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Alok Dhawan and Mahima Bajpayee (eds.), Genotoxicity Assessment: Methods and Protocols, Methods in Molecular Biology, vol. 1044, DOI 10.1007/978-1-62703-529-3_12, © Springer Science+Business Media New York 2013

Chapter 12

Fluorescence In Situ Hybridization (FISH) Technique for the Micronucleus Test

Ilse Decordier and Micheline Kirsch-Volders

Abstract

In recent years, cytogenetics in combination with molecular methods has made rapid progress, resulting in new molecular cytogenetic methodologies such as fl uorescence in situ hybridization (FISH). FISH is a molecular cytogenetic technique used for the detection of specifi c chromosomal rearrangements and appli-cable to many different specimen types. It uses fl uorescently labeled DNA probes complementary to regions of individual chromosomes. These labeled DNA segments hybridize with the cytological targets in the sample and can be visualized by fl uorescence microscopy in interphase nuclei or on metaphase chro-mosomes. Here, we describe the FISH methodology with centromeric probes for human cells, which is used in combination with the cytokinesis-block micronucleus assay and which allows discrimination between mutagens inducing DNA breakage (clastogens) or chromosome loss (aneugens).

Key words DNA probe , Hybridization , Immunodetection , Micronucleus , Clastogens , Aneugens

1 Introduction

Fluorescence in situ hybridization (FISH) with DNA probes allows the visualization of defi ned nucleic acid sequences in particular cellular or chromosomal sites by hybridization of complementary fl uorescently labeled probe sequences within intact metaphase or interphase cells. FISH is used for various purposes, including anal-ysis of chromosomal damage, gene mapping, clinical diagnostics, molecular toxicology, and cross-species chromosome homology. FISH is usually applied to standard cytogenetic preparations on microscope slides, but it can be used on slides of formalin-fi xed tissue, blood or bone marrow smears, and directly fi xed cells or other nuclear isolates. The basic principle of the method is that single-stranded DNA will bind or anneal to its complementary DNA sequence. Thus, a DNA probe for a specifi c chromosomal region will recognize and hybridize to its complementary sequence on a metaphase chromosome or within an interphase nucleus. Both have to be in single-strand conformation; therefore the DNA

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probe and the target DNA must be denatured (Fig. 1 ). Typical probe length is between 250 bp and 1 kb, depending on the application.

The standard FISH protocol includes the following steps, each of which is crucial for obtaining a successful FISH result: sample preparation and fi xation, denaturation of probe and sample, hybridization of a fl uorescently labeled probe to sample (anneal-ing), post-hybridization washing, and detection. The probe signal can then be seen through a fl uorescent microscope and the sample DNA can be scored for the presence or the absence of the signal. There are many types of probes which can be used for in situ hybridization. For molecular cytogenesis, three different types of probes are generally used: unique sequence probes which bind to single-copy DNA sequences in a specifi c chromosomal region or gene; probes for repetitive DNA sequences, centromeres (alpha satellite DNA), or telomeric sequences; and whole chromosome paints which are cocktails of unique sequence probes that recognize the unique sequences spanning the length of a particular chromo-some. Previously, probes could be labeled by the researcher either directly with nucleotides coupled to a fl uorochrome or indirectly with nucleotides coupled to a reporter molecule, which subse-quently can be detected by conventional immunochemical meth-ods. Nowadays, FISH is usually performed with pre-labeled commercially available probes.

Fig. 1 Schematic illustration of the fl uorescence in situ hybridization principle (adapted from Rautenstrauβ and Liehr, 2002)

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A widely applied application of the FISH in genetic toxicology is its combination with the in vitro cytochalasin-B block micronu-cleus (CBMN) methodology that allows discrimination between mutagens inducing DNA breakage (clastogens) or chromosome loss (aneugens) (Fig. 2 ). Combination of these methodologies allows the characterization of the genetic contents of MN, thereby distin-guishing between MN originating from chromosome loss or break-age and determining the involvement of specifi c chromosomes and chromosome fragments in MN formation [ 1 ]. The simultaneous use of the CBMN assay and FISH allows achievement of a higher sensitivity for the adequate hazard assessment of mutagens and a better understanding of the biological mechanisms involved and the possibility to address thresholds for induction of aneuploidy versus clastogenicity. In addition, FISH with centromeric chromo-some-specifi c probes also allows an accurate analysis of nondisjunc-tion (unequal distribution of unique homologous chromosome pairs in the daughter nuclei Fig. 2 ). This is very helpful to perform risk assessment of compounds with threshold type of dose–responses. FISH with centromeric and chromosome- specifi c centromeric probes has been widely applied in genetic toxicology to elucidate the genotoxic potential of various mutagenic compounds and for the detection of chromosome damage induced in vitro and in vivo by chemical and physical agents (reviewed in refs. 2 , 3 ).

Fig. 2 Detection of chromosome loss and chromosome nondisjunction in the cytokinesis-block micronucleus assay combined with FISH

Pancentromericprobe

Chromosomespecific probe

Chromosomespecific probe

Pancentromericprobe

Chromosomebreakage

Chromosomenon-disjunction

Chromosome loss Chromosome loss

Chromosome loss

Chromosomebreakage

Chromosome loss

Chromosomenon-disjunction

+ cytochalasin B - cytochalasin B

Add aneugen or clastogen

FISHFISH

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Our laboratory used the MN assay in combination with FISH for the in vitro demonstration of thresholds for microtubule inhibitors, aneugenic compounds binding specifi cally to alpha- tubulin and inhibiting tubulin polymerization such as nocodazole, a chemother-apeutic drug, or carbendazim, a pesticide [ 4 , 5 ]. The two endpoints refl ecting aneuploidy were studied in vitro in human lymphocytes: chromosome loss and nondisjunction. To assess chromosome loss, the detection of centromere-positive versus centromere- negative MN by FISH with a general alphoidcentromeric probe was performed on cytochalasin-B-blocked binucleates, resulting from cultures exposed to the spindle poisons. For chromosome nondis-junction, the same compounds were investigated on cytokinesis-blocked binucleated lymphocytes in combination with FISH using chromosome-specifi c centromeric probes for chromosome 1 and chromosome 17. This allowed the accurate evaluation of nondis-junction, since artifacts were excluded from the analysis, as only binucleates with the correct number of hybridization signals were taken into account. We demonstrated dose dependency of the aneu-genic effects and the existence of thresholds for the induction of chromosome nondisjunction and chromosome loss by these spindle inhibitors (lower for nondisjunction than for chromosome loss).

Recently, Lindberg et al. [ 6 ] used directly labeled pan- centromeric and pan-telomeric DNA probes to assess the content of MN in cultured binucleated lymphocytes of unexposed, healthy subjects. The authors suggested the combined centromeric and telomeric FISH methodology as a practical method to enhance the specifi city of the MN assay allowing detection of MN harboring terminal/interstitial fragments, acentric/centric fragments, chromatid- type/chromosome-type fragments, and entire chroma-tids/chromosomes. The specifi city of the assay should further be evaluated in vitro using model mutagens such as inducers of chro-matid breaks and exchanges, chromosome breaks, and aneugens.

In conclusion, the MN assay combined with FISH allows to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. It permits to discriminate between aneugenic and clastogenic effects and has been applied in many studies elucidating mecha-nisms of genomic instability and mode of action of various geno-toxic agents.

2 Materials

1. 20× Saline-sodium citrate buffer (SSC): 3 M NaCl, 0.3 M sodium citrate, pH 7, fi lter through Whatman 41 fi lter and store at room temperature.

2. RNase A: 100 μg/mL RNAse A in 2× SSC. Aliquot the stock RNAse A solution in volumes of 500 μL and store at −20 °C.

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3. Pepsin: 40 units/mL pepsin prepared in 10 mM HCl, aliquot stock in volumes of 50 μL and store at −20 °C.

4. Ethanol: 100 % ethanol, also prepare 50 and 75 % dilution series with water.

5. 2 μg/mL DAPI in antifade mounting medium and/or 5 μg/mL ethidium bromide in antifade medium.

6. 0.075 M KCl. 7. Fixative: 3:1 methanol/acetic acid mixture. Freshly prepared

just before use. 8. Formaldehyde. 9. Fluorescence microscope, fi lters, and optional triple-band-pass

fi lter. 10. Glass slides. 11. Plastic coverslips for incubation and hybridization steps. 12. Heat block. 13. Coplin jars for washing steps.

3 Methods

1. Transfer cells into 15 mL Falcon tubes ( see Note 1 ). 2. Centrifuge cell suspension at 129.5 × g for 8 min at room

temperature. 3. Discard supernatant with water pump till only 500 μL remains. 4. Hypotonic shock: Add 3 mL of cold (4 °C) 0.075 M KCl to

the cells on vortex (1000 rpm) or with dispenser. 5. Immediately centrifuge at 129.5 × g for 8 min at room

temperature. 6. Discard supernatant with water pump till 500 μL remain, and

resuspend by patting. 7. Fixation: Add the volume of a full Pasteur pipette of 3:1 freshly

prepared methanol/acetic acid mixture, drop by drop (slowly at the start, a little faster afterwards), on vortex (1,000 rpm) to the cell suspension, followed by four drops of formaldehyde ( see Note 2 ).

8. Centrifuge at 129.5 × g for 8 min at room temperature. 9. Discard supernatant with water pump till 500 μL of supernatant

remains, and resuspend by patting. 10. Repeat fi xation twice (slowly at the start, a little faster after-

wards) without formaldehyde (if necessary for time manage-ment, tubes can rest overnight after second fi xation).

11. Centrifuge the cells at 129.5 × g for 8 min at room temperature.

3.1 Sample Preparation and Fixation

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12. Discard the supernatant till 100–200 μL of the supernatant (dependant on pellet size) remains.

13. Resuspend the pellet in 600 μL of methanol/acetic acid. 14. Drop the fi xed cells onto slides using a Pasteur pipette, leave to

dry (overnight), and store at −20 °C if not immediately used.

1. Incubate the slides in a 100 mL coplin jar with 0.05 % RNase A solution in 2× SSC buffer for 60 min at 37 °C ( see Note 4 ).

2. Wash the slides twice in 2× SSC for 5 min at room temperature in a coplin jar (100 mL).

3. Incubate the slides with 0.005 % pepsin prepared in 10 mM HCl for 10 min at 37 °C in a coplin jar (100 mL) ( see Note 5 ).

4. Wash the slides in 1× PBS for 5 min at room temperature in a coplin jar.

5. Dehydrate the slides in an ethanol series: 50 %, then 75 %, and then fi nally in 100 % ethanol, in a coplin jar, for 5 min each at room temperature.

6. Air-dry the slides.

1. Put the probe, at the volume cited by the supplier, onto the slide and cover with a coverslip.

2. Denature the probe and sample (cells on the slides) simultane-ously for 4 min at 90 °C on a heat block.

3. Hybridize at 37 °C overnight in a humidity chamber ( see Note 7 ).

1. Wash the slides in 2× SSC at room temperature in a coplin jar (100 mL) to remove the coverslip ( see Note 11 ).

2. Wash the slides in 1× PBS for 5 min at room temperature in a coplin jar.

3. Dehydrate slides in an ethanol series: 50 %, 75 %, and then 100 % ethanol, in a coplin jar, for 5 min each at room temperature.

4. Counterstain the slides either with two to three drops of 5 μg/mL ethidium bromide in antifade mounting medium or with two to three drops of 2 μg/mL DAPI in antifade mounting medium and cover with a coverslip ( see Note 12 ).

5. Leave the slides for at least 1 h at 4 °C before analyzing.

1. Analyze the slides with a fl uorescence microscope. 2. Per culture and per concentration at least 500 cytochalasin-B-

blocked binucleated cells are scored with a maximum of 1,000 binucleate cells per culture.

3. The standard scoring criteria for MN (1/3 diameter, no overlap, shape) are used [ 7 ].

3.2 Slide Preparation for FISH ( See Note 3 )

3.3 Hybridization ( See Note 6 )

3.4 Post- hybridization Wash and Counterstaining ( See Notes 8 – 10 )

3.5 Analysis ( See Notes 13 and 14 )

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4. The MN in binucleated cells are examined for the presence of one or more spots and classifi ed as centromere positive (MNCen+) or centromere negative (MNCen−), the latter showing no centromeres [ 5 , 8 ].

5. For chromosomal nondisjunction, the scoring is restricted to binucleated cells having the diploid number for the analyzed chromosomes (two spots in for each of the two probes) to avoid artifacts in scoring of cells where the staining is not accu-rate. The distribution of the signals for both probes between the binucleated cells is scored as 2/2, 1/3, and 0/4. The events involving in both chromosomes analyzed are scored independently of each other but recorded in parallel per cell.

4 Notes

1. FISH technique for the micronucleus test can be performed in vitro in lymphocytes and cell lines. In this assay using human lymphocytes, the cells are cultured in the presence of phytohe-magglutinin to stimulate mitosis. After 24 h of stimulation, the test compound is added for an appropriate time. At 44 h of culture, cytochalasin-B is added and at 72 h the cells are har-vested. In case of cell lines cytochalasin-B should be added during the fi rst cell cycle following the start of the treatment and the cells should be harvested prior to the second mitosis. For cell lines in suspension, cells are allowed to grow for 24 h. After 24 h, the medium is replaced with fresh medium and the test compound is added; cytochalasin-B should be added dur-ing the fi rst cell cycle following the start of the treatment and the cells should be harvested prior to the second mitosis. For adherent cell lines, cells are seeded into 25 cm 2 fl asks and are allowed to attach for 24 h. After 24 h, the medium is replaced with fresh medium and the test compound is added; cytocha-lasin- B should be added during the fi rst cell cycle following the start of the treatment and the cells should be harvested prior to the second mitosis by trypsinization. A detailed protocol and a summary of the key steps in preparation of lymphocytes/cell lines for the cytokinesis-block assay can be found in [ 1 ].

2. Formaldehyde in the fi xation is used for the preservation of the general organelle structure of the cell.

3. The protocol described above can be applied to human lym-phocytes/cell lines. Depending on the type of cells used, the concentrations of RNase and pepsin may differ and should be tested to obtain an optimal penetration of the probe.

4. RNase treatment serves to remove endogenous RNA and may improve the signal-to-noise ratio in DNA–DNA hybridizations.

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5. Pepsin can signifi cantly improve probe penetration and serves to increase target accessibility by digesting the protein that surrounds the target nucleic acid.

6. From the hybridization step on, protect the slides and probes from light, as the probes are fl uorescently labeled.

7. Prepare a humidity chamber by inserting a piece of Whatman paper on the bottom of a beaker and tissue papers at the sides, soak them with water, put a coplin jar with the slides in hori-zontal position into the beaker, cover with parafi lm before putting into the incubator, and protect from the light.

8. The protocol described here is a standard protocol for FISH. Depending on the brand and supplier of the probe of interest used, the protocol can be modifi ed as indicated by the supplier. This is especially the case for the post-hybridization washing.

9. Do not allow the slides to dry out at any time during the wash or the detection procedures.

10. The temperature and buffer concentrations (stringency) of hybridization and washing are important steps, as lower strin-gency may result in nonspecifi c bonding of the probe to other sequences, and higher stringency may result in a lack of signal.

11. Coverslips should fall off during this wash. If not, gently help them to fall off with a pincet.

12. Be careful when putting the coverslip and avoid air bubbles in order to have a clear image under the microscope.

13. The probes will display a single fl uorescent spot at the location of the centromere of the chromosome.

14. If many aspecifi c signals are observed, include one or more post-hybridization washing steps.

References

1. Kirsch-Volders M, Plas G, Elhajouji A et al (2011) The in vitro MN assay in 2011: origin and fate, biological signifi cance, protocols, high throughput methodologies and toxicological relevance. Arch Toxicol 85:873–899

2. Norppa H, Falck GC (2003) What do human micronuclei contain? Mutagenesis 18:221–233

3. Hovhannisyan GG (2010) Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicol-ogy. Mol Cytogenet 3:1–11

4. Elhajouji A, Van Hummelen P, Kirsch-Volders M (1995) Indicationsfor a threshold of chemically- induced aneuploidy in vitro inhu-man lymphocytes. Environ Mol Mutagen 26:292–304

5. Elhajouji A, Tibaldi F, Kirsch-Volders M (1997) Indication forthresholds of chromosome non- disjunction versus chromosomelagging induced

by spindle inhibitors in vitro in humanlympho-cytes. Mutagenesis 12:133–140

6. Lindberg HK, Falck GC, Jarventaus H et al (2008) Characterizationof chromosomes and chromosomal fragments in humanlymphocyte micronuclei by telomeric and centromeric FISH. Mutagenesis 23:371–376

7. Fenech M, Chang WP, Kirsch-Volders M et al (2003) Human MicronNucleus project. HUMN project: detailed description of the scoring criteria for thecytokinesis-block micro-nucleus assay using isolated human lymphocyte cultures. Mutat Res 534:65–75

8. Kirsch-Volders M, Elhajouji A, Cundari E et al (1997) The in vitro micronucleus test: a multi- endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chro-mosome loss and non-disjunction. Mutat Res 392:19–30

Ilse Decordier and Micheline Kirsch-Volders