Chromosomal changes: induction, detection methods and applicability in human biomonitoring

Biochimie 88 (2006) 1515–1531 www.elsevier.com/locate/biochi

Chromosomal changes: induction, detection methods and applicability in human biomonitoring R. Mateuca*, N. Lombaert, P.V. Aka, I. Decordier, M. Kirsch-Volders Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium Received 2 March 2006; accepted 10 July 2006 Available online 04 August 2006

Abstract The objective of this state of the art paper is to review the mechanisms of induction, the fate, the methodology, the sensitivity/specificity and predictivity of two major cytogenetic endpoints applied for genotoxicity studies and biomonitoring purposes: chromosome aberrations and micronuclei. Chromosomal aberrations (CAs) are changes in normal chromosome structure or number that can occur spontaneously or as a result of chemical/radiation treatment. Structural CAs in peripheral blood lymphocytes (PBLs), as assessed by the chromosome aberration (CA) assay, have been used for over 30 years in occupational and environmental settings as a biomarker of early effects of genotoxic carcinogens. A high frequency of structural CAs in lymphocytes (reporter tissue) is predictive of increased cancer risk, irrespective of the cause of the initial CA increase. Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase. The cytokinesis-block micronucleus (CBMN) assay is the most extensively used method for measuring MN in human lymphocytes, and can be considered as a “cytome” assay covering cell proliferation, cell death and chromosomal changes. The key advantages of the CBMN assay lie in its ability to detect both clastogenic and aneugenic events and to identify cells which divided once in culture. Evaluation of the mechanistic origin of individual MN by centromere and kinetochore identification contributes to the high sensitivity of the method. A number of findings support the hypothesis of a predictive association between the frequency of MN in cytokinesis-blocked lymphocytes and cancer development. Recent advances in fluorescence in situ hybridization (FISH) and microarray technologies are modifying the nature of cytogenetics, allowing chromosome and gene identification on metaphase as well as in interphase. Automated scoring by flow cytometry and/or image analysis will enhance their applicability. © 2006 Elsevier Masson SAS. All rights reserved. Keywords: Chromosomal aberration; Micronuclei; Fluorescence in situ hybridization; Genetic polymorphisms

1. Introduction The genotoxic effects of a potential mutagen depend on its cellular target(s). Some chemicals need to be metabolized

Abbreviations: BFB cycle, breakage-fusion-bridge cycle; CA assay, chromosomal aberration assay; CAs, chromosomal aberrations; CBMN assay, cytokinesis-block micronucleus assay; CSAs, chromosome-type aberrations; CREST, Calcinosis, Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly and Telangiectasia; CTAs, chromatid-type aberrations; FISH, fluorescence in situ hybridization; mBANDs, multicolor banding technique; mFISH, multiplex FISH; MN, micronuclei; NBUD, nuclear budding; NPB, nucleoplasmic bridge; PBLs, peripheral blood lymphocytes; SCEs, sister chromatid exchanges; SKY, spectral karyotyping. * Corresponding author. Tel.: +32 2 629 1326; fax: +32 2 629 2759. E-mail address: [email protected] (R. Mateuca). 0300-9084/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2006.07.004

before acquiring their mutagenic capacity. Mutagens can induce genomic changes by targeting DNA directly or/and indirectly, by binding to proteins involved in the maintenance of genome integrity (Fig. 1) [1]. For example, ionizing radiation has DNA as a direct target and gives rise to single and double-strand breaks (DSBs). The indirect effects of ionizing radiation on DNA occur after a photon interacts with a water molecule leading to hydrolysis and production of free radicals which subsequently attack DNA and proteins [2,3] (Fig. 2). Tubuline disrupting chemicals like nocodazole and carbendazim [4–6] induce malsegregation of chromatids/chromosomes by interfering with the accurate functioning of the mitotic and meiotic spindle (for review [7]). Metals form a particularly complex class of mutagens, due to the fact that they have multiple cellular targets. The most important mutagenic processes described so far for metals are summarized in Fig. 3 and

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Fig. 1. Mechanisms of action of DNA and non-DNA interacting genotoxicants. Mutagens can induce genotoxic effects and cancer either by targeting DNA directly or indirectly, by binding to proteins involved in the maintenance of genome integrity (e.g. tubulins, DNA repair enzymes, proteins involved in the control of the cell cycle etc.). Non-genotoxic compounds are also capable to induce cancer by increasing cell proliferation rate (e.g. mitogens), by changing the DNA methylation status or by triggering cytotoxicity. Cell proliferation can be a primary effect of the carcinogen or a secondary effect consequent to cell toxicity. Apoptosis can be induced by several types of genotoxicants. Excessive elimination of cells by apoptosis can induce compensatory cell proliferation to restore homeostasis. Carcinogenesis is therefore the result of the balance between mutations, epigenetic changes, cell proliferation and cell death.

Fig. 2. Mechanisms of ionising radiation effect. Ionising radiation acts directly on DNA producing DNA adducts, DNA strand breaks and CSAs. Direct action on proteins results in the formation of protein adducts and crosslinks which, if not repaired, will give rise to genome mutations. Radiolysis of H2O by ionising radiation (indirect mutagenic effect) results in the ejection of an electron (e–) and the formation of an ionized water molecule (H2O+●). Trapping of the e– by polarizing water molecules produces a highly reactive hydrated electron (e–aq). The hydrated electron causes the formation of negatively charged ion (H2O–●). H2O+● and H2O–● are unstable and both can dissociate to generate a stable ion and free radical. MNcen+ are centromere positive micronuclei resulting from chromosome loss, MNCen– are micronuclei containing acentric chromosome/chromatid fragments, and are probably the result of chromosome breakage.

Table 1. The consequences of mutagen–target interactions may lead to different types of DNA damage (DNA adducts, alkalilabile sites, strand breaks) and mutations going from single nucleotide changes (gene mutations) to structural (chromosome mutations) or numerical chromosome changes (genome mutations). The fate of the cell is finally determined by whether or not the various lesions inflicted on the genome are repaired or eliminated by apoptosis [6].

The first objective of this state of the art paper is to review the mechanisms of induction, the fate, the methodology, the sensitivity/specificity and predictivity of the two major cytogenetic endpoints applied for genotoxicity studies and biomonitoring purposes: chromosome aberrations (CAs) and micronuclei (MN). In addition, recent evolution of cytogenetic techniques combining cytological approaches with molecular technologies and/or automated scoring will be considered.

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Fig. 3. Mechanisms of action of metals. Metals interact in different ways with the cellular machinery: by competition with other metals, by binding to DNA, to specific amino-acids (e.g. histidine) or to specific sites (e.g. SH groups). The genotoxic effects of a given metal are therefore the consequences of its relative affinity for these binding sites. Balance between direct DNA mutagenicity, indirect mutagenicity (inhibition of DNA repair or spindle formation) and induction of cell death (apoptosis) will define the specific genotoxic profile of each metal. Table 1 Mechanisms of action of metals

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the two chromatids of a chromosome or several chromosomes [9,10]. Generation of structural CAs requires one or several DNA DSBs, but the mechanisms of CSAs and CTAs formation appear to differ with the mutagen (ionizing radiation versus chemicals) and involve specific DNA repair mechanisms [10]. CSAs result from incompletely repaired or unrepaired DSBs mostly generated in vivo in G0–G1 lymphocytes by S-phaseindependent clastogens (e.g. ionizing radiation). After DNA synthesis and chromosome duplication, the aberrations formed in G0–G1 are doubled and chromosome-type breaks and exchanges (e.g. dicentric and ring chromosomes, balanced translocations) are seen in metaphase. CTAs (e.g. chromatid type breaks and exchanges) arise predominantly in vitro during the S-phase of the cultured lymphocytes, in response to base modifications and single-strand breaks (SSBs) induced in vivo by S-phase-dependent clastogens (e.g. chemicals) [9,10]. Fig. 4 shows some examples of possible mechanisms involved in the formation of structural CAs [11]. Since structural CAs may be induced via DNA breakage, their survival depends on the fate of the DNA breaks. DNA breaks may either rejoin such that the chromosome is restored to its original state, rejoin incorrectly or not rejoin at all. These last two cases may be observable on microscopic preparations of metaphase cells. The type of chromosomal aberration will be decisive for the fate of the cell. Cells bearing unstable aberrations such as dicentrics, rings and chromosome fragments can be eliminated by apoptosis in a p53-dependent way [12]. Stable aberrations, such as balanced translocations, on the other hand, may have deleterious consequences for the organism since they are much less effective in causing apoptotic cell death. Numerical CAs refer to changes in normal chromosome number (i.e. aneuploidy, polyploidy) which occur due to abnormal chromosome segregation [9]; they may arise either spontaneously or as a result of aneugen treatment. 2.2. The chromosome aberration assay (http://www.crios.be)

2. Chromosomal aberrations 2.1. Mechanisms of chromosomal aberration formation Chromosomal aberrations (CAs) are changes in normal chromosome structure or number that can occur spontaneously or as a result of chemical/radiation treatment [8]. Structural CAs may be induced by direct DNA breakage, by replication on a damaged DNA template, by inhibition of DNA synthesis and by other mechanisms (e.g. topoisomerase II inhibitors) [9]. Based on morphological criteria, structural CAs can be divided into two main classes: chromosome-type aberrations (CSAs), involving both chromatids of one or multiple chromosomes, and chromatid-type aberrations (CTAs) involving only one of

Structural CAs in peripheral blood lymphocytes (PBLs), as assessed by the chromosome aberration (CA) assay, have been used for over 30 years in occupational and environmental settings as a biomarker of early effects of genotoxic carcinogens [10]. Moreover, they are used routinely for the assessment of genotoxicity both in vitro, in human primary lymphocytes and cell lines, and in vivo, in rodent bone marrow. The CA assay has a key position in the test battery for genotoxic compounds and its protocol is defined by OECD guidelines (http://www. ecb.jrc.it/testing-methods). Structural CAs are most commonly scored in metaphasearrested cells that have been fixed, spread on microscope slides, and Giemsa stained [13] (Fig. 5). However, this method is not suitable for estimation of numerical CAs as artefactual chromosome loss may occur and its use for this purpose will not be considered here. The advantages of the CA assay are related to the cell by cell approach, an accurate identification

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Fig. 5. In vitro chromosome aberration assay: methodology (http://www.crios. be). The in vitro chromosome aberration assay is most often performed on human PBLs and allows detection of structural CAs in metaphase-arrested proliferating cells. As peripheral lymphocytes are in the resting G0 stage of the cell cycle, they are stimulated to divide by an aspecific antigen (e.g. phytohaemagglutinin). After 46.5 hours, a spindle inhibitor (e.g. colcemid, colchicine) is added to block the cells in the (pro)metaphase of the first mitosis. After a subsequent hypotonic treatment, fixation (at 48 hours), even spreading of the chromosomes in a single plane on the microscope slides, and classically Giemsa staining, the metaphases can be analyzed for structural CAs under the microscope [13].

specific rearrangements and/or loss by the fluorescence in situ hybridization (FISH) methods (see Sections 3 and 4). 2.3. Sensitivity/specificity of the CA assay in human lymphocytes

Fig. 4. Examples of CA formation by different mechanisms [11]. Upper panel (1) and (2): CA such as dicentrics and translocations may result by homologous recombination (HR) between homologous DNA sequences located on different chromosomes (ectopic HR). Lower panel: (3) formation of exchange type CA (e.g. dicentrics) by nonhomologous end joining (NHEJ) requires the presence of two DSB and is independent of sequence homology; (4) a single DSB occurring between two direct repeat sequences can be repaired by homology-dependent single-strand annealing (SSA), which leads to the intrachromosomal deletion of one repeat unit and of the intervening sequence.

of all the different chromosome mutation types (discrimination between chromatid and chromosome-type), a possible codetection of mitotic indices and the semi-automated scoring by image analysis. The major drawback of the traditional chromosome aberration test is its labor intensiveness and requirement for specifically skilled and experienced staff (Table 2). Recently, advanced cytogenetic techniques have been introduced, which allow the detection of both chromosome-

The sensitivity (lowest detectable dose/concentration) of the CA assay is dependent on the type of mutagen. For the particular case of ionizing radiation exposure, structural CAs can be detected at doses as low as 0.5 Gy in the case of dicentrics and 1 Gy in the case of translocations [14,15]. The specificity of the CA assay lies in its ability to identify a specific type of mutagen and is rather limited. CA assay detects indeed mutagens which are capable to induce DNA strand breaks, but does not allow identification of the clastogen class. However, information on the types of aberrations induced [S-phase-independent (CSAs) versus S-phase-dependent (CTAs)] following occupational and/or environmental exposure, gives some indication on the nature of the clastogenic damage produced (i.e. strand breaks versus base damage, respectively) [9]. The use of FISH chromosome painting methods to detect structural and numerical CAs may provide an increased efficiency and specificity for identifying certain kinds of CAs induced in vivo [9] (e.g. translocations, stable symmetrical rearrangements derived from CTAs, hyperploidy). For biomonitoring of cumulative exposure, stable structural CAs (e.g. translocations), which are moreover found in tumors, are the most important biomarkers. Dicentric chromosomes which are easy to identify also with classic Giemsa staining, represent an attractive biomarker, especially for assessment of recent exposure.

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Table 2 Advantages and disadvantages of CBMN assay as compared to CA assay (http://www.crios.be)

2.4. Chromosomal aberrations as a biomarker for aging, genetic instability, and individual susceptibility Chromosomal damage as measured by the frequency of translocations, acentric fragments, telomere shortening, nondisjunction, chromosome loss, aneuploidy, and MN formation has been shown to increase progressively with age [16]. However, most studies have used the cytokinesis-block micronucleus (CBMN) assay (see section 3) and not the CA assay to assess chromosomal damage in relationship to age and genomic instability because of the advantages of the former technique. As far as individual susceptibility is concerned, Norppa [17] has reviewed the influence of genetic polymorphisms in xenobiotic metabolism and DNA repair enzymes on the baseline level of structural CAs. With respect to xenobiotic metabolism polymorphisms, several studies have shown an increased baseline frequency of lymphocyte CAs in subjects with the NAT2 slow acetylator genotypes, with no association to known exposures [18–20]. Tuimala et al. [21] also found a higher frequency of chromosome-type breaks in non-smokers carrying the NAT2 slow acetylator genotype. The GSTT1 null genotype was shown to be associated with an increase in spontaneous CAs in lymphocytes [22,23]. Moreover, a higher frequency of chromosome-type breaks was observed in GSTT1 null smokers [21]. Sorsa et al. [24] found an elevated mean frequency of CAs in controls carrying the GSTM1 positive genotype; high CAs levels in the lymphocytes of GSTM1 positive nonsmokers were also reported [21]. Control subjects carrying the EPHX low activity genotype were shown to have an increased frequency of CAs [25,26]. In addition, a recent study by Migliore et al. [27] found a moderate decrease in CAs in controls carrying the fast activity EPHX genotype. An increase in translocations in new-borns with the CYP1A1 MspI heterozygous genotype [28], and in CAs in control subjects with the CYP2E1*5B (PstI) heterozygous genotype [29] were also reported. As far as DNA repair polymorphisms are concerned, the XRCC1399 Gln/Gln genotype was associated with a statistically significant reduction in chromatid gaps in non-smokers [21]. Individuals carrying variant alleles for XRCC1 codons 280

and 194 (showed a decreased level of chromosome-type breaks, while an elevated frequency of chromatid-type breaks was seen in XRCC3241 heterozygotes [21]. Decreased CAs frequencies in individuals with homozygous genotype for the XPD 751Gln variant allele were also observed [30], the effect of XPD genotype on CAs being particularly apparent in smokers. XPD variant 312Asn allele was associated with an increase in the risk of elevated CAs frequencies, the risk being more pronounced in smokers [31]. Folate metabolism polymorphisms are expected to influence the background level of chromosomal damage by affecting fundamental cellular processes responsible for maintaining the genomic integrity. However, most studies have assessed the link between folate metabolism polymorphisms and background chromosomal damage by use of the micronucleus (MN) assay. 2.5. Chromosomal aberrations as a biomarker for smoking The influence of smoking on the level of structural CAs has been addressed in both large- and small-scale population studies using chromosome banding and molecular cytogenetic techniques (FISH) (for review see [32]). However, no clear trend has emerged. Some studies showed that smoking did not increase the frequencies of structural CAs [33,34], but did increase the frequency of hyperploidy [34]. Other studies found that smoking triggered a 10-20% increase in CAs frequency [35], or caused a significant 1.5-fold increase in stable aberrations in newborns whose mothers smoked during pregnancy [36]. Although several studies have found increased frequencies of CAs in lymphocytes from smokers as compared to non-smokers [37–39], no increase in CAs in the PBLs of second-hand smokers was seen [40] (for review see [41]). The observation that smokers have lower folate levels in their red blood cells compared to non-smokers may explain the higher frequency of CAs detected in the former ones [42]. Several studies have addressed the link between tobacco smoke exposure, CAs frequency and cancer risk (for review see [32]). In a recent large international study, Bonassi et al. [43] showed that an elevated frequency of structural CAs in human lymphocytes predicts the risk for cancer independently of car-

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cinogen exposure, including cigarette smoke. However, studies performed on normal bronchial epithelium and human lymphocytes showed an elevated loss of heterozygosity (LOH) [44] as well as an elevated expression of fragile sites at cancer-specific breakpoints [45] in smokers. In addition, a much higher frequency of structural CAs was observed in lung tumors from smokers as compared to non-smokers (48% vs. 11%), suggesting that lung cancer in smokers results from genetic alterations distinct from those in non-smokers [46]. 2.6. Predictivity of chromosomal aberrations for cancer The extensive use of the CA assay over the last 30 years has resulted in the accumulation of analytical data in many European laboratories and has enabled the examination of the potential association between previously measured structural CA frequency and subsequent cancer outcome [47]. Nordic and Italian cohort studies revealed that a high frequency of structural CAs, but not sister chromatid exchanges (SCEs) or MN, in PBLs is predictive of increased cancer risk [48–51]. However, these studies did not have sufficient power and /or follow up time for a conclusive result with respect to the cancer predictivity of MN. A further case–control study nested within the cohort indicated that the association between CAs and cancer was not explained by tobacco smoking or known occupational exposure to carcinogens [43], suggesting that a high frequency of structural CAs as such is predictive of an increased cancer risk, irrespective of the cause of the initial CA increase. The impact of various types of CAs on human cancer risk has been studied in a small group (22 cancer cases and 22 controls) of residents of an endemic blackfoot area in Taiwan [52]. The results suggested that CSAs but not CTAs predict cancer risk. Recently, a large Nordic and Italian cohort study aimed at evaluating whether CSAs and CTAs have different cancer risk predictivity [10]. A significantly elevated cancer risk was observed in the Nordic cohorts for subjects with both high CSAs and high CTAs at test, while the results of the Italian cohort did not indicate any clear-cut difference in cancer predictivity between the CSA and CTA biomarkers. 3. Micronuclei 3.1. Mechanisms of micronuclei formation Micronuclei are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase [53]. Micronuclei harboring chromosomal fragments may result from direct double-strand DNA breakage, conversion of SSBs into DSBs after cell replication, or inhibition of DNA synthesis. Misrepair of two chromosome breaks may lead to an asymmetrical chromosome rearrangement producing a dicentric chromosome and an acentric fragment. Frequently, the centromeres of the dicentric chromosomes are pulled to opposite poles of the cells at anaphase resulting in the forma-

tion of a nucleoplasmic bridge (NPB) between the daughter nuclei and an acentric fragment that lags behind to form a MN [54,55]. Micronuclei harboring whole chromosomes are primarily formed from defects in the chromosome segregation machinery such as deficiencies in the cell cycle controlling genes, failure of the mitotic spindle, kinetochore, or other parts of the mitotic apparatus or by damage to chromosomal substructures, mechanical disruption [9] and hypomethylation of centromeric DNA [56]. Micronuclei can also arise by gene amplification via breakage-fusion-bridge (BFB) cycles when amplified DNA is selectively localized to specific sites at the periphery of the nucleus and eliminated via nuclear budding (NBUD) during the S-phase of the cell cycle [57]. Fig. 6 (adapted from Ref. [57,58]) shows possible mechanisms for the formation of MN. The fate of MN after their formation in the micronucleated cell is poorly understood. Their post-mitotic fate includes: ● elimination of the micronucleated cell as a consequence of apoptosis [6]; ● expulsion from the cell (when the DNA within the MN is not expected to be functional or capable of replication owing to the absence of the necessary cytoplasmic components) [59]; ● reincorporation into the main nucleus (when the reincorporated chromosome may be indistinguishable from those of the main nucleus and might resume normal biological activity) [59]; ● retention within the cell’s cytoplasm as an extra-nuclear entity (when MN may complete one or more rounds of DNA/chromosome replication) [59]. 3.2. The CBMN assay (http://www.humn.org) (http://www.crios.be) Scoring of micronuclei can be performed relatively easily, without an extra in vitro cultivation step and on different cell types relevant for human biomonitoring: lymphocytes, fibroblasts and exfoliated epithelial cells. MN observed in exfoliated cells are not induced when the cells are at the epithelial surface, but when they are in the basal layer. Although MN may already be present in cells in vivo, the induction of DNA damage and/or DNA misrepair in post-mitotic cells as MN requires ex vivo nuclear division. Therefore, Fenech and Morley [53] developed a now widely applied methodology: the cytokinesis-block MN assay. An ex vivo or an in vitro analysis of lymphocytes in the presence of cytochalasin-B (an inhibitor of actins), added 44 hours after the start of cultivation, allows to distinguish between mononucleated cells, which did not divide, and binucleated cells, which completed one nuclear division during in vitro culture. Indeed, under these conditions, the frequencies of mononucleated cells provide an indication of the background level of chromosome/genome mutations accumulated in vivo, while the frequencies of binucleated cells with MN represent a

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Fig. 6. Mechanisms of MN formation. MN can mainly arise from: acentric chromosome/chromatid fragments resulting from DNA breakage events (a) (adapted from Ref. [58]); and whole chromosomes/ chromatids that lag behind in anaphase due to: misattachment of tubulin fibers on kinetochore (adapted from Ref. [58]) (b); tubulin depolymerization (adapted from Ref. [58]) (c); defects in centromeric DNA, in kinetochore proteins or in kinetochore assembly (adapted from Ref. [58]) (d); late replication, peripheral location in the nucleus and epigenetic modifications of histones (adapted from Ref. [58]) (e). MN can also arise as a result of NPB formation/breakage f (1) and f (2) (adapted from Refs. [57,58]). Misrepair of two chromosome breaks f (1) may lead to an asymmetrical chromosome rearrangement producing a dicentric chromosome and an acentric fragment; alternatively, dicentric chromosomes may also arise by telomere end fusions f (2). Centromeres of the dicentric chromosomes are pulled to opposite poles of the cells at anaphase resulting in the formation of a NPB between the daughter nuclei [f (1) and f (2)]. In the first case f (1), the lagging acentric fragment accompanying the dicentric chromosome will form a MN. In both of the above cases, MN could also arise by breakage of the NPB [f (1) and f (2)]. Micronuclei can also arise by gene amplification via BFB cycles when amplified DNA is selectively localized to specific sites at the periphery of the nucleus and eliminated via NBUD during the S-phase of the cell cycle (g) (adapted from Refs. [57,58]).

measure of the damage accumulated before cultivation plus mutations expressed during the first in vitro mitosis (Fig. 7; legend adapted from Ref. [7]). Moreover, it was shown by us [60] that in the presence of spindle inhibitors, cells can go to

metaphase arrest followed by mitotic slippage and formation of tetraploid cells with or without micronuclei. Therefore, MN scoring is also recommended in the mononucleated cells found in the CBMN assay [61]. The criteria for scoring were

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Fig. 6. (continued)

established and validated by the Human MicroNucleus (HUMN) international collaborative project (http://www. humn.org) and can also be found on http://www.crios.be. The combination of the micronucleus assay and the fluorescence in situ hybridization (FISH) assay with probes labeling the pan (peri-)centromeric region of the chromosomes provides the methodology to distinguish between micronuclei contain-

ing a whole chromosome (centromere positive micronucleus, C+MN) and an acentric chromosome fragment (centromerenegative micronucleus, C-MN) (Fig. 8). Detailed recommendations and protocols for biomonitoring are described in [61]. As far as genotoxicity studies are concerned, the in vivo MN assay in rodent bone marrow plays a crucial role in the test battery aiming at hazard identification for mutagens. The

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Fig. 6. (continued)

in vitro MN assay, since its modification with the cytochalasinB block, was promoted as an alternative test for the in vitro CA assay. Protocols for human primary lymphocytes and cell lines

were validated and harmonized [62,63], being now in the final phase of validation by ECVAM (http://www.ecvam.jrc.cec.eu. int/index.htm) and of acceptance in the OECD guidelines.

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Fig. 7. In vitro cytochalasin-B micronucleus assay: methodology (http://www. crios.be). The standard in vitro cytochalasin-B micronucleus assay is usually performed on human lymphocytes but has also been adapted to various cell lines of different origin. In a classical test, human lymphocytes are cultured in the presence of phytohaemagglutinin to stimulate mitosis. After 44 hours, cytochalasin-B is added to the culture. The use of this inhibitor of actin polymerization will block cytokinesis allowing the distinction between binucleated cells (cells that have divided once in culture) and mononucleated cells (cells that did not divide or escaped the cytokinesis-block). At 72 hours the cells are harvested onto microscopic slides, fixed and stained [7].

Fig. 8. In vitro micronucleus test combined with FISH (http://www.crios.be). Combination of FISH with pan(peri)centromeric probes and micronucleus scoring allows discrimination between clastogenic events (inducing chromosome breakage) and aneugenic events (inducing chromosome loss).

3.3. Sensitivity/specificity of CBMN assay in human lymphocytes The key advantage of the CBMN assay lies in its ability to detect both clastogenic and aneugenic events, leading to structural and numerical CAs, respectively [64,65]. The distinction between the two phenomena can be achieved even at low doses of mutagen exposure; for the particular case of ionizing radiation exposure, the sensitivity of the CBMN assay is 0.2 Gy for micronuclei (clastogenic and aneugenic events) and 0.1 Gy when probing with pancentromeric FISH (clastogenic events only) is applied to the MN [14,15]. Evaluation of the mechanistic origin of individual MN by centromere and kinetochore identification contributes to the high sensitivity and specificity of the method. Micronuclei arising from lagging chromosomes can be identified by the pre-

sence of a kinetochore using anti-kinetochore antibodies derived from the serum of scleroderma Calcinosis, Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly and Telangiectasia (CREST), or by the presence of centromerespecific DNA using FISH. Micronuclei that do not contain kinetochore proteins or centromeric DNA sequences are interpreted as harboring acentric chromosomal fragments [9]. However, MN formed from entire chromosomes with disrupted or detached kinetochore may result in MN with no kinetochore signal. Since a considerable proportion of MN harboring the centromere of the X or Y chromosome seem to be kinetochore negative (K–), the use of kinetochore identification in human biomonitoring studies could be a problem [66]. Moreover, when potential genotoxic compounds are assessed, their interference with the production of kinetochore proteins might lead to false negatives. Therefore, centromeric FISH should be recommended for both biomonitoring studies and genotoxicity testing. Discrimination between mutagens inducing DNA breakage (clastogens) or chromosome loss/non-disjunction (aneugens) contributes to the high specificity of the MN assay, without allowing structural identification of the compound. The assay has been shown to be an indicator of chromosome damage at least as sensitive as classical metaphase chromosome analysis [64]. In comparison with CAs, the scoring of MN is simpler, requires shorter training and is less time consuming [66]. Moreover, a big advantage of the CBMN assay is the statistical power obtained from scoring larger numbers of cells (thousands) than are typically used for metaphase analysis (a hundred or a few hundred). These large numbers of binucleated cells are achieved because it is possible to cytokinesisblock cultures for 24–48 hours; however, colchicine blocking for metaphase analysis can only be performed for 1–4 hours as longer times cause chromosome condensation making metaphases unscorable. Automation of MN scoring will further increase the statistical power of the assay by eliminating inter-individual variation in assessing this type of chromosomal damage. Besides its capacity to detect micronuclei (a marker of chromosome breakage and loss), the CBMN assay can provide additional measures of genotoxicity and cytotoxicity: NPB (a marker of chromosome rearrangement), NBUD (a marker of gene amplification) [67], cell division inhibition (by estimation of the nuclear division index) [65] necrosis and apoptosis [68] (for review see [54]). For this reason, the CBMN test can be considered as a “cytome” assay covering chromosome instability, mitotic dysfunction, cell proliferation and cell death. However, it is known that MN frequency measured by the CBMN method may not identify all chromosome damage events; e.g. aberrations such as symmetrical reciprocal translocations are not expressed as MN, but asymmetrical translocations, such as dicentric chromosomes, and their associated acentric fragments may be observed as NPBs and MN, respectively [64]. The advantages and disadvantages of the MN-assay as compared to CA assay are presented in Table 2.

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3.4. The CBMN assay as a biomarker for aging, genetic instability, and individual susceptibility In populations not exposed to occupational or environmental toxicants, the major contributors to differences in MN frequencies were shown to be age and gender. Several studies have reported that MN frequencies increase with age in adults, and women have higher MN frequencies than men [51,69,70]. Identification of centromere DNA and kinetochore proteins in MN has indicated that the well-known age-dependent increase in lymphocyte MN frequency primarily reflects an increase in MN harboring whole chromosomes and is mostly due to excessive micronucleation of the X and Y chromosomes. A gender increase in MN frequency has also been described [71,72], with X chromosome micronucleation being prevalent in women (for review see [66]). The high frequency of X micronucleation in women is still not fully understood but it has been suggested that it is primarily due to the inactive X chromosome, which was observed to show a higher age-dependent telomere shortening than the active homologue or all chromosomes in metaphases of cultured lymphocytes from new-born, middle-aged and elderly females [72]. Hando et al. [73] and Nath et al. [74] suggested that faulty kinetochores or centromeres may also cause lagging of the inactive X chromosome during anaphase. For instance, the inactive X chromosome has been shown to be more prone to premature centromere division (PCD) as compared to the active X chromosome centromere, which divides normally [75]. However, several studies demonstrated no significant differences in the micronucleation of the active and inactive X chromosomes, indicating that both homologues are prone to micronucleation [70]. This explains the fact that men also show a high micronucleation frequency of the only one, active, X chromosome [76]. Moreover, kinetochore defects may also be responsible for this phenomenon of X chromosome micronucleation [73,77]. A recent extensive meta-analysis by Neri et al. [78] clearly showed an age-related increase in MN frequencies in children and adolescents (age range 0–18 years), with significantly lower values in newborns. Additional parameters that can influence MN frequencies are inherited (or acquired) genetic polymorphisms (or mutations) in genes responsible for the metabolic activation and detoxification of clastogens, for the fidelity of DNA replication (mismatch repair), DNA repair and/or chromosome segregation. A pooled analysis of eight studies on the influence of GSTM1 and GSTT1 [79] polymorphisms on MN frequencies in human lymphocytes in vivo was recently undertaken within the frame of the HUMN and CancerRiskBiomarker1 projects. Although the analysis indicated that GSTT1 null subjects had lower micronucleus frequencies than their positive counterparts in the total population, no significant influence of genotypes on 1

Cytogenetic Biomarkers and Human Cancer Risk (CancerRiskBiomarkers), available at [May 2006] http://www.ec.europa.eu/research/quality-oflife/ka4/pdf/report_cancerriskbiomarkers_en.pdf.

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the baseline level of MN was observed in the control population. A recent study by Iarmarcovai et al. [80] found an increased frequency of C-MN in GSTM1 positive controls as compared to their null counterparts. Migliore et al. [27] showed that the fast activity EPHX genotype was associated with a moderate decrease in MN in controls. Ishikawa et al. [81] showed that CYP2E1(*)3 polymorphism may have the potential to influence the baseline frequency of MN, subjects with the CYP2E1(*)3 variant allele having lower MN frequencies than subjects with the CYP2E1(*)1/(*)1 wild-type. Scarpato et al. [82] did not find a significant influence of the NAT2 genotype on the baseline frequency of MN in human lymphocytes. Several recent studies showed no significant influence of the XRCC1194 [83,84], XRCC1280 [83,84], XRCC1399 [80,83–85], XRCC3241 [80,83–86] and XPD312 [86] genotypes on the baseline MN level. However, MN frequency was shown to be lower in controls carrying the variant hOGG1326 genotype as compared to wild-type carriers [83]. The influence of folate metabolism polymorphisms on the baseline MN frequencies has been addressed by several studies [67,87,88]. An in vitro study performed by Crott et al. [87] in human lymphocytes showed that the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism did not influence the levels of chromosome damage as assessed by the CBMN assay. In contrast, Kimura et al. [67] found a higher MN level in the lymphocytes of variant MTHFR TT genotypes as compared with the wild-type MTHFR CC genotypes, and a lower NBUD level in TT homozygotes relative to CC homozygotes for the MTHFR C677T mutation. Zijno et al. [88] observed a significant association between the methionine synthase reductase (MTRR) 66GG variant genotype and higher micronucleus rates, after correction for age, gender and GSTM1 genotype. Because acentric chromosomal fragments resulting from DSBs can form MN, the proportion of MN harboring acentric fragments is expected to be influenced by genetic polymorphisms involved in the repair of DNA strand breaks. The proportions of MN harboring whole chromosomes could also be expected to be influenced by the lack of essential cofactors (e.g. magnesium and calcium) required for kinetochore and spindle assembly [56] and by genetic polymorphisms of enzymes controlling the reactivity of aneugens (e.g. inhibitors of tubulins, topoisomerases and cyclins) or the activity of cell cycle check points (such as hCDC4, whose dysfunction increases MN frequency [89]), although the presence and importance of such polymorphisms are still unexplored issues. 3.5. The CBMN assay as a biomarker of nutritional deficiency The comprehensive CBMN assay has also been used for assessing the effect of micronutrients on genomic stability and cell death [67]. Several studies [90,91] showed a clear increase in the level of MN, NPB and NBUD with a decrease in the folic acid concentration from 120 to 12 nmol/l, which coincides with the physiological range in the serum of individuals consuming an unsupplemented diet (8–35 nmol/l) [67].

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Cross-sectional studies performed on lymphocytes of vegetarians and non-vegetarians [92], older men [90,93] and young adults [91,94] indicated that MN frequency was negatively correlated with plasma folate and vitamin B12, positively correlated with homocysteine and vitamin C and unrelated to vitamin E status [54]. A depletion–repletion study in nine post-menopausal women [95] and placebo-controlled dietary intervention studies [90,91,93] have demonstrated that supplementation with specific micronutrients can lead to a reduction in MN frequency. It has been shown that there is an optimal level of micronutrient intake that can minimize genome damage. However, this optimal micronutrient concentration may exceed normal intake levels from diet (for review see [54]). Therefore, the sensitivity of the MN index to small variations in micronutrient status within the physiological range makes it an excellent biomarker for identifying dietary factors that are essential for genome stability, and for defining their optimal intake levels [96]. One exciting area of research is the interaction between genotype and diet in modulating cancer risk. Gene mutations (e.g. polymorphisms) resulting in a decreased binding affinity of a gene product for its cofactor have the potential to influence the induction of DNA damage and subsequently, the cancer outcome. Therefore, cofactor supplementation from diet may be an effective way of modulating cancer risk. It has been suggested [54] that a more useful approach for the prevention of diseases caused by genome damage would take into consideration the genotype of individuals, with a focus on common genetic polymorphisms that alter the bioavailability and the metabolism of micronutrients and/or the affinity of key enzymes involved in DNA metabolism for their micronutrient cofactor. 3.6. The CBMN assay as a biomarker for smoking Although the influence of tobacco smoking on the frequency of MN in human lymphocytes has been examined in many population studies (for review see [97]), mixed results have been obtained. In most studies, no effect of smoking on MN frequency has been observed, while in many instances smokers had lower MN frequencies as compared to nonsmokers. However, none of the published studies were specifically and optimally designed to detect the effect of smoking on MN. Aiming at understanding the influence of smoking habit on MN frequency, Bonassi et al. [97] performed a pooled reanalysis of 24 databases from the HUMN international collaborative project. This analysis showed that many smokers do not experience an overall increase in MN frequency when compared to non-smokers. In addition, a small decrease in MN frequencies was found in current and former smokers (all smoking levels combined) as compared to non-smokers, among non-exposed subjects. However, when stratifying by level of smoking, a significant increase of MN was observed in non-exposed heavy smokers (>30 cigarettes per day). In contrast, MN frequency was not influenced by the number of cigarettes smoked per day among occupationally-exposed sub-

jects. As an explanation for this finding, the authors suggested that occupational exposure to genotoxins may have stimulated the expression of DNA repair genes or detoxification mechanisms that are important in attenuating the genotoxic effects of chemicals in cigarette smoke. As a general conclusion, Bonassi et al. [97] recommended that quantitative data about smoking habit should always be collected when designing biomonitoring studies, since the simple comparison of smokers versus non-smokers could lead to misleading results. 3.7. Predictivity of micronuclei for cancer The link between MN induction and cancer development was previously addressed by the earlier Nordic and Italian cohort studies [48–51], which found that high MN frequencies in peripheral lymphocytes were not predictive of an increased cancer risk. However, at that time, the size of the cohort was too small and the material too heterogeneous to provide reliable findings. Moreover, most of the data had not been obtained by using the more sensitive ex vivo/in vitro cytokinesis-block methodology. A recent analysis of new results from the European cohorts (CancerRisk Biomarkers and HUMN projects) indicates that subjects who had higher MN frequencies were more likely to develop cancer 12– 15 years after the test was done (Bonassi et al., submitted)2. The hypothesis of a predictive association between the frequency of micronuclei in cytokinesis-blocked lymphocytes (reporter tissue) and cancer development is supported by a number of findings: ● An association between MN frequency and cancer risk was inferred from mechanistic similarities with CAs, which were shown to be predictive for cancer [48–51]. ● In vitro, a high concordance is observed between the CA and MN assays [98–102]. ● An increase in MN frequency is observed in lymphocytes of cancer patients and in patients with syndromes that make them cancer prone such as Bloom syndrome and ataxia telangiectasia [103–105]. ● Micronucleus frequency is significantly associated with the blood concentration of vitamins such as folate, whose deficiencies are associated with increased risk for some cancers [90–92,106]. ● A direct link between MN frequencies and early stages of carcinogenesis, was recently described by Olaharski et al. [107] who found that the frequencies of MN increase significantly in both the low-grade and high grade diagnostic categories of cervical carcinogenesis in women. 2 Bonassi, S., Znaor, A., Ceppi, M., Lando, C., Chang, W.-P., Holland, N., Kirsch-Volders, M., Zeiger, E., Ban, S., Barale, R., Bigatti, M.P., Bolognesi, C., Cebulska-Wasilewska, A., Fabianova, E., Fucic, A., Hagmar, L., Joksic, G., Martelli, A., Migliore, L., Mirkova, E., Scarfi, M.R., Zijno, A., Norppa, H., Fenech, M. (submitted for publication). An increased micronucleus frequency in PBLs predicts the risk of cancer in humans.

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4. Evolution of cytogenetic techniques for genotoxicity and biomonitoring studies Recent advances in FISH and microarray technologies are modifying the nature of cytogenetics, allowing chromosome and gene identification on metaphase as well as in interphase. The high resolution that is achieved by these techniques is blurring the traditional distinction between cytogenetics and molecular biology (for review, [108,109]). FISH with whole chromosome paints has been the primary method to quantify and characterize chromosome damage from environmental or occupational exposures. The advantages of chromosome painting are the speed of the assay and the ability to identify relatively stable events such as translocations in parallel with the enumeration of unstable dicentrics. Currently, most painting is performed with just one color of paint, but sometimes with two or three. Each additional probe in the cocktail increases the proportion of the genome in which aberrations can be observed and also increases the fraction of all exchanges that can be detected. Some years ago, spectral karyotyping (SKY) [110] and multiplex FISH (mFISH) [111] made it possible to paint each of the 24 human chromosomes in a unique color, thereby enabling the identification of every interchromosomal exchange in each cell. Although research applications have been more limited, mFISH has been successfully employed to decipher complex chromosome rearrangements. However, this approach requires expensive probes and the analysis time per cell is substantially longer than when only a few chromosomes are painted. Besides interchromosomal exchanges commonly detected by chromosome painting, intrachromosomal exchanges such as pericentric and paracentric inversions occur and may form an important component of risk evaluation. These are not detectable by chromosome painting and require the use of chromosome bands. The bands may be natural, e.g. G-bands, or synthetic, i.e. based on region-specific partial chromosome paints that are hybridized simultaneously and labeled in multiple colors with the multicolor banding technique (mBANDs) [112,113]. This technology is very accurate, well validated but labor and cost demanding. Automation can be the best alternative for this problem. Some progress has been made, for example, in the area of karyotyping and aberration detection [114,115]. However, fully automatic analysis of chromosomes in metaphase has yet to be accomplished. Even the best automated system may never replace a skilled observer, but a system that could count the normal cells and conservatively eliminate most of them from consideration as normal cells could reduce the human effort involved in scoring by as much as 90%. With such a system, the observer would view a preselected set of cells that had been enriched for abnormal cells and score those cells in the usual manner. An even more promising field is the automation of MN scoring, which has resulted in the ability to evaluate large numbers of cells quickly and efficiently. Flow cytometry and slidebased image analyses have both been used with considerable success. Micronuclei can easily be identified by flow cytometry in erythrocytes [116] (which lost their macronucleus) but

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distinction between macronucleus and micronuclei is difficult in other cell types with this approach. Image analysis systems for scoring micronuclei in mammalian cell lines as well as cultured human lymphocytes [117–121] described a laserscanning system that combines the analytical capabilities of flow and image cytometry. Validation of these automated facilities to score micronuclei is in progress. 5. Conclusion Due to its potential to detect early effects of mutagens/carcinogens, its capacity to identify inheritable changes and its predictivity for cancer, cytogenetics will become more and more important in the future. Furthermore, combining cytogentics with molecular biology provides increasing sensitivity. In addition, the high level of international validation reached by the most valuable biomarkers and the perspective of high throughput applicability by implementation of automated systems also demonstrates the importance of cytogenetics. This will undoubtedly enhance our ability to pre-screen efficiently new chemicals for genotoxic effects and to improve risk assessment of human populations exposed to environmental and/or occupational mutagens. Acknowledgements This work was partly supported by ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: "Food Quality and Safety" (Contract No 513943) and partly supported by NewGeneris (Newborns and Genotoxic Exposure Risks), an integrated research project operating within the same EU Framework Program, Priority 5 (Contract No 016320- 2). References [1]

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