Radiation Environment and Medicine 2022 Vol.11, No.2 82–90

ReportCytogenetic Biodosimetry in Radiation Emergency Medicine:3. The Basics of Chromosomes for Biodosimetry
Akifumi Nakata1 *, Kentaro Ariyoshi2 , Yu Abe3, Yohei Fujishima4, Valerie Goh Swee Ting5,
Ryo Nakayama4, 6, Kai Takebayashi4, 6, Mai Tran Thanh7, Kosuke Kasai6,
Mitsuaki A. Yoshida4, 8 and Tomisato Miura4
Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 15-4-1, Maeda 7-jo, Teine-ku, Sapporo,
Hokkaido 006-8585, Japan
2
Center for Integrated Science and Humanities, Fukushima Medical University,
1 Hikariga-oka, Fukushima City, Fukushima 960-1295, Japan3
Department of Radiation Biology and Protection, Atomic Bomb Disease Institute, Nagasaki University,
1-12-4 Sakamoto, Nagasaki, Nagasaki 852-8523, Japan
4
Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine, Hirosaki University,
66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
5
Department of Radiobiology, Singapore Nuclear Research and Safety Initiative, National University of Singapore,
1 Create Way, Singapore 138602, Singapore
6
Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences,66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
7
Biodosimetry Group, Centre of Radiation Technology and Biotechnology, Dalat Nuclear Research Institute,
1 Nguyen Tu Luc, Ward 8, Dalat City, Lamdong Province, Vietnam
8Institute of Chromosome Life Science, 11-5-409, Fukuokachuo 2-Chome, Fujimino-shi, Saitama 356-0031, Japan
1

Received 24 March 2022; revised 17 May 2022; accepted 20 May 2022

Chromosomes are observed during the mitotic phase of the cell-cycle. As organisms have a
species-specific chromosome number and morphology, any changes in the number or morphology
can be considered as chromosome aberrations. It is well known that radiation exposure causes
chromosome aberrations, and multiple studies have reported on the mechanisms of radiationinduced chromosome aberrations and the effects of radiation dose and quality on chromosome
aberrations. Radiation-induced chromosome aberrations can be divided into 2 categories: stable
chromosome aberrations which are inherited by daughter cells in cell division (e.g. translocations,inversions, partial deletions and duplications) and unstable chromosome aberrations which cause
cell death and eventually disappear (e.g. dicentrics, rings and acentric fragments). In acute external
exposure, unstable chromosome aberrations are usually used as indicators for accurate dose
assessment to facilitate radiation emergency medical care. On the other hand, stable aberrations
are generally used for retrospective dosimetry caused by past exposures. This article summarizes
the essential information on chromosomes and chromosome aberrations for cytogenetic
biodosimetry.
Key words: cytogenetic biodosimetry, the morphology of chromosome, chromosome aberration,
cell-cycle*Akifumi Nakata: Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 15-4-1, Maeda 7-jo, Teine-ku, Sapporo, Hokkaido 006-8585, Japan.
E-mail: nakata_a@hus.ac.jp†
These authors contributed equally to this workhttps://doi.org/10.51083/radiatenvironmed.11.2_82
Copyright © 2022 by Hirosaki University. All rights reserved.

Akifumi Nakata et al./ Radiation Environment and Medicine 2022 Vol.11, No.2 82–90

83Fig. 1. Representative image of a metaphase spread with diverse radiation-induced chromosome aberrations. Solid arrow: acentric fragment,
white arrow: dicentric chromosome, black triangle: ring chromosome, D: Chromosome belonging to group D, G: Chromosome belonging togroup G,＊: Marker chromosome (larger chromosome than normal group D chromosome caused by translocation), ictg: Isochromatid gap.

Fig. 2. Examples of chromosome aberrations.

1. Introduction
Depending on the type of radiation exposure, theoptimal cytogenetic assay is selected for radiation dose
assessment (biodosimetry). Such assays include thedicentric chromosome (Dic) assay1) (DCA), cytokinesisblock micronucleus (CBMN) assay 1-4), prematurechromosome condensation (PCC) assa y 1, 5-9) andfluorescence in situ hybridization based translocation
assay1, 10). These assays depend on chromosome aberrations
formed during the cell division. In Dic and translocation
assays, aberrations are evaluated on chromosomes
observed during the mitotic (M) phase of the cell-cycle.
As organisms have a species-specific chromosome

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number and morphology, any changes in the number
or morphology can be considered as chromosome
aberrations and may also induce genetic changes.
Radiation is known to cause chromosome aberrations,and multiple studies have reported on the mechanisms ofradiation-induced chromosome aberrations and the effects

Fig. 3. Direct and indirect effects of radiation on DNA.

of radiation dose and quality on chromosome aberrations.
As seen in Figure 1 and 2, chromosome aberrations
induced can be diverse.
Radiation-induced chromosome aberrations and DNA
damage can be caused directly by radiation or indirectly
by reactive oxygen species such as hydroxyl radicals(・OH) generated in water molecules by radiation (Fig. 3).
During DNA repair of a radiation-induced doublestrand break, the broken DNA strand is expected to becorrectly repaired with the original DNA strand serving
as a template. However, if the double-stranded DNA isdamaged more than once and is accidentally bound todifferent DNA strands, such DNA rejoining can lead tovarious chromosome aberrations such as a Dic with twocentromeres on one chromosome, a ring chromosome
with one chromosome broken end joined together toform a doughnut-shaped circular chromosome andtranslocation, in which parts from the same or different
chromosomes are interchanged1) (Fig. 4).
Cytogenetics is a field of study that integrates themethods and findings of cytology and genetics, focusing
on chromosomes and their behavior in the cell. To date,cytogeneticists have revealed that the frequency ofchromosome aberrations correlates with radiation quality,
dose and dose-rate11-15). Therefore, exposure dose canbe estimated from dose-response curves established inprevious studies.
In cytogenetic biodosimetry, Dic and ring chromosomes

Fig. 4. Various radiation-induced chromosome aberrations formed after DNA repair and chromosome rejoining at different sites.

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85

chromosome morphology and the types of chromosome
aberrations.
In this article, we review the f undamentals ofchromosomes and chromosome aberrations as theunderstanding of chromosomes is required when
performing cytogenetic biodosimetry.

Fig. 5. Different levels of chromosome condensation affectedby humidity and temperature during slide preparation. (a) Overly
condensed and (b) elongated chromosomes.

Fig. 6. Phases of the cell-cycle.

are commonly used as endpoints because they can beeasily distinguished from other normal chromosomes
based on their morphological features. However,even for trained and highly skilled scorers, Dic andring identification can be quite challenging as thedetection is generally based on morphological analysisof chromosome images using a single-color stain with
Giemsa. In addition, other factors such as colcemid
treatment for metaphase arrest during cell culture16, 17),
hypotonic treatment18, 19) during cell harvest and thetemperature and humidity during slide preparation 20)
also affect the optimal chromosome morphology foreasy aberration scoring (Fig. 5). Thus, scorers musthave considerable experience in preparing chromosome
specimens and identifying chromosome morphology withmetaphase images or under a microscope. Moreover,
several guidelines have been published to facilitate Dic
identification21, 22) and prepare appropriate chromosome
specimens 23, 24). These guidelines can greatly facilitate theobservation of chromosomes in metaphase spreads byhelping scientists with limited experience to understand2. Chromosomes in the cell-cycle
Figure 6 depicts the different cell-cycle phases. Many cell
types are usually in G0 phase, which is a resting state asno cell division is taking place. When a cell divides andproliferates under the appropriate stimulation, the cyclefrom the initial cell division to the next division is known
as the cell-cycle. In the cell-cycle, the period during whichchromosomes are observed is called M phase, and theperiod between mitosis and the next mitosis is calledinterphase. Cells then enter the cell-cycle as needed andwill divide and proliferate through interphase and mitosis.
The interphase of the cell-cycle is composed of G1, S,
and G2 phases, in which DNA is replicated for cell divisionin S phase. Chromatin is found in the cell nucleus during
interphase, which then condenses into chromosomes thatcan be easily observed with a light microscope during
M phase. The M phase in human lymphocytes occupies
a short period, about 30 minutes to an hour within theentire cell-cycle of about 24 hours 25).
The M phase can then be subdivided into prophase,prometaphase, metaphase, anaphase and telophase.
Chromosome morphology and its arrangement in
the nucleus differ in each phase. During prophase toprometaphase, the nuclear envelope breaks down anda chromosome composing of two sister chromatids
emerges. In addition, spindle microtubules from eachcentriole that have moved to each cell pole of the cellextend into the center of the cell and attach to thecentromeres at the kinetochore (spindle assembly).
During metaphase, the spindle-associated chromosomesalign along the equator planes (metaphase plate) withequal tension from both poles. In anaphase, each pair ofsister chromatids separates and begins to move towards
opposite poles of the cell. From late anaphase to telophase,the separated daughter chromosomes migrate to the poleof the cell, during which two daughter cells are formed bycytokinesis, a process in which the cytoplasm is divided
into two parts. Finally, in telophase, the nuclear envelope
reforms and the condensed daughter chromosomes thendecondenses and return to the interphase state.
In order to transmit accurate genetic information todaughter cells after cell division, cells have cell-cycle
checkpoints where various cell conditions are assessed26, 27).
If any defects are found, the cell-cycle is arrested until thedamage is repaired. The G1/S checkpoint is necessary
to prevent replication of damaged DNA. The G 2/M

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Fig. 7. Human, rat and mouse chromosome karyotypes.

Fig. 8. Structural characteristics of a chromosome.

Fig. 9. Classification of chromosomes based on centromere position.
M: metacentric, SM: submetacentric, ST: Subtelocentric, and A:
acrocentric chromosomes.

checkpoint ensures that DNA replication is complete
and prevents damaged DNA from entering mitosis. In
the spindle checkpoint, all chromosomes must attachwith microtubules and align on the middle of the cell in
metaphase in order to move on to anaphase (Fig. 6).

As the genetic material was subsequently shown toexist within chromosomes, the relationship between
chromosome structure31, 32), species and chromosome
number 19, 33, 34), and cancer and chromosome aberrations35, 36)
has become increasingly clear.
Eukaryotes have a species-specific chromosome
formula (karyotype), which are sorted and orderedbased on the differences in chromosome numbers (Fig.
7). The chromosomes are symmetrical in shape on thelongitudinal axis and have a central constriction dividing
the entire chromosome arm length to a short arm (petite:
p arm) and a long arm (queue: q arm). The centromere
is located at the central/primary constriction while
telomeres occupy the chromosome ends (Fig. 8).
Chromosome can be classified morphologically basedon the arm ratio, which is the ratio of the length of theshort arm and the length of long arm, and the centromere
index (CI), which is presented as the ratio of the lengthof the short arm to the total length of the chromosome 37).
According to the centromere position as defined by thesevalues, chromosomes can be categorized into metacentric

3. Structural characteristics of the chromosome
Chromosomes were initially discovered as filamentous
structures that appear during cell division and were
thought to be just one of the many structures in the cell.
However, it took additional years of research to realizethat chromosomes also serve as genetic materials. W.
S. Sutton28) and T. H. Boveri29) proposed for the firsttime in 1902 and 1904, respectively, that the mode ofinheritance is due to the nature of the chromosome. The“chromosome theory of inheritance”, in which genes
reside on the chromosome, was not yet readily accepted.
This theory was ultimately confirmed by T. H. Morgan
and colleagues, who found that genes are arranged ineach chromosome30).

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87

Table 1. Classiﬁcation of human chromosomes based on morphology using Giemsa staining
Group Chromosomes
A

1~3

Type

Description

metacentric and submetacentric

B

4, 5

submetacentric

Chromosomes 1 and 3 are metacentric. Chromosome 3 is slightly smaller than 1.
Chromosome 1 has a secondary constriction at the proximal region of long arm.
Chromosome 4 is slightly longer.

C

6 ~ 12, X

submetacentric

Chromosome 9 has a secondary constriction at the proximal region of long arm.

D

13 ~ 15

telocentric

These chromosomes have satellites at the short arms.

E

16 ~ 18

metacentric and submetacentric

Chromosome 16 has a secondary constriction at the proximal region of long arm.

F19, 20metacentricG

21, 22, Y

telocentric

Chromosomes 21 and 22 have satellites at the short arm.

Fig. 10. Different types of radiation-induced chromosome aberrations.

(nearly median centromere), submetacentric (submediancentromere), subtelocentric (subterminal centromere)
and acrocentric (terminal centromere) (Fig. 9).
The centromeres contain specialized regions calledkinetochores that attach chromosomes to spindle fibers,
which are also often referred to as the centromere inchromosome analysis. One chromosome in mitosis
consists of two chromatids. These two chromatids
are replicated in the S phase of the cell-cycle and are
genetically identical, thus are known as sister chromatids.
Telomeres protect the end of the chromosome, andchromosome fragments with missing telomeres are
more likely to form Dic and ring chromosomes because
their broken ends are easily fused with other breaks.
In some cases, regions of the chromosome arms may

become thinner and constricted in regions other thanthe centromere. This constriction is termed secondary
constriction (SCT) and is observed as a satellite at
the chromosome tips. Such chromosomes are notchromosome aberrations and should be noted (Fig. 8).
In human somatic cells including lymphocytes, there
are 46 chromosomes composing of 22 pairs of autosomes
and two sex chromosomes (XX or XY). They are thenclassified into seven groups based on chromosome
morphology (Fig. 7) and the characteristics of eachchromosome are shown in Table 1. In particular for Dic
assay, if groups D and G chromosomes are involved in theformation of the dicentric chromosome, it is important toverify the number of chromosomes in groups D and G toensure a reliable analysis38).

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4. Chromosome aberrations formed due to misrepair
during interphase
In general, most cells are in G0 phase and the durationof mitosis is also short in mitotic cells. Thus, radiationinduced damage is thought to occur in interphase andmost chromosome aberrations generated are likely
the result of DNA misrepair. When radiation-induced
DNA double-strand breaks (DSBs) occur during G1,
this usually triggers cell-cycle checkpoints and induce
cell-cycle arrest. If incomplete G1/S checkpoints allow
incorrect genome replication at S phase, chromosometype aberrations are seen at metaphase as DNA breaks
and exchanges occur at the same locations of duplicatedchromatids (Fig. 10). On the other hand, when DSBs arise
from late S to G2 phase after DNA replication, chromatidtype aberrations with DNA breaks or exchanges in only
one chromatid are instead observed (Fig. 10). Therefore,
depending on the cell-cycle phase in which radiation
exposure occurs, the chromosome aberration seen indamaged cells is either a chromosome or chromatid-type
aberration. For example, as peripheral blood lymphocytes
at the time of exposure are in G0 phase, chromosome-type
aberrations are usually seen.
5. Chromosome aberrations in human peripheral
blood lymphocytes
To prepare chromosome specimens from G0 peripheral
blood lymphocytes for cytogenetic biodosimetry, it isnecessary to stimulate cell division in peripheral blood
lymphocytes with a mitogen, such as phytohemagglutinin(PHA), a type of lectin39-41). Cells entering the cell-cycle arethen treated with microtubule polymerization inhibitors,
such as colcemid, which limits microtubule formation andinactivates the spindle fiber and leads to activation of thespindle checkpoint and metaphase arrest in mitosis. As aresult, a large number of metaphase cells can be acquiredfor chromosome aberration analysis.
Radiation-induced chromosome-type aberrations
can be classified into stable chromosome aberrations
that are inherited by daughter cells in cell division(e.g. translocations, inversions, partial deletions andduplications) (Fig. 7) and unstable chromosome
aberrations that cause cell death and eventuallydisappear with each cell-cycle (e.g. Dic, ring, and acentricchromosome fragments). Thus, aberration stability
is dependent on the presence of centromeres andtelomeres as chromosomes require one centromere for
equal distribution during cell division and telomeres forstructural maintenance at the ends of both arms.
Acentric ring chromosome and chromosome fragmentsthat do not have centromeres are unable to migrate toeither cell pole during mitosis and therefore likely todisappear during anaphase42, 43). Dic, on the other hand,forms a chromosome bridge when the two centromeres
segregate to the opposite poles at anaphase, which either
inhibits cell division or physically cleaves the chromosome
on itself 44). Cells harboring unstable chromosome
aberrations without centromeres and telomeres often
die during cell division. Even if they can divide, they
may be impaired in further cell division. Therefore, thehalf-life of cells with unstable chromosome aberrations
in vivo is relatively short, and it has been reported from4 months to 1 year and a half 45-47). In contrast, as cells
carrying stable chromosome aberrations can slip throughcell-cycle checkpoints, the half-lives of cells with stable
chromosome aberrations are usually much longer, from 2
years to even beyond the average human life span47-49).
Based on the above, unstable chromosome aberrations(e.g. Dic, rings) are scored for dose assessment tofacilitate radiation emergency care after acute external
exposure, while stable aberrations (e.g. translocations) are
generally used for retrospective dosimetry caused by past
exposures.
6. Summary
When chromosome aberrations are used for dose
assessment, it is very important to prepare optimal
chromosome specimens which can easily distinguishchromosome aberrations. The chromosome quality isaffected by a variety of factors, including the number of
metaphases in the sample, the degree of chromosome
condensation, the extent of metaphase spreading andthe condition of dye staining. In addition, the effects
of reagents such as mitogens and colcemid could vary
among individuals, depending on the different genetic
backgrounds caused by the age and gender. Therefore, itis necessary to routinely check the standardized protocol
for chromosome preparation and evaluate scoring criteriaof chromosome aberrations in order to reduce individual
differences among scorers and reduce the evaluation
errors such that more accurate biodosimetry can be
performed.
Chromosome analysis is often regarded as a highly
specialized skill due to the difficulty of experimental
techniques and morphological observations. However,in recent years, manuals of chromosome analysis havebeen developed which facilitates the sharing of various
information. As chromosome aberration classifications,test protocols, and results criteria can subtly differ fromeach cytogenetic laboratory, the use of international
standardized protocols has been recommended.
It is our hope that this article will serve as a concise
summary of chromosomes and their structure to facilitate
medical professionals involved in radiation emergency
medicine in understanding cytogenetic biodosimetry.

Akifumi Nakata et al./ Radiation Environment and Medicine 2022 Vol.11, No.2 82–90

Point 1. Introduction of a chromosome• A chromosome is a structure observed under lightmicroscopy during the mitotic phase of the cell-cycle.
• As chromosomes contain DNA and other genetic
material, DNA misrepair induces chromosome
aberrations.

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Point 2. C hromosome structure and morphology

Structural characteristics of a chromosome consistsof a centromere at central/primary constriction, andtelomeres at the chromosome ends. The short arm isthe p arm while the long arm is the q arm.
• Chromosomes can be subdivided into different
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Point 3. Classification of chromosome aberrations• Peripheral blood lymphocytes at the time of
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• Radiation-induced chromosome-type aberrations can
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