The in vitro micronucleus technique

Abstract

The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.

Introduction

The observation that chromosome damage can be caused by exposure to ionising radiation or carcinogenic chemicals was among the first reliable evidence that physical and chemical agents can cause major alterations to the genetic material of eukaryotic cells [1]. Although our understanding of chromosome structure is incomplete, evidence suggests that chromosome abnormalities are a direct consequence and manifestation of damage at the DNA level — for example, chromosome breaks may result from unrepaired double strand breaks in DNA and chromosome rearrangements may result from misrepair of strand breaks in DNA [2]. It is also recognised that chromosome loss and malsegregation of chromosomes (non-disjunction) are an important event in cancer and ageing and that they are probably caused by defects in the spindle, centromere or as a consequence of undercondensation of chromosome structure before metaphase [3], [4], [5].

In the classical cytogenetic techniques, chromosomes are studied directly by observing and counting aberrations in metaphases [6]. This approach provides the most detailed analysis, but the complexity and laboriousness of enumerating aberrations in metaphase and the confounding effect of artefactual loss of chromosomes from metaphase preparations has stimulated the development of a simpler system of measuring chromosome damage.

It was proposed independently by Schmid [7] and Heddle [8] that an alternative and simpler approach to assess chromosome damage in vivo was to measure micronuclei (MNi), also known as Howell–Jolly bodies to haematologists, in dividing cell populations such as the bone-marrow. The micronucleus assay in bone-marrow and peripheral blood erythrocytes is now one of the best established in vivo cytogenetic assays in the field of genetic toxicology, however, it is not a technique that is applicable to other cell populations in vivo or in vitro and methods have since been developed for measuring MNi in a variety of nucleated cells in vitro.

MNi are expressed in dividing cells that either contain chromosome breaks lacking centromeres (acentric fragments) and/or whole chromosomes that are unable to travel to the spindle poles during mitosis. At telophase, a nuclear envelope forms around the lagging chromosomes and fragments, which then uncoil and gradually assume the morphology of an interphase nucleus with the exception that they are smaller than the main nuclei in the cell, hence the term “micronucleus” (Fig. 1). MNi, therefore, provide a convenient and reliable index of both chromosome breakage and chromosome loss. Because MNi are expressed in cells that have completed nuclear division they are ideally scored in the binucleated stage of the cell cycle [9], [10]. Occasionally nucleoplasmic bridges between nuclei in a binucleated cell are observed. These are probably dicentric chromosomes in which the two centromeres were pulled to opposite poles of the cell and the DNA in the resulting bridge covered by nuclear membrane (Fig. 1). Thus, nucleoplasmic bridges in binucleated cells provide an additional and complementary measure of chromosome rearrangement, which can be scored together with the micronucleus count.

It is evident from the above that MNi can only be expressed in dividing eukaryotic cells. In other words, the assay cannot be used efficiently or quantitatively in non-dividing cell populations or in dividing cell populations in which the kinetics of cell division is not well understood or controlled. Consequently, there was a need to develop a method that could distinguish between cells that are not dividing and cells that are undergoing mitosis within a cell population. Furthermore, because of the uncertainty of the fate of MNi following more than one nuclear division it is important to identify cells that have completed one nuclear division only. These requirements are also necessary because cells divide at different rates in vivo and in vitro depending on the various physiological, genetic and micronutrient conditions.

Several methods have been proposed based on stathmokinetic, flow cytometric and DNA labelling approaches but the method that has found most favour due to its simplicity and lack of uncertainty regarding its effect on base-line genetic damage is the cytokinesis-block micronucleus (CBMN) assay [9], [10], [11].

In the CBMN assay, cells that have completed one nuclear division are blocked from performing cytokinesis using cytochalasin-B (Cyt-B) and are consequently readily identified by their binucleated appearance (Fig. 1). Cyt-B is an inhibitor of actin polymerisation required for the formation of the microfilament ring that constricts the cytoplasm between the daughter nuclei during cytokinesis [12]. The use of Cyt-B enables the accumulation of virtually all dividing cells at the binucleate stage in dividing cell populations regardless of their degree of synchrony and the proportion of dividing cells. MNi are then scored in binucleated cells only, which enables reliable comparisons of chromosome damage between cell populations that may differ in their cell division kinetics. The method was initially developed for use with cultured human lymphocytes [9], [10], but has now been adapted to various cell types such as solid tumour and bone-marrow cells [13], [14]. Furthermore, new developments have also occurred that allow (a) MNi originating from whole chromosomes to be distinguished from MNi originating from chromosome fragments [15], [16], [17], [18], [19], [20], (b) the conversion of excision-repaired sites to MNi within one cell division [21], (c) the use of molecular probes to identify non-disjunction events in binucleated cells [22], [23], [24] and (d) the integration of necrotic and apoptotic cells within the CBMN assay [25], [26].

It has recently been proposed that the micronucleus assay be used instead of metaphase analysis for genotoxicity testing of new chemicals. A recent special issue of Mutation Research has been dedicated to this topic [27]. The current methodologies and data for the in vitro micronucleus test were reviewed at the Washington International Workshop on Genotoxicity Test Procedures which was held in 1999 [28].

The standard CBMN assay and its various modifications are described in detail in the next sections. The methods described are mainly applicable to cultured human lymphocytes, however, modifications of the assay for application to other cell types are included.

In this technique MNi are scored only in those cells that have completed one nuclear division following phytohaemagglutinin (PHA) stimulation. These cells are recognised by their binucleated appearance after they are blocked from performing cytokinesis by Cyt-B which should be added before the first mitotic wave. Optimal culture conditions should yield 35–60% or more binucleates as a proportion of viable cells (i.e., all cells excluding necrotic and apoptotic cells) at 72 h after PHA stimulation. All equipment should have biosafety features to protect the operator and solutions used in this procedure should be filter sterilised.

(1) Fresh blood is collected by venepuncture in tubes with heparin as anticoagulant and stored at 22°C for less than 4 h prior to lymphocyte isolation.

(2) The blood is then diluted 1:1 with isotonic (0.85%) sterile saline and gently inverted to mix.

(3) The diluted blood is overlaid gently on Ficoll Paque (Pharmacia) density gradients using a ratio of approximately 1:3 (e.g., 2 ml Ficoll Paque to 6 ml of diluted blood), being very careful not to disturb the interface.

(4) The gradient is then spun in a centrifuge at 400×g for 25–40 min at 22°C after carefully balancing the tubes.

(5) The lymphocyte layer at the interface of Ficoll Paque and diluted plasma is collected with a sterile plugged pasteur pipette and added to 3–5 times volume of Hanks balanced salt solution (HBSS) at 22°C. The resulting cell suspension is centrifuged at 280–400×g for 5–10 min depending on the volume.

(6) The supernatant is discarded, the cells resuspended in 2–5 times volume HBSS and centrifuged at 180–400×g for 5 min depending on the volume.

(7) The supernatant is discarded and the cells resuspended in 1 ml RPMI 1640 culture medium.

(8) Cell concentration is then measured using a Coulter Counter or haemocytometer and the concentration adjusted by the percentage of viable cells measured using trypan blue exclusion assay.

(9) The cells are resuspended in RPMI 1640 medium containing 10–15% heat inactivated foetal calf serum at 0.5–1.0×10⁶ cells/ml and cultured in 0.75–1.0 ml volume in round–bottom tissue culture tubes (10 mm width).

(10) Lymphocytes are then stimulated to divide by adding phytohaemagglutinin (PHA) (Glaxo Wellcome HA15) to each culture tube at 10 μl/ml (from a stock solution in H₂O of 2.25 mg/ml) and incubated at 37°C with loose lids in a humidified atmosphere containing 5% CO₂. The concentration of PHA used has to be optimised depending on the purity and source of the reagent to ensure maximum number of binucleated cells after Cyt-B block.

(11) Forty-four hours after PHA stimulation, 4.5 μg Cyt-B is added to each milliliter of culture [USE GLOVES AND FUME HOOD]: a 100 μl aliquot of Cyt-B stock solution in DMSO (600 μg/ml) is thawed, 900 μl culture medium added and mixed. Seventy-five microliters of the mixture is added to each 1 ml of culture to give a final concentration of 4.5 μg Cyt-B/ml (other laboratories have successfully used 6.0 μg Cyt-B/ml in their cultures). Culture tubes are then re-incubated with loose lids.

(12) Twenty-eight hours after adding Cyt-B, cells are harvested by cytocentrifugation (Shandon Elliot). One hundred microliters of the culture medium is removed without disturbing the cells and then cells are gently resuspended in their tubes. 100–120 μl of cell suspension is transferred to cytocentrifuge cups (Shandon Elliot) and centrifuged to produce 2 spots per slide [Set the cytocentrifuge as follows — time: 5 min, speed: 600 rpm]. Slides are removed from the cytocentrifuge and allowed to air dry for 10–12 min only and then fixed for 10 min in absolute methanol.

(13) The cells can be stained using a variety of techniques that can clearly identify nuclear and cytoplasmic boundaries. In our experience, the use of “Diff Quik” (Lab-Aids, Australia), a commercial ready-to-use product, provides rapid and optimal results.

(14) After staining, the slides are air-dried and coverslips placed over the cells using Depex (DPX) mounting medium. This procedure is carried out in the fume hood and the slides are left to set in the fume hood and then stored indefinitely until required.

Important note: Duplicate cultures of control or genotoxin-treated cells should be set up and slides from each culture should be prepared. This is essential to obtain a measure of experimental variation, i.e., coefficient of variation, which should be quoted with each set of duplicate cultures. This experimental design is summarised in Fig. 2.

For fluorescence microscopy staining with acridine orange (40 μg/ml in Sorensen's phosphate buffer pH 6.9) is recommended. If a cytocentrifuge is not available, slides can be prepared using the procedure, described below, for whole blood cultures.

Slides are best examined at 1000× magnification using a light or fluorescence microscope. Slides should be coded before analysis so that the scorer is not aware of the identity of the slide. A score should be obtained for slides from each duplicate culture. The number of cells scored should be determined depending on the level of change in the MN index that the experiment is intended to detect and the expected standard deviation of the estimate. For each slide the following information should be obtained:

• The number of micronuclei (MNi) in at least 1000 binucleate [BN] cells should be scored and the frequency of MNi per 1000 BN cells calculated. The criteria for scoring MNi in BN cells are detailed below.

• The distribution of BN cells with zero, one or more MNi; the number of MNi in a single binucleated cell normally ranges from 0 to 3 in lymphocytes of healthy individuals but can be greater than 3 on occasion depending on genotoxin exposure and age.

• The frequency of micronucleated BN cells in at least 1000 BN cells.

• The frequency of nucleoplasmic bridges in 1000 BN cells. Scoring criteria for nucleoplasmic bridges are described below.

• The proportion of mononucleated, binucleated, tri-nucleated and tetra-nucleated cells per 500 cells scored. From this information, the Nuclear Division Index (explained below) can be derived.

• The number of dead or dying cells due to apoptosis or necrosis per 500 cells may also be scored on the same slide (scoring criteria for these cells are detailed below) while scoring the frequency of viable mono-, bi- and multi-nucleated cells.

It is important to note that it is best to skip scoring a cell if one is uncertain on how to classify it. The basic elements of a typical score sheet are listed in Table 1.

The cytokinesis-blocked cells that may be scored for MN frequency should have the following characteristics:

(a) The cells should be binucleated;

(b) The two nuclei in a binucleated cell should have intact nuclear membranes and be situated within the same cytoplasmic boundary;

(c) The two nuclei in a binucleated cell should be approximately equal in size, staining pattern and staining intensity;

(d) The two nuclei within a BN cell may be attached by a fine nucleoplasmic bridge which is no wider than 1/4th of the nuclear diameter.

(e) The two main nuclei in a BN cell may touch but ideally should not overlap each other. A cell with two overlapping nuclei can be scored only if the nuclear boundaries of each nucleus are distinguishable.

(f) The cytoplasmic boundary or membrane of a binucleated cell should be intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells.

Examples of the type of binucleated cells that may or may not be scored are illustrated diagrammatically in Fig. 3. The cell types that should not be scored for micronucleus frequency include mono-, tri-, quadr- and multi-nucleated cells, and cells that are necrotic or apoptotic (illustrated in Fig. 4).

MNi are morphologically identical to but smaller than nuclei. They also have the following characteristics:

(a) The diameter of MNi in human lymphocytes usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei which corresponds to 1/256th and 1/9th of the area of one of the main nuclei in a BN cell, respectively.

(b) MNi are non-refractile and they can therefore be readily distinguished from artefact such as staining particles;

(c) MNi are not linked or connected to the main nuclei;

(d) MNi may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary;

(e) MNi usually have the same staining intensity as the main nuclei but occasionally staining may be more intense.

Examples of typical MNi that meet the criteria set above are shown in Fig. 5. Examples of cellular structures that resemble MNi but should not be classified as MNi originating from chromosome breakage or loss are illustrated in Fig. 6. Induction of gene amplification may lead to extrusion of amplified genes into nuclear buds (e.g., Fig. 6c and d) during S phase that are eventually detached from the nucleus to form a micronucleus (Shimizu et al., 1998); it may be necessary to quantify the frequency of nuclei with nuclear bud formation if gene amplification is suspected.

Nucleoplasmic bridges are sometimes observed in binucleated cells following exposure to clastogens. They are a continuous link between the nuclei in a binucleated cell and are thought to be due to dicentric chromosomes in which the centromeres were pulled to opposite poles during anaphase. The width of a nucleoplasmic bridge may vary considerably but usually does not exceed 1/4th of the diameter of the nuclei within the cell. The nucleoplasmic bridge should have the same staining characteristics of the main nuclei. On very rare occasions, more than one nucleoplasmic bridge may be observed within one binucleated cell. A binucleated cell with a nucleoplasmic bridge often contains one or more micronuclei. Examples of binucleated cells with nucleoplasmic bridges are illustrated in Fig. 1, Fig. 5.

Fig. 7 describes the various pathways and events that may be expected to occur in cultured lymphocytes exposed to a toxic agent. Cytogenetic genotoxicity assays that require hypotonic treatment for the preparation of interphase cells (for whole blood micronucleus assay) or metaphase plates for chromosome analysis are not usable for cytotoxicity assays because hypotonic treatment may destroy necrotic cells and apoptotic cells making them unavailable for assay. Inclusion of necrosis and apoptosis is important for the accurate description of mechanism of action and measurement of cellular sensitivity to a chemical or radiation. Isolated lymphocyte culture assay or culture of cell lines does not require hypotonic treatment of cells for slide preparation, thus making it possible to preserve the morphology of both necrotic and apoptotic cells. The use of Cyt-B, should make it easier to score apoptotic cells because it is expected to inhibit the disintegration of apoptotic cells into smaller apoptotic bodies. The latter process requires microfilament assembly [29], which is readily inhibited by Cyt-B [12].

The following guidelines for scoring necrotic and apoptotic cells are recommended: (a) cells showing chromatin condensation with intact cytoplasmic and nuclear boundaries or cells exhibiting nuclear fragmentation into smaller nuclear bodies within an intact cytoplasm/cytoplasmic membrane are classified as apoptotic; (b) cells exhibiting a pale cytoplasm with numerous vacuoles and damaged cytoplasmic membrane with a fairly intact nucleus or cells exhibiting loss of cytoplasm and damaged/irregular nuclear membrane with a partially intact nuclear structure are classified as necrotic. These criteria and results for these measures with hydrogen peroxide have been recently reported elsewhere [26].

Fig. 4, Fig. 7 illustrate typical examples of necrotic and apoptotic cells.

NDI is often calculated according to the method of Eastmond and Tucker [30]. Five hundred viable cells are scored to determine the frequency of cells with 1, 2, 3 or 4 nuclei and calculate the NDI using the formula:

$$\text{NDI}\text{=(}\text{M}\text{1+2(}\text{M}\text{2)+3(}\text{M}\text{3)+4(}\text{M}\text{4))/N,}$$

where M1–M4 represent the number of cells with one to four nuclei and N is the total number of viable cells scored. The NDI and the proportion of binucleated cells are useful parameters for comparing the mitogenic response of lymphocytes and cytostatic effects of agents examined in the assay.

A more accurate assessment of nuclear division status is obtained if necrotic and apoptotic cells are included in the total number of cells scored because at higher toxic doses of chemicals tested one can expect a very large proportion of cells becoming non-viable. It is therefore important to note that both binucleate ratio and the NDI are overestimated if necrotic and apoptotic cells are not included when scoring cells. A more accurate estimate of nuclear division status and cell division kinetics can be obtained using the following modified equation which takes account of viable as well as necrotic and apoptotic cells:

$$\text{NDCI}\text{=(}\text{Ap}\text{+}\text{Nec}\text{+}\text{M}\text{1+2(}\text{M}\text{2)+3(}\text{M}\text{3)+4(}\text{M}\text{4))/N}{}^{\mathrm{*}}\text{,}$$

where NDCI = nuclear division cytotoxicity index, Ap = number of apoptotic cells, Nec = number of necrotic cells, M1–M4 = number of viable cells with 1–4 nuclei and N^* = total number of cells scored (viable and non-viable).

After assessing the MN response in human G₀ lymphocytes following exposure to a variety of genotoxins it became evident that the extent of micronucleus formation in relation to cytotoxicity was low for chemicals and ultraviolet radiation which mainly induce base-lesions and adducts on DNA rather than strand breakage or spindle damage [21]. We hypothesised that this was due to either efficient repair of the lesions or that such sites, if left unrepaired, do not convert to a double stranded break in DNA following one round of DNA synthesis. Furthermore, we reasoned that inhibition of excision repair by cytosine arabinoside (ARA) would result in the conversion of such base lesions to a single stranded break which would become a double stranded break following DNA synthesis leading to the production of an acentric fragment which would then be expressed as a MN within one division cycle [21], [31]. Using this concept (illustrated in Fig. 8) we showed that addition of ARA during the first 16 h of lymphocyte culture (i.e., before DNA synthesis) did result in a dramatic increase (10-fold or greater) in the MN dose–response following UV or MNU treatment. However, the ARA-induced increase following X-ray exposure was only 1.8-fold as would be expected from the proportion of DNA adducts or base lesions relative to the induction of DNA strand breaks. This method has since been used to identify pesticides that induce excision repair and to distinguish between genotoxic agents that do or do not induce excision repair [32]. The ARA protocol is an important adjunct to the basic CBMN assay and should be attempted particularly if strong cytotoxic effects are observed in conjunction with weak MN induction. Precise measurement of excision-repaired DNA lesions using the ARA method is only possible using the CBMN assay because (a) the conversion of excision-repaired DNA lesions to MN occurs only in cells that have completed nuclear division and (b) the addition of ARA may also result in significantly altered cell division kinetics which could confound results in MN assays without Cyt-B.

ARA inhibition of DNA polymerase may cause DNA strand breaks in cells undergoing replicative DNA synthesis. Therefore, it is only possible to use this method in PHA-stimulated G₀ lymphocytes with ARA exposure occurring during the G₁ phase and prior to S-phase, because excision repair is activated during G₁. In practice, this means that cells are cultured in the presence of ARA during the first 16–20 h after PHA stimulation, following which the cells are washed to remove ARA and incubated in culture medium containing deoxycyidine to reverse ARA inhibition of DNA polymerase; after these steps the standard CBMN protocol is followed. For more procedure details and typical results refer to Fenech and Neville [21] and Surrales et al. [32].

The CBMN assay in human lymphocytes can also be performed using whole blood cultures. Typically 0.4–0.5 ml of whole blood is added to 4.5 ml of culture medium (e.g., RPMI 1640) supplemented with fetal calf serum containing l-glutamine, antibiotics (optional) and PHA. Cyt-B is added at 44-h post PHA stimulation. The recommended optimal concentration of Cyt-B for accumulating binucleated cells in whole blood cultures is 6 μg/ml [33]. The binucleated lymphocytes are harvested 28 h after adding Cyt-B, hypotonically treated with 0.075 M KCl to lyse red blood cells and fixed with methanol:acetic acid prior to transfer to slides and staining (it is also possible to smear the cells on the slides first and then fix them after air-drying). As an alternative it is also possible to isolate the binucleated lymphocytes directly from the whole blood culture using Ficoll gradients and then transfer cells to slides by cytocentrifugation prior to fixation and staining (unpublished observation) which precludes the requirement for hypotonic treatment and enables optimal preservation of the cytoplasm.

Lymphocytes are isolated either from the spleen or peripheral blood and cultured according to the procedures described by Fenech et al. [34]. Because murine lymphocytes have shorter cell division cycles than human lymphocytes it is essential to add Cyt-B no later than 18 h after stimulation by mitogen and to harvest the cells 20 h later. Depending on the culture conditions, it is possible to obtain good binucleate ratios even at 72-h post mitogen stimulation.

The CBMN assay can be readily adapted to other primary cell types to assess DNA damage induced in vitro, in vivo or ex-vivo. The most important points to remember are (a) to ensure that MNi are scored in the first nuclear division following the genotoxic insult and (b) to perform preliminary experiments to determine the concentration of Cyt-B at which the maximum number of dividing cells will be blocked at the binucleate stage. It is also important to remember that Cyt-B may take up to 6 h before it starts to exert its cytokinesis-blocking action (unpublished observation). When using established or primary cell lines from dividing cell populations it is usual to add Cyt-B shortly after exposure to genotoxin to capture all cells undergoing their first nuclear division as binucleated cells — this usually requires an incubation period of about 24 to 48 h, depending on the cell cycle time, before harvesting the cells. Attached cells can be trypsinised and then prepared by cytocentrifugation as described for human lymphocytes. Specific methods have been described for use with nucleated bone-marrow cells [14], lung fibroblasts [35], skin keratinocytes [36] and primary tumour cell cultures [13]. It is generally more practical to assess in vivo induction of micronuclei by blocking cytokinesis in dividing cells after the cells have been isolated from the animal and placed in culture medium in the presence of Cyt-B; this approach has proven to be successful with a variety of cell types including fibroblasts, keratinocytes and nucleated bone-marrow cells.

There is some debate that Cyt-B, used to accumulate binucleated cells, may interfere with the expression of MN [28]. Studies with normal cells do not show an induction of MNi by Cyt-B or a dose–response effect of Cyt-B with MN frequency in binucleated cells at doses that are usually used to block cells in cytokinesis [10], [37], [38], [39]. A recent study suggests that MN expression induced by spindle poisons may be less than expected in the cytokinesis-blocked BN cells because of pole-to-pole distance shortening which may increase the probability of re-inclusion of lagging chromosome fragments or whole chromosomes back into a nucleus but this did not diminish the effectiveness of the CBMN assay [40].

There has been an increased interest in exploring further the possibility of performing the in vitro MN assay without Cyt-B to minimise the possible confounding effect of Cyt-B while running the potential risk of obtaining a false negative result because of inadequate control of cell division kinetics, i.e., inhibition of nuclear division inhibits micronucleus expression. While the evidence of obtaining a false positive result with the CBMN assay in normal cells is lacking, there is already adequate evidence that performing the MN assay in a manner that does not account for inhibition of nuclear division can lead to false negative results or an underestimate of MN induction in human lymphocyte cultures [10], [11], [41] and an example of this defect of MN assays without Cyt-B is shown in Fig. 9. Nevertheless, recent studies comparing the micronucleus assay with or without Cyt-B suggest that if cell lines with good growth characteristics are used and culture and nuclear division conditions are optimal it is possible to obtain comparable results between the CBMN assay and the MN assay without Cyt-B when strong clastogens are tested [42], [43]. A mathematical model of MN expression predicts (1) that scoring MN in BN cells is the most reliable way of determining micronucleus frequency and (2) scoring MN in mononucleated cells in cultures without cytokinesis-block is likely to generate false negative results when nuclear division is significantly inhibited by the chemical tested or the culture conditions do not allow an optimal number of dividing cells [44]. Consequently, results for micronucleus frequency obtained by scoring micronuclei in mononucleated cells in cultures without Cyt-B cannot be considered conclusive and that a negative result with this system should be confirmed using the CBMN assay.