Mutagenesis vol. 26 no. 1 pp. 3–10, 2011
1093/mutage/geq085
Advance Access Publication 27 October 2010
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,
2
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
*
To whom correspondence should be addressed. Tel: +1-410-975-0480;
Fax: +1-410-975-0481;Email: jtmacgregor@earthlink.net
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
Ó The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.
doi:10.
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
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
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 pro-
ducing 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
3
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).
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 Country-
man and Heddle’s conventional MN assay results in lympho-
cytes 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
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 poly-
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
merisation 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 mam-
malian 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
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).

radiation exposure was then verified for the first time in
cancer patients undergoing radiotherapy (25). Using kinet-
ochore antibodies, it was demonstrated that the great major-
ity of MN induced by ionising radiation were kinetochore
negative while those induced by spindle poisons were ki-
netochore positive, as one would expect given that radiation
causes chromosome breakage leading to acentric chromo-
some 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 even-
tually 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 Environ-
mental 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,
Development of MN assays
nucleoplasmic bridges and nuclear buds indicates that these
biomarkers of genomic instability are mechanistically related
to one another. They could be explained by the breakage-
fusion-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
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
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 normochro-
matic 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 distin-
guishing 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
5
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 evalu-
ation 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 ten-
dency. 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
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
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

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 suc-
cessfully 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, cyclophospha-
mide 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 MN assays
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
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
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, non-
human 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 toxicolo-
gy 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 improve-
ment 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.
References
1. Drake, J. W., Abrahamson, S., Crow, J. F. et al. (1975) Environmental
mutagenic hazards. Science, 187, 503–514.
2. Wassom, J. S. (1989) Origins of genetic toxicology and the Environmental
Mutagen Society. Environ. Mol. Mutagen., 14, 1–6.
3. Evans, H. J. and Scott, D. (1969) The induction of chromosome
aberrations by nitrogen mustard and its dependence on DNA synthesis.
Proc. R. Soc. Lond. B. Biol. Sci., 173, 491–512.
4. McLeish, J. (1954) The consequences of localised chromosome breakage.
Heredity, 8, 385–407.
5. Evans, H. J., Neary, G. J. and Williamson, F. S. (1959) The relative
biological efficiency of single doses of fast neutrons and gamma-rays on
Vicia faba roots and the effect of oxygen. Part II. Chromosome damage:
the production of micronuclei. Int. J. Rad. Biol., 1, 216–229.
6. Heddle, J. A. and Bodycote, D. J. (1970) On the formation of
chromosomal aberrations. Mutat. Res., 9, 117–126.
7. Wolff, S. and Luippold, H. E. (1965) Mitotic delay and the apparent
synergism of far-red radiation and x-rays in the production of
chromosomal aberrations. Photochem. Photobiol., 4, 439–445.
8. Heddle, J. A. (1973) A rapid in vivo test for chromosomal damage. Mutat.
Res., 18, 187–190.
9. Schmid, W. (1975) The micronucleus test. Mutat. Res., 31, 9–15.
10. Matter, B. and Schmid, W. (1971) Trenimon-induced chromosomal
damage in bone-marrow cells of six mammalian species, evaluated by the
micronucleus test. Mutat. Res., 12, 417–425.
11. Maier, P. and Schmid, W. (1976) Ten model mutagens evaluated by the
micronucleus test. Mutat. Res., 40, 325–337.
12. Yamamoto, K. I. and Kikuchi, Y. (1980) A comparison of diameters of
micronuclei induced by clastogens and by spindle poisons. Mutat. Res.,
71, 127–131.
13. Tinwell, H. and Ashby, J. (1991) Micronucleus morphology as a means to
distinguish aneugens and clastogens in the mouse bone marrow
micronucleus assay. Mutagenesis, 6, 193–198.
14. Salamone, M., Heddle, J., Stuart, E. and Katz, M. (1980) Towards an
improved micronucleus test: studies on 3 model agents, mitomycin C,
cyclophosphamide, and dimethylbenzanthracene. Mutat. Res., 74,
347–356.
8
15. Salamone, M. F. and Heddle, J. A. (1983) The bone marrow micronucleus
assay: rationale for a revised protocol. In Hollaender, A. (ed), Chemical
Mutagens, Vo1. 8. Plenum, New York, pp. 111–149.
16. Heddle, J. A., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J. T.,
Newell, G. W. and Salamone, M. F. (1983) The induction of micronuclei
as a measure of genotoxicity. A report of the U.S. Environmental Agency
Gene—Tox Program. Mutat. Res., 123, 61–118.
17. Nichols, W. W., Moorhead, P., Brewen, J. G. et al. (1972) Chromosome
methodologies in mutagen testing. Tox. Appl. Pharm., 22, 269–275.
18. Heddle, J. A. and Carrano, A. V. (1977) The DNA content of micronuclei
induced in mouse bone marrow by X-irradiation: evidence that micro-
nuclei arise from acentric chromosomal fragments. Mutat. Res., 44, 63–69.
19. Countryman, P. I. and Heddle, J. A. (1976) The production of micronuclei
from chromosome aberrations in irradiated cultures of human lympho-
cytes. Mutat. Res., 41, 321–332.
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
20. Heddle, J. A., Lue, C. B., Saunders, E. F. and Benz, R. D. (1978)
Sensitivity to five mutagens in Fanconi’s anemia as measured by the
micronucleus method. Cancer Res., 3, 2983–2988.
21. Krepinsky, A. B., Heddle, J. A. and German, J. L. (1979) Sensitivity of
Bloom’s syndrome lymphocytes to ethyl methanesulphonate. Hum.
Genet., 50, 151–156.
22. Carter, S. B. (1967) Effects of cytochalasins on mammalian cells. Nature,
213, 261–264.
23. Fenech, M. and Morley, A. A. (1985) Measurement of micronuclei in
lymphocytes. Mutat. Res., 147, 29–36.
24. Fenech, M. and Morley, A. A. (1986) Cytokinesis-block micronucleus
method in human lymphocytes: effect of in vivo ageing and low dose X-
irradiation. Mutat. Res., 161, 193–198.
25. Fenech, M., Denham, J., Francis, W. and Morley, A. (1990) Micronuclei
in cytokinesis-blocked lymphocytes of cancer patients following fraction-
ated partial-body radiotherapy. Int. J. Radiat. Biol., 57, 373–383.
26. Fenech, M. and Morley, A. A. (1989) Kinetochore detection in
micronuclei: an alternative method for measuring chromosome loss.
Mutagenesis, 4, 98–104.
27. Eastmond, D. A. and Tucker, J. D. (1989) Identification of aneuploidy-
inducing agents using cytokinesis-blocked human lymphocytes and an
antikinetochore antibody. Environ. Mol. Mutagen., 13, 34–43.
28. Parry, J. M., Parry, E. M., Bourner, R. et al. (1996) The detection and
evaluation of aneugenic chemicals. Mutat. Res., 353, 11–46.
29. Bonassi, S., Fenech, M., Lando, C. et al. (2001) HUman MicroNucleus
project: international database comparison for results with the cytokinesis-
block micronucleus assay in human lymphocytes: I. Effect of laboratory
protocol, scoring criteria, and host factors on the frequency of micro-
nuclei. Environ. Mol. Mutagen., 37, 31–45.
30. Bonassi, S., Neri, M., Lando, C. et al. (2003) HUMN collaborative group:
effect of smoking habit on the frequency of micronuclei in human
lymphocytes: results from the Human MicroNucleus project. Mutat. Res.,
543, 155–166.
31. Bonassi, S., Znaor, A., Ceppi, M. et al. (2007) An increased micronucleus
frequency in peripheral blood lymphocytes predicts the risk of cancer in
humans. Carcinogenesis, 28, 625–631.
32. Fenech, M., Holland, N., Chang, W. P., Zeiger, E. and Bonassi, S. (1999)
The HUman MicroNucleus Project—an international collaborative study
on the use of the micronucleus technique for measuring DNA damage in
humans. Mutat. Res., 428, 271–283.
33. Fenech, M., Chang, W. P., Kirsch-Volders, M., Holland, N., Bonassi, S.
and Zeiger, E. (2003) Human MicroNucleus project. HUMN project:
detailed description of the scoring criteria for the cytokinesis-block
micronucleus assay using isolated human lymphocyte cultures. Mutat.
Res., 534, 65–75.
34. Bonassi, S., Biasotti, B., Kirsch-Volders, M. et al. (2009) HUMNXL
Project Consortium. State of the art survey of the buccal micronucleus
assay—a first stage in the HUMN(XL) project initiative. Mutagenesis, 24,
295–302.
35. Thomas, P., Holland, N., Bolognesi, C., Kirsch-Volders, M., Bonassi, S.,
Zeiger, E., Knasmueller, S. and Fenech, M. (2009) Buccal micronucleus
cytome assay. Nat. Protoc., 4, 825–837.
36. Fenech, M. (2007) Cytokinesis-block micronucleus cytome assay. Nat.
Protoc., 2, 1084–1104.
37. Fenech, M., Crott, J., Turner, J. and Brown, S. (1999) Necrosis, apoptosis,
cytostasis and DNA damage in human lymphocytes measured simulta-
neously within the cytokinesis-block micronucleus assay: description
of the method and results for hydrogen peroxide. Mutagenesis, 14,
605–612.
38. Umegaki, K. and Fenech, M. (2000) Cytokinesis-block micronucleus
assay in WIL2-NS cells: a sensitive system to detect chromosomal damage

induced by reactive oxygen species and activated human neutrophils.
Mutagenesis, 15, 261–269.
39. Crott, J. W., Mashiyama, S. T., Ames, B. N. and Fenech, M. (2001) The
effect of folic acid deficiency and MTHFR C677T polymorphism on
chromosome damage in human lymphocytes in vitro. Cancer Epidemiol.
Biomarkers Prev., 10, 1089–1096.
40. Thomas, P., Umegaki, K. and Fenech, M. (2003) Nucleoplasmic bridges
are a sensitive measure of chromosome rearrangement in the cytokinesis-
block micronucleus assay. Mutagenesis, 18, 187–194.
41. Kimura, M., Umegaki, K., Higuchi, M., Thomas, P. and Fenech, M.
(2004) Methylenetetrahydrofolate reductase C677T polymorphism, folic
acid and riboflavin are important determinants of genome stability in
cultured human lymphocytes. J. Nutr., 134, 48–56.
42. Fenech, M. (2010) The lymphocyte cytokinesis-block micronucleus
cytome assay and its application in radiation biodosimetry. Health Phys.,
98, 234–243.
43. Bull, C. and Fenech, M. (2008) Genome-health nutrigenomics and
nutrigenetics: nutritional requirements or ’nutriomes’ for chromosomal
stability and telomere maintenance at the individual level. Proc. Nutr.
Soc., 67, 146–156.
44. MacGregor, J. T., Wehr, C. M. and Langlois, R. G. (1983) A simple
fluorescent staining procedure for micronuclei and RNA in erythrocytes
using Hoechst 33258 and pyronin Y. Mutat. Res., 120, 269–275.
45. Hayashi, M., Sofuni, T. and Ishidate, M., Jr (1983) An application of
Acridine Orange fluorescent staining to the micronucleus test. Mutat. Res.,
120, 241–247.
46. OECD (1997) Test No. 474: Mammalian Erythrocyte Micronucleus Test,
OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects,
OECD Publishing, Paris, France.
47. International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (1995) ICH Harmonised
Tripartite Guideline. S2A. Guidance on Specific Aspects of Regulatory
Genotoxicity Tests for Pharmaceuticals. http://www.ich.org (accessed
October 7, 2010).
48. Hayashi, M., Morita, T., Kodama, Y., Sofuni, T. and Ishidate, M., Jr
(1990) The micronucleus assay with mouse peripheral blood reticulocytes
using acridine orange coated slides. Mutat. Res., 245, 245–249.
49. The Collaborative Study Group for the Micronucleus Test (CSGMT)
(1992) Micronucleus test with mouse peripheral blood erythrocytes by
acridine orange supravital staining: the summary report of the 5th
collaborative study by CSGMT/JEMS
MMS. Mutat. Res., 278, 83–98.
50. Sato, S. M., Taketomi, M., Nakajima, M. et al. (1995) Effect of aging on
spontaneous micronucleus frequencies in peripheral blood of nine mouse
strains: the results of the 7th collaborative study organized by CSGMT/
MMS. Mutat. Res., 338, 51–57.
51. The Collaborative Study Group for the Micronucleus Test (CSGMT)
(1986) Sex difference in the micronucleus test. Mutat. Res., 172,
151–163.
52. The Collaborative Study Group for the Micronucleus Test (CSGMT)
(1988) Strain difference in the micronucleus test. Mutat. Res., 204,
307–316.
53. Hayashi, M., Sutou, S., Shimada, H., Sato, S., Sasaki, Y. F. and
Wakata, A. (1989) Difference between intraperitoneal and oral gavage
application in the micronucleus test: the 3rd collaborative study by
CSGMT/JEMS.MMS. Mutat. Res., 223, 329–344.
54. Wakata, A., Miyamae, Y., Sato, S., Suzuki, T., Morita, T., Asano, N.,
Awogi, T., Kondo, K. and Hayashi, M. (1998) Evaluation of the rat
micronucleus test with bone marrow and peripheral blood: the summary of
the 9th collaborative study by CSGMT/MMS
JEMS. Environ. Mol.
Mutagen., 32, 84–100.
55. Hamada, S., Sutou, S., Morita, T. et al. (2001) Evaluation of the rodent
micronucleus assay by a 28-day-treatment protocol: summary of the 13th
collaborative study by CSGMT/JEMS
MMS. Environ. Mol. Mutagen., 37,
93–110.
56. Morita, T., Asano, N., Awogi, T. et al. (1997) Evaluation of the rodent
micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A,
and 2B). The summary report of the 6th collaborative study by CSGMT/
JEMS
MMS. Mutat. Res., 389, 3–122.
57. Romagna, F. and Staniforth, C. D. (1989) The automated bone marrow
micronucleus test. Mutat. Res., 213, 91–104.
58. Asano, N., Katsuma, Y., Tamura, H., Higashikuni, N. and Hayashi, M.
(1998) An automated new technique for scoring the rodent micronucleus
assay: computerized image analysis of acridine orange supravitally stained
peripheral blood cells. Mutat. Res., 404, 149–154.
Development of MN assays
59. Huetter, K. and Stöhr, M. (1982) Rapid detection of mutagen induced
micronucleated erythrocytes by flow cytometry. Histochemistry, 75,
353–362.
60. Tometsko, A. M. (1991) An automated peripheral blood micronucleus
assay using flow cytometry. Environ. Mol. Mutagen., 17 (Suppl. 19), 74.
61. Hayashi, M., Norppa, H., Sofuni, T. and Ishidate, M., Jr (1992) Flow
cytometric micronucleus test with mouse peripheral erythrocytes.
Mutagenesis, 7, 257–264.
62. Dertinger, S. D., Torous, D. K. and Tometsko, K. R. (1996) Simple and
reliable enumeration of micronucleated reticulocytes with a single-laser
flow cytometer. Mutat. Res., 371, 283–292.
63. Dertinger, S. D., Torous, D. K., Hall, N. E., Tometsko, C. R. and
Gasiewicz, T. A. (2000) Malaria-infected erythrocytes serve as biological
standards to ensure reliable and consistent scoring of micronucleated
erythrocytes by flow cytometry. Mutat. Res., 464, 195–200.
Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018
64. Torous, D., Dertinger, S., Hall, N. and Tometsko, C. (2000) Enumeration
of micronucleated reticulocytes in rat peripheral blood: a flow cytometric
study. Mutat. Res., 465, 91–99.
65. Miller, E. C. and Miller, J. A. (1971) The mutagenicity of chemical
carcinogens: correlations, problems, and interpretations. In Hollaender, A.
(ed), Chemical Mutagens, Vol. 1. Plenum, New York, pp. 83–119.
66. Boller, K. and Schmid, W. (1970) Chemische mutagenese beim Säuger.
Das knochenmark des Chinesischen hamsters als in vivo-Testsystem.
Hämatologische befunde nach behandlung mit trenimon. Humangenetik, 11,
35–54.
67. Schmid, W., Arakaki, D. T., Breslau, N. A. and Culbertson, J. C. (1971)
Chemical mutagenesis. The Chinese hamster bone marrow as an in vivo
test system. I. Cytogenetic results on basic aspects of the methodology,
obtained with alkylating agents. Humangenetik, 11, 103–118.
68. Schmid, W. (1976) The micronucleus test for cytogenetic analysis. In
Hollaender, A. (ed), Chemical Mutagens, Vo1. 4. Plenum, New York, pp.
31–53.
69. Matter, B. and Grauweiler, J. (1974) Micronuclei in mouse bone marrow
cells. A simple in vivo model for the evaluation of drug-induced
chromosomal aberrations. Mutat. Res., 23, 239–249.
70. von Ledebur, M. and Schmid, W. (1973) The micronucleus test.
Methodological aspects. Mutat. Res., 19, 109–117.
71. MacGregor, J. T., Wehr, C. M. and Gould, D. H. (1980) Clastogen-
induced micronuclei in peripheral blood erythrocytes: the basis of an
improved micronucleus test. Environ. Mutagen., 2, 509–514.
72. Choy, W. N. and MacGregor, J. T. (1985) A semi-automated scoring
method that markedly improves the statistical power of the peripheral
blood micronucleus test. Environ. Mutagenesis, 7(Suppl. 3), 51.
73. MacGregor, J. T., Schlegel, R., Choy, W. N. and Wehr, C. M. (1983)
Micronuclei in circulating erythrocytes: a rapid screen for chromosomal
damage during routing toxicity testing in mice. In Hayes, A. W., Schnell,
R. C. and Miya, T. S. (eds), Developments in the Science and Practice of
Toxicology. Elsevier, Amsterdam, pp. 555–558.
74. Schlegel, R. and MacGregor, J. T. (1984) The persistence of micro-
nucleated erythrocytes in the peripheral circulation of normal and
splenectomized Fisher 344 rats: implications for cytogenetic screening.
Mutat. Res., 127, 169–174.
75. MacGregor, J. T., O’Loughlin, K. G. and Hill, J. R. (1992) Micro-
nucleated erythrocytes in bone marrow and peripheral blood of the beagle
dog. Environ. Mol. Mutagen., 19 (Suppl. 20), 38.
76. Schlegel, R., MacGregor, J. T. and Everson, R. B. (1986) Assessment of
cytogenetic damage by quantitation of micronuclei in human peripheral
blood erythrocytes. Cancer Res., 46, 3717–3721.
77. Everson, R. B., Wehr, C. M., Erexson, G. L. and MacGregor, J. T. (1988)
Association of marginal folate depletion with increased human chromo-
somal damage in vivo: demonstration by analysis of micronucleated
erythrocytes. J. Natl Cancer Inst., 80, 525–529.
78. Smith, D. F., MacGregor, J. T., Hiatt, R. A. et al. (1990) Micronucleated
erythrocytes as an index of cytogenetic damage in humans: demographic
and dietary factors associated with micronucleated erythrocytes in
splenectomized subjects. Cancer Res., 50, 5049–5054.
79. MacGregor, J. T., Wehr, C. M., Hiatt, R. A. et al. (1997) Spontaneous
genetic damage in man: evaluation of interindividial variability, relation-
ship among markers of damage, and influence of nutritional status. Mutat.
Res., 377, 125–135.
80. Blount, B. C., Mack, M., Wehr, C. M., MacGregor, J. T., Hiatt, R. A.,
Wang, G., Wickramasinghe, S. N., Everson, R. B. and Ames, B. N. (1997)
Folate deficiency increases uracil misincorporation into human DNA and
chromosome breakage: implications for cancer and neuronal damage.
Proc. Natl Acad. Sci. USA, 94, 3290–3295.
9
J. A. Heddle et al.

81. MacGregor, J. T., Bishop, M. E., McNamee, J. P. et al. (2006) Flow
cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An
efficient method of monitoring chromosomal damage in the rat. Toxicol.
Sci., 94, 92–107.
82. Dertinger, S. D., Bishop, M. E., McNamee, J. P. et al. (2008) Flow
cytometric analysis of micronuclei in peripheral blood reticulocytes: I.
Intra- and interlaboratory comparison with microscopic scoring. Toxicol.
Sci., 94, 83–91.
83. Hayashi, M., MacGregor, J. T., Gatehouse, D. G. et al. (2007) In vivo
erythrocyte micronucleus assay: III. Validation and regulatory acceptance
of automated scoring and the use of rat peripheral blood reticulocytes,
with discussion of non-hematopoietic target cells and a single dose-level
limit test (IWGT Expert Working Group Report). Mutat. Res., 627, 10–30.
84. Harper, S. B., Dertinger, S. D., Bishop, M. E., Lynch, A. M., Lorenzo, M.,
Saylor, M. and MacGregor, J. T. (2007) Flow cytometric analysis of
100. Thybaud, V., Aardema, M., Clements, J. et al. (2007) Strategy for
genotoxicity testing: hazard identification and risk assessment in relation
to in vitro testing. Mutat. Res., 627, 41–58.
101. Thybaud, V., MacGregor, J. T., Müller, L. et al. (2010) Strategies in case
of positive in vivo results in genotoxicity testing (IWGT Expert Working
Group Report). Mutat. Res., (in press).
102. Parvinen, M., Lähdetie, J. and Parvinen, L. M. (1984) Toxic and
mutagenic influences on spermatogenesis. Arch. Toxicol. Suppl., 7,
128–139.

Downloaded from https://academic.oup.com/mutage/article-abstract/26/1/3/1065999 by ARN user on 12 September 2018

micronuclei in peripheral blood reticulocytes III. An efficient method of
monitoring chromosomal damage in the beagle dog. Toxicol. Sci., 100,
406–414.
85. Torous, D., McKeon, M., Schmuck, G., Xu, Y., Burgess, S.,
Avlasevich, S., Dertinger, S. and Kirkland, D. (2008) Cyclophosphamide
and etoposide canine studies demonstrate the cross-species potential of the
peripheral blood micronucleated reticulocyte endpoint. Meeting abstract.
Environ. Mol. Mutagen., 49, 569.
86. Hotchkiss, C. E., Bishop, M. E., Dertinger, S. D., Slikker, W., Jr,
Moore, M. M. and MacGregor, J. T. (2008) Flow cytometric analysis of
micronuclei in peripheral blood reticulocytes IV: an index of chromo-
somal damage in the Rhesus monkey (Macaca mulatta). Toxicol. Sci.,
102, 352–358.
87. Morris, S. M., Dobrovolsky, V. N., Shaddock, J. G. et al. (2009) The
genetic toxicology of methylphenidate hydrochloride in non-human
primates. Mutat. Res., 673, 59–66.
88. Abramsson-Zetterberg, L., Zetterberg, G., Bergqvist, M. and Grawé, J.
(2000) Human cytogenetic biomonitoring using flow-cytometric analysis
of micronuclei in transferrin-positive immature peripheral blood retic-
ulocytes. Environ. Mol. Mutagen., 36, 22–31.
89. Dertinger, S. D., Torous, D. K., Hall, N. E., Murante, F. G.,
Gleason, S. E., Miller, R. K. and Tometsko, C. R. (2002) Enumeration
of micronucleated CD71-positive human reticulocytes with a single-laser
flow cytometer. Mutat. Res., 515, 3–14.
90. Dertinger, S. D., Chen, Y., Miller, R. K. et al. (2003) Micronucleated
CD71-positive reticulocytes: a blood-based endpoint of cytogenetic
damage in humans. Mutat. Res., 542, 77–87.
91. Dertinger, S. D., Miller, R. K., Brewer, K. et al. (2007) Automated human
blood micronucleated reticulocyte measurements for rapid assessment of
chromosomal damage. Mutat. Res., 626, 111–119.
92. Grawé, J., Biko, J., Lorenz, R., Reiners, C., Stopper, H., Vershenya, S.,
Vukicevic, V. and Hempel, K. (2005) Evaluation of the reticulocyte
micronucleus assay in patients treated with radioiodine for thyroid cancer.
Mutat. Res., 583, 12–25.
93. Offer, T., Ho, E., Traber, M. G., Bruno, R. S., Kuypers, F. A. and
Ames, B. N. (2005) A simple assay for frequency of chromosome breaks
and loss (micronuclei) by flow cytometry of human reticulocytes. FASEB.
J., 19, 485–487.
94. Stopper, H., Hempel, K., Reiners, C., Vershenya, S., Lorenz, R.,
Vukicevic, V., Heidland, A. and Grawé, J. (2005) Pilot study for
comparison of reticulocyte-micronuclei with lymphocyte-micronuclei in
human biomonitoring. Toxicol. Lett., 156, 351–360.
95. Flanagan, J. M., Howard, T. A., Mortier, N., Avlasevich, S.,
Smeltzer, M. P., Wu, S., Dertinger, S. D. and Ware, R. E. (2010)
Assessment of genotoxicity associated with hydroxyurea therapy in
children with sickle cell anemia. Mutat. Res., 698, 38–42.
96. Witt, K. L., Cunningham, C. K., Patterson, K. B., Kissling, G. E.,
Dertinger, S. D., Livingston, E. and Bishop, J. B. (2007) Elevated
frequencies of micronucleated erythrocytes in infants exposed to
zidovudine in utero and postpartum to prevent mother-to-child trans-
mission of HIV. Environ. Mol. Mutagen., 48, 322–329.
97. MacGregor, J. T., Wehr, C. M., Henika, P. R. and Shelby, M. D. (1990)
The in vivo erythrocyte micronucleus test: measurement at steady state
increases assay efficiency and permits integration with toxicity studies.
Fund. Appl. Toxicol., 14, 513–522.
98. MacGregor, J. T., Tucker, J. D., Eastmond, D. A. and Wyrobek, A. J.
(1995) Integration of cytogenetic assays with toxicology studies. Environ.
Mol. Mutagen., 25, 328–337.
99. Blakey, D., Galloway, S. M., Kirkland, D. J. and MacGregor, J. T. (2008)
Regulatory aspects of genotoxicity testing: from hazard identification to
risk assessment. Mutat. Res., 657, 84–90.

10