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-     

Use of a centromere-specific DNA probe (p82H) in nonisotopic in situ hybridization for classification of micronuclei.

The DNA probe p82H was used to visualize centromeric DNA in micronuclei (MN) of human cells. Slides prepared from cultures treated by the aneugen (causing aneuploidy) colcemid showed significantly more MN with centromeric signals than those treated by the clastogen (causing chromosome breakage) bleomycin. These results indicate that in situ hybridization with the alphoid p82H DNA probe is a suitable method with which to distinguish between MN containing whole chromosomes and acentric fragments, and hence allows one to discriminate between the clastogenic and aneugenic effects in MN formation.     

Chromosomal composition of micronuclei in mouse NIH 3T3 cells treated with acrylamide, extract of Tripterygium hypoglaucum (level) hutch, mitomycin C and colchicine, detected by multicolor FISH with centromeric and telomeric DNA probes

The chromosomal composition of micronuclei (MN) induced by the model mutagens mitomycin (MMC) and colchicine (COL) as well as by acrylamide (AA) and the traditional Chinese medicine Tripterygium hypoglaucum (level) hutch (THH) in NIH 3T3 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using DNA probes for the centromere repeated minor satellite DNA and the telomeric hexamer repeat (TTAGGG). The majority of MN (78.6%) from treatment with MMC (0.1 microg/ml) did not show centromeric signals, reflecting the clastogenic action of MMC. Following treatment with COL (0.1 microg/ml), 74.5% of the MN showed centromeric signals and several telomeric signals, indicating that MN induced by this well-known aneugen were mainly composed of whole chromosomes. After treatment with AA (100, 200 and 400 microg/ml) both MN containing whole chromosomes and MN containing acentric fragments were found to increase in a dose-dependent manner, demonstrating that AA is not only a clastogen but also an aneugen. THH induced a high frequency of MN harboring whole chromosomes at all concentrations tested (5, 10 and 20 microl/ml) and produced a dose-dependent increase in fragment-containing MN, indicating that THH has both aneugenic and clastogenic potential.     

Evaluation of a suitable DNA damage biomarker for human biomonitoring of exposed workers

The objective of this study was to identify a sensitive and noninvasive biomarker of early genotoxic effects, for health risk assessment of workers exposed to mixtures of low doses of xenobiotics. We studied 30 workers exposed to antineoplastic drugs, 57 workers exposed to different mixtures of polycyclic aromatic hydrocarbons (PAHs) (41 airport workers and 16 paving workers) and 76 controls. Comet and micronucleus (MN) tests were performed on lymphocytes and exfoliated buccal cells. The MN assay on lymphocytes did not show significant differences between exposed and controls, while the MN assay on exfoliated buccal cells showed higher values in workers exposed to antineoplastics as compared with controls (0.85 vs. 0.48, P = 0.042). The comet assay on lymphocytes showed a higher comet percentage value (18.11 vs. 11.24 in controls, P = 0.001) and mean tail moment (TM) value (21.84 vs. 16.72 in controls, P = 0.003) in individuals exposed to PAHs as compared with controls; no significant differences were found in exposed to antineoplastics. The comet assay on exfoliated buccal cells did not show significant differences between exposed and control groups for comet percentages, whereas the TM value was higher in workers exposed to PAHs (55.1 vs. 32.31 for controls, P < 0.001). These results show that exfoliated buccal cells, obtained by a noninvasive procedure, represent robust target cells to assess the occupational exposure to inhalable mixture of chemicals at low doses. The comet assay seems to be suitable to promptly evaluate the genotoxic effects of PAHs mixtures that also contain volatile substances. The MN test is suitable to evaluate the effects of antineoplastics. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

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     

Flow cytometric analysis of genetic damage, effect on cell cycle progression, and apoptosis by thiophanate-methyl in human lymphocytes

Flow cytometric technique was used to study the effects of the fungicide Thiophanate-methyl on cell proliferation, micronucleus induction, and apoptosis in human peripheral blood lymphocytes treated in vitro. In particular, a combined approach of flow cytometry and fluorescence in situ hybridization (FISH) with a pancentromeric alpha-satellite probe was used to evaluate the mechanism of micronucleus induction by Thiophanate-methyl. Flow sorted micronuclei (MN) induced in human lymphocytes by Thiophanate-methyl were analyzed by FISH and the results were compared with results from FISH analysis on MN in binucleated cells. It could be shown that most MN induced by Thiophanate-methyl did not reveal any centromeric signal, thus demonstrating clastogenic action of this fungicide. Moreover, it was found that as a function of the concentration of Thiophanate-methyl, cellular proliferation was delayed and the frequency of apoptotic cells was increased.     

Detection of centromeres in vinblastine- and radiation-induced micronuclei of human lymphocytes using FISH with an α satellite pancentromeric DNA probe

Fluorescence in situ hybridisation (FISH) with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in micronuclei of human lymphocytes induced by γ-irradiation and by Vinblastine sulfate. In a cytokinesis-block micronucleus assay a dose-dependent increase of micronuclei was detected for both agents. 72–89% of Vinblastine-induced micronuclei, but only 7–48% of radiation-induced micronuclei showed centromere-positive fluorescence signals. Vinblastine treatment frequencies of centromere-negative micronuclei did not increase compared to control values, nor did frequencies of centromere-positive micronuclei in irradiated lymphocytes. Since FISH with an α satellite DNA probe allows the direct detection of centromeric DNA sequences the spindle damaging or clastogenic effectiveness of a compound can be easily and reliably examined in a cytokinesis-block micronucleus assay in human lymphocytes. © 1996 Wiley-Liss, Inc.     

Aneugenic activity in human cultured lymphocytes. An overall study with colchicine using the micronucleus assay and fluorescence in situ hybridization techniques

The effects induced by aneugenic agents on chromosome segregationare manifold. The biological relevance of these effects hasled to the development of assays specifically detecting aneugens.In this context, the micronucleus (MN) assay in bmucleated humanlymphocytes along with FISH has been considered a pertinenttool for detecting aneugenic and clastogenic activity. However,the MN assay is insensitive in detecting aneugenic effects otherthan chromosome loss. By using the aneugenic model compoundcolchicine and X chromosome centromere-specific FISH, we haveshown that besides chromosome loss in binucleated cells, othereffects such as MN in mononucleated cells, cells arrested atmetaphase, polyploidy and non-disjunction are also consistentlyinduced by aneugenic agents. A chromosome 1 centromeric probewas used simultaneously with X chromosome centromeric labelingin mononucleated cells in order to distinguish polysomy frompolyploidy. It is concluded that all these effects should beconsidered for a comprehensive evaluation of aneugenic activity. ¹To whom correspondence should be addressed. Tel: +34 3 581 2052; Fax: +34 3 581 2387; Email: rmd{at}cc.uab.es     

Molecular cytogenetic evaluation of the aneugenic effects of teniposide in somatic and germinal cells of male mice

The ability of topoisomerase II inhibitor, teniposide, to induce aneuploidy and meiotic delay in somatic and germinal cells of male mice was investigated by fluorescence in situ hybridisation (FISH) assay using labelled DNA probes and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, respectively. Colchicine and mitomycin C were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. Using FISH assay with centromeric and telomeric DNA probes for erythrocyte, micronuclei (MN) showed that teniposide is not only clastogenic but also aneugenic in somatic cells in vivo. The assay also showed that chromosomes can be enclosed in the MN before and after centromere separation. By using the BrdU incorporation assay, it could be shown that the meiotic delay caused by teniposide in germ cells was ～48 h. Disomic and diploid sperms were shown in epididymal sperm hybridised with DNA probes specific for chromosomes 8, X and Y after teniposide treatment. The prevalence of autodiploid (XX88 and YY88) sperm and disomic XX8 or YY8 sperm indicates that the second meiotic division was more sensitive to teniposide than the first meiotic division. The results also suggest that earlier prophase stages contribute relatively less to teniposide-induced aneuploidy. Both the clastogenic and the aneugenic potential of teniposide can give rise to the development of secondary tumours and abnormal reproductive outcomes in cured cancer patients and medical personnel exposing to drug regimens that include teniposide. Thus, genetic counselling of these patients should take place before the start of chemotherapy and should take the present results into consideration.     

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     

Genotoxicity of hydrochlorothiazide in cultured human lymphocytes. I. Evaluation of chromosome delay and chromosome breakage

Hypertension is often treated with diuretics, like hydrochlorothiazide (HCTZ). Previous results on the in vitro genotoxicity of HCTZ are equivocal. In the present study, we have evaluated the genotoxicity of HCTZ in cultured human lymphocytes using the Cytokinesis Blocked Micronucleus (CBMN) assay. In addition, micronucleus (MN) induction was analyzed by Fluorescence In Situ Hybridization (FISH) with an alpha-satellite DNA centromeric probe to distinguish between clastogenic and aneugenic effects. Lymphocyte cultures from 32 healthy adults were exposed to 5 and 40 microg/ml HCTZ. Age, gender, and smoking were evaluated as factors affecting the MN analysis. We found that HCTZ increased MN frequencies. FISH analysis revealed that HCTZ exerts its genotoxicity more strongly at the 40 microg/ml concentration, and principally through chromosome delay (aneugenicity). Multiregression analysis of our results confirmed the known effect of age and gender on MN induction in human lymphocytes. Smoking was also a confounding factor for MN induction, especially for centromere-negative MN frequencies. Under the experimental conditions used, only age had a clear positive effect on the response of lymphocytes to HCTZ. These data indicate that HCTZ produces micronuclei in cultured human lymphocytes by a mechanism that involves chromosome delay and to a lesser extent through chromosome breakage.     

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.     

Centromere detection in vinblastine- and radiation-induced micronuclei of cytokinesis-blocked mouse cells by using in situ hybridization with a mouse gamma (major) satellite DNA probe.

Non-isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis-blocked micronucleus assay a dose-dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere-positive hybridization signals in an order of magnitude of 70-90%, but after radiation exposure the magnitude was only 10-20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis-blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy-inducing and clastogenic agents.     

Analysis of micronuclei induced by 2-chlorobenzylidene malonitrile (CS) using fluorescence in situ hybridization with telomeric and centromeric DNA probes, and flow cytometry

Micronuclei (MN) induced in NIH 3T3 cells by the tear gas 2-chlorobenzylidenemalonitrile (CS) were studied in detail using fluorescence insitu hybridization (FISH). The chromosomal composition of CS-inducedMN was analysed by simultaneous use of DNA probes for the telomerichexamer repeat (TTAGGG) and for mouse major satellite DNA. Themajority of CS-induced MN, 63–73% of all CS-induced MNat doses from 10 to 30 µM CS, revealed centromeric signalsand several telomeric signals suggesting their origin from wholechromosomes. Almost 50% of all CS-induced MN showed one centromericsignal and were assumed to contain one single chromosome. Only4.5% of all MN did not show any signal and 23–28%showedtelomeric signals only, thus containing acentric fragments.Based on the experimental data from FISH the distribution ofthe DNA content of CS-induced MN was calculated assuming randombreakage of chromosomes, and random combination of chromosomesand chromosome fragments. Good agreement between calculatedMN distributions and distributions measured by flow cytometrywas obtained. By sorting MN with distinct DNA content and hybridizationof the sorted MN with the centromeric probe, regions in theMN distribution containing mainly MN with single whole chromosomescould be demonstrated.     

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     

Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C

Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using two protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standard compounds, whereas in protocol B (Prot. B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concentrations tested (7.5 and 15 microg/ml). At the highest dose (30 microg/ml) the aneugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher ( approximately 3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change when different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, the greater clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B, approximately 3-fold higher than Prot. A at the most effective concentration and approximately 2-fold with 24 h treatment at 0.17 microg/ml MMC. With GF, the FISH data in human liver fibroblasts (80% C+MN) were fairly consistent with those obtained in the rodent cell lines. In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C+MN. FISH analysis showed that MMC induced mainly MN containing acentric fragments rather than whole chromosomes. In conclusion we have demostrated that chemically induced genetic effects are strongly dependent on the cell culture employed, treatment schedule and intra- and post-treatment experimental conditions.     

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.     

Cytogenetic and oxidative damage induced in human lymphocytes by platinum,rhodium and palladium compounds

This study of soluble compounds of platinum, palladium and rhodium investigated the genotoxic properties of (NH(4))(2)PtCl(4), PtCl(2), PtCl(4), (NH(4))(2)PdCl(4), PdCl(2) and RhCl(3) using the human lymphocyte micronucleus (MN) assay coupled with fluorescence in situ hybridization (FISH). A pancentromeric DNA probe was used to detect both centromere-positive micronuclei (C+ MN) as well as centromere-negative micronuclei (C- MN). A modified alkaline single cell gel electrophoresis (SCGE) assay was used to evaluate the possible role of oxidative damage in genotoxicity of the Pt, Pd and Rh compounds tested. Two enzymes, endonuclease III and formamidopyrimidine glycosylase, were used to recognize and subsequently cut oxidized pyrimidines and purines, respectively. A significant induction of MN by Pt and Rh compounds was observed compared with controls, while (NH(4))(2)PdCl(4) and PdCl(2) displayed weak significant MN induction. The FISH technique revealed no significant difference in the frequency of C+ MN and C- MN for all compounds tested. These findings suggest that MN induction is due both to a clastogenic and an aneuploidogenic mechanism. SCGE detected an increase in the level of DNA oxidative damage for the Rh compound and for Pt(IV) which was also capable of inducing an increase in primary DNA damage at all the tested doses. This work highlights the stronger genotoxicity, likely mediated by oxidative damage induction, of Pt and Rh compounds compared with Pd salts.     

Characterization of chromosomes and chromosomal fragments in human lymphocyte micronuclei by telomeric and centromeric FISH

Micronuclei (MN), used as a biomarker of effect in exposure to genotoxic carcinogens, derive from chromosomes and chromosomal fragments lagging behind in anaphase. The two types of MN are usually distinguished from each other by centromeric fluorescence in situ hybridization (FISH), centromere-positive (C(+)) MN representing entire chromosomes and centromere-negative (C(-)) MN chromosomal fragments. The incorporation of various types of chromosomal fragments and chromosomes and chromatids to MN is still poorly understood. We used directly labelled pancentromeric and pantelomeric DNA probes to examine the contents of MN in cultured binucleate lymphocytes of four unexposed, healthy subjects (two men and two women) 35-56 years of age. The presence and number of telomeric and centromeric signals was evaluated in 200 MN (50 MN per subject). These data were used to estimate the proportion of MN harbouring terminal/interstitial fragments, acentric/centric fragments, chromatid-type/chromosome-type fragments and entire chromatids/chromosomes. The majority of the C(+) MN (96% in men and 86% in women) found contained telomeric (T(+)) sequences. Most of the C(+) T(+) MN had one centromere and two or one telomere signals, suggesting that single chromatids were more frequently involved in MN than both sister chromatids. Among the C(-) MN, telomere signals were found in 91% (men) and 79% (women), showing that fragments in MN were mostly terminal. Most C(-) T(+) MN had one telomere signal, indicating higher prevalence for chromatid-type than chromosome-type terminal fragments. Combined centromeric and telomeric FISH is expected to increase the sensitivity of detecting exposure-related effects, when the exposure induces specific types of MN and its effect is low. This approach could particularly have use in discriminating between MN harbouring chromatid- and chromosome-type fragments in studies of human exposure to chemical clastogens and ionizing radiation.     

What do human micronuclei contain?

As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between these phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of genotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. A specific analysis of the induced type of MN may considerably improve the sensitivity of detecting the exposure effect. MN harbouring chromosomes can be distinguished from those harbouring acentric fragments by the presence of a centromere. The proportion of centromere-positive MN in human lymphocytes increases with age, which primarily reflects an age-dependent micronucleation of the X and Y chromosomes. The X chromosome especially tends to lag behind in female lymphocyte anaphase, being micronucleated more efficiently than autosomes. There is some evidence for an enhanced prevalence of fragments from chromosome 9 in spontaneous human lymphocyte MN and from chromosomes 1, 9 or 16 in MN induced in vitro by some clastogens; the breakage appears to occur in the heterochromatic block of these chromosomes. Although there are indications that centromere identification can improve the detection of clastogenic effects in humans in vivo, smokers have not shown an increase in centromere-negative MN in their cultured lymphocytes, although smoking is known to produce chromosomal aberrations. This may suggest that fragment-containing MN and chromosomal aberrations cover partly different phenomena. Understanding the mechanistic origin and contents of MN is essential for the proper use of this cytogenetic end-point in biomarker studies, genotoxicity testing and risk assessment.