Mutation Research, 31 (1975) g-15 0 Elsevier Scientific Publishing Company,

Amsterdam-Printed

in The Netherlands

THE

MICRONUCLEUS

TEST

W. SCHMID Division of Medical Genetics, Department (Received September roth, 1974) of Pediatrics, University of Ziirich, Ziirich (Switeerland)

INTRODUCTION

The micronucleus

test (m.t.) in vivo is a method devised primarily

for screening

chemicals for chromosome-breaking effects. The test substances are normally applied sub-acutely to small mammals, and the effect is read in direct smears from bone marrow. This testing procedure, developed by SCHMID and coworkers1~3~5~6has a number of important advantages over the analysis of bone-marrow metaphase chromosomes. In technique of preparation as well as the reading of the slides, it is simpler and faster than chromosome analysis in the same material, but not at the expense of accuracy. In the monitoring of chromosome breakage, the test is at least as sensitive as the metaphase method; in addition it includes effects on the spindle apparatus. All these properties render the m.t. highly suitable for routine toxicological screening. The method is based on the following principles and observations: in anaphase, acentric chromatid and chromosome fragments lag behind when the centric elements move towards the spindle poles. After telophase the undamaged chromosomes, as well as the centric fragments, give rise to regular daughter nuclei. The lagging elements are included in the daughter cells, too, but a considerable proportion is transformed into one or several secondary nuclei which are, as a rule, much smaller than the principal nucleus and are therefore called micronuclei. Similar events occur if the functioning of the spindle apparatus is impaired, e.g. under the influence of colchicine; in this event, however, the main nucleus is often replaced by a whole group of small nuclei, which, in general, are considerably larger than typical micronuclei. In bonemarrow smears from mammals treated with chromosome-breaking agents, micronuclei are found in numerous cell types, always provided that these cells have completed-under the influence of the mutagen-one or a few mitoses. Micronuclei can be found e.g. in myeloblasts, myelocytes and erythroblasts. In cell types with little cytoplasm they are not always easily distinguishable from normal nuclear lobes or projections (Fig. If). An analysis restricted to nucleated bone-marrow cells is rather tedious and unreliable if the effects are small. Fortunately, the great majority of micronuclei present themselves in a cell type much more suitable for analysis, namely the class of the youngest erythrocytes. A few hours after completion of the last mitosis, erythroblasts expel their nucleus; for unknown reasons, micronuclei remain in the cytoplasm of the young erythrocyte and there they are easily recognizable. As an adAbbreviation: m.t., micronucleus test.

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W. SCHMID

ditional advantage for the micronucleus test, young erythrocytes stain differently from older forms. For the duration of their adolescence, lasting approx. 24 h, they stain not red but bluish; to use a technical term: they are polychromatic. If scoring of micronuclei is restricted to this cell type, then it is known that the anomalies were bound to arise mostly, if not exclusively, during the course of the immediately preceding mitosis and under the influence of the temporally limited action of the mutagen. The morphology were mitotically of all other types of bone marrow cell does not reveal whether active during the test period. they

Micronuclei in polychromatic erythrocytes are characteristic and obtrusive elements which can be scored by personnel without special training in cytogenetics. In hematological routine these micronuclei have long been known as Howell-Jolly bodies. Their differentiation from occasional artifacts will be dealt with in a later paragraph. Typically, micronuclei are round with a diameter of about I/ZO to I/S of an erythrocyte. (Fig. ra-c) Even after treatment with high doses of chromosomebreaking agents, most of the micronucleated cells contain just one micronucleus. There are, however, always some cells with two or more of these bodies as well as some with almond-shaped micronuclei (Fig. Id). If the findings deviate from the picture described above, the observations must be considered as aty$ical and have to be clarified by additional means. A particular virtue of the m.t. resides in its permitting a fast comparison of a test result in any number of different mammalian species. Number and morphology of the chromosomes do not pose limitations to this techniques. In cases of particular importance, bone marrow from primates or man can be studied as well. On the sensitivity of the method, compared with the analysis of metaphase chromosomes, the following can be said: experienced investigators agree that the spontaneous aberration rate for chromatid-type aberrations in direct preparations of bone marrow from numerous mammalian species is very low, generally below half a percent. This situation is different from that often found in tissue culture cells where the spontaneous aberration rate is often in the region of several percent and is influenced by numerous uncontrollable factors acting in cell cultures. In practice, however, the reliability of metaphase chromosome analyses in bone marrow all too often proves to be unsatisfactory. Many results suffer from too small numbers of metaphases and those few chromosome spreads that are analyzed often exhibit a technical quality which simply does not permit a satisfactory evaluation of the karyotype. Difficulties stemming from too small numbers are not met in the micronucleus test : the number of polychromatic erythrocytes is almost unlimited and the spontaneous rate of micronucleated cells is low and consistent, in the region of three per thousand. The test reacts positively to very low dosages of standard chromosome-breaking mutagens4y7. As mentioned above, the m.t., in contrast with metaphase scoring, is capable of indicating loss of entire chromosomes due to disturbances of the spindle apparatus. One thing the test cannot provide is the characterization of the nature of the nuclear disturbance that has led to a positive score. Therefore, it is clear that any unexpected test result obtained with the m.t. must be clarified with appropriate studies in V~VO or in vitro. This is the place where metaphase chromosome studies come into their right.

MICRONUCLEUS

TEST

II

Fig. r. (a) Typical morphology of micronuclei in polychromatic erythrocytes: a single, round (Feulgen-positive) body with a diameter of I /zo to I /5 of an erythrocyte. (b) and (c) Even after treatment with high doses of chromosome-breaking agents, multiple micronuclei are present in a minority only of all micronucleated cells. (d) A small proportion of the micronuclei are almond- or ring-shaped. (e) A small micronucleus in an erythrocyte and a lagging element in an anaphase. (f) Micronuclei in nucleated bone marrow cells are difficult to distinguish from normal nuclear lobes or projections; therefore, they are not evaluated quantitatively in this test,
THE TECHNICAL PROCEDURE

Normally, test substances are applied intraperitoneally or per OS.It should not be overlooked that the i.p. injections, especially in rats, sometimes end up unnoticed in the cecum. The consequence is that the test substance gets into the blood to a very reduced extent, at best. This is one more reason not to work with too small numbers

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W. SCHMID

of animals. Experience with known chromosome-breaking substances suggests that, when compounds of unknown mutagenic activity are tested, a subacute treatment over about 30 h should be chosens. The intention is to submit a great proportion of the cell population to the tested compound during two cell cycle9. Whether this expectation can be fulfilled with two applications, e.g. 30 and 6 h before the animals are killed, depends on the pharmacodynamic properties of the test compound. The dose should always cover a wide range, from the usage level up to the highest tolerable dose. Before a test is done, problems of solubility, mode of application and dosage must be clarified with the aid of experts (a) Extraction in toxicology. small animals

of bone mawow from

Before the animals are killed, a 5-ml centrifuge tube is filled with fetal calf serum for each individual. This admittedly expensive fluid has proved superior to all other rinsing solutions or cheaper sera tried so far. With substitutes the cells were often damaged or the erythrocytes were partially agglutinated resulting in preparations that were unusable or consumed too much time for analysis. From a single mouse one can easily obtain enough bone marrow cells for several slides. For the m.t. the animals are, of course, not pretreated with metaphase-blocking agents. From the freshly killed animal both femora are removed in toto, which means that one is cutting through pelvis and tibia. The bones are then freed from muscle by the use of gauze and fingeis. By gentle traction the distal epiphyseal portion is torn off together with the rest of the tibia and the surrounding muscle. The proximal end of the femur is carefully shortened with scissors until a small opening to the marrow canal becomes visible. With the needle of appropriate size mounted, about 0.2 ml serum is pulled from the tube into a disposable plastic syringe. Then the needle is inserted a few mm into the proximal part of the marrow canal which is still closed at the distal end. Next, the femur is submerged completely in the serum and squeezed against the tube to prevent the bone from slipping off the needle. Subsequently, the marrow is aspirated; should the needle have become obstructed, the serum in the syringe is first pressed out. After several gentle aspirations and flushings, the process is repeated from the distal end of the femur. The bone marrow cells should get into the serum as a fine suspension and not in the form of gross particles. (b) Preparation of the smears The tube is centrifuged at IOOO rev./mm for 5 min. The supernatant is removed with a Pasteur pipette. If the sediment is large, half a drop of serum is left; if it is minute, all the supernatant is drawn off. The cells in the sediment are carefully mixed by aspiration into the capillary part of a fresh, siliconized Pasteur pipette. A small drop of the viscous suspension is put on the end of a slide and spread by pulling the material behind a polished cover glass held at an angle of 45 degrees. The size of the droplet is chosen so that all material is used up at a distance of 2-3 cm. The preparations are then air dried.

(c) Staining
The best results are obtained if staining takes place the day following preparation. If staining is done immediately, the slides must be flamed shortly. Staining is carried out in ordinary vertical staining jars according to the following procedure:

MICRONUCLEUS

TEST

I3

stain for 3 min in undiluted May-Gruenwald solution; stain for 2 more min in MayGruenwald diluted with distilled water I : I ; stain for IO min in Giemsa diluted with distilled water I : 6 ; rinse in distilled water; blot dry with filter paper; clean back side of slide with methanol; clear in xylene for 5 min, and mount with cover glass. (Note: contrary to hematological routine it is necessary to mount these preparations so that they are analyzable at high and medium magnifications.) (d) Analyzing the slides First the slides are screened, at medium magnification, for regions of suitable technical quality, where the cells are well spread, undamaged and perfectly stained. Such regions are normally located in a zone close to the end of the smear. A perfect morphology of the nucleated cells serves as criterion for good quality, even though the nucleated cells are not evaluated in the test. The erythrocytes must be well spread, neither globular nor having slurred contours. Their staining has to be vigorous, red in mature erythrocytes (anulocytes) and with a strong bluish tint in the immature forms (polychromatic In control preparations erythrocytes). the proportion of erythrocytes in the regions described

is in the order of 8%, and roughly half of these are polychromatic. At medium to high magnification one thousand polychromatic erythrocytes are screened for the presence of micronuclei. The scored elements are the micronucleated cells and not the number of micronuclei. As an important control it is necessary to register, separately, the number of micronucleated mature red erythrocytes. If their incidence exceeds a limit of five in the fields containing one thousand polychromatic erythrocytes the suspicion arises that the preparation contains artifacts resembling micronuclei. Such a suspicion has to be substantiated or repudiated by careful study. Although a single preparation contains thousands of analyzable erythrocytes it is, for many reasons, preferable to increase the number of animals investigated instead of the number of cells studied per animal.
REMARKS CONCERNING SPECIFIC POINTS OF THE METHOD

Test aninzals To ensure good quality preparations it is advisable to use adolescent animals, e.g. outbred mice 7-12 weeks old. Fat tissue in the bone marrow of older animals has a deleterious effect on the smears. In very young animals the percentage of polychromatic erythrocytes is higher, reflecting a higher turn-over. Real differences in the sensitivity between animals of different age groups have not been demonstrable, nor were differences found between the two sexes (SCHMID AND SUTTER, unpublished results). In the classical method the entire content of the femora is used for the preparations; therefore bone-marrow cells and peripheral blood are always in a given relation to each other which is influenced only by the treatments the animal received. If, in larger animals or in man, bone marrow is obtained by puncture, then the admixture of peripheral blood varies greatly, owing to unpredictable lesions of blood vessels. Again, large amounts of fat render it difficult to obtain satisfactory slides. The technique of preparation For good reasons the technical steps of preparation have been described explicitly. Despite its simplicity the method demands ample practice, not only with con-

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W. SCHMID

trols but especially with animals treated with strong mutagens. In these, the amount of bone-marrow cells can be reduced drastically, even within 30 h, and preparation becomes much more demanding. Trying to analyze preparations of unsatisfactory quality wastes time and is likely to produce unreliable results. Artifacts The micronucleus test is a relatively simple method yet it was not devised for evaluation by fools. The test offers numerous possibilities for checking the validity of the results obtained. If, e.g., there should be a mechanism producing inclusions in erythrocytes by acting on the expulsion of the nuclei from erythroblasts instead of by breaking chromosomes or causing non-disjunction, a thinking investigator would immediately recognize this hypothetical mechanism by the mere observation that micronuclei are missing from the nucleated cell types on the same slides. Artifacts that are sometimes barely distinguishable from micronuclei do occur, although rarely. They can be formed by residues of stain, or, especially in the bone marrow of rats, by basophilic granules of destroyed precursors of basophilic leucocytes. The presence of such artifacts can be recognized by their pattern of distribution in the preparation. In contrast with real micronuclei, which under the conditions of the experiment are bound to be restricted to the polychromatic forms, artifacts of the origins mentioned are distributed indiscriminately over all types of erythrocyte. This is why the analyst has to register micronuclei even when they appear in red, mature erythrocytes. A different kind of observation was reported by JENSSEN~ and confirmed in our laboratory. After treatment of animals with aminoacridines, a high proportion of polychromatic erythrocytes exhibit multiple dust- or thread-like, basophilic inclusions (Fig. 2). The morphological picture of this phenomenon, however, is completely different from that of micronuclei produced by chromosome-breaking agents or inhibitors of mitosis. Furthermore, they are Feulgen-negative and, being fragments of nucleoli, they stain with KNA-specific reagents (MAIER AND SCHMID, in preparation). The protocol of analysis In the section on analysis

the routine

procedure

consisting

of evaluation

of

Pig. 2. Multiple, dust- or thread-like, basophilic inclusions in polychromatic erythrocytes treatment of the animals with aminoacridines. Their morphology is completely different micronuclei produced by chromosome-breaking agents or inhibitors of mitosis.

after from

MICRONUCLEUS

TEST erythrocytes per animal was described. A second possibility

I.5 may

IOOO polychromatic

be considered, depending on the nature of the substances to be tested. This second procedure is based on the following considerations. The transition from polychromatic to mature erythrocytes is a short but nevertheless continuous process. Whether an erythrocyte in transition should be scored as polychromatic or as red will always be a more or less subjective decision made by the investigator. In controls, as well as after treatments not markedly affecting the proliferation of the bone-marrow cells, the relation of polychromatic to mature erythrocytes is fairly consistent, in the region of I : I. Analysis of 2000 total erythrocytes in place of IOOO polychromatic erythrocytes reduces the number of subjective decisions; furthermore, the results of different investigators are easier to compare. If this protocol is restricted to the use in tests where there is no marked reduction in the proportion of young erythrocytes it is perfectly acceptable in toxicological screening. As soon as dose-effect relationships are studied on chromosome-breaking compounds, or if the normal proliferation of bone marrow cells is disturbed for other reasons, e.g. by antimetabolites, scoring has to be restricted to the population of polychromatic erythrocytes. Under such influences the bone marrow regularly becomes flooded with peripheral blood and the mentioned I : I relation becomes badly distorted in favor of the irrelevant mature red cells. If the m.t. is carried out only occasionally, and on a wide spectrum of substances, the laboratory should use only the classical method of analysis; if the test is used frequently and on substances from which it is known in advance that they have no drastic hematological effects, then the second mode of evaluation is preferable. Time conswn$dion for the m.t. Apart from the training of the investigator, time consumption for the m.t. depends, to a great extent, on the quality of the preparations. An unproblematical test using four different dosages and five animals per dose keeps the tester busy for about a week. For preparation and staining he needs about a day and half, and the analysis of IOOO erythrocytes per animal takes from 30 to 60 min. The test can take much longer when low positive results ask for repeats, additional dose ranges or other modes of application. REFERENCES
BOLLER, K., AND W. SCHMID, Chemische Mutagenese beim Sauger. Das Knochenmark des Chinesischen Hamsters als in viva-Testsystem. Hlmatologische Befunde nach Behandlung mit Trenimon. Humangenetik, I I (1970) 35-54. JENSSEN, D. S., Chromosomal damage induced by some aminoacridines in mouse bone marrow cells, (Abstract), 4th Annual Meeting of the European Environmental Mutagen Society, Heidelberg, Germany, 1974. MATTER, B., AND W. SCHMID, Trenimon-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test, Mutation Res., 12 (1971) 4r7425. MATTER, B. E., AND J. GRAUWILER, Micronuclei in mouse bone marrow cells. A simple in viva model for the evaluation of drug-induced chromosomal aberrations, Mutation Res., 23 (1974) SCHMID W., D. T. ARAKAKI, N. A. BRESLAU AND J. C. CULBERTSON, Chemical mutagenesis. The Chinese hamster bone marrow as an in viva test system, I. Cytogenetic results on basic aspects of the methodology, obtained with alkylating agents, Humangenetik, II (1971) 103-118. SCHMID, W., Chemical mutagen testing on in viva somatic mammalian cells, Agents and Actions, VON LEDEBUR, M., AND

239-249.

3 (1973) 77-85.

W. SCHMID, The micronucleus

test. Methodological

aspects, Mutation

Res., 19 (1973) Iog-117.