Mutagenesis vol. 26 no. 1 pp.

310, 2011 Advance Access Publication 27 October 2010

doi:10.1093/mutage/geq085

COMMENTARY

Reections on the development of micronucleus assays

John A. Heddle1, Michael Fenech2, Makoto Hayashi3 and James T. MacGregor4,*

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada, Commonwealth Scientic 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: jtmacgregor@earthlink.net 1

Received on June 17, 2010; revised on July 16, 2010; accepted on August 27, 2010

These are personal reections 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 eld 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 nding them takes longer than analysing them. When I was rst 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 rst 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 efciency (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 Schmids 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

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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.

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 specically but neither happened. Our work was designed to establish that MN originated from chromosome aberrations and that the method was more efcient than chromosomal analysis, besides being easier, which we did. The nal evidence was the DNA content of MN (18). In any case, I was planning to move on and suggested to Paul Countryman, my rst 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 denition 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 xed 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 rst time the development of a MN assay in cultured lymphocytes. My initial efforts veried Countryman and Heddles 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 aw 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 Lehningers Principles of Biochemistry undergraduate text book disclosed a brief sentence mentioning cytochalasins as inhibitors of cytokinesis by blocking polymerisation of actin into the microlament ring required for binucleate cell ssion. It was evident from a key paper by Carter (22) that cytochalasin-B was the form that could inhibit cytokinesis most efciently 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 efcacy of the lymphocyte CBMN technique in terms of reproducibility and sensitivity for detecting low dose exposures to ionising radiation (540 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

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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 veried for the rst 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 Parrys laboratory in Swansea (Wales). Jim had started coordinating an aneuploidy research program funded by the European Union that eventually identied 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-specic 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 eld (2933). 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 identied 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 deciency (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 (3740,42). 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 efcient 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 classication of cells, however, required a subjective decision by the investigator and that was sometimes difcult 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 specic chemicals. To overcome these problems, uorescent staining methods with more specic 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 specically. Acridine orange stains both RNA and DNA but they uoresce differently, i.e. RNA uoresces red and DNA uoresces 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 uoresce because they lack RNA. The MN is the only element that contains DNA in the mammalian erythrocyte and it can therefore be identied clearly and specically. This DNA-specic 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 uorescence in contrast to the green/yellow uorescence 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 uorochrome in the MN assay using rodent peripheral blood as well as bone marrow (Figure 2C) (48). Acridine orange can penetrate into unxed cells and 5

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J. A. Heddle et al.

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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 unxed peripheral blood erythrocytes are stained with acridine orange, a reticulum is formed in reticulocytes (immature erythrocytes) and uoresces 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 classication 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 signicant increase in mean spontaneous MNRET frequency in their last month of the life, suggesting the possibility of strain-specic 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 inuence results of the assay. The rst 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 efciently 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 ow cytometry (5964). Regulatory applications and human health and safety studies (J.T.M.) I began my scientic 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 rst 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 sufcient to assess the risk associated with given levels of exposure and that an in vivo assay was necessary to ll that need. Luckily, the rst descriptions by Schmid et al. (9,10,6669) 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 eld 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 ow cytometry were attempted (60,61,72,73). The

development of simple and effective ow cytometric scoring techniques, applicable to small blood samples from the species of most scientic 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 (7680) 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 ow 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 ow 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 sufcient 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 ow cytometric scoring and led to the demonstration that peripheral blood sampling with ow cytometric scoring could in fact be applied successfully even to species with efcient splenic selection, including the beagle dog (84,85), non-human primate (86,87) and, most importantly, the human (8896). 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 dened 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

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J. A. Heddle et al.

has enabled the eld of genetic toxicology to move from its previous reliance on hazard screening assays toward a more quantitative evaluation of in vivo risks associated with specic exposures to potentially genotoxic agents (99101). 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 eld 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
Conict of interest statement: None declared.

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