Analysis of micronuclei induced in mouse early spermatids by mitomycin C, vinblastine sulfate or etoposide using fluorescence in situ hybridization

Non-radioactive in situ hybridization with mouse centromerespecific (major) gamma satellite DNA probe was used to analyzethe mechanism of induction of spermatid micronuclei (MN) causedby the alkylating agent mitomycin C (MMC), the spindle poisonvinblastine sulfate (VBL) or the DNA topoisomerase II inhibitoretoposide (VP-16). Male mice were treated with a single i.p.injection of 25 mg/kg VP-16, 5 mg/kg MMC or 2 mg/kg VBL, respectively.After 24 h (VP-16, VBL) or 13 days (MMC) stage I spermatid slideswere prepared and in situ hybridization was performed usinga polymerase chain reaction amplified mouse (major) gamma satelliteDNA probe. The observed MN frequencies for VP-16 and MMC, 6.2/1000and 7.5/1000 round spermatids, respectively, show a strong mutageniceffect on mouse germ cells compared with controls (1.4/1000spermatids). VBL, on the contrary, induced a much lower totalfrequency of MN (2.8/1000 spermatids) compared with previousresults on mouse somatic cells. Of MN in controls, 24% carrieda FISH signal. After correcting for background, MMC induced38.6% signal-positive MN, consistent with a predominantly clastogenicmode of action, while VBL induced 67.9% signal-positive MN,consistent with a mainly aneugenic mechanism. VP-16 induced65.5% signal-positive MN, indicating that its MN-inducing capacityis mainly due to whole chromosome lagging.     

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe

The DNA content distributions of micronuclei induced in mouse3T3 cells by ionizing radiation and chemicals was measured byflow cytometry. For a quantitative understanding of these distributions,micronuclei with increasing DNA contents were sorted and analysedfor the presence of centromeric signals using fluorescent insitu hybridization (FISH) with a mouse centromeric gamma satelliteprobe. Radiation-induced micronuclei were found to be producedmainly by chromosome fragments, whereas micronuclei inducedby the tear gas chlorobenzylidene malonitrile (CS) were foundto be produced mainly by whole chromatids. In contrast, micronucleiinduced by vinblastine (VBL) were, according to the shape oftheir DNA content distributions, produced mainly by whole chromosomesand by combinations of two or more whole chromosomes. With increasingDNA content, micronuclei induced by ionizing radiation alsocontained one or more whole chromosomes, whereas micronucleiinduced by CS or VBL were found to contain several whole chromatidsor chromosomes respectively. Computerized random breakage ofchromosomes and random combination of chromosome fragments,whole chromatids and whole chromosomes were used according tothe FISH results to simulate the measured DNA content distributionsof micronuclei. A good agreement was obtained between measuredand simulated distributions of micronuclei as well as betweenresults of the measured frequency of micronuclei showing centromericsignals as a function of their DNA content and those predictedby the simulations. These results demonstrate the usefulnessof flow cytometry and sorting combined with the FISH techniqueand computer simulations for producing a more detailed analysisof mechanisms of micronucleus induction. ⁵To whom correspondence should be addressed     

Micronuclei induced by alachlor, mitomycin-C and vinblastine in human lymphocytes: presence of centromeres and kinetochores and influence of staining technique

Antikinetochore antibodies and fluorescence in situ hybridizationwith an alphoid centromeric probe were applied to the cytokinesis-blockmicronucleus (MN) assay to study the suitability of these methodologiesto detect clastogenic/aneugenic activity in isolated human lymphocytes.The chemicals selected for this study were the herbicide alachlor,the clastogen mitomycin-C (MMC), and the aneugen vinblastinesulphate (VBL). Futhermore, MN frequencies obtained from slidesstained with May–Grünwald–Giemsa (MGG) andwith the DNA fluorochrome 4', 6'diamidino-2-phenylindole (DAPI)were compared to check if the DNA-specific DAPI facilitateda more accurate recording of MN than the unspecific MGG. Theresults showed that the detection of kinetochores (KC) or centromeres(CM) within MN are equally reliable and sensitive techniquesto study the mode of action of clastogenic and aneugenic agents.The comparison of CM and KC detection in control cultures suggestedthat up to 17% of spontaneous chromosomecontaining MN may bedue to KC disruption, whereas the majority are caused by dysfunctionin other components of the mitotic apparatus. Alachlor (7.5–20µg/ml) and MMC (0.6 µM) acted as pure clastogenswithout aneugenic activity, inducing exclusively KC- and CM-negativeMN. VBL produced primarily KC- and CM-positive MN, in accordancewith its known mechanism of action. A comparison between CMand KC data in the VBL treatment suggested that some 7% of KC-containingMN may not be detected by the probe. The frequencies of MN weregenerally higher in slides stained with DAPI than in those stainedwith MGG, especially in controls and clastogen-treated cultures.This finding probably reflects an underestimation with MGG ofsmall, light MN indistinguishable from the cytoplasmic background. ⁴To whom correspondence should be addressed     

Differential effects of cordycepin on the induction of sister-chromatid exchange and chromatid breaks in BALB/Mo mouse lymphocytes treated with mitomycin C

BALB/Mo mice lymphocytes, carrying endogenous Moloney murineleukaemia virus (M-MuLV), show significantly higher in vitrobaseline frequencies of sister chromatid exchange (SCE) thanlymphocytes from control (M-MuLV free) BALB/c mice. In vitrotreatment of lymphocytes with cordycepin (10 µg/ml), anantiviral substance, lowers the level of SCE in BALB/Mo cellsto the same value of BALB/c cells. The drug also reduces thehigher sensitivity of BALB/Mo compared to BALB/c lymphocytesto the induction of SCE by mitomycin C (MMC) administered eitherin vitro (3 x 10^–8, 10^–7 M) or in vivo (0.3, 3 mg/kg).BALB/Mo lymphocytes treated in vivo with a high dose of MMC(10 mg/kg) show reduced susceptibility to the induction of SCEbut increased frequencies of chromatid breaks and micronuclei.In this situation, cordycepin increases the level of SCE inBALB/Mo lymphocytes to exactly the level seen in BALB/c cells,but it does not affect the frequency of chromosomal aberrations.Since cordycepin is known to inhibit poly(A) synthesis, thusblocking RNA maturation, it is suggested that M-MuLV proviralintegration is not the sole factor responsible for the alteredsusceptibility of BALB/Mo lymphocytes to SCE induction, butthat it is most likely viral gene expression that is neededfor this effect to occur. On the contrary, the high susceptibilityof BALB/Mo lymphocytes to the induction of chromatid aberrationsby a high dose of MMC administered in vivo seems to be independentof viral maturation. ²To whom correspondence should be addressed     

Analysis of chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent damage resulting from low dose exposure to spindle poisons

The chromosome malsegregation pattern produced by the spindlepoisons vinblastine (VBL) and colchicine (COL) in human lymphocyteswas investigated. For this purpose, the fluorescence in situhybridization with centromeric DNA probes for chromosomes Xand 1 was applied to cell cultures treated with cytochalasinB, a cytokinesis-blocking agent. With this method, chromosomesegregation in daughter nuclei—retained in the same cellenvelope—can be easily analysed, simultaneously determiningthe loss and non-disjunction of specific chromosomes. Preliminaryexperiments demonstrated that the aneugenic effects elicitedby low dose exposure to spindle poisons were effectively detectedwith treatments from the S/G₂ phase (43 h) to harvest of cellcultures (60 or 72 h), with no drugfree medium recovery. Thisexposure protocol was used in subsequent experiments, whereCOL and VBL were applied at concentrations which had no effecton the cell cycle ranging to producing marked mitotic block.To account for sex differences in chromosome X instability,lymphocyte cultures from both male and female donors were usedto study X chromosome malsegregation. Chromosome 1 malsegregation,however, was analysed in female lymphocytes only. VBL inducedreproducible, significant increases of micronuclei in cytokinesis-blockedcells at 5 ng/ml and over. In female lymphocytes, chromosomeX loss was observed at 5 ng/ml whereas chromosome 1 loss wasonly observed at 10 ng/ml. In male lymphocytes, no significantchromosome loss was observed. On the other hand, nondisjunctionof both chromosomes X and 1 was effectively induced in femalelymphocytes even at 1.25 ng/ml, the lowest dose tested. In malelymphocytes, non-disjunction of chromosome X was observed at5 ng/ml. Treatments with COL produced a significant increaseof micronucleated cells only at the highest dose tested (20ng/ml). No significant increase in the incidence of either chromosomeX or chromosome 1 loss was observed. With cell cultures fromdonors of both genders, a significant increase in nondisjunctionof chromosome X was observed at 5 ng/ml. At the same dose, chromosome1 non-disjunction significantly increased. These results suggestthat in cytokinesis-blocked human lymphocytes, non-disjunctionis the prevalent error in chromosome segregation induced bylow dose exposure to spindle poisons. Interestingly, non-disjunctionwas effectively induced even at doses which did not producedetectable detrimental effects on the cell cycle. ¹To whom correspondence should be addressed     

A cytogenetic approach to evaluate in vivo somatic aneuploidy. Effects of diethylstilboestrol on mouse bone marrow cells

A cytogenetic approach to estimate in vivo mitotic non-disjunctionis proposed, based on chromosomal counting of mouse bone marrowmetaphases differentially stained by bromodeoxyuridine (BrdU)incorporation. The method allows the simultaneous assessmentof cell cycle delay, aneuploidy and SCE induction. Male mice,implanted with agar-coated BrdU tablets, were injected i.p.with diethylstilboestrol-diphosphate (DES-dp) in the dose range10–300 mg/kg and killed 18 or 24 h later. To investigatepossible sex differences a group of female mice of the samestrain and age was injected with 100 mg/kg. As positive controlssix males were injected with 1.8 mg/kg of vinblastine(VBL) sulphate.The induction of cell cycle delay was estimated by the relativefrequency of first, second and third mitoses after treatment.In spite of a large biological variability, a dose-dependentdelay of cellular proliferation kinetics was observed in DES-treatedmale mice. Treatment with VBL strongly delayed cell cycle progression,according to its antimitotic activity. Hyperploidy was assessedby chromosome counting of second generation metaphases only.After VBL injection, 10.2% of second mitoses were hyperploid,which is a frequency significantly higher than the 0.2% seenin control mice. No significant effect was detected at any DESdose. SCE induction was estimated in the same cells. A significantincrease over the control frequency was observed after 200 and300 mg/kg of DES. By analysis of variance (MANOVA) the dose—effectrelationship was fitted by a quadratic model. A sex differencewas observed only for spontaneous frequency of SCE with femalesshowing higher levels probably due to their lower weight andrelatively higher BrdU in vivo concentration. ¹Present address: Laboratory of Comparative Toxicology and Ecotoxicology,Istituto Superiore di Sanità, Viale Regina Elena 299,Rome, Italy ²To whom correspondence should be addressed      

Differentiation of micronuclei in mouse bone marrow cells: a comparison between CREST staining and fluorescent in situ hybridization with centromeric and telomeric DNA probes

Immunofluorescent staining (CREST) of kinetochore proteins andin situ hybridization (FISH) with centromeric DNA probes areable to distinguish between micronuclei (MN) containing laggingchromosomes or acentric fragments. Different frequencies ofsignal-positive MN induced by mitomycin C (MMC) were obtainedby Miller et al. (Mutagenesis, 6, 297–302, 1991) betweenCREST labelling and FISH with the mouse major-     

Gene amplification and gene deletion are induced at different rates in V79/AP4 Chinese hamster cells

The effect of mitomycin C (MMC) treatment on gene amplificationand gene deletion induction in a V79/AP4 Chinese hamster cellline was investigated. Spontaneous and induced cellular variantsresistant to N-phosphonacetyl-L-aspartate (PALA), which selectsfor carbamyl-P-synthetase, aspartate transcarbamylase and dihydroorotase(CAD) gene amplification events, and to intermediate concentrationsof 2,6-diaminopurine (DAP), which selects for adenine phosphoribosyltransferase (aprt) gene deletion events, were isolated. Themolecular analysis of spontaneous and induced PALA- and DAP-resistantclones showed that while in the former the CAD gene was amplified,in the latter both mutations and deletions occurred at the aprtgene. These results indicate that MMC favored the rise of geneamplification rather than gene deletion events, and suggestthat aberrant DNA replication processes were responsible forthe observed gene amplification. ¹To whom correspondence should be addressed     

Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X-irradiation analysed by fluorescence in situ hybridization. III. Chromosome malsegregation/aneuploidy

Transgenic mice with foreign DNA inserted into three pairs ofchromosomes were exposed to 2 Gy X-rays in order to study theinduction and persistence of chromosome malsegregation and aneuploidyup to 28 days after exposure. By tracing the marker chromosomesin cytokinesis–blocked binucleated splenocytes using fluorescencein situ hybridization (FISH), reciprocal products of chromosomemalsegregation in the daughter nuclei were analysed. FISH withmurine minor satellite DNA was employed to detect chromosomeloss (MN with a centromere) in binucleated splenocytes. In additionto its clastogenic effects, X- irradiation also showed aneugenicactivity, which was observed as centromere positive micronuclei(C + MN) and malsegregated marker chromosomes detected by FISH.The initial frequency of micronuclei (MN) analysed by AcridineOrange staining immediately after X-ray exposure was found tobe 42.3 per 100 binucleated cells. The MN frequency declinedin an exponential manner and at day 14, reached about half thevalue observed immediately after irradiation and 14% MN weredetected at day 28. Of these MN, 25% were centromere positiveat day 0 as detected by minor satellite signal after FISH. Thepercent-age C + MN increased further at day 3 and declinedat day 14 to the level observed at day 0. There were 7.6% malsegregatedcells immediately after X-irradiation as analysed by two colourFISH. This value increased further during later intervals andremained stable until day 28. A combination of the AcridineOrange staining and FISH with minor satellite DNA and markerDNA to detect aneuploidy and chromosome malsegregation, wasutilized in the present study to demonstrate the induction andpersistence of aneugenic and clastogenic damage in trans-genicmice irradiated in vivo. ¹To whom correspondence should be addressed     

Classification of micronuclei in murine erythrocytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA.

Micronuclei (MN) in erythrocytes of mouse bone marrow cells were induced in vivo by the spindle poisons colchicine (COL) and vinblastine (VBL), by hydroquinone (HQ) and by the alkylating agent mitomycin C (MMC). Two different methods were applied to detect whole chromosomes with centromeric proteins or chromatin in MN to discriminate between spindle damaging or clastogenic activity of these chemicals. One method determined the fraction of MN with centromeric chromatin by immunofluorescent staining using antikinetochore antibodies (CREST staining). The other method applied non-radioactive in situ hybridization with a novel DNA probe. The fractions of MN that showed positive signals by either technique thus indicating with a high probability the presence of whole chromosomes instead of acentric fragments, were in good agreement for COL, VBL and HQ. After application of MMC, however, 4.5% of the MN were CREST-positive, while 29% gave a positive hybridization signal. The results suggest, that kinetochores may have lost certain centromeric antigens due to treatment with MMC so that MN containing whole chromosomes appear CREST-negative. The presented in situ hybridization scheme using satellite DNA is a more direct detection and is advantageous to the CREST staining technique in that it is unaffected by damage of kinetochore or centromeric function.     

DNA content determination of micronucleated polychromatic erythrocytes induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood

The frequencies and DNA distributions of micronuclei (MN) inpolychromatic erthrocytes (PCEs) from bone marrow (BM) and peripheralblood (PB) of mice after four different treatments were determinedby flow cytometry. PCEs were differentiated from normochromaticerythrocytes (NCEs) using the fluorescent RNA stain Thiazoleorange, while MN in erythrocytes were detected with the DNAstain Hoechst 33342. The treatments were X-irradiation (1 Gy)cyclophosphamide (CPA; 30 mg/kg) vincristine sulphate (VCR;0.08 mg/kg) and cholchicine (COL; 1 mg/kg). All treatments showedincreased frequencies of micronucleated PCEs at 30 h after treatmenthi BM and at 50 h in PB. The clastogens(X-irradiation and CPA)and the spindle poisons (VCR and COL) could be grouped accordingto the fluorescent characteristics of the induced MN as wellas the relative frequency of small (0.5—2% of the diploidDNA content) and large (2—10%) MN. In PB the relativefrequency of large MN was lower than in BM indicating that theywere partly eliminated before entrance into the peripheral circulation. ³To whom correspondence should be addressed     

Antigenotoxicity of coffee in the Drosophila assay for somatic mutation and recombination

The somatic mutation and recombination test (SMART) in Drosophilamelanogaster was carried out to investigate whether or not coffeecan modulate the genotoxicity of the well-established mutagenic/carcinogenicchemicals cyclophosphamide (CPH), diethylnitrosamine (DEN),mitomycin C (MMC), procarbazine (PRO) and urethane (URE). Forthis purpose, 3-day-old larvae, trans-heterozygous for the winghair markers mwh (multiple wing hairs) and flr³ (flare³), wereraised on instant medium containing either the genotoxin aloneor in combination with instant coffee. From the results obtained,it was evident that the chronic co-administration of coffeewas effective in significantly reducing the frequencies of singleand twin spots induced by CPH, DEN, MMC and URE but not PRO.The maximum reduction was observed in the frequencies of twinspots (produced by mitotic recombination) after feeding larvaeon medium containing coffee in combination with the compoundsCPH or URE. ¹C-4/48 (second floor), Safdarjung Development Area, New Delhi - 110 016, India     

Detection of aneugenic and clastogenic potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe

In human hepatoma (Hep G2) cells and peripheral blood lymphocytes(HPBL) the cytokinesis-blocked micronuclei (MN) and fluorescentin situ hybridization (FISH) assays were applied to study aneugenicand clastogenic potentials of X-rays, directly and indirectlyacting chemicals. Induction of MN was studied in vitro followingtreatment with X-rays, directly acting chemicals, such as methylmethanesulphonate(MMS), colchicine (COL), vincristine sulphate (VCS) and vinblastinesulphate (VBS), and indirectly acting agents, such as cyclophosphamide(CP), hexamethylphosphoramide (HMPA), 2-acetylaminofluorene(2-AAF) and 4-acetylaminofluorene (4-AAF). Depending on thepresence of the fluorescent signal in the MN following FISHwith a human DNA centromeric probe, MN in the binucleated HepG2 cells and lymphocytes were scored as centromere-positiveor centromere-negative, representing an aneugenic and clastogenicevent respectively. In the controls     

Extruded micronuclei induced by colchicine or acrylamide contain mostly lagging chromosomes identified in paintbrush smears by minor and major mouse DNA probes

In the mouse bone marrow micronucleus assay, it was studiedwhether micronuclei (MN) could be expelled from polychromaticerythrocytes (PCE) in a similar way to the main nucleus. Toavoid the disrupting centrifugation step of the conventionalbone marrow preparation procedure, the paintbrush techniquewas used in the present experiments. With May–Grünwald–Giemsastaining of paintbrush slides, 5% of the colchicine (COL)-inducedMN were found attached to the outside membranes of PCE and wereregarded as extruded. Of the acrylamide (AA)-induced MN, 22%were extruded. After fluorescence in situ hybridization (FISH)of a total of 300 MN per chemical treatment with the mouse minorand major satellite DNA probes, 9.7% MN were extruded in theCOL group and 8.3% MN were extruded in the AA group. FISH showedthat 76% of the retained COL-induced MN were signal-positive,indicating that they contained entire chromosomes. With A A,29% minor-positive and 28.3% major-positive retained MN werefound, confirming its known clastogenicity. However, the observedfrequency of signal-positive MN (1.7 MNPCE_pos/ 1000 PCE) inthe AA group was about three times higher than in the control(0.5 MNPCE_pos/1000 PCE) which indicates that AA has aneugenicpotential. FISH analysis of the extruded MN showed 72–100%major as well as minor signals. It is concluded that expelledMN contain mostly entire chromosomes. ³To whom correspondence should be addressed     

Detection of chromosome malsegregation to the daughter nuclei in cytokinesis-blocked transgenic mouse splenocytes

Most of the recently developed tests for detecting aneugenic activity of chemicals are based on the induction of micronuclei (MN) in cytokinesis-blocked (CB) binucleated cells. In such a test, aneugens can be discriminated from clastogens by checking for the presence of centromeres in the MN, indicating the loss of whole chromosomes. Tracing particular chromosomes in interphase nuclei using fluorescencein situ hybridization (FISH) with chromosome-specific DNA probes is another method used for detecting numerical chromosome aberrations. Here, we describe a method using a cytokinesis-blocked MN assay in combination with identifying specific chromosomes of mice. For this purpose transgenic mice with foreign DNA inserted in three pairs of their chromosomes were generated. Splenocytes of these mice were cultured and treatedin vitro with vinblastine (VBL) or X-rays, followed by recovery in medium containing cytochalasin B. By tracing the marker chromosomes in binucleated splenocytes, reciprocal products of chromosome malsegregation to the daughter nuclei could be easily traced. The results showed that besides clastogenic activity, X-rays also exhibited aneugenic activity. Treatment with vinblastine showed a close relationship between micronuclei induction and chromosome malsegregation, although at higher doses malsegregation processes became more prominent. Simultaneous malsegregation of more than one chromosome was observed frequently, but the three marker chromosomes were found to be randomly involved in this process.     

The micronucleus assay in mouse peripheral blood reticulocytes demonstrates the transmission of chromosomal instability induced by mitomycin C and benzo[a]pyrene

The frequency of micronuclei (MN) in mouse peripheral bloodreticulocytes supravitally stained with acridine orange wasassessed at different time intervals after treatment in vivowith two dose levels of mitomycin C (MMC) and benzo[a]pyrene.Increased frequencies were observed for many days after treatment,indicating that MN may be produced as a consequence of chromosomalinstability transmitted by proliferating erythroblasts. Theseresults confirm previous evidence of the persistent cytogeneticeffects of MMC and suggest that clastogenic agents acting bydifferent primary lesions may have a similar ability to inducethis effect.     

Analysis of micronucleus induction of pyrimethamine in in vitro CHL cells and in in vivo mouse bone marrow cells

In vivo and in vitro mutagenicity of pyrimethamine were examinedin the micronucleus test. Pyrimethamine strongly induced micronucleiin a dose-dependent manner in the in vitro micronucleus testusing the Chinese hamster lung (CHL) cell line, when treatedat 0.2–1.6 µg/ml for 48 h. The in vivo micronucleustest was carried out in mice after the first, second, thirdand fourth administration of doses up to 40 mg/kg p.o. The resultsshowed no increased frequency of micronuclei after any treatment,though pyrimethamine was shown to persist at levels >2 µ/mlin plasma after a single oral administration of 50 mg/kg. ¹To whom correspondence should be addressed     

Combination effects of clastogens in the mouse peripheral blood micronucleus assay

In order to study how two chemicals interact to induce micronuclei,simple ethylating agents [ethyl methanesulfonate (EMS), ethylethanesulfonate (EES) and N-ethyl-N-nitrosourea (ENU)], spindlepoisons [vincristine sulfate (VINC) and colchicine (COL)] andan oxidizing agent [potassium bromate (KBrO₃)] were used asmodel chemicals for combination treatments. The frequency ofmicronucleated reticulocytes (MNRETs) was evaluated in micetreated with two of these chemicals at a time. The combinationsof ethylating agents (EMS and EES; EMS and ENU) and of spindlepoisons (VINC and COL) induced more micronuclei than those expectedon an additive basis. The apparent synergism was due to a ‘combineddose’ which could be calculated by the dosimetric conversionof one chemical to the other, when damage induced by each chemicalwas ‘equivalent’ in the induction of MNRETs. Incontrast, no apparent synergism in induction of micronucleiwas observed when two chemicals with different modes of clastogenicaction (EMS and KBrO₃ or EMS and VINC) were combined. ¹Present address: Department of Drug Safety Research, EisaiCo. Ltd, 1-3 Tokodai 5-Chome, Tsukuba-shi, Ibaraki 300-26, Japan ²Present address: Department of Biological Sciences, RiversState University, PMB 5080, Portharcourt, Nigeria ³To whom correspondence should be addressed      

Chromosome content and ultrastructure of radiation-induced micronuclei

Unrepaired or misrepaired radiation damage in mammalian chromosomescan result in micronucleus formation at the first cell division.This represents loss of genomic information which may causecell death. To improve our understanding of the mechanism ofradiation-induced micronucleus formation, we characterized micronucleusultrastructure and identified the origin of micronucleus DNA.Immunofluorescence microscopy showed that micronuclei were structurallysimilar to main nuclei since they contained nuclear lamins Aand C and were encapsulated by a network of vimentin intermediatefilaments. The contents of radiation-induced micronuclei werecharacterized using fluorescence in situ hybridization to probefor DNA originating from chromosomes 2, 7, 11 and 16. We postulatedthat if incorporation of DNA into micronuclei were random, thenthe probability of chromosomal DNA in micronuclei would be relatedto the target, i.e. chromosome size. Our results demonstratedthat incorporation of DNA from smaller chromosomes (11 and 16)was not different from expected values but incorporation ofDNA from the larger chromosomes (2 and 7) was significantlygreater than expected. Not all chromosomes in the human genome,therefore, were equally susceptible to genomic loss by micronucleusencapsulation. In conclusion, radiationinduced micronuclei havesimilar structural characteristics to main nuclei, chromosomedamage and/or repair after ionizing radiation may be non-random,and micronucleus formation may reflect this variability. ³To whom correspondence should be addressed     

Mitotic indirect non-disjunction in phytohemagglutinin stimulated human lymphocytes

In a previous publication we demonstrated that in cells of Viciafaba micronuclei derived from whole lagging chromosomes or chromatidsmay perform DNA synthesis and mitotic condensation in synchronywith main nuclei and be regained by main nuclei at the nextmitosis, giving rise to trisomic cells together with diploids.This process was called ‘mitotic indirect non-disjunction’(MIND). In the present work the occurrence of MIND was studiedin human lymphocytes cultivated in vitro. Human lymphocyteswere treated with low colcemid concentrations until fixation;BrUdR was supplied together with colcemid to distinguish thenumber of mitoses performed by the cells (M₁ M₂ and M₃ cells).The frequencies of M₁ ana-telophases with single lagging chromosomes/chromatidsand of M₂⁺ prophases with single micronuclei in synchronousmotitic condensation with main nuclei were evaluated. On thisbasis the expected frequencies of both monosomic and trisomicM₂ cells were calculated, according to the hypothesis of MIND.Their observed frequencies were very close to those expected.These results support the hypothesis of the occurrence of MINDin human lymphocytes. ⁵To whom correspondence should be addressed