Mutagenesb vol.12 no.4 pp.201-207, 1997

Extruded micronuclei induced by colchicine or acrylamide containmostly lagging chromosomes identified in paintbrush smears byminor and major mouse DNA probes

Gerlinde Schriever-Schwemmer1, Ulf Kliesch2 andIlse-Dore Adler2^

'Ludwigs-Maximillians-Universitfit MUnchen, TierSrztliche Fakultflt,Lehrstuhl fllr Allgemeine Pathologic und Neuropathologie,VeterinarstraBe 13, D-80539 MUnchen and ^SF-Forschungszentrum fUrUmwelt und Gesundheit, Institut filr Saugetiergenetik, I>85764 Neuherberg,Germany

'To whom correspondence should be addressed

In the mouse bone marrow micronucleus assay, it wasstudied whether micronuclei (MN) could be expelled frompolychromatic erythrocytes (PCE) in a similar way to themain nucleus. To avoid the disrupting centrifugation stepof the conventional bone marrow preparation procedure,the paintbrush technique was used in the present experi-ments. With May-Grtinwald-Giemsa staining of paint-brush slides, 5% of the colchicine (COL)-induced MN werefound attached to the outside membranes of PCE and wereregarded as extruded. Of the acrylamide (AA)-lnduced MN,22% were extruded. After fluorescence in situ hybridization(FISH) of a total of 300 MN per chemical treatment withthe mouse minor and major satellite DNA probes, 9.7%MN were extruded in the COL group and 83% MN wereextruded in the AA group. FISH showed that 76% of theretained COL-induced MN were signal-positive, indicatingthat they contained entire chromosomes. With AA, 29%minor-positive and 283% major-positive retained MN werefound, confirming its known clastogenicity. However, theobserved frequency of signal-positive MN (1.7 MNPCEpo,/1000 PCE) in the AA group was about three times higherthan in the control (0.5 MNPCEp /̂lOOO PCE) which indi-cates that AA has aneugenic potential. FISH analysis ofthe extruded MN showed 72-100% major as well as minorsignals. It is concluded that expelled MN contain mostlyentire chromosomes.

IntroductionDuring erythrocyte maturation, the main nucleus is expelled.It is possible that large micronuclei (MN) undergo expulsionby the same mechanism as the main nucleus. To verify theexistence of extruded MN, the preparation method was changedfrom the common smear method to the paintbrush methoddescribed by Styles et al. (1983). This paintbrush techniqueavoids the centrifugation step and, therefore, may enable theobservation of MN which are in the process of leaving theerythrocyte or have just left the erythrocyte and are stillattached to the outer membrane. The question was 'do extrudedMN exist and do they contribute significantly to the MNyield?' Parton et al. (1991) described extruded MN inducedby demicolcine which were mostly large in size. The secondquestion then was whether these large extruded micronucleicontained entire chromosomes or acentric fragments.

In the past decade, centromere detection methods have beenused to distinguish MN formed by acentric fragments from

© UK Environmental Mutagen Society/Oxford University Press 1997

those formed by whole chromosomes (Miller et al., 1991,1992; Salassidis et al, 1992; Miller and Nlisse, 1993; Farooqiet al., 1993; Schriever-Schwemmer and Adler, 1994). Formice, the major DNA probe was used primarily; it hybridizesto the pericentric heterochromatic block. The major probelabels all chromosomes, except the Y chromosome, in a one-dot pattern (Pietras et al, 1983; Miller et al, 1991; Weieret al, 1991). Another centromere probe is the minor DNAprobe which labels all chromosomes except the Y at or nearthe centromere in the kinetochore region with two dots, similarto the labelling by CREST antibodies (Broccoli et al, 1990;Moens and Pearlman, 1990; Schriever-Schwemmer and Adler,1993). The safest way to determine centromere-containing MNis to use double labelling with the major and the minor DNAprobe (Eastmond et al, 1993; Chen et al, 1994; Schriever-Schwemmer and Adler, 1994).

Currently, colchicine (COL) is used as a typical aneugenand as a positive control (Miller and Adler, 1989; Adler et al,1991; Schriever-Schwemmer and Adler, 1994). Acrylamide(AA) was tested for its possible aneugenicity. One of the bestdocumented effects of AA is its clastogenicity in somatic andgerminal cells. Chromosomal aberrations (chromatid gaps,breaks and exchanges) were observed in mammalian cellcultures (Moore et al, 1987; Tsuda et al, 1993), °as well asin mouse bone marrow cells and in mouse spermatogonia(Shiraishi, 1978; Adler et al, 1988) after AA treatment.Furthermore, AA induced dominant lethal mutations, heritabletranslocations and specific locus mutations in male mousegerm cells (Shelby et al, 1987; Ehling and Neuhauser-Klaus,1992; Gutierrez-Espeleta et al, 1992; Adler et al, 1994).Aneugenicity data of AA are scarce, however, some studiesin mouse bone marrow and spermatocytes indicate that AAhas aneugenic potential (Gassner and Adler, 1995, 1996).

The present experiments were carried out to verify theexistence of extruded MN, to analyse whether these extrudedMN contain lagging chromosomes and to analyse the aneugen-icity of AA in the mouse micronucleus assay in vivo. Toanswer these questions, both chemicals were used at only onedose which previously had been shown to induce a highfrequency of MN (Adler et al, 1988, 1991).

Materials and methods

Chemicals and animal treatment

Male (lCH/ElxCSH/El)?! mice from the GSF animal colony were used at theage of 10-14 weeks weighing 25-29 g. Mice were injected i.p. at a volumeof 0.1 ml/10 g body weight with 1 mg/kg of COL and 125 mg/kg of AA.COL and AA were purchased from Sigma (TJeisenhofen, Germany). Bothcompounds were dissolved in bidistilled water. Control animals were injectedi.p. with equal volumes of bidistilled water.

Slide preparation

The mice were killed 24 h after treatment and the femurs were extracted. Thefemur was opened at the knee end, so that a paintbrush of size 0 or 00 couldbe inserted. The bone marrow was sampled by gently turning the paintbrush,which had been wetted with fetal calf serum (FCS), in the opening of thebone. The bone marrow was then brushed onto a silane-coated slide whichhad been wetted before with a drop of FCS. Five or six slides were made for

201

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022

CSchriever-Schwemmer, U.Kllesch and I.-D.Adler

each animal; these were dried at room temperature, fixed for 20 min in 90%methanol at -20°C and air-dried overnight The slides were stored in thefreezer at -20°C until use. All slides were used for fluorescence in situhybridization (FISH) with the exception of one slide per animal, which wasstained with May-Grflnwald-Giemsa solution for conventional assessment ofthe MN frequencies (Adler, 1984)

DNA probes

The murine -^satellite DNA probe (Saiki et al. 1988. Vissel and Choo. 1989),a gift from H Zitzelsberger (GSF-Institut fUr Strahlenbiologie) was amplifiedand biotinylated as described elsewhere (Weier and Rosette, 1988, 1990;Weier et al, 1991). The biotinylated DNA probe was stored without furtherpurification at -20°C until use.

The munne minor satellite DNA probe, pMKB6 (Wong and Rattner, 1988),a 273 bp fragment that represents approximately two tandem repeats, inplasmid pTZI9U, was a gift from B.Vig (Reno, USA). It was propagated inEschenchia coli JM101 and separated with Midi-columns for DNA preparation(Diagen, Hilden, Germany). The plasmid DNA was biotinylated or labelledwith digoxygenin by nick translation (Sambrock et al, 1989) The use ofbiolin and digoxygenin as haptens allowed the use of two DNA probes at thesame time due to the possibility of different detection systems for the haptens.

Hybridization procedure

FISH was carried out according to Pinkel et al. (1986) with slight modifications.Frozen slides were incubated for 8 min at 37°C on a warming plate andrehydrated in 2x 0.3 M sodium chloride/0.03 M sodium citrate (SSC, pH 7.0)for 5 min at room temperature prior to treatment with 0.1% Tnton X-100 in2X SSC for 3 min in a moist chamber. After washing the slides in 2X SSC,they were fixed in 1 % paraformaldehyde for 5 min at room temperature

The slides were denatured for 10 min in 70% formamide, 2X SSC, pH 7.0at 78°C and dehydrated in an ice-cold 70, 90 and 100% ethanol series Afterair-drying, the slides were brought to 37°C for <3 min on a wanning plate

The hybridization mix contained seven parts of the master mix 1 0 (50%formamide, 10% dextransulphate), one part of two different hapten-markedDNA probes (-20 ng/probe/slide) and one part of herring sperm DNA(500 |ig/ml). The hybridization mix was denatured for 5-10 min at 78°C andchilled on ice. Aliquots of 20 nl of the hybridization mix were added to theslides. The slides were covered with 24X50 mm coverslips and sealed withrubber cement. Hybridization was carried out overnight in a moist chamberat 37°C.

Signal detection

The slides were washed in 50% formamide, 2X SSC, pH 7.0 for 40 min at44°C, followed by two washes for 15 min in PN buffer (0 1 M sodiumphosphate, pH 8 0. 0.1% Nonidet P-40) at 37°C.

After incubation with 40 [i\ PNBR (PN buffer plus 5% blocking reagentand 0.02% sodium azide. Boehringer Mannheim, Germany) for 10 min thebiotin-labelled probe was detected with 40 |il Cy3-streptavidin (1.33 |ig/ml.Dianova. Hamburg. Germany) in PNBR buffer for 20 min at room temperaturein a moist chamber. Thereafter, the slides were washed twice in PN bufferfor 10 mm each When necessary, the signals were amplified by up totwo rounds of biolinylated anti-strcptavidin (5 (ig/ml. Vector Laboratories.Burlinghame. CA, USA) in PNBR buffer and Cy3-streptavidin. Betweenevery detection step the slides were washed twice in PN buffer for 10 min each.

The digoxygenin-labelled DNA probe was visualized with 20-40 (ilfluorescein isothiocyanate (FITC)-conjugated sheep anti-digoxygenin antibody(1:20 diluted in PNBR. Boehnnger Mannheim). These signals were enhancedby FITC-conjugated rabbit anti-sheep antibody (5 ng/ml in PNBR; Sigma)and when necessary by FITC-conjugated sheep anti-rabbit antibody (5 Hg/mJin PNBR: Sigma).

After the end of the detection procedure, the slides were counterstainedwith 0.1-0.5 ng/ml 4'.6-diamidino-2-phenylin-doledihydrochloride (DAPI)for 3 min at room temperature and coverslipped in Vectorshield (Sigma).Slides were stored in the dark at 4°C and analysed by fluorescence microscopy(Zeiss Axiophot: 100 W mercury lamp) within a few days. The individualfillers had the following characteristics- for FTTC- HQ 480/40. HQ535/5O. forCy3: HQ 535/50, HQ 610/75, (both Chroma Technology, Brauleboro, USA)and for DAPI: BP365. LP 397 (Zeiss, Germany). The microscopic images ofthe micronuclei were digitized with the computer program ISIS3 (MelaSystems.Altlussheim. Germany).

Microscopic and statistical analysis

The conventional micronucleus scoring and the signal analysis after double-colour FISH libelling Mas performed in parallel on slides from throe animalsper chemical treatment and two animals per .solvent control.

The con\entionall\ stained slides were microscopically anahsed at v125Omagnification for the presence of MN inside of or attached to polychromaticerythrocvtes (PCEi. Debns and possible nuclear fragments of ruptured

apoptotic nucleated cells were clearly distinguished from true MN by shapeand colour of PCE as well as MN. At least 1000 PCE were scored per animal.The PCE/NCE ratio was also evaluated. For signal detection 100 MN peranimal were scored at XI250 magnification. The number of signals per MNwere counted.

The x2 test was used to determine significant differences in the MN data.

ResultsPaintbrush technique and conventionally analysed micronucleiInstead of the widely used bone marrow smear technique thepaintbrush technique described by Styles et al. (1983) wasused to preserve cells undergoing enucleation and especiallythe expulsion of the MN from the erythrocytes. Figure 1illustrates the expulsion process of micronuclei at the differentstages: micronucleated PCE (a and b), PCE with MN stillattached to the plasmalemma (extruded MN; c-e) and totallyextruded MN (f).

The frequencies of retained and extruded MN, the PCE/NCE ratios and the MNPCEp^lOOO PCE obtained in thepresent experiments are shown in Table I. In the two solvent-control animals, the average of 1.0 MN/1000 PCE is in thenormal range for solvent controls in our laboratory and noextruded MN were found. At 24 h after treatment with 1 mg/kgCOL, 7.3 retained MN/1000 PCE and 0.4 extruded MN/1000PCE were found. Thus, 4.4% of all COL-induced MN wereextruded. At 24 h after AA treatment, 4.7 retained MN/1000PCE and 1.3 extruded MN/1000PCE were observed witha dose of 125 mg/kg. Thus, 22.2% of all AA-induced MNwere extruded. The MN frequencies in the treated groups(retained or total) were significantly higher than the control.No significant difference could be detected comparing thefrequency of total (retained + extruded) MN to the retainedMN frequency in either group.

To correlate the FISH data with the conventional MN datawe calculated a MNPCEpog/1000 PCE frequency. For example,after treatment with COL, 7.7 MN/1000 PCE were found inthe conventional MNT and 76% MN were centromere-positive,thus 5.9 MN/1000 PCE were calculated to be centromere-positive. For the control, 0.5 MN/1000 PCE and for AA 1.7MN/1000 PCE were centromere-positive. The frequency ofcentromere-positive MN in the AA group was more than threetimes higher than in the control.

The MNNCE frequencies were between 0.25 and 0.5/1000NCE in all groups (data not shown). Thus, no discriminationbetween MN induced in PCE and NCE was required for theFISH analysis since the NCE only contributed minimally tothe total number of MN. No significant reduction of the PCE/NCE ratio was found in either of the two treatment groupscompared with the solvent control.

FISH double labelling with the minor and major DNAprobesExamples of retained and extruded MN with FISH using majorand minor DNA probes are shown in Figure 2.

The results of the total of 300 scored MN after doublelabelling with the minor and major probes are shown in TableII. In the control, 80 (53.3%) of the 150 MN analysed showedno signal and 70 MN (46.7%) were double-labelled with theminor and the major probe. After treatment with 1 mg/kg ofCOL, 72 out of 300 (24%) MN showed no signal and 228 MN(76%) were major- as well as minor-positive. With 125 mg/kgAA, 215 MN (71.7%) of 300 MN analysed had no signal, twoMN (0.7%) were minor-positive only and 85 MN (28.3%)were double-labelled with the minor and major DNA probe.

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022

Lagging chromosomes in extruded mkronuclei induced by colchlcine or acryiamide

Fig. 1. Different stages of MN expulsion (May-GrUnwald-Giemsa staining), fa and b) Polychromatic erythrccytes (PCE) with micronuclei (MN); (c, d and e)PCE with MN still attached to the erythrocyte membrane (extruded MN) and (f) totally extruded MN.

The distribution of the numbers of signals in the minor- ormajor-positive MN is shown in Table HI. In the control, 10MN (14.3%) out of 70 minor-positive MN had one signal, 57MN (81.4%) showed two signals, tvvo MN (2.9%) showedthree signals and one MN (1.4%) showed four signals. Aftertreatment with 1 mg/kg of COL, 65 (28.5%) of 228 minor-

positive MN had one signal, 135 MN (59.2%) showed twosignals, nine MN (4%) had three signals and 21 MN (9.2%)had four signals. With 125 mg/kg of AA, 23 MN (26.4%) of87 minor-positive MN had one signal, 49 MN (56.3%) showedtwo signals, eight MN (9.2%) had three signals and six MN(7%) had more than three signals. With the major DNA probe

203

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022

CSchriever-Schwemmer, U.Kliesch and I.-D.Adler

Table I. Frequencies of retained and

Chemical

ControlCOLAA

Dose(mg/kg)

01125

No ofanimals

233

extruded MN in

Individual

total MN

2,06, 8, 94, 7, 7

polychromatic erythrocytes of

animal scores per 1000 PCE

extruded MN

0,00, 0, 10. 2, 2

bone marrow after I

Mean MNPCE

total MN

1.0 ± 1.07.7 ± 1.5"6.0 ± 1.7b

.p. treatment of male mice with COL

(% ± SD)

retained MN

1.0 ± 1.07.3 ± 2.1"4.7 ± 0.6b

PCE/NCE- (± SD)

0.97 ± 0.030.89 ± 0.060.98 ± 0.03

and AA

MNPCEpo, per1000 PCE

05591.7

'P < 0.01; bP < 0.05 (x2 test)MNPCE, micronucleated polychromatic erythrocytes; NCE, normochromauc erythrocytes; PCE, polychromatic erythrocytes.

Fig. 2. Examples of mouse erythrocytes with MN labelled by FISH with minor (CY3-red) and major (FITC-yellow) satellite DNA probes. The MN arecounterstained with DAPI (blue). Erythrocytes show autofluorescence in green The pictures were digitized using the ISIS program (MetaSystems.Altlussheim. Germany) (a) Minor- and major-negative retained MN; (b) minor- and major-negative MN that is just about to be expelled; (c) minor- (red) andmajor- (yellow) positive retained MN; (d) minor- and major-positive extruded MN still attached to the membrane of the erythrocyte. The overlap of red,yellow and blue colour signals results in a white spot, however, the individual colours were always seen with the respective individual filters.

the following frequencies were found: in the control 62 MN(88.6%) out of 70 major positive MN had one signal and 8MN (11.4%) showed two signals. After treatment with 1 mg/kgof COL, 191 MN (83.8%) of 228 major-positive MN hadone signal and 37 MN (16.2%) showed two signals. With125 mg/kg of AA, 63 MN (74.1%) of 85 major-positive MNhad one signal. 19 MN (22.4%) showed two signals and threeMN (3.5%) had three signals.

The numbers of extruded MN labelled with the minor and

major DNA probe are shown in Table IV. In the control, sixMN (4%) out of 150 MN were extruded and all six (100%)had minor and major signals. After treatment with 1 mg/kgCOL, 29 MN (9.7%) of 300 MN were extruded and onlyfour (13.8%) of the extruded MN had no signals while 25(86.2%) of the extruded MN had minor and major signals.With 125 mg/kg of AA. 25 MN (8.3%) of 300 MN wereextruded and se\en (28%) of the extruded MN had no signalwhereas 18 MN (72%) showed minor and major signals.

204

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022

Lagging chromosomes in extruded micronudei induced by colchicine or acrylamide

Table n. Double labelling of MN with minor and major DNA probes by FISH

Chemical Dose(mg/kg)

No. of MN MN without anyscored signal

MN without minor MN with majorsignal signals

MN with minorsignals

MN with major andminor signals

ControlCOLAA

Table m.

01

125

Distribution

150300300

of the signal

total

8072

215

frequencies among

Tc

53.324.071.7

total

8072

213

MN after double

53.324.071.0

labelling with

total

70228

85

minor and

46.776.028.3

major DNA

total

70228

87

probes by

46.776.029.0

FISH

total

70228

85

%

46.776.028.3

Chemical Dose(mg/kg)

MN withminorsignals

One minorsignal(SO

Twominorsignals(SO

Threeminorsignals(SO

Fourminorsignals(SO

More thanfive minorsignals(SO

MN withmajorsignals

One majorsignal

Two major Threesignals major(%) signals

Control

COL

AA

0

1

125

70

228

87

10(14.3)65

(28.5)23

(26.4)

57(81.4)135(59.2)49

(56.3)

2(2.9)9

(4.0)8

(9.2)

1(1.4)21(9.2)5

(5.8)

0

0

1(1.2)

70

228

85

62(88.6)191(83.8)63

(74.1)

8(11.4)37

(16.2)19

(22.4)

0

0

3(3.5)

Table IV.

Chemical

ControlCOLAA

Number of extruded

Dose(mg/kg)

01

125

MN labelled

No. of MNscored

150300300

with minor and major DNA

No of MN extruded

total

62925

%

4.09.78.3

probes by

Extruded

total

047

FISH

MN without any signals

%

0.013.828.0

Extruded MN

total

62518

with major and minor signals

%

100.086.272.0

Discussion

The purpose of the present study was to verify whether:(i) extruded MN could be detected; (ii) extruded MN containedlagging chromosomes; and (iii) AA had an aneugenic effect.

The present results using the paintbrush method for theconventional micronucleus staining yielded MN frequenciesfor all groups which were a little lower than the publisheddata (Adler et al, 1988, 1991; Schriever-Schwemmer andAdler, 1994). For the control, the MN frequency of 1.0/1000PCE agreed well with the published data. For COL, Schriever-Schwemmer and Adler (1994) found 10.5 MN/1000 PCE andAdler et al. (1991) found 12.3 MN/1000 PCE compared withthe 7.3 retained MN in the present study. For AA, Adler et al.(1988) found 8.8 MN/1000 PCE compared with 4.7 retainedMN/1000 PCE in the present study. These lower MN frequen-cies could be due to a better preservation of all erythrocytesso that no loss of erythrocytes occurred as it may when acentrifugation step is involved or to inter-animal variation andthe small number of animals in the present study.

Parton et al. (1991) analysed the amount of extruded MNafter treatment of mice with demicolcine and cyclophosph-amide using the paintbrush method to preserve cells undergoingenucleation. Treatment with demicolcine, a classical aneugen,caused relatively high frequencies of extruded MN (15—40%of all MN), however, no extruded MN were observed aftertreatment with cyclophosphamide, a classical clastogen.

In this study, using May-Grtlnwald-Giemsa staining, noextruded MN were found in the control, however, after treamentwith COL, 4.4% of all MN were extruded and after treatment

with AA, 22.2% of all MN were extruded (Table I). UsingFISH labelling, 4.0% MN were extruded in the control, 9.7%in the COL group and 8.3% in the AA group (Table IV).The results indicate that scoring only retained MN mayunderestimate the true level of MN induction, however, thedifference between total and retained MN in our experimentswas not significant. Extrusion of MN is not only a propertyof demicolcine, as argued by Parton et al. (1991), but theextrusion of MN can be observed with other chemicals, as inthe present study with COL and AA. Thus, the extrusion ofMN may be a common phenomenon.

To verify whether the extruded MN contain lagging chromo-somes or acentric fragments, FISH analysis with the minorand major DNA probes to detect the centromeres was carriedout. This technique makes it possible to discriminate betweenMN formed by clastogens and by aneugens (Miller et al.,1991; Chen et al., 1994; Schriever-Schwemmer and Adler,1994). The discrimination is particularly important for chemicalclastogens which may also have aneugenic potential. Suchchemicals may interact with the targets for chromosomalmissegregation on the chromosomal level, i.e. with kinetochoreproteins, centromeric DNA or telomeres.

In control animals, the yield of MN with minor and majorsignals was identical, i.e. 46.7% of the 150 MN analysed(Table II). This result was in the same range as for controlsof other in vivo bone marrow MN studies (Vanderkerkenet al., 1989; Miller et al, 1991; Schriever-Schwemmer andAdler, 1994).

After treatment with the classical spindle poison COL, the

205

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022

G.Schriever-Schwemmer, U.KIiesch and I.-DAdler

FISH labelling with the minor and major probe gave compar-able results (Table II), i.e. 76% of the MN contained entirechromosomes as evidence by concurrent minor and majorsignals. This result was slightly higher than that of previousstudies when between 67% major-positive MN and 75% minor-positive MN were found (Miller et ai, 1991; Schriever-Schwemmer and Adler, 1994).

AA is a well-known clastogen (Shelby et ai, 1987; Adleret ai, 1988, 1994; Gutierrez-Espeleta et ai, 1992) and asuspect aneugen (Adler et ai, 1993; Gassner and Adler, 1996).In the present study, 29% MN were minor-positive and 28.3%MN were major-positive. Thus, AA-induced MN containingacentric fragments were 2.5 times more frequent than AA-induced MN containing lagging chromosomes (Table II).However, the frequency of positive MN was more than threetimes higher after AA treatment than in the control, i.e. 1.7MNPCEpos/1000 PCE versus 0.5 MNPCE^/IOOO PCE (TableII). Therefore, AA has to be regarded as having an aneugenicpotential in addition to its clastogenic activity.

The distribution of the signal frequencies among the double-labelled MN show 82-95% of the double-labelled MN haveone or two minor signals and 96-99% have one or two majorsignals, indicating that these MN contained whole chromatidsor chromosomes (Table III). These distribution results are verysimilar to earlier observations in experiments with COL andmitomycin C in our laboratory (Schriever-Schwemmer andAdler, 1994).

The MN ratios found using the FISH technique also includedextruded MN, which were still attached to the erythrocytemembranes. In the control, 4% of the analysed MN wereextruded. After COL treatment, 9.7% MN were extruded, andafter AA treatment, 8.3% were extruded (Table IV). In thecontrols, 100% of these extruded MN showed major as wellas minor signals. In the COL group, 86% and in the AAgroup, 72% of the extruded MN showed major as well asminor signals. Thus, only 0-28% of the extruded MN werewithout any signals. The signal negative extruded MN fre-quency was higher in the AA group which may be due to thestronger clastogenicity of AA.

We conclude that: (i) extruded MN do exist; (ii) mostly MNformed by lagging chromosomes stand a risk of expulsion,however, also MN containing acentric fragments can beexpelled from the erythrocytes: and (iii) A A has aneugenicpotential.

AcknowledgementsThe authors appreciate the efficient technical assistance of Gillian Blenkinsop.We thank Dr Baldev Vig for providing the mouse minor satellite DNA probeand Dr Horst Zitzelsberger for providing the mouse major satellite DNAprobe. The work was supported by CEC contract EV5V-CT94-O403

ReferencesAdler.I.-D. (1984) Cytogenetic tests in mammals In Venitt.S and Parry J M

(eds). Mutagemcity Testing. A Practical Approach. IRL. Oxford, pp.275-306.

Adler.I.-D. Ingwersen.I., Kliesch.U and EI-Tarras.A (1988) Clastogeniceffects of acrylamide in mouse bone marrow cells. Mutai Res., 206, 379-385.

Adler.I.-D.. Kliesch.U.. van Hummelen.P. and Kirsch-Volders.M. (1991)Mouse micronucleus tests with known and suspect spindle poisons: resultsfrom two laboratories. Mutagenesis. 6, 47-53

Adler.I-D . Zhouh.R and Schmid.E (1993) Perturbation of cell di\ision h\acrylamide in vitrv and in vivo. Mutat. Res.. 301, 249-254

Adler.I D . Reitmeir.R Schmoller.R and Schne\er-Schuemmer.G (I994iDose response for heritable trunslocations induced b> acnlamide inspermatids of mice Mutai Res . 309, 285-291

Broccoli.D , Miller.O J. and Miller.D.A (1990) Relationship of mouse minorsatellite DNA to centromere activity. Cvtogenet. Cell Genet., 54,182-186

Chen.H.W., Tomar.R. and Eastmond.D.A. (1994) Detection of hydroquinone-induced nonrandom breakage in the centromenc heterochromatin of mousebone marrow cells using multicolor fluorescence in situ hybridization withthe mouse major and minor satellite probes Mutagenesis, 9, 563-569.

Eastmond.D A., Rupa,D S., Chen.H.W and HasegawaX. (1993) Muticolorfluorescence in situ hybridization with centromenc DNA probes as a newapproach to distinguish chromosome breakage from aneuploidy in interphasecells and micronuclei In Vig.B K. (ed). Chromosome Segregation andAneuploid\ NATO ASI Senes Vol. H72. Springer Verlag, Berlin, Heidelberg,pp. 377-390

Ehling.U H and Neuhiuser-Klaus.A. (1992) Reevaluation of the induction ofspecific-locus mulauons in spermatogonia of the mouse by acrylamideMutat. Res., 283, 185-191.

Farooqi.Z., Darroudi.F. and Natarajan.A.T (1993) The use of fluorescence insitu hybridization for the detection of aneugens in cytokinesis-blockedmouse splenocytes Mutagenesis, 8, 329-334

Gassner.P. and Adler,I.-D. (1995) Analysis of chemically induced spindleaberrations in male mouse germ cells' comparison of differential andimmunofiuorescent staining procedures Mutagenesis, 10, 243-252.

Gassner.P. and Adler.I.-D (1996) Induction of hyperploidy and cell cycledelay by acrylamide in somatic and germinal cells of mals mice Mutat.Res, 367, 195-202.

Gutierrez-Espeleta,G.A., Hughes.L.A., Piegorsch.W.W., Shelby.M D andGeneroso.W.M (1992) Acrylamide dermal exposure produces geneticdamage in male mouse germ cells. Fund Appl. Toxicol, 18, 189-192.

Miller.B.M. and Adler.I.-D (1989) Suspect poisons, analysis of c-mitoticeffects in mouse bone marrow cells Mutagenesis, 4, 208-215

Miller.B.M. and NUsse.M (1993) Analysis of micronuclei induced by 2-chlorobenzyhdene malonitnle (CS) using fluorescence in situ hybridizationwith telomeric and centromeric DNA probes, and flow cytometry.Mutagenesis, 8, 35-41.

Miller.B.M.. Zitzelsberger.H., Weier.H.-U and Adler.I-D (1991)Classification of micronuclei in murine erythrocytes immunofiuorescentstaining using CREST antibodies compared to in situ hybridization withbiotinylated gamma satellite DNA Muiagenesis, 6, 297-302.

Miller.B M , Wemer.T. Weier.H U and NUsse.M (1992) Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH)simultaneously using telomenc and centromenc DNA probes. Radial. Res.,131, 177-185

Moens.P.B and Pearlman.R E. (1990) Telomere and centromere DNA areassociated with the cores of meiotic prophase chromosomes. Chromosoma,100, 8-14.

Moore.M.M.. Amtower.A . Doerr.C . Brock.K.H. and Dearfield.K.L (1987)Mutagemcity and clastogenicity of acrylamide in L5178Y mouse lymphomacells. Environ. Mulagen.. 9, 261-267.

PartonJ.W., Gamot.M L. and Beyers.J E (1991) Expulsion of Democolcine-induced rrucronuclei from mouse bone marrow polychromatic erythrocytes.Environ Mmagen , 17, 79-83.

Pietras.D.F.. Bennett.K L . Siracusa.L D.. Woodworth-Gutai.M.. ChapmanV.M.. Gross.K.W.. Kane-Haas.C. and Hastie.N.D. (1983) Construction of asmall Mus musculus repetitive DNA library identification of a new satellitesequence in Mus musculus. Nucleic Acids Res . 11, 6965—6983.

Pinkel.D., Straume.T. and GrayJ.W. (1986) Cytogenetic analysis usingquantitative, high-sensitivity, fluorescence hybridization Proc. Natl. Acad.Sci USA, 83, 2934-2938.

Russo.A.. Gabbani.G. and Simoncini.B (1994) Weak genotoxicit) ofacrylamide on premeiotic and somatic cells of mouse. Mulai. Res . 309,263-272

Saiki.R.K., Gelfand.D.H . Stoffel.S.. Scharf.S J.. Higuichi.R . Hom.G.T.Mullis.K.B. and Erlich.H.A (1988) Primer-denved enzymatic amplificationof DNA with a thermostable DNA polymerase. Science, 239, 487-491.

Salassidis.K.. Huber.R., Ziuelsberger.H. and BauchingerAl. (1992)Centromere detection in vinblastine- and radiation-induced micronuclei ofcytokinesis-blocked mouse cells by using in situ hybridization with a mousegamma (major) satellite DN'A probe. Emiron Mol. Mutagenesis. 19, 1-6.

SambrookJ.. Frilsch.E.F and Manialis.T. (1989) Molecular Cloning ALaboratory Manual. Cold Spnng Harbor Laboratory Press. Cold SpringHarbor. NY. USA.

Schrrud.W. (1975) The micronucleus test Mutat Res . 31. 9-15Schrie\er-Schuemmer.G and Adler.I-D 11993) A mouse stock with 38

chromosomes den\ed from the reciprocal translocation T(7.15l33AdCuonenet. Cell Genet.. 64. 122-127

206

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022

Lagging chromosomes in extruded micronudei induced by colchicine or acryiamide

Schriever-Schwemmer.G. and AdlerJ.-D. (1994) Differentiation of micronucleiin mouse bone marrow cells: a comparison between CREST staining andfluorescent in situ hybridization with centromeric and telomeric DNAprobes. Mutagenesis, 9, 333-340.

ShelbyJVl.D., Chain,K.T., Cornen,C.V. and Generoso.W.M. (1987) Acryiamide:induction of heritable translocations in male mice. Environ, Mutagen., 9,363-368.

Shiraishi.Y. (1978) Chromosome aberrations induced by monomericacryiamide in bone marrow and germ cells of mice. Muxat. Res., 57,313-324.

StylesJ.A., Richardson.C.R. and BurlinsonJB- (1983) A comparison of theincidence of micronuclei in blood and bone marrow in 3 strains of mousedosed with cyclophosphamide or hexamethylphosphoramide (HMPA).Muxat. Res., 122, 143-147.

Vanderkerken.K., Vanparys.P.H., VerschaeveX. and Kirsch-Volders,M. (1989)The mouse bone marrow micronucleus assay can be used to distinguishaneugens from clastogens. Mutagenesis, 4, 6-11.

VisseUB. and Choo,K.H. (1989) Mouse major (gamma) satellite DNA is highlyconserved and organized into extremely long tandem arrays: implicationsfor recombination between nonhomologous chromosomes. Genomics, 5,407-414.

Weier,H.-U. and Rosette.C. (1988) Generation of labelled RNA probes fromenzymatically amplified DNA templates. Nucleic Acids Res., 16, 11836.

Weier,H.-U. and Rosette.C. (1990) Generation of clonal DNA templatesfor in vitro transcription without plasmid purification. BioTechniques, 8,252-257.

Weier,H.-U., Zitzelsberger,H.F. and GrayJ.W. (1991) Non-isotopical labelingof murine heterochromatin m situ by hybridization with in vivo synthesizedbiotinylated mouse major satellite DNA. BioTechniques, 10, 498-505.

Wong,A.K.C. and RattnerJ.B. (1988) Sequence organization and cytologicallocalization of the minor satellite of mouse. Nucleic Acids Res., 16,11645-11661.

Yamamoto.K.I. and Kikuchi,Y. (1980) A comparison of diameters ofmicronuclei induced by clastogens and by spindle poisons. Mutat. Res., 71,127-131.

Received on November 4, 1996; accepted on February 10, 1997

207

Dow

nloaded from https://academ

ic.oup.com/m

utage/article/12/4/201/1059410 by National C

heng Kung University Library user on 29 Septem

ber 2022