Mutagenesis vol. 26 no. 1 pp. 3–10, 2011
Advance Access Publication 27 October 2010
doi:10.1093/mutage/geq085
COMMENTARY
Reflections on the development of micronucleus assays
John A. Heddle1, Michael Fenech2, Makoto Hayashi3 and
James T. MacGregor4,*
1
Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada,
Commonwealth Scientific and Industrial Research Organisation, Food and
Nutritional Sciences Division, Nutritional Genomics and DNA Damage
Diagnostics, Adelaide BC SA 5000, Australia, 3Biosafety Research Center,
Foods, Drugs and Pesticides, 582-2, Shioshinden, Iwata, 437-1213, Japan and
4
Toxicology Consulting Services, 201 Nomini Drive, Arnold, MD 21012, USA
2
*
To whom correspondence should be addressed. Tel: +1-410-975-0480;
Fax: +1-410-975-0481;Email: [email protected]
Received on June 17, 2010; revised on July 16, 2010;
accepted on August 27, 2010
These are personal reflections on the development of
methods to use micronuclei as a measure of genetic damage
and their use in research and in toxicology by four people
who have been intimately involved with this work, a
personal rather than a comprehensive history. About 6000
papers have been published using such methods in many
tissues in vivo or in cultured cells of many organisms from
plants to humans, but the majority of the work has been on
mammalian erythrocytes and human lymphocytes, the
areas in which we have worked primarily. Although this is
by no means a complete history, those working in the field
may be interested in some of the personal events that lie
behind the development and acceptance of methods that
are now standard.
The beginnings (J.A.H.)
Chromosomal aberrations and gene mutations are each critical
manifestations of DNA damage, so assays are needed for both
(1,2). The analysis of chromosomal aberrations was, and is,
a time-consuming craft, not easy to learn well but easy to do
badly. Furthermore, metaphase cells are required, so sometimes
finding them takes longer than analysing them.
When I was first developing the micronucleus (MN) assay in
mice, Anton Marshak came to visit me at the University of
California in San Francisco (UCSF) and showed me his
proposal for a rapid assay which was ‘Just count the number of
chromosomal bodies’. Clearly this would be quick and easy,
though it did require metaphases. It would have worked quite
well for ionising radiations that produce chromosome-type
aberrations but not for chromatid-type aberrations, the type
produced by most mutagens (3).
Long before, there was a MN test, it was well known that
acentric chromosomal fragments formed micronuclei (MN) in
plant cells. At that time, mammalian cytogenetics was in its
infancy and the structure of chromosomes was unknown.
Chromosomal aberrations and MN were of interest primarily
because of what they revealed about chromosomes, nuclei and
their structures and functions. Even the fact that an acentric
fragment could apparently form or attract a nuclear membrane
was interesting. McLeish (4) showed that some MN could
decondense and reform an acentric fragment when the cell
again entered mitosis and that MN with nucleoli could
replicate. The first use of MN as an index of chromosomal
damage was by Evans et al. (5), who were studying the
cytogenetic effects of neutrons in root tips.
Although not a large number of chemicals had been tested,
both experience and theory indicated that no agent could
produce heritable chromosomal aberrations without also producing acentric fragments and, therefore, MN (6). The issues
were where to look for them and what exposure and sampling
protocols to use. Both of the groups who initiated this work
chose mouse bone marrow. My work began at the ‘Rad Lab’ at
the UCSF, where I had advice from Mary Mahoney, an expert in
radiation effects and bone marrow. As time after treatment is the
paramount variable in studies of chromosomal aberrations (7),
we did a time course and found no increase in MN for 14 h
after x-irradiation and a large increase thereafter. Judy Bodycote
and I compared the MN with the frequency of aberrations to
estimate efficiency (8). As the slides were Feulgen stained to
avoid the granules found in many white blood cells, we detected
only DNA positive structures and related these to the nearby
nuclei. It was only when we treated with cyclophosphamide and
found many more MN than nuclei that we used phase contrast
microscopy to discover that most MN were in red blood cells. I
do not know how the Schmid group came to discover this or
whether they were knowledgeable enough to expect this. We did
know from our work, however, that the protocol proposed by
Schmid of treatments 24 and 6 h before sampling (9), although
based on studies of chromosomal aberrations, would not work
well as no MN were evident within 12 h of treatment. Hence,
the second treatment could only interfere with, not help, the
assay. With his colleagues, however, Schmid showed that MN
could be detected in six mammalian species and by several well
known mutagens (10,11). One of these was colcemid, an
aneugen. Further work has shown that aneugens produce
a distinctively larger spectrum of MN (12,13).
When Michael Salamone joined the group, now at York
University, we were interested in the effect of combinations of
chemicals and for this to be studied properly multiple time
points for sampling would be required (14). It was this
approach together with the use of controls and two treatments
with the same chemical that led to a proposal for a treatment
protocol (15). The Gene-Tox program of the Environmental
Protection Agency under the direction of Fred de Serres
brought people together from all over the world and started the
evolution of the protocol to its modern form (16).
At the time I began our work, I was unaware of Schmid’s
work but Ilse-Dore Adler told me about it when we were both
at a meeting in Cambden, New Jersey, in 1970 on how to test
for chromosomal damage. I wanted a mention of MN in the
report though our work was unpublished and so she told me
Werner Schmid was working on a MN assay but with no
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J. A. Heddle et al.
details. Our two original publications were delayed by my move
to York University in 1971. I thought that the report (17) would
be published quickly (rather than in 1972) and that our MN
work would be referred to specifically but neither happened.
Our work was designed to establish that MN originated from
chromosome aberrations and that the method was more efficient
than chromosomal analysis, besides being easier, which we did.
The final evidence was the DNA content of MN (18).
In any case, I was planning to move on and suggested to
Paul Countryman, my first MSc student, that an assay could be
developed in human lymphocytes that would be robust,
sensitive and quick, which he showed to be true (19). One of
the complications that arose early was the definition of an MN.
There were three main problems: the presence of cells that we
would now recognize as apoptotic, the presence of some
staining artefacts that appeared here and there on the slides and
the size of the MN, as there were occasional strange cells that
were almost binucleate. A few simple rules were tested on
samples from different doses and any objects that might have
been MN but that did not increase with dose were noted. After
the rules were developed, they were tested in a like manner,
the most fundamental rule being that a MN must resemble the
nucleus of the cell in which it is found, i.e. it must look like
nuclear material. Benz joined the group and quickly brought
precision to the work when we began to use the lymphocyte
method to study the chromosome breakage syndromes. When
we said that the cells were fixed at 72 h (20), we meant 72 h 1
min. To do this with large numbers of cultures, Dan had us
initiate the cultures at 7 min intervals and we worked in an
assembly line to make the preparations. The most interesting
result from this work, though never published, was an indication
that Bloom Syndrome was sensitive to ethylmethane sulphonate,
a fact later established by Krepinsky using the more sensitive
sister chromatid exchange (SCE) method in our laboratory (21).
methods was satisfactory because one could not be certain
whether the labelled cells had actually completed nuclear
division or had, in fact, completed only one division.
After considerable frustration, the ‘eureka’ moment occurred
very early one summer morning in 1984 when it occurred to
me that the optimal point at which to identify once-divided
cells is the short-lived binucleated stage in telophase. I
wondered how one could block cytokinesis so that the cell,
having completed nuclear division, would no longer be able to
complete cellular division. A quick search that morning of the
term ‘cytokinesis’ in Lehninger’s ‘Principles of Biochemistry’
undergraduate text book disclosed a brief sentence mentioning
cytochalasins as inhibitors of cytokinesis by blocking polymerisation of actin into the microfilament ring required for
binucleate cell fission. It was evident from a key paper by
Carter (22) that cytochalasin-B was the form that could inhibit
cytokinesis most efficiently in lymphocytes and other mammalian cells. That same day, I added cytochalasin-B at 3 lg/ml
to ongoing lymphocyte cultures, and the following morning
observed that a very large proportion of the lymphocytes were
blocked as binucleated cells. Thus, the cytokinesis-block MN
assay was born. Figure 1A shows the concept of the CBMN
assay and Figure 1B shows examples of cytokinesis-blocked
binucleated cells containing MN.
The following year was spent verifying the efficacy of the
lymphocyte CBMN technique in terms of reproducibility
and sensitivity for detecting low dose exposures to ionising
radiation (5–40 cGy) and demonstrating that the rate of
increase of MN frequency with age was underestimated in the
conventional method relative to the CBMN method (23,24).
The utility of the assay for biological dosimetry of in vivo
The cytokinesis-block micronucleus assay emerges and
evolves into a comprehensive cytome assay of DNA
damage, cell death and cytostasis (M.F.)
The cytokinesis-block micronucleus (CBMN) assay was
developed in the early 1980s during my PhD studies under
the supervision of Prof. Alec Morley. He suggested exploring
the method of Countryman and Heddle (19), who had
described for the first time the development of a MN assay
in cultured lymphocytes. My initial efforts verified Countryman and Heddle’s conventional MN assay results in lymphocytes for ionising radiation. It soon became apparent, however,
that the results obtained varied depending on harvest time, and
false-negative results could be generated under conditions of
strong inhibition of mitosis as often occurs with chemical
exposure. Furthermore, it was evident that a greater proportion
of lymphocytes respond to mitogen in cultures of lymphocytes
from young subjects than in those from elderly individuals. I
hypothesised that the MN assay in its then-current form had
a major flaw due to the fact that the observed MN frequency
depended on the proportion of lymphocytes that responded to
mitogen as well as the number of divisions that occurred during
the culture period prior to harvesting cells. I predicted that the
assay would only be robust if a method to accumulate and
identify once-divided cells was developed and so set about
devising a variety of methods to identify such cells based on
DNA synthesis labelling or parallel cultures blocked in mitosis
to correct for the proportion of dividing cells. Neither of these
4
Fig. 1. The CBMN assay. (A) MN and nucleoplasmic bridge (NPB) formation
in cells undergoing nuclear division. MN originate from either lagging whole
chromosomes or acentric chromosome fragments. NPBs originate from
dicentric chromosomes that may be caused by mis-repair of double-strand
DNA breaks or telomere end fusions. These events can only be observed in
cells completing nuclear division that are recognised by their binucleated
appearance after cytokinesis blocking with cytochalasin-B. (B)
Photomicrograph of a binucleated cells with one MN (left) and a binucleated
cell with a nucleoplasmic bridge and one MN (right).
Development of MN assays
radiation exposure was then verified for the first time in
cancer patients undergoing radiotherapy (25). Using kinetochore antibodies, it was demonstrated that the great majority of MN induced by ionising radiation were kinetochore
negative while those induced by spindle poisons were kinetochore positive, as one would expect given that radiation
causes chromosome breakage leading to acentric chromosome fragments (26,27).
During my postdoctoral training at the MRC Cell Mutation
Unit in Sussex, UK, in 1988, I visited Jim Parry’s laboratory in
Swansea (Wales). Jim had started coordinating an aneuploidy
research program funded by the European Union that eventually identified the in vitro CBMN assay (in combination with
centromere probes) as a better method for assessing aneuploidy
events in mammalian cells because, apart from MN containing
whole chromosomes, it was possible to measure chromosome
malsegregation (non-disjunction) events by counting the
number of chromosome-specific centromere probe signals
within the daughter nuclei of a cytokinesis-blocked binucleated
cell even if it did not contain a MN (28). This led to
establishing aneuploidy testing as compulsory for regulatory
purposes and further development of the draft Organisation for
Economic Cooperation and Development guidelines for the in
vitro MN assay. This was a major change as compared to early
days when only gene mutations and chromosomal damage
needed to be assessed for in vitro genetic toxicology tests.
The year 1997 proved to be one of the most important in the
further validation and development of the CBMN assay
because it was then that Bonassi and I, after a year of email
correspondence, decided to launch the HUMN project
(www.humn.org) at the International Conference on Environmental Mutagenesis in Toulouse. We were overwhelmed by
interest internationally in the HUMN project concept that had
as its primary aims the collation of data worldwide to
determine the main variables affecting lymphocyte MN
frequency, the establishment of scoring criteria for this assay,
the performance of an inter- and intra-laboratory slide scoring
exercise and a prospective study to test the hypothesis that MN
frequency in lymphocytes predicts cancer risk. All these
objectives were met successfully within the following 10 years,
and the resulting publications are among the most cited in the
MN field (29–33). The prospective study did in fact show that
a mid- or high-tertile level of MN frequency predicted an
increased cancer risk (31). Currently, the HUMN project is
focused on the validation of the buccal MN cytome assay and
the association of its multiple biomarkers with disease
outcome, which is a sub-project now known as the HUMNxl
project (34,35).
From 2000 onwards, my laboratory embarked on research to
create and validate a new way to perform the cytokinesis-block
MN assay using a ‘cytome’ approach (36). In this approach,
not only MN but also nucleoplasmic bridges (Figure 1) and
nuclear buds are scored. Cells undergoing cell death by
necrosis and apoptosis are also identified and counted and the
extent of cell proliferation among viable cells measured using
the ratios of mono-, bi- and multinucleated cells to determine
the nuclear division index.
The MN assay cytome approach was validated by the studies
of Keizo Umegaki, Michiyo Kimura, Philip Thomas, Jimmy
Crott, Shauna Brown, Will Greenrod, Bianca Benassi, Sasja
Beetstra and other scientists and students in my laboratory
using models of oxidative stress and/or folate deficiency (37–
41). The observed strong positive correlations between MN,
nucleoplasmic bridges and nuclear buds indicates that these
biomarkers of genomic instability are mechanistically related
to one another. They could be explained by the breakagefusion-bridge cycle model when the nutritional or exposure
conditions generated double-strand breaks in DNA and led to
the formation of dicentric chromosomes (37–40,42).
The possibility of scoring nucleoplasmic bridges in the
CBMN cytome assay is of particular importance given that it is
a potentially reliable assay for dicentric chromosome formation
and therefore relevant to radiation biodosimetry (40,42). It also
allows a possible functional assay for telomere dysfunction
when combined with telomere probes (36), given that telomere
end fusions lead to dicentric chromosome formation and
nucleoplasmic bridge formation. This has opened a possible
new approach for studying the role of nutrition and other
environmental factors on telomere dysfunction, which is
virtually unexplored as yet (43).
Improvements in the in vivo MN assay (M.H.)
One of the technical challenges of the erythrocyte MN assay
was the development of an efficient staining method to identify
newly formed erythrocytes [polychromatic erythrocyte (PCE)]
unequivocally (Figure 2). Giemsa staining has been used
successfully for this assay and with this stain, PCEs were
distinguished from normochromatic (mature) erythrocytes by
the colour, i.e. PCEs showed bluish colour and normochromatic ones showed pinkish. The classification of cells,
however, required a subjective decision by the investigator
and that was sometimes difficult to make. Moreover, there were
potential artefacts not easily distinguished from MN stained by
Giemsa, for example the granules of mast cells in rat bone
marrow and the aggregated RNA inclusions induced by some
specific chemicals. To overcome these problems, fluorescent
staining methods with more specific staining characteristics
were introduced. Two methods were introduced at almost the
same time, one was pyronin Y and Hoechst 33258 by
MacGregor et al. (44) and the other was acridine orange
which we introduced (45). Pyronin Y stains RNA and Hoechst
33258 stains DNA specifically. Acridine orange stains both
RNA and DNA but they fluoresce differently, i.e. RNA
fluoresces red and DNA fluoresces green-yellow. The acridine
orange method was simpler and more convenient for routine
scoring, and it has become the standard for routine microscopic
scoring of MN (Figure 2A).
Immature erythrocytes contain RNA in their cytoplasm and
can be distinguished easily from mature erythrocytes, which do
not fluoresce because they lack RNA. The MN is the only
element that contains DNA in the mammalian erythrocyte and
it can therefore be identified clearly and specifically. This
DNA-specific staining has overcome the problem of distinguishing true MN from artefactual MN-like particles and is
now recommended for use in worldwide regulatory guidelines
(46,47). Importantly, the fact that acridine orange staining
clearly distinguishes MN from mast cell granules (Figure 2B),
which contains mucopolysaccharide and have strong red
fluorescence in contrast to the green/yellow fluorescence of
MN, has enabled the MN assay in rats that are more commonly
used in toxicological studies than mice.
Several years later, we introduced acridine orange as
a supravital staining fluorochrome in the MN assay using
rodent peripheral blood as well as bone marrow (Figure 2C)
(48). Acridine orange can penetrate into unfixed cells and
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J. A. Heddle et al.
Fig. 2. Micronucleated immature erythrocytes. (A) Micronucleated mouse bone marrow cells. Giemsa staining (upper) and acridine orange staining (lower). (B)
Micronucleated rat bone marrow cells with scattered mast cell granules. Arrows show true MN. Giemsa staining (upper) and acridine orange staining (lower). (C)
Micronucleated mouse peripheral reticulocyte using acridine orange supravital staining, showing red reticulum and yellow-green MN.
interact with RNA and DNA. When unfixed peripheral blood
erythrocytes are stained with acridine orange, a reticulum is
formed in reticulocytes (immature erythrocytes) and fluoresces
red and MN yellow/green. Only a tiny amount of peripheral
blood is necessary for this method, so one can take a sample for
the MN assay repeatedly from the same animal. It also allows
classification of reticulocytes into different age categories,
which can give more mechanistic and expression kinetics
information. This method was evaluated and validated by the
international collaboration organized by the Collaborative
Study Group on the Micronucleus Test (CSGMT), which is
a study group in the Mammalian Mutagen Study (MMS)
group, which is a sub-organization belonging to the Japanese
Environmental Mutagen Society (49). Because it is not
necessary to kill animals to evaluate the micronucleated
reticulocyte (MNRET) frequency, repeat sampling and evaluation of spontaneous MNRET from the same animals
throughout the entire life span is possible (50). Using this
method, it has been shown that both sexes of the BDF1 strain
and females of the A/J strain showed a statistically significant
increase in mean spontaneous MNRET frequency in their last
month of the life, suggesting the possibility of strain-specific
age-dependent chromosomal instability. SAMP6/Tan, an
accelerated senescence-prone strain, showed the same tendency. The other strains studied, ddY, CD-1, B6C3F1, SAMR1
and MS/Ae, did not show age-related differences.
To standardize the methodology of the erythrocyte MN assay,
extensive collaborative studies have been conducted by the
MMS group, of which I was a part. The group organised
extensive collaborative studies on the factors that might influence
results of the assay. The first objective was the evaluation of
differences between male and female mice. They showed that
there was no essential qualitative difference but there were
several quantitative differences. The differences, however,
diminished when dose levels were adjusted based on the area
of body surface rather than being based on the body weight (51).
In the second collaborative study, mouse strain differences
were evaluated (52). All four strains used gave positive results
with all six model positive control chemicals. Unfortunately,
the mutagen-sensitive strain, which seemed superior at high
6
dose levels, is no longer available (as MS/Ae) commercially.
This group also evaluated the route of administration. Of 17
chemicals, only 2 chemicals gave contradictory results between
intraperitoneal (i.p.) injection and oral gavage administration
[per os (p.o.)]: potassium chromate (VI) gave a positive response
by the i.p. but not by the p.o. route and benzene p.o. gave
positive results but i.p. gave only a marginal response (53).
The peripheral blood of rat was also evaluated with 40
chemicals although spleens of rat as well as human eliminate
micronucleated erythrocytes efficiently from peripheral blood
(54). The concordance between bone marrow and peripheral
blood in rats was 92% and that between rat and mouse
erythrocytes was 88%; thus, it was concluded that the rat
peripheral blood can be used as well as mouse.
CSGMT/MMS engaged the study on the evaluation of the
rodent MN assay by a 28-day repeat treatment protocol (55).
Fifteen model mutagens were tested in rats mainly by p.o.
administration daily for 28 days and were evaluated in bone
marrow cells and peripheral blood. Thirteen chemicals were
positive within the estimated dose range of a general
toxicology test, suggesting the possibility of integration of
the MN end point into general toxicology tests. These data
were used in the revision of the mutagenicity guideline of
International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human
Use. The group also studied .100 chemicals that were
evaluated by the International Agency for Research on Cancer
as human carcinogens (Group 1), probable human carcinogens
(Group 2A) and possible human carcinogens (Group 2B),
using the rodent MN assay, and concluded that if information
about the structure of the chemical was taken into account, the
positive rates of the rodent MN assay were 90%, 65% and 60%
for Group 1, Group 2A and Group 2B, respectively (56).
There are several advantages of the erythrocyte MN assay
using rodent haematopoietic cells in comparison with those of
metaphase analysis in evaluation of clastogenicity/aneugenicity
of chemicals. One of the important advantages was the simple
end point, namely the small nucleus-like inclusion in the
erythrocyte from which the main nucleus has been expelled.
Therefore, the MN is the only DNA-containing structure in the
Development of MN assays
erythrocyte. This characteristic made it feasible for us and
others to develop automated scoring of MN frequencies by
image analysis (57,58) and flow cytometry (59–64).
Regulatory applications and human health and safety
studies (J.T.M.)
I began my scientific career in the early 1970s, a time at which
the recent recognition that chemical exposures could induce
inheritable genetic alterations responsible for human disease,
including cancer (2,65), converged with the introduction of two
new simple laboratory assays that permitted the detection and
monitoring of important genetic changes. These assays were
the Ames Salmonella mutagenicity assay and the rodent in vivo
MN assay, and they provided a means to explore the
relationships between chemical exposures, genetic damage
and disease.
I learned of the Salmonella assay in 1971 during my
postdoctoral training when Bruce Ames described it in
a seminar at the University of California, San Francisco, and
when I began my first job the next year in the food safety
research group at the USDA Western Regional Research
Center near Berkeley, California, I began using the Salmonella
assay to study food and dietary constituents. However, as
a toxicologist, I realized that a bacterial screening assay would
not in itself be sufficient to assess the risk associated with given
levels of exposure and that an in vivo assay was necessary to
fill that need. Luckily, the first descriptions by Schmid et al.
(9,10,66–69) and Heddle (8) of a new and simple in vivo
cytogenetic assay, the rodent bone marrow MN assay, appeared
at this same time. This assay provided a simple means of
monitoring cytogenetic damage in vivo that could be easily
adopted even by toxicologists not formally trained in genetic
techniques, such as myself. The power and simplicity of this
assay has served me well throughout my career, as it has served
others in the general field of genetic toxicology. It was soon
adopted as one of the ‘core’ regulatory assays throughout the
world (46,47).
In one of the landmark publications describing the bone
marrow MN assay, von Ledebur and Schmid (70) emphasized
that this assay was limited to bone marrow, concluding that ‘ . . .
the MN test in acute or subacute experiments cannot successfully be performed on samples of peripheral blood’.
(Emphasis is that of the original publication.) I did not question
this conclusion, until serendipity proved that it was incorrect.
This occurred a few years later when we were assessing
whether a MN assay in liver tissue was feasible and my senior
technician, Carol Wehr, brought some histological sections
from the liver of mutagen-treated mice to me and pointed out
that many of the red blood cells within blood vessels in the
sections appeared to contain MN. We immediately evaluated
the response of micronucleated polychromatic erythrocytes in
the blood of mice exposed to nitrogen mustard, cyclophosphamide and dimethylbenzanthracene and found that the responses
in peripheral blood were very similar to those in bone marrow
(with the expected temporal delay as newly formed cells
moved from bone marrow into peripheral blood (71). The
potential to conduct the assay using only microlitre quantities
of blood offered the promise of a minimally invasive assay and
the uniform cell population in peripheral blood promised to
facilitate automated scoring and thereby to simplify the assay
and greatly improve its statistical sensitivity. Both image
analysis and flow cytometry were attempted (60,61,72,73). The
development of simple and effective flow cytometric scoring
techniques, applicable to small blood samples from the species
of most scientific interest, was eventually successful and is
discussed in the article by Dertinger et al. in this special issue
of Mutagenesis.
Initially, it was known that many species, including the rat
(74), beagle dog (75) and human (76), actively remove
micronucleated cells from the peripheral circulation, and it
was believed that this would preclude the use of peripheral
blood samples in these important species. In the human, initial
studies showed that microscopic scoring of MNRET and red
blood cells in the peripheral blood of splenectomised
individuals could be used as an index of damage in bone
marrow (76–80) but because the human exhibited the strongest
splenic selection against micronucleated cells among the
species considered important for genetic toxicology safety
studies, it was not considered likely that peripheral blood
would be a suitable tissue for studies in normal humans with
intact functioning spleens.
However, careful evaluations of the relative responses of
MNRET frequencies in peripheral blood relative to bone
marrow in the rat showed that peripheral blood responses were
a reliable index of those occurring in bone marrow (54,81).
Further, application of flow cytometric scoring of peripheral
blood greatly reduced intra- and inter-laboratory variability and
increased statistical power relative to the historical microscopic
evaluation of bone marrow (81,82). Automated scoring has
now become an acceptable alternative to the classical
methodology for regulatory studies (46,83), and the advantages
of automated methodologies will likely make these the
predominant methods used in the future.
With the advent of reliable flow cytometric scoring
techniques, it became feasible to evaluate MN responses in
the newly formed RBCs of species with normally low
reticulocyte counts and with relatively strong splenic selection
against micronucleated cells, including beagle dogs, nonhuman primates and humans. The low spontaneous frequencies
of both reticulocytes and of MN reticulocytes in these species,
especially the primate species, makes it prohibitively laborious
to use manual microscopic scoring to obtain sufficient cell
counts to reliably estimate frequencies. These studies that
demonstrated that rat blood could be used successfully
encouraged more careful assessments of other higher species
using flow cytometric scoring and led to the demonstration that
peripheral blood sampling with flow cytometric scoring could
in fact be applied successfully even to species with efficient
splenic selection, including the beagle dog (84,85), non-human
primate (86,87) and, most importantly, the human (88–96).
With the establishment of methods that allow the monitoring
of cytogenetic damage in the erythrocyte lineage in bone
marrow cells and in circulating lymphocytes via the analysis
of small blood samples, it has become possible to integrate
studies of genetic damage into routine laboratory toxicology and safety evaluation studies and also to monitor rates
of genetic damage in human populations. Studies of the
kinetics of micronucleated cell formation and elimination
(14,71,81,84,86,91,97) have defined the appropriate sampling
times associated with maximum responses to acute exposures
and the times necessary to achieve steady-state frequencies
during repeated exposures in various species of interest,
facilitating optimal study designs for monitoring genotoxic
damage. The ability to integrate these and other end points of
genetic damage into routine toxicology and safety studies (98)
7
J. A. Heddle et al.
has enabled the field of genetic toxicology to move from its
previous reliance on hazard screening assays toward a more
quantitative evaluation of in vivo risks associated with specific
exposures to potentially genotoxic agents (99–101). This
integrated assessment of genetic and other health risks, together
with appropriate human studies, promises a major improvement in our approaches to assessing those factors that affect
human health and safety.
Concluding comment and acknowledgements
We hope that this historical perspective will be of interest to
those in the field of genetic toxicology research and that it will
convey an appreciation of the rewards and satisfaction of
discovering new knowledge relevant to understanding the role
of genetic alterations in human health and population genetics.
This is not a comprehensive review, lacking 6000 references
to work in plants, other organisms and other mammalian cell
types, including toxicological studies in meiotic cells (102) but
is an account of work in which we have been involved
personally. We recognize the contributions of many others not
mentioned in this paper but who nevertheless contributed to the
rich tapestry of MN assay history. Many of these individuals
are authors or co-authors of papers in this special issue.
Acknowledgements
Conflict of interest statement: None declared.
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