetic assays have been extensively used
for biodosimetry purpose since the 1960’s. The cytogenetic Fluorescent In Situ
Hybridization assay (FISH) is a powerful tool aiding in the identification and
enumeration of both stable and unstable chromosomal aberrations. In light of this, the
present study aimed to validate and establish its usefulness for biodosimetry purpose.
Hence the specific objectives of the study comprised of:
a) Construction of in vitro dose response curves for Co-60 gamma rays by the
FISH assay using dicentrics and translocations as endpoints.
b) Validation of in vitro FISH based dicentric yields using:
i) In vitro dicentric yields for Cobalt-60 gamma rays by Fluorescence Plus
Giemsa assay.
ii) In vitro micronucleus yields for Cobalt-60 gamma rays.
c) Validation of in vitro FISH based translocation yields using:
i) In vitro translocation yields for Cobalt-60 gamma rays by G-banding.
ii) In vitro translocation yields for Cobalt-60 gamma rays by mFISH.
iii) To check if radiation induced DNA damage is random or non random.
d) Validation of in vivo FISH based translocation frequency using G-banding
e) Impact of radiation on certain genes and search for newer biodosimetry
techniques at the gene level.
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