CARYOLOGIA

*Corresponding author: phone: +421908241672; fax: 055/6711674; e-mail: galdikova@uvm.sk

Use of FISH technique with whole chromosome painting probes for the detection of chromosomal aberrations induced by positive mutagens in bovine peripheral lymphocytes

Galdíková* Martina, Katarína Šiviková, Beáta Holečková, Ján Dianovský and Martin Mesarč

University of Veterinary Medicine and Pharmacy, Department of Veterinary Genetics, Komenského 73, 04181 Košice, Slovak Republic, Europe.

Abstract — The effects of mitomycin C (MMC) and ethyl methanesulfonate (EMS) on the frequency of struc-tural and numerical chromosomal aberrations were evaluated in bovine peripheral lymphocytes in vitro using fluorescence in situ hybridization (FISH) technique. Three whole chromosome painting probes (WCPs), BTA 1 for the bovine chromosome 1 (red colour), BTA 5 for chromosome 5 (green colour) and BTA 7 for chromo-some 7 (red colour) were used in experiments. Under conditions of our experiment, mostly unstable structural aberrations were observed. The detection of unstable chromosomal aberrations of the type of chromatid and chromosome breaks, and chromatid exchanges was statistically significant (p<0,001) that corresponded to the results of conventional cytogenetic analysis. Several cells with polyploidy and aneuploidy were also detected; the damage was preferentially associated with the bovine chromosome 5. Chromosomal instability and aneuploidy represent two critical steps in the process of carcinogenesis.

Key words: Bovine peripheral lymphocytes, chromosome aberrations, ethyl methanesulfonate, mitomycin C, fluorescence in situ hybridization, whole chromosome painting probes.

Vol. 64, no. 4: 427-434, 2011

INTRODUCTION

Chromosome aberrations, sister chromatid exchanges and micronuclei are widely used in genotoxic/cytogenetic studies. Cytogenetic bi-omarkers provide the information about the environmental contamination with chemical agents that could be useful in the assessment of genetic risk increase for humans. Primary at-tention is focused on chromosome aberrations (CAs) that were recognised as a biomarker of the effect; they have been standardized and validat-ed in relation to the cancer induction (Albertini et al. 2000; bonAssi et al. 2000). CAs were also described in association with the appearance of developmental failures in offspring of animals or

in their foetuses during pregnancy (VAnroose et al. 2000), and in human new-borns, as well (nA-tArAjAn 2002).

Mitomycin C (MMC) and ethyl methanesul-fonate (EMS) are well known alkylating agents used for many years in mutagenic and recombi-nation studies as positive mutagens (sAnderson et al. 1996; lundin et al. 2005). MMC acts by the formation of DNA interstrand cross-links pref-erentially (Verweij and Pinedo 1990), which, when unrepaired, left to the replication block in mitosis and subsequently to induction of cyto-toxicity (Abdel-HAlim et al. 2005). Ethyl meth-anesulfonate (EMS) as a monofunctional ethyl-ating agent is known to induce DNA damage by a direct mechanism (Gocke et al. 2009).

Biological effects after the exposure to MMC were extensively demonstrated in mammalian cells or in microorganisms; e.g., selective inhi-bition of DNA synthesis, recombination, and DNA repair were studied in bacteria, chromo-some damage, and induction of sister chromatid

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exchanges were demonstrated in human lym-phocytes (Giri and cHAtterjee 1998; milić and koPjAr 2004).

On the chromosome level, the induction of micronuclei after the treatment with different agents, namely pesticides, was the most fre-quently examined. Using an antikinetochore antibody technique, cHAnnArAyAPPA and onG (1992) reported clastogenic and aneuploidog-enic effects of MMC in Chinese hamster V79 cells after cytochalasin B-blocked mitotic divi-sion. Similarly, increases in the frequency of mi-cronuclei in mouse splenocytes were described by benninG et al. (1992) and in lacZ transgenic mice in vivo by suzuki et al. (1993). kAtoH et al. (1981) reported elevated frequency of dominant lethal mutations in early spermatids of mice. The persistence of high frequency of chromosome aberrations after the treatment with MMC was found in mouse bone marrow cells (russo et al. 1992).

Numerous studies were concerned with EMS toxicity (GonzAles-beltrAn and morAlez-rAm-irez 2003; wAGner et al. 2003). Genotoxic ef-fects were observed in the EMS-exposed virus-es/phages, bacterial, fungal, plant, insect, and mammalian cells. Treatment with the mutagen during embryogenesis caused a reduction of foe-tal weight, and induction of malformations in rat and mouse offspring (PlAtzek et al. 1995).

We report here the induction of chromosome aberrations after the exposure to both positive mutagens, MMC and EMS, in bovine periph-eral lymphocytes in vitro using conventional chromosome analysis and fluorescence in situ

hybridizations (FISH) technique after the ap-plication of three bovine fluorescent-labelled whole chromosome painting probes (WCP) - BTA1, BTA5 and BTA7 complementarily to the corresponding bovine chromosome. Conven-tional cytogenetic analysis was performed with the purpose to verify the optimal time of expo-sure to detect the higher frequency of induced chromosomal damage in bovine peripheral lym-phocytes. The FISH seems to be a promising method making possible to detect chromosomal aberrations more precisely than those seen at the conventional cytogenetic analysis. The purpose of our study was applying the FISH technique for detection of stable and/or unstable structural chromosomal aberrations, and aneuploidy/poly-ploidy induced by positive mutagens in bovine peripheral lymphocytes, as well.

MATERIALS AND METHODS

Cell cultivation - 0,5 ml of heparinised whole blood of 2 healthy young bulls (Slovak spot-ted cattle, 6-8 months old) was added to 5 ml of chromosome medium RPMI 1640 supple-mented with L- glutamine, 15 µmol/L HEPES (Sigma, St. Louis, MO, USA), 15% foetal calf serum (BOFES, Sigma, Chemical Co. St. Louis, MO, USA), antibiotics (penicillin 250 U/mL and streptomycin 250 µg/mL and phytohaemag-glutinin (PHA, 180 µg/mL, Welcome, Dartford, England). Lymphocyte cultures were incubated at 37oC for 72 h. Mitomycin C (MMC, Sigma, St. Louis, MO, USA, 0,4 µg/mL), and ethyl meth-

tAble 1 — Chromosomal aberrations in bovine peripheral lymphocytes detected by the conventional chromosomal analysis and induced by EMS and MMC.

DoseTypes of chromosomal

aberration % breaks(± SD )

% aberrantcells

% MIG CB IB CE IE

Control 4 2 - - - 1.0 ± 0.10 1.0 ± 0.10 7.7

24 h Treatment

EMS ( 250 µg/mL) 14 40 - 18 - 38.0 ± 0.79*** 24.0 ± 0.43*** 3.5***

MMC (0,4µg/mL) 10 18 - - - 9.0 ± 0.28** 9.0 ± 0.28** 6.6a

48 h Treatment

EMS ( 250 µg/mL) 8 14 - - - 7.0 ± 0.25* 7.0 ± 0.25* 6.5a

MMC (0,4µg/mL) 16 32 - 2 - 18.0 ± 0.43*** 16.0 ± 0.37*** 4.4**

CB, IB - chromatid, isochromatid breaks, CE, IE – chromatid, isochromatid exchanges 200 metaphases were examined in each control and experimental groups (EMS and MMC after 24h and after 48 h of treatment). a - statistically non significant data. * (p<0,05),** (p<0,01), *** (p<0,001), c2 test.

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anesulfonate (EMS, Sigma, St. Louis, MO, USA) at a concentration of 250 µg/mL, were added to the cultures for 48 and 24 hours of the incuba-tion. One and half hour before the end of cul-tivation, colchicine (Merck, Darmstadt, Germa-ny) was added at the final concentration of 5 µg/mL. For the standard cytogenetic analysis the slides were stained with Giemsa solution. One hundred well-spread metaphases were analysed for the CA including chromatid, isochromatid breaks (CB, IB) and chromatid, isochromatid exchanges (CE, IE). Gaps (G) were examined separately. The mitotic index (MI) was calculat-ed as the ratio of metaphases to the total number of 2000 cells.

Fluorescence in situ hybridization - Orange-red labelled whole chromosome painting probes (WCPs), specific for the bovine chromosome 1 and 7, and green labelled WCP, specific for the bovine chromosomes 5 were used for hybridiza-tion, simultaneously. The probes were acquired in cooperation with Veterinary Research Insti-tute, Brno, Czech Republic, where they had been prepared by laser microdissection (kubičkoVá et al. 2002). Three chromosome painting probes

were selected to cover the highest proportion of the bovine painted genome. The painting probe in hybridization mixture (50% forma-mid in 2xSSC, 10% dextran sulphate, salmon sperm DNA, competitor DNA) was denatured at 72˚C for 10 min and reannealed at 37˚C for 90 min. The denaturation of slides was performed in 70% formamide in 2xSSC (pH 7.0) at 72˚C for 2 min and followed by a dehydration pro-cedure (70, 80, 90% ethanol, -20˚C and 96% ethanol, Room Temperature) for 2 min. After overnight hybridization at 37˚C, the slides were washed in 2xSSC (3-5 min, room temperature), in 0, 4 xSSC with 0, 3% Igepal at 72˚C for 2 min and again in 2xSSC at room temperature from 5 sec to 2 min. The slides were counterstained in DAPI/Antifade (4´, 6´-diamidino-2-phenylin-dole, Q-BIOgene, UK).

One thousand of well spread metaphases were analysed for each and all experimental cultures and controls. Aberrations were scored according to the PAINT nomenclature (tucker et al. 1995). A fluorescent microscope NIKON Labophot 2A/2, equipped with dual band pass filter FITC/TRITC, was used for probe visualization.

Fig. 1 — Metaphase with triradial exchange of the chromosome 5. after treatment for MMC for 48 h of incubation. A fluorescent microscope NIKON Labophot 2A/2, equipped with dual band pass filter FITC/TRITC, was used for probe visualization. Cat-tle metaphase spread hybridized with painting probes for bovine chromosome 1 (red), 5 (green) and 7 (red). CE (CH.5) represents triradial exchange between marked chromo-some 5 and another unmarked one.

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Statistical analysis was performed using the c2 test for the estimation of CAs and MI in both experimental conditions.

RESULTS

The frequency of chromosomal aberrations induced by positive mutagens estimated by the conventional cytogenetic analysis and FISH technique in bovine lymphocyte cultures are shown in Table 1 and 2. The mean value ± S.D. in each group are given with respect to the fact that no apparent differences between calf do-nors were seen.

For the conventional analysis, statistically sig-nificant elevations in the mean of chromosomal aberrations (CAs), and significant inhibition of mitotic activity were observed after the expo-sure to EMS for the last 24 h and for MMC after the 48 h incubation. The common types of CAs were chromatid breaks. Several cells with chro-matid exchanges were also detected. Statistical significance in comparison to the controls was evaluated using the c2 test (Table 1, p<0.001).

FISH technique was selected for the inves-tigation of stable/unstable structural chromo-somal aberrations and numerical aberrations of the type aneuploidy/polyploidies. The frequen-cy of chromosome aberrations after the use of the FISH technique is summarised in Table 2. From the structural aberrations, especially un-stable chromosomal damage was found (Fig. 1). We also detected some putative non-reciprocal translocations, which involved exchanges be-tween painted chromosomes and unpainted material. However, their presence was without statistical significance. Identically with the con-ventional chromosomal analysis results, MMC induced chromatid, isochromatid breaks and chromatid exchanges, while after the exposure to EMS, mainly chromatid-type of breaks have

been detected (p<0.001, χ2 test). From numeri-cal aberrations, several cells with polyploidy were obtained. Monosomy and trisomy on bo-vine chromosome 5 were the most frequent type of aneuploidies (Fig. 2). After the exposure to MMC, trisomy and monosomy were detected at the same rate while EMS induced preferentially monosomy (p<0.001, and p<0.05, c2 test).

DISCUSSION

MMC and EMS have induced wide spectra of chromosomal damage; chromatid/ chromosome breaks, and chromatid/chromosome exchanges. These agents were selected because of the extent use in conventional cytogenetic studies (šiVikoVá and diAnoVský 1999, 2006; rencüzoğullAri and toPAktAş 2000; HolečkoVá et al. 2009; diler and toPAktAs 2010).

The higher frequency of chromosomal aber-rations induced by the MMC was observed after the prolonged time of incubation of the mutagen (48 h). The MMC is known to induce not only chromatid but also chromosome breaks, thus the lesion induced by the agent can be transformed to the chromosome aberration after entering an S-phase of mitosis (Giri and cHAtterjee 1998).

On the contrary, in the cultures treated with the EMS the high frequency of chromosomal damage was obtained at the last 24 h of incu-bation. Clastogenic ability of the EMS was well established in in vivo and in vitro mutagenic experiments (odeiGAH and osAnyiPeju 1995; Gocke et al. 2009). Highly damaged cells or the cells with a total fragmentation of chromosomes induced by the EMS could be seen just in the first cell cycle; than they failed to occur after the prolonged time of exposure to mutagen (Table 1). The EMS was known as a teratogen and brain carcinogen from numerous experimental works (iArc 1987).

tAble 2 — The frequency of chromosome aberrations in bovine peripheral lymphocytes exposed to positive muta-gens after treatment for EMS of 24 and for MMC for 48 h of incubation evaluated by WCP in vitro from two donors.

DoseAneuploidy with WCP

Polyploidy (4n)Type of unstable aberrations

% of breaksTotal 1 5 7 CB IB CE IE

Control 5 1 2 2 4 - - - - 0,0±0,0

MMC(0,4 µg/mL) 26*** 4 18 4 10a 4 5 2 - 1,3±0,11***

EMS (250 µg/mL) 15* 2 8 5 9a 16 1 1 - 1,9±0,14***

Two thousand metaphases in each group were determined. A mean value of chromosomal aberrations/donor was included. a - statistically non significant data. * (p<0, 05), *** (p<0,001), c2 test.

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The introduction of FISH in cytogenetic studies has also lead to a better understanding of the mechanisms in the formation of chromo-some aberrations that was extensively studied after the treatment of humans with ionizing radiation (nAtArAjAn and boei 2003). Experi-ments were usually performed using three paint-ed probes marking the longest chromosomes (GArciA-sAGredo 2008). In animal cytogenetics several studies were carried out to construct a physical map and subsequently hundreds loci have been determined (iAnuzzi 1998; di meo et al. 2006). The chromosomal rearrangements on reproduction were reported, as well (di meo et al. 2000; iAnuzzi et al., 2001; molteni et al. 2007; switonski et al. 2008).

Our interest was directed towards to the de-tection of stable chromosomal aberrations by FISH technique, which does not result in the loss of chromosome material and are believed to be heritable. Using bovine WCP for chromo-somes 1 and 7 and porcine WCP for chromo-somes 1 and 13, ŘezáčoVá et al. (2001) reported that frequency of stable chromosomal aberra-tions in cow lymphocytes was lower (0.38 ±0.60) than seen in pigs (0.790±0.81). The authors suggested that cattle have a reduced sensitivity to chromosome mechanisms that could cause structural chromosomal aberrations.

Under conditions of our experiments, no stable aberrations of the type of the reciprocal translocations were detected. We assume that it

Fig. 2 — Bovine metaphase with aneuploidy of the chromosome 5 after treatment for EMS of 24 incubation. Cattle metaphase spread hybridized with painting probes for bovine chromosome 1 (red), 5 (green) and 7 (red). Monosomy of chromosome 5 (green) was detected.

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could be caused by some limitation of our ex-periments. When three available WCPs were used, stable chromosome aberrations could be analyzed only in three pairs of marked chromo-somes that represent a relatively low proportion of the bovine genome (fries and PoPescu 1999). In our work, we have also recorded only non-reciprocal exchanges between painted and un-painted chromosomes.

There are several reports about the basic frequency of stable aberrations in humans. The spontaneous frequency of translocations (0.35 per 100 cells) was described by finnon et al. (1995) after the hybridization of human met-aphases with tree human painting probes for chromosomes 2, 5 and 5. In other studies, the mean in the genomic frequency, in cells obtained from healthy donors, of reciprocal and total translocations was (0.58±0.08 and 0.90±0.12 per cells) (ciGArrán et al. 2001). The frequency of translocations in C57BL/6 mice was 0.24 per 100 cells (tucker et al. 1999).

The MMC as a crosslinker agent effectively induces triradial and quadriradial chromosome exchanges that were shown by the multicolour FISH technique on Chinese hamster and human cells. sAtoH et al. (2002) demonstrated various types of exchanges, especially triradials, after the treatment of human lymphoblastoid cell line WTK-1 with the MMC for 48 h. Of the total 650 aberrant cells, the author found 4,6% triradial and 14,2% quadriradial exchanges. Further au-thors, sontAkke and fulzele (2009), reported that the MMC does not induce chromosomal aberrations randomly. They described that chro-mosomes 1, 9 and 16 in humans were affected more frequently after the exposure to the mu-tagen. They suggested that the MMC acts com-monly on heterochromatin at secondary con-strictions of chromosome 1, 9 and 16.

In our experiments, bovine chromosome 5 was more frequently damaged, that corresponds with finding of PAuciulo et al. (2010) in Italian autochtonous cattle breeds. Unstable structural aberrations of the triradial exchange type, ane-uploidy of the type monosomies and trisomies were associated with the damage on this chro-mosome.

Using RBG-banding rodriGuez et al. (2002) found aphidicolin-induced break points on chromosomes 1 and X in Holstein-Friesian cat-tle. Several reports documented chromosomal fragility in association with teratogenic effects in sheep exposed to dioxins (IAnuzzi et al. 2004; PerucAtti et al. 2006). Moreover szczerbAl et al.

(2006) described chromosomal aberrations asso-ciated with congenital malformations of limb in a calf.

Identification of chromosome aberrations by the FISH seems to be a suitable technique in cy-togenetic studies. Chromosomal aberrations are also associated with numerous developmental failures not only in humans but also in domestic animals.

FISH technique could complete and precise the data of micronucleus assay in genotoxic/cytogenetic studies, the assay that was gener-ally appropriated for the detection of aneuploi-dy. Chromosomal instability and aneuploidy are considered for two critical steps in human carcinogenesis (duesberG and rAsnick 2000; louzAdA et al. 2010).

Acknowledgements — This work was supported by the Slovak Research and Development Agency under the contract no. APVV-0010-07, the Minis-try of Education and Science of the Slovak Republic VEGA 1/0334/10 and VEGA1/0125/11.

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Received March 14th 2011; accepted September 20th 2011