WO2002014859A1

WO2002014859A1 - Micronucleus assay

Info

Publication number: WO2002014859A1
Application number: PCT/AU2001/001007
Authority: WO
Inventors: Michael Felix Fenech; Dean Raymond Hewish; Heddy Zola
Original assignee: Diatech Pty Ltd
Priority date: 2000-08-16
Filing date: 2001-08-14
Publication date: 2002-02-21
Also published as: AUPQ943700A0

Abstract

The present invention relates to methods for determining the frequency of micronuclei events in cell populations. These methods are useful for detecting genetic damage in human and animal cells.

Description

Micronucleus Assay

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

A micronucleus assay is a means of detecting genetic damage in human and animal cells. It makes use of the property of the cells whereby chromosomes that have been damaged or fragmented will frequently be handled abnormally by the cell machinery during nuclear division and packaged into small bodies separate from the daughter nuclei. This can result in damaged cells having one or more discrete small bodies that appear under the microscope, after appropriate staining, like minute cell nuclei. However these bodies (micronuclei) are clearly smaller than the cell nuclei and are physically separate from them. Normal cells do not possess micronuclei and damaged cells can therefore be easily distinguished from undamaged cells by methods that score micronucleus presence.

The micronucleus assay is based on a measurement of the proportion of cells that contain micronuclei in a cell or tissue sample taken from an individual. Peripheral blood lymphocytes and mammalian cell lines are frequently used because they provide a readily available sample of a rapidly dividing tissue that would be expected to show a high sensitivity to genotoxic effects. These types of cells are readily available and convenient to use.

The assay provides an important measure of chromosome breakage/repair and chromosome malsegregation/loss, two key events in carcinogenesis, ageing and developmental defects. The assay in human lymphocytes has become an important diagnostic tool in the following areas: (i) genetic toxicology testing of new pharmaceuticals (Kirsch-Volders et al., 2000, Fenech M., 2000a); (ii) biomonitoring of populations exposed to environmental carcinogens (Fenech et al., 1999a; Albertini et al. 2000); (iii) identifying dietary factors that prevent DNA damage (Fenech M. et al. 1998); and (iv) determining the sensitivity of cells to ionising radiation (Scott et al., 1998, Rothfus et al, 2000). Because the appearance of micronuclei as distinct bodies within a cell will only occur after the cell has undergone a division, it is important to be able to score the presence of micronuclei only in once-divided cells, in order to obtain a precise index of damaged cells. The cytokinesis-block micronucleus (CBMN) assay (Fenech and Morley, 1985) makes use of a chemical, cytochalasin-B, that prevents cells from separating into daughter cells after the chromosomes have divided and segregated into daughter nuclei. Thus, after cells have been incubated for some time in the presence of cytochalasin-B, those cells which have attempted to divide will have two nuclei (binucleate) and will be clearly identifiable by microscopy. The proportion of these cells containing micronuclei is then scored to give an estimation of the proportion of cells with genetic damage.

Using the cytokinesis-block micronucleus (CBMN) assay it has been recently shown that radiosensitivity of lymphocytes is an important marker of breast cancer risk with 50% of cases showing this phenotype compared to 7% in the general population (Scott et al., 1998) and 95% of cases with BRCA1 mutation showing this sensitivity (Rothfus et al, 2000). However BRCA1 and BRCA2 mutation only accounts for 7-10% of breast cancer cases indicating that there are several other radiation sensitivity genes associated with breast cancer that are yet to be identified, making the phenotypic radiosensitivity a more practical and cost-effective approach. Elevated malsegregation of chromosome 21 identified with the CBMN assay has also been used to distinguish Alzheimer's disease cases from unaffected controls (Migliore et al, 1999). The CBMN assay is currently the standard method of micronucleus scoring. The assay has been further developed by workers in the CSIRO Division of Health Sciences and Nutrition (Fenech et al., 1999b, Fenech, 2000a) and is being used world-wide as a visual microscopy scoring procedure. Its successful application in the various fields of environmental toxicology, in vitro toxicology, nutrition and genomic stability, ageing, cancer risk assessment and effects of radiotherapy have been demonstrated.

The micronucleus assays used to date, which depend on visual scoring of the cells containing micronuclei, suffer from being time consuming and subjective. Because of the strong diagnostic potential of this method it is important to develop objective and automated scoring procedures. Prototypes of software for automated image analysis of the CBMN have been described in the literature since the early 1990s and developed by Becton-Dickinson and Loats Associates, Inc (LAI Automated Micronucleus Assay System;. 201 East Main St. Westminster, MD 21157 410-876-8055). However, the recognition software to date has not proven to be significantly more rapid than visual scoring and still requires visual verification of cells. Furthermore it is currently not tailored to internationally acceptable scoring criteria that have been recently described (Fenech 2000a). Automated image analysis systems are expensive and purpose-built, requiring a high capital investment in a system that is capable of only a single assay procedure. A simplified and more efficient micronucleus assay is therefore desirable.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the present invention provides a method for quantifying micronuclei events in a cell population, the method comprising:

(i) incubating a population of cells for a sufficient time to allow a substantial portion of the cells to complete at least one cellular division;

(ii) determining the proportion of once divided cells in a first sample of cells from step (i);

(iii) lysing a second sample of cells from step (i) to release nuclei and micronuclei;

(iv) analysing the nuclei and micronuclei from step (iii) in order to determine the frequency of micronuclei; and (v) correcting the frequency determined in step (iv) for the proportion of once divided cells as determined in step (ii).

In one embodiment of the first aspect, the method further comprises labelling the cells with a dye prior to step (i). Preferably, the dye is a fluorescent dye. More preferably, the fluorescent dye is selected from the group consisting of carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series.

In another embodiment of the first aspect, step (i) involves incubating the cells in the presence of a cytokinesis blocking agent. Preferably, the cytokinesis blocking agent is a cytochalasin. More preferably, the cytochalasin is cytochalasin-B. In a preferred embodiment of the first aspect, the proportion of once divided cells in step (ii) is determined by flow cytometry.

In a further preferred embodiment of the first aspect, the nuclei and micronuclei in step (iii) are analysed by flow cytometry. In a further preferred embodiment of the first aspect, the nuclei and micronuclei are stained with a dye prior to flow cytometry. Preferably, the dye is propidium iodide, Hoechst 33342 or 4'-6'-diamino-2-phenylindole (DAPI).

In a second aspect, the present invention provides a method for quantifying micronuclei events in a cell population, the method comprising:

(ii) sorting the cells from step (i) to obtain a substantially pure population of cells which have undergone one cellular division; (iii) lysing the sorted cells from step (ii) to release nuclei and micronuclei; and

(iv) analysing the nuclei and micronuclei from step (iii) in order to determine the frequency of micronuclei.

In one embodiment of the second aspect, the method further comprises labelling the cells with a dye prior to step (i). Preferably, the dye is a fluorescent dye. More preferably, the fluorescent dye is selected from the group consisting of carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series.

In another aspect of the second invention, step (i) involves incubating the cells in the presence of a cytokinesis blocking agent. Preferably, the cytokinesis blocking agent is a cytochalasin. More preferably, the cytochalasin is cytochalasin-B.

In a preferred embodiment of the second aspect, the cells are sorted in step (ii) by flow cytometry. In a further preferred embodiment of the second aspect, the nuclei and micronuclei in step (iv) are analysed by flow cytometry. Preferably, the nuclei and micronuclei are stained with a dye prior to flow cytometry. More preferably, the dye is propidium iodide, Hoechst 33342 or 4'-6'-diamino-2- phenylindole (DAPI). In a third aspect, the present invention provides a kit for quantifying micronuclei events in a cell population, the kit comprising a micronuclei standard. Preferably, the kit further comprises a dye for labeling cells and/or an agent for lysing cells.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Schematic representation of micronucleus expression and the use of CFSE dye to identify once-divided cells. The latter have half the CFSE concentration of non-divided cells.

Figure 2: An example of flow cytometry detection of cell generations using carboxyfluorescein diacetate succinimidyl ester (CFSE) staining.

Figure 3: Schematic representation of membrane lysis of flow-sorted once divided cells.

Figure 4: Identification of micronuclei by flow cytometry (Nusse & Marx, 1997).

Figure 5: Steps involved in a preferred micronucleus assay of the present invention, (a) Step 1: location of cell peak with low propidium iodide fluorescence (gate 1); (b) Step 2: location of intact cell population (gate 2); (c)

Step 3: identification of CFSE histogram (excluding high propidium positive and including healthy cells: those cells simultaneously in gates 1 and 2) representing lx divided cells (gate 3). These are the cells that are sorted, collected and fixed for subsequent micronucleus frequency measurements;

(d) Step 4: doublets are gated out using this plot to identify population (R ) for micronucleus analysis; (e) Step 5: micronucleus population (R2) and nuclear population (R3) are identified on these plots which only include events from Rl in step 4. Micronucleus frequency = no of events in R2/no of events in R3 x 100%. Results are shown for [A] control cultures and [B] cultures treated with hydrogen peroxide. Figure 6: Estimation of micronucleus frequency in tetraploid (binucleate ) cells following cytochalasin-B treatment. Cells were treated with cytochalasin-B for 24 hours as described and stained with Hoechst 33342 dye to identify tetraploid cells. The tetraploid cells were sorted then lysed to allow analysis of micronuclei. A: light scatter plot of cytochalasin B treated cells Gated area eliminates debris and cell aggregates. B: DNA profile of whole cells in gate 1. Tetraploid cells are identified in gate 2. C: Nuclei and micronuclei from sorted tetraploid cells corresponding to Gate 1 and Gate 2. Percentage micronuclei calculated from relative number of events in region NN (normal nuclei) and MN (micronuclei).

Figure 7: Estimation of micronucleus frequency in a whole cell population without cell sorting. Cells were stained with CFSE as described and lysed to yield nuclei and micronuclei. The number of cells that had undergone a single division is estimated by that population of whole nuclei with decreased fluorescence (Gate 1) and the proportion of micronuclei estimated by the number of events in gate 2. A: Scattergram of peak vs integral propidium iodide signals used to eliminate doublets from the analysis as previously described (Gate 3). B: DNA profile of CFSE stained cells used to determine number of once-divided cells (Gate 1). C: Scattergram of light scatter vs PI fluorescence to identify and count number of micronuclei (Gate 2).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed improved micronucleus assays that do not rely on visual scoring of micronuclei in once divided cells. Instead, these assays involve the release of genetic material from cells that have undergone one cellular division, and a determination of the frequency of micronuclei per nuclei within the released genetic material.

Accordingly, in a first aspect the present invention provides a method for quantifying micronuclei events in a cell population, the method comprising: (i) incubating a population of cells for a sufficient time to allow a substantial portion of the cells to complete at least one cellular division;

(iv) analysing the nuclei and micronuclei from step (iii) in order to determine the frequency of micronuclei; and

(v) correcting the frequency determined in step (iv) for the proportion of once divided cells as determined in step (ii) . In a second aspect, the present invention provides a method for quantifying micronuclei events in a cell population, the method comprising:

(i) incubating a population of cells for a sufficient time to allow a substantial portion of the cells to complete at least one cellular division; (ii) sorting the cells from step (i) to obtain a substantially pure population of cells which have undergone one cellular division;

(iii) lysing the sorted cells from step (ii) to release nuclei and micronuclei; and

(iv) analysing the nuclei and micronuclei from step (iii) in order to determine the frequency of micronuclei. In one embodiment of the first and second aspects, cells that have undergone one cellular division are identified or sorted from cells that have not divided by the use of cell tracking dye technology. Preferably, the cells are labeled with a non toxic dye that binds irreversibly to cell components such as membranes and gives a strong fluorescent signal. As cell division occurs, new cell components are synthesised that do not carry the fluorescent label and the fluorescence of the cells decreases in proportion to the number of cell division cycles that have occurred (see Figure 1). This method allows unambiguous identification and/or sorting of those cells that have undergone one or more divisions, preferably by flow cytometry (see, for example, Figure 2). Various dyes that are suitable for this procedure have been described. Preferred dyes include Carboxyfluorescein diacetate succinimidyl ester (CFSE) which labels cell proteins, and dyes of the PKH series which label cell membranes and which are marketed expressly for this purpose (Shapiro 1994).

In an alternative embodiment of the first and second aspects, the starting population of cells is incubated with a cytokinesis blocking agent. The cytokinesis blocking agent prevents cells from separating into daughter cells after the chromosomes have divided and segregated into daughter nuclei. Thus, after cells have been incubated for some time in the presence of a cytokinesis blocking agent, those cells that have attempted to divide will have two nuclei. This method therefore also allows unambiguous identification and/or sorting of those cells that have undergone one or more divisions.

Lysis of cells to release nuclei and micronuclei as described in step (iii) of the first and second aspects of the present invention may be achieved using procedures such as those described in Nusse and Marx, 1997.

Analysis of nuclei and micronuclei as described in step (iv) of the first and second aspects of the present invention may be performed by flow cytometry as described in Nusse and Marx, 1997. The analysis may involve staining the nuclei and micronuclei with a DNA specific dye prior to flow cytometry. The DNA specific dye may be, for example, propidium iodide, Hoechst 33342 or 4'-6'-diamino-2-phenylindole (DAPI). It will be appreciated by those skilled in the art that a number of alternative nucleic acid dyes may also be used. Some of these dyes require destruction of RNA before accurate estimation of DNA can be carried out. In this embodiment, nuclei may be distinguished from micronuclei by their greater light scatter and greater DNA content.

In the first aspect of the present invention, isolation or sorting of once- divided cells is not required. Rather, the method defined in this aspect involves two separate analyses of the population of cells after incubation in step (i). A first sample of the incubated cell population is analysed to determine the proportion of once-divided cells in the total population. A second sample of incubated cell population is used to lyse cells and analyse the nuclei and micronuclei. The micronucleus frequency per nucleus is then determined, and this figure is then corrected for the proportion of once- divided cells, preferably using the mathematical equation described below:

MNf(corr) = MNf(Ntot/Nodc)

where

MNf(corr) = micronucleus frequency per nucleus corrected for frequency of once-divided cells;

MNf = micronucleus frequency per nucleus measured without correcting for proportion of once-divided cells;

Ntot = number of total cells analysed;

Node = number of once-divided cells among total cells analysed.

This mathematical equation is based on the principles of the mathematical model for micronucleus expression recently described by

Fenech (2000b).

In the method of the second aspect of the present invention, the once- divided cells are sorted, preferably by flow cytometry procedures, prior to lysis in order to obtain a substantially pure population of cells which have undergone one cellular division. By "substantially pure population of cells which have undergone one cellular division" we mean a population of cells that is substantially free of (a) cells which have not completed nuclear division, (b) cells which have not completed cell division, (c) cells which have completed more than one cell division, (d) cell debris and (e) cells with degraded nucleic acid (apoptotic or necrotic cells).

In a preferred embodiment, the method of the second aspect involves labeling cells with suitable fluorescent membrane tracking dye (eg CFSE) and allowing sufficient time for a substantial portion of the cells to complete one nuclear /cell division. Cells may then be flow sorted on the basis of cellular fluorescence levels. Once-divided cells, which exhibit half the fluorescence of non-divided cells, may be separated from other cells in the population.

Alternatively, cells are incubated in the presence of a cytokinesis blocking and are then flow sorted on the basis of cellular nucleic acid levels. The cell sorting step in the method of the second aspect eliminates non-divided cells as well as necrotic and apoptotic cells. The sorted once- divided cells are then lysed to release nuclei and micronuclei using published procedures (Nusse and Marx, 1997) (see Figure 3). Nuclei and micronuclei are then analysed to determine the frequency of micronuclei in the sorted once divided cells. Accordingly, one advantage of this embodiment is that micronuclei detection is not confounded by dead cells and cellular debris.

Preferred embodiments of the present invention relate to micronucleus assays which are based on the cell analysis procedure known as flow cytometry. Flow cytometry is an analytical procedure for measuring the characteristics of individual cells (Shapiro 1994). In the flow cytometry procedure, cells are injected into a fluid stream such that they are lined up in single file by hydrodynamic forces and carried through the focus of an intense light source such as a laser beam. Light scattering from the cells is measured by light detectors in two directions. Light scattered in the direction of the light beam is termed forward light scatter (FSC). This scatter generally increases in intensity with the size of the cells, although other factors such as refractive index can also influence the scatter. Light scattered at right angles to the light beam, or side scatter (SSC), is scattered from structures within the cells and is a measure of cell internal granularity. It is particularly influenced by the size and shape of the cell nucleus. In addition, the light can excite fluorescence from cell components or from dyes added to the cells either to stain cell components or attached to antibodies that recognise specific cell components. As with side scatter, the fluorescence emission is measured by light detectors placed at 90° to the illuminating light beam. Optical filters are used in the light path to discriminate between fluorescence emission from different dyes that emit distinct wavelengths of light. Therefore several different dyes can be measured simultaneously, provided that their spectral characteristics are sufficiently separate. The electrical pulses from the light detectors are processed electronically and analysed by computer software that allows the collected data to be displayed and cell populations with unique characteristics to be identified and analysed in isolation from other cells in the sample. Because flow cytometry measures the characteristics of individual cells, it gives quantitative data on a sample of the cell population that can give a statistical measurement of the characteristics of the population. It can also identify sub-populations based on their light scatter or fluorescence properties. Flow cytometry is very rapid, as data rates in excess of 1000 cells per second can be processed by modern machines. After cell sub-populations have been identified by flow cytometry, it is possible to physically sort them into tubes using a flow cytometer with sorting capability. Cell sorting may be accomplished by electrostatic separation of fluid droplets containing the cells or by fluidic/mechanical sorting, depending on the machine design and manufacturer. Flow cytometry can also be used to analyse sub-cellular components after cells have been deliberately lysed. In this way, cell nuclei can be distinguished from micronuclei by their greater light scatter and greater DNA content (greater fluorescence of DNA binding dye) (Figure 4). This procedure has been described in the literature (Nusse & Marx 1997). The limitation of this method alone is that, although it detects and measures the presence of micronuclei in a cell population, it does not identify the cells from which the micronuclei have come and can not distinguish the proportion of micronuclei relative to the number of cells that have undergone division. The methods of the present invention overcome this limitation. The methods of the first and second aspects of the present invention may also involve further analysis of the nuclei and micronuclei. For example, oligonucleotide or PNA probes which hybridise specifically to centromeres or telomeres may be used to distinguish micronuclei originating via chromosome breakage from those arising through chromosome loss and may provide information on telomere shortening status in the nuclei. Chromosome-specific centromeric probes can also be used to assess abnormal chromosome number in nuclei of once-divided cells which is indicative of chromosome malsegregation during the preceding mitosis. The detection of centromere and telomere sequences in nuclei and micronuclei may be achieved by cell or nuclei suspension hybridisation methods such as those described by Rufer et al. (1998). Thus there is the potential for identifying different classes of genetic damage.

The methods of the present invention may also comprise one or more processes for identification of cellular sub-populations using known cell surface markers. These markers can be used to identify those sub- populations of cells that are more or less susceptible to genetic damage. In a third aspect, the present invention provides a kit for quantifying micronuclei events in a cell population, the kit comprising a micronuclei standard. Micronuclei standards are preferably in the form of (a) isolated and fixed micronuclei from human or mammalian cells, (b) synthetic microbeads with the same scatter and fluorescence properties as pre-stained micronuclei, or (c) eukaryotic cells with DNA contents similar to that of micronuclei in human cells.

Preferably, the kit of the third aspect further comprises a dye for labeling cells. Preferred dyes include carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series.

In a further preferred embodiment, the kit further includes an agent for lysing cells. Preferably, the agent is a mild detergent such as Triton X100, Tween 20 or Nonidet NP40.

In a another embodiment the kit further comprises molecular probes for the specific identification of unique DNA sequences and DNA adducts in nuclei and micronuclei together with appropriate reagents for successful detection during flow cytometry.

It is envisaged that the methods of the present invention will be applicable to human peripheral blood lymphocytes and any other eukaryotic or mammalian cells. Within the context of the present invention, it is also possible to identify different sub-populations of peripheral blood lymphocytes using well characterised cell surface markers. These markers can be used to identify those sub-populations that are more or less susceptible to genetic damage. As will be appreciated by those skilled in the art, the methods of the present invention provide means for detecting genetic damage in cells. It is envisaged that these methods will have wide ranging applicability. For example, the methods of the present invention may be useful for genotoxicity testing of new pharmaceuticals, and for the biomonitoring of exposure to radiation and carcinogens in humans. Other potential applications include use of the assays in the determination of the risk of cancer, infertility or Alzheimer's disease; in tests for marine and aquatic toxicology (eg., by monitoring genetic damage in the cells of dolphins or other marine animals); in the assessment of DNA damage rate as a health indicator and in the identification of diets and dietary supplements that reduce the rate of DNA damage. The following examples are offered for illustration purposes, and are not intended to limit or define the invention in any manner.

Example 1: Methods for flow cytometric micronucleus assay utilising cell tracking dye

This procedure uses carboxyfluorescein diacetate succinimidal ester (CFSE) or an equivalent cell lineage tracking dye (e.g. PKH2) to identify cells that have carried out a single replication cycle. These cells are distinguished by having one half of the average fluorescence of the original labeled cell population (Lyons and Doherty, 1998). The once-divided cells are sorted by flow cytometry and their micronucleus content estimated by flow cytometry after cell lysis and staining with a DNA specific dye.

(i) CFSE staining

Materials β Single cell suspension of lymphoid cells (cultured WIL2 NS) in phosphate buffered saline (PBS)

• 5mM 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes LTD Eugene Oregon USA)

• RPMI 1640/10% fetal bovine serum (FBS)

• Culture medium

• Additional reagents and equipment for cell culture and harvesting

Method

Cells were resuspended at a final concentration of 5xl0^β cells/ml PBS. 2μl of 5mM CFSE was added to a millilitre of cells in a tube >6x the volume of the cells. Cells were incubated for 10 mins at 37°C. Staining was quenched by adding 5 volumes of ice cold RPMI/10% FBS and then kept on ice for 5 min. Cells were washed three times in culture medium (RPMI/2.5% FBS). After washing cells were resuspended at 2-10xl0⁵ cells/ml in culture medium and incubated for 6 hours at 37°C.

The method described above was per standard protocol (Lyons and Doherty, 1998). In this method the standard amount of dye was lOμM per 5x10^s cells. Other concentrations used were 0.1, 0.25, 0.5 and lμM. (ii) PKH2 staining

Materials

« Single cell suspension of lymphoid cells (cultured WIL2 NS) » PKH2 green fluorescent cell linker kit (Sigma Biosciences) composed of PKH2 dye stock and diluent C

• RPMI 1640/10% foetal bovine serum (FBS)

• Culture medium without serum

• serum • Additional reagents and equipment for cell culture and harvesting

Method

Cells were harvested and washed once in RPMI without serum. The loose pellet was at approximately 2xl0⁷ per sample. This was resuspended in 1 ml diluent C. Immediately prior to staining PKH2 dye was prepared by adding 2.5μl per ml Diluent C. 1 ml cells was added to 1 ml of dye preparation and incubated at RT for 5 mins with frequent inversions of the tube. The staining reaction was stopped by adding 2 ml FBS. This was incubated for a further minute before the addition of 4 ml RPMI/FBS. Cells were centrifuged for 10 min at 400g at 25°C. After removing supernatant cells were washed a further three times in complete medium before culturing in the same manner as the CFSE cells.

(Hi) Induction of micronucleus formation with hydrogen peroxide

Materials β Single cell suspension of WIL2 NS in culture. These cells were divided into CFSE, PKH2 or unstained. • Hydrogen peroxide (H₂0₂) (Sigma)

Method

H₂0₂ was added to cell culture flasks to achieve a concentration of 30uM. Incubation was for 6h before harvesting. (iv) Sorting first generation of CFSE stained cells

After staining with CFSE, WIL2 NS cells were cultured for 6 hours and were resuspended in a small volume of culture medium (about 2 ml). 20μl of propidium iodide (lmg/ml) (Sigma) or 20μl of 7-aminoactinomycin D (7- AAD) (lmg/ml) (Sigma) was added to the tube containing the cells. The mixture was filtered through nylon mesh prior to sorting.

Cells were run through the Epics Elite Flow Cytometer using the 488- nm argon laser. The sort gate was set by the following criteria:

• Propidium iodide (PI) or 7-AAD negative cells. This procedure (a) distinguishes between dead and necrotic cells which take up the PI or 7-

AAD dyes and viable cells which do not and (b) eliminates the presence of dead and necrotic cells that will contribute DNA fragments indistinguishable from micronuclei in subsequent analyses (Figure 5 a, Step 1) «» Light scattering characteristics expected for viable mononuclear cells. The light scatter further distinguishes live cells from necrotic cells, cellular debris and aggregated cells (Figure 5b, Step 2).

• Lower CFSE green fluorescence peak which represents 1st generation of cells because first generation cells have half of the average fluorescence of undivided cells (Figure 5c, Step 3).

Approximately lxlO⁶ cells were collected into culture medium.

(v) Micronucleus assay on first generation cells.

Materials

• 70% ethanol

• PBS

• Cells (1st generation sorted or pre-treated with mitomycin C) • Propidium iodide (PI) staining solution (10ml PBS containing 0.1%

Triton X-100, 5μl lmg/ml propidium iodide with 2 mg RNase A) » Alternative DNA stain - Hoechst 33342 (Molecular Probes) staining solution (10ml PBS containing 0.1% Triton x-100, 5μl 1 mg/ml Hoechst

33342) • Flow cytometer equipped with 488-nm argon laser or, if Hoechst 33342 dye is used, a UN laser • Additional reagents and equipment for preparing cell suspensions

Method

Cells to be analysed were collected in centrifuge tubes and were centrifuged. Approximately 10^β-10⁷ cells were used per test. Cell pellets were resuspended in PBS and washed in at least 5ml. After discarding supernatant each cell pellet was resuspended in 0.5ml PBS. 4.5ml ice cold 70% ethanol was added to each tube. Cells suspended in 70% ethanol can be kept at 0° to -40°C for several months. Fixation occurs after 2 hours. Ethanol was removed by centrifugation and aspiration of supernatant.

The cell pellet was resuspended in PBS, incubated for 1 min at 22°C, then washed once. Cells were resuspended in 1 ml of either propidium iodide or Hoechst 33342 staining solution. Cells in propidium iodide were incubated for 15 mins at 37°C in the dark and cells in Hoechst solution were incubated for at least 30 mins at RT in the dark.

Ribonuclease (RNAse A) was used with propidium iodide staining to avoid staining of RNA. Hoechst 33342 does not stain RNA and therefore ribonuclease treatment is not required when using this dye.

The fluorescence signals from propidium iodide and CFSE (or similar green fluorescent lineage tracking dye) have some spectral overlap and electronic or software compensation is preferably applied to remove the contribution of CFSE fluorescence to the propidium iodide signal. The fluorescence signals from CFSE and Hoechst 33342 and related dyes overlap completely and optical and/or electronic procedures are preferably in place if the two fluorochromes are to be measured simultaneously.

The parameters set on the flow cytometer were as follows:

Log forward scatter Log side scatter

Linear DNA (Hoechst 33342 or PI)

DNA peak (Hoechst 33342 or PI)

Log DNA (Hoechst 33342 or PI)

Time (to ensure DNA signal stability) Data was collected for 5 mins for each sample. Estimation of micronuclei was performed in the Rl population selected after gating to exclude debris and doublets (Figure 5d, Step 4). The micronucleus (MN) population (R2) is defined as those events in Rl having fluorescence between one tenth and one hundreth of the fluorescence of the main nuclear DNA population (R3) (Figure 5e, Step 5). The micronucleus (MN) frequency was determined by the ratio of micronucleus events (R2) relative to the number of events in the main nuclear DNA peak (R3). Figure 5e shows results for both control and H₂0₂-treated cultures demonstrating a six-fold increase in MN frequency in the latter relative to control.

Doublet discrimination may be carried out during signal acquisition to eliminate aggregated nuclei and micronuclei that will interfere with estimations of statistics. This may be accomplished by the well established procedure whereby the linear integrated DNA fluorescence signal is plotted against the peak height fluorescence signal. A diagonal gate defining the main population of events, eliminating any events that lie off the diagonal, excludes doublets and higher order aggregates from the analysis.

Alternatively, the ratio of integrated signal to peak height signal can be set as a parameter and plotted against integrated signal. Doublets and higher aggregates form clearly defined populations in this plot and can be gated out of subsequent analyses. These techniques would be familiar to any person skilled in the art and variations of the technique must be applied in the case of those cytometer apparatus that may collect different signal parameters. For example, fluorescence signal peak width may be substituted for integrated fluorescence. The use of specialised electronic signal processing techniques such as Pulse Pile-Up discrimination and similar pulse analysis is also included as a contributory doublet discrimination method.

Example 2: Method for Micronucleus assay on supravitally stained cells treated with Cytochalasin B

This procedure uses cytochalasin B to accumulate once-divided cells as binucleated cells. These cells are distinguished by their nuclear content which is tetraploid or greater than tetraploid. These once-divided cells are sorted by flow cytometry and their micronucleus content estimated by flow cytometry after cell lysis. Materials

• Single cell suspension of lymphoid cells (cultured WIL2 NS) o RPMI 1640/10% foetal bovine serum (FBS)

• Cytochalasin B (Sigma) at 0. lmg/ml in DMSO Hoechst 33342 (Molecular Probes)

*» Additional reagents and equipment for cell culture and harvesting

• 70% ethanol β PBS

• DNA stain - Hoechst 33342 (Molecular Probes) staining solution (10ml PBS containing 0.1% Triton x-100, 5μl 1 mg/ml Hoechst 33342)

• Flow cytometer with UN light illumination source

Method

Supravital staining of DΝA offers the possibility of sorting live cells on the basis of differences in their DΝA content. WIL2 ΝS cells were suspended at 1 x 10^β cells/ml RPMI/FBS. Cytochalasin B at 4.5 μg/ml medium was added to the culture. The cells were incubated at 37°C for 24h. The next day Hoechst 33342 was added at 2μg/ml medium and cells were incubated at 37°C for a further 20 mins. The cells were harvested, resuspended in a small volume and sorted on the flow cytometer on the basis of light scattering properties and tetraploid DΝA as seen by in the linear DΝA plot (see Figure 6A,B).

The sorted cells were then fixed overnight at -20°C in 70% ethanol. The next day cells were washed in PBS and treated with DΝA stain for 30 mins. The sorted and lysed cells were then analysed for the ratio of micronuclei (MΝ) and nuclei (ΝΝ) (figure 6C) as previously described (Example 1, section (v)). MΝ frequency is expressed as MΝ/ΝΝ as previously. Example 3: Micronucleus assay on unsorted CFSE stained cells

In this method the number of once-divided cells and the number of micronuclei are determined separately.

Mateήals

• Single cell suspension of lymphoid cells (cultured WIL2 NS) in phosphate buffered saline (PBS)

• 5mM 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes LTD Eugene Oregon USA) • RPMI 1640/10% fetal bovine serum (FBS)

• RPMI 1640/2.5% fetal bovine serum (FBS)

• Culture medium

• Additional reagents and equipment for cell culture and harvesting

• 70% ethanol « PBS

• Propidium iodide (PI) staining solution (10ml PBS containing 0.1% Triton X-100, 5μl lmg/ml propidium iodide with 2 mg RNase A)

Method Cells were stained as previously and incubated at 37°C for 6 hours.

After incubation they were fixed overnight in 70% ethanol at -20°C. The next day the cells were washed in PBS and resuspended in PI staining solution for

15 mins at 37°C. Analysis was performed on flow cytometer with 488nm argon laser. The following parameters were collected:

Log forward scatter

Log side scatter

Log CFSE

Linear DNA (PI) DNA peak (PI)

Log DNA (PI)

Time (to ensure DNA signal stability)

Doublet discrimination (figure 7 A) was performed as previously described. The number of 1st generation nuclei were estimated from the CFSE histogram (Figure 7B). Total micronuclei were estimated from the scattergram (log PI vs log side scatter) (Figure 7C).

All publications referred to in the description above are incorporated in their entirety herein by this reference.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

REFERENCES

Albertini R.J., Anderson D., Douglas G.R., Hagmar L., Hemminki K., Merlo F., Natarajan A.T., Norrpa H„ Shuker D.E., Tice R., Waters M.D., Aitio A. (2000) IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans, (in press).

Fenech M. (2000)a. The in vitro micronucleus technique. Mutation Res. (in press).

Fenech M. (2000)b. Mathematical model of in vitro micronucleus assay predicts false negative results if micronuclei are not scored specifically in binucleated cells or cells that have completed one nuclear division. Mutagenesis (in press) .

Fenech M. Aitken C, Rinaldi J.(1998) Folate, vitamin B12, homocysteine status and DNA damage in young Australian adults. Carcinogenesis 19(7) 1329-1336 - Accelerated paper.

Fenech M. and Morley A. A., 1985. Measurement of micronuclei in human lymphocytes. Mutation. Res. 148:29-36

Fenech M., Holland N., Chang W.P., Zeiger E. and Bonassi S. 1999a The HUMN Project - An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutation Res. 428: 271-283.

Fenech M., Crott J., Turner J. and Brown S., 1999b. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide. Mutagenesis 14(6):605-612

Kirsch-Volders M., Sofuni T., Aardema M., Albertini S., Eastmond D., Fenech M., Ishidate M., Lorge E., Norrpa H., Surrales J., von der Hude W., Wakata A., 2000. Report from the in vitro micronucleus assay working group. International Workshop on Genotoxocity Test Procedures (Washington DC, March 1999) Mutation Res. (in press).

Lyons, A.B. and Doherty, K.V, 1998. Current Protocols in Cytometry. John Wiley & Sons. Inc. 9.11.1-9.11.9

Lyons A.B. 2000. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Imm. Methods. 243: 147- 154.

Migliore L., Botto N., Scarpato R., Petrozzi L., Cipriani G., and Bonucelli U., (1999) Preferential occurrence of chromosme 21 malsegregation inperipheral blood lymphocytes of Alzheimer's disease patients. Cytogen. Cell Genet. 87: 41-46.

Nusse M. and Marx K. (1997) Flow cytometric analysis of micronuclei in cell cultures and human lymphocytes: advantages and disadvantages. Mutation Res. 109-115.

Rothfus A. , Schutz P., Bochum S., Volm T., Eberhardt E., K eienberg R., Vogel W., Speit G. (2000) Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of BRCAl mutation in breast cancer families. Cancer Res. 60:390-394.

Rufer N., Dragowska W., Thornbury G., Rossnek E., Lansdorp P.M. (1998) Telomere length dynamics in human lymphocyte sub-populations measured by flow cytometry. Nature Biotechnology 16: 743-747.

Scott D., Barber J.B.P., Levine E.L., Burrill W., Roberts SA (1998) Radiation- induced micronucleus induction in lymphocytes identifies a high frequency of radiosensitive cases among breast cancer patients: a test for predisposition? Br. J. Cancer 77(4): 614-620.

Shapiro H.M.(1994) Practical Flow Cytometry, 3rd Ed., Wiley-Liss New York

Claims

Claims:

1. A method for quantifying micronuclei events in a cell population, the method comprising: (i) incubating a population of cells for a sufficient time to allow a substantial portion of the cells to complete at least one cellular division;

(v) correcting the frequency determined in step (iv) for the proportion of once divided cells as determined in step (ii).

2. A method as claimed in claim 1 in which the method further comprises labelling the cells with a dye prior to step (i).

3. A method as claimed in claim 2 in which the dye is a fluorescent dye.

4. A method as claimed in claim 3 in which the fluorescent dye is selected from the group consisting of carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series.

5. A method as claimed in claim 1 in which step (i) involves incubating the cells in the presence of a cytokinesis blocking agent.

6. A method as claimed in claim 5 in which the cytokinesis blocking agent is a cytochalasin.

7. A method as claimed in claim 6 in which the cytochalasin is cytochalasin-B.

8. A method as claimed in any one of claims 1 to 7 in which the proportion of once divided cells in step (ii) is determined by flow cytometry.

9. A method as claimed in any one of claims 1 to 8 in which the nuclei and micronuclei in step (iii) are analysed by flow cytometry.

10. A method as claimed in claim 9 in which the nuclei and micronuclei are stained with a dye prior to flow cytometry.

11. A method as claimed in claim 10 in which the dye is propidium iodide, Hoechst 33342 or 4'-6'-diamino-2-phenylindole (DAPI).

12. A method for quantifying micronuclei events in a cell population, the method comprising:

13. A method as claimed in claim 12 in which the method further comprises labelling the cells with a dye prior to step (i).

14. A method as claimed in claim 12 in which the dye is a fluorescent dye.

15. A method as claimed in claim 14 in which the fluorescent dye is selected from the group consisting of carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series.

16. A method as claimed in claim 12 in which step (i) involves incubating the cells in the presence of a cytokinesis blocking agent.

17. A method as claimed in claim 16 in which the cytokinesis blocking agent is a cytochalasin.

18. A method as claimed in claim 17 in which the cytochalasin is cytochalasin-B.

19. A method as claimed in any one of claims 12 to 18 in which the cells are sorted in step (ii) by flow cytometry.

20. A method as claimed in any one of claims 12 to 19 in which the nuclei and micronuclei in step (iv) are analysed by flow cytometry.

21. A method as claimed in claim 20 in which the nuclei and micronuclei are stained with a dye prior to flow cytometry.

22. A method as claimed in claim 21 in which the dye is propidium iodide, Hoechst 33342 or 4'-6'-diamino-2-ρhenylindole (DAPI).

23. A kit for quantifying micronuclei events in a cell population, the kit comprising a micronuclei standard.

24. A kit as claimed in claim 23, the kit further comprising a dye for labeling cells and/or an agent for lysing cells.