Chapter 1 Introduction - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/4972/6/06_chapter...

Evaluation of ionizing radiation exposure for biodosimetry: Validation of Whole Chromosome

Painting by Cytogenetic and Molecular techniques.

xiv

Chapter 1

Introduction



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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



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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



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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



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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.



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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



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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



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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



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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



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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



14

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.



21

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



22

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



23

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



24

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.



25

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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27

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,



28

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.



29

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.



30

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