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.     

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.     

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 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.     

Comparison of the aneugenic effect of vinorelbine and vincristine in cultured human lymphocytes.

Vinca alkaloids are used clinically against a variety of hematological and solid tumors. These compounds interact with tubulin subunits to prevent microtubule assembly, inducing abnormal chromosome segregation in dividing cells and causing aneuploidy. The vinca alkaloid vincristine sulfate (VCR) and the semisynthetic analog vinorelbine (VRB) were studied by analysis of micronuclei (MN) in cultured human lymphocytes using the cytokinesis block method. Furthermore, fluorescence in situ hybridization with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in isolated MN of VRB- or VCR-treated lymphocytes. At all the doses tested, both chemicals induced a significant increase in MN frequencies in binucleated (BN) cells (P < 0.001). The maximal effect was reached at a dose of 0.50 microgram/ml. At this dose, VRB produced an approximately 5-fold increase with respect to the control frequency of MN, while with VCR, this frequency increased 10-fold. Both drugs produced a slowing of the cell cycle, causing a decrease in the percentage of BN cells. This effect was lower with VRB. The percentages of centromere-positive MN were 89.79 and 87.60% in VRB- and VCR-treated cultures, respectively (control 27.03%). The high percentage of positive-signals in treated cultures (P < 0.001) indicates that the MN contained whole chromosomes. Our results confirm the aneugenic mode of action of these chemicals, VRB having less genetic effect.     

Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells

The exfoliated cell micronucleus (MN) assay using fluorescent in situ hybridization (FISH) with a centromeric probe is a rapid method for determining the mechanism of MN formation in epithelial tissues exposed to carcinogenic agents. Here, we describe the use of this assay to detect the presence or absence of centromeric DNA in MN induced in vivo by radiation therapy and chronic arsenic (As) ingestion. We examined the buccal cells of an individual receiving 6,500 rads of photon radiation to the head and neck. Exfoliated cells were collected before, during, and after treatment. After radiation exposure a 16.6-fold increase in buccal cell MN frequency was seen. All induced MN were centromere negative (MN −) resulting from chromosome breakage. This finding is consistent with the clastogenic action of radiation and confirmed the reliability of the method. Three weeks post-therapy, MN frequencies returned to baseline. We also applied the assay to exfoliated bladder cells of 18 people chronically exposed to high levels of inorganic arsenic (In-As) in drinking water (average level, 1,312 μg As/L) and 18 matched controls (average level, 16 μg As/L). The combined increase in MN frequency was 1.8-fold (P = 0.001, Fisher's exact test). Frequencies of micronuclei containing acentric fragments (MN −) and those containing whole chromosomes (MN+) both increased (1.65-fold, P = 0.07, and 1.37-fold, P = 0.15, respectively), suggesting that arsenic may have both clastogenic and weak aneuploidogenic properties in vivo. After stratification on sex, the effect was stronger in male than in female bladder cells. In males the MN-frequency increased 2.06-fold (P = 0.07) while the frequency of MN+ increased 1.86-fold (P = 0.08). In addition, the frequencies of MN − and MN+ were positively associated with urinary arsenic and its metabolites. However, the association was stronger for micronuclei containing acentric fragments. By using FISH with centromeric probes, the mechanism of chemically induced genotoxicity can now be determined in epithelial tissues. © 1996 Wiley-Liss, Inc.     

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-     

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.     

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.     

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.     

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     

Detection and analysis of origin of i(12p), a diagnostic marker of human male germ cell tumors, by fluorescence in situ hybridization.

The i(12p) chromosome marker has been shown to be a diagnostic and prognostic marker of human male germ cell tumors (GCTs). An analysis of the i(12p) and chromosome 12 aneuploidy was performed in five primary cell cultures and three established cell lines derived from human male GCTs by fluorescence in situ hybridization (FISH) with a chromosome 12 centromere-specific alpha-satellite DNA probe. Distinct differences in the centromeric signals originating from the i(12p) and normal chromosome 12 were detected, which were found to be useful for unambiguous distinction between the i(12p) and normal chromosomes 12 at interphase as well as at metaphase in these cultures. This method can be used for rapid screening of large numbers of interphase cells, eliminating the main limitation of conventional karyotypic analysis, namely, frequent inability to obtain sufficient numbers of dividing cells in direct preparations or in short-term culture of fresh biopsies. Our analysis of chromosome 12 centromeric signal size along with karyotypic data and results of analysis of restriction fragment length polymorphisms (RFLPs) on 12q in four GCTs suggested that the i(12p)s are formed by nonreciprocal centromeric interchanges between nonsister chromatids of homologous chromosomes.     

Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

The mechanism of action of the topoisomerase II inhibitor etoposide(VP-16) was investigated in male mouse meiosis using the spermatidmicronucleus (MN) test and two molecular cytogenetic approaches:(i) fluorescence in situ hybridization (FISH) with a mouse centromerespecific minor satellite DNA probe; and (ii) immunolabellingof kinetochore proteins with CREST autoimmune serum. VP-16 causedsignificant increases in the frequencies of MN at all meioticstages studied. VP-16 induced MN showed significantly elevatedfrequencies of centromeric hybridization signals compared tothe controls. Similarly, after CREST immunostaining the majorityof MN induced by the drug showed kinetochore signals when meioticS phase and diplotene-diakinesis were treated. This would suggestthat most induced MN were due to lagging of whole chromosomes.However, more than 80% of the small MN observed were signal-positiveand a large pool of minute MN almost exclusively (92%) containeda kinetochore or centromere-DNA signal. This indicates thatVP-16 causes chromosome fragmentation at centromeres. In addition,arrested first division (MI) anaphase figures with stretchedbivalent(s) at the spindle equator were observed when diplotene-diakinesisand MI were targeted. Moreover, many small and medium size MNhad two centromere or kinetochore signals at opposite sides,suggesting that inhibition of topo II at MI causes lagging ofwhole bivalents. Together, these results indicate that VP-16acts by several genotoxic mechanisms at male meiosis: (i) fragmentationof centromeres possibly as a result of inhibition of the DNAstrand religation reaction in a topoisomerase II mediated decatenationprocess of sister centromeres; and (ii) the induction of aneuploidyas a result of failures in separation of homologous chromosomearms possibly due to disturbances of chiasma resolution anddecatenation processes during MI. Our results indirectly suggestthat topoisomerase II plays an important role in male meiosisand its activity is needed at the metaphase-anaphase transitionof both meiotic divisions for proper chromosome disjunction. ¹To whom correspondence should be addressed     

Detection of whole chromosomes in micronuclei of cytokinesis-blocked human lymphocytes by antikinetochore staining and in situ hybridization

The effect of cytochalasin B (Cyt-B; 3 and 6 µg/ml; forthe last 28 h) on micronuclei (MN) was studied in 72-h purifiedlymphocyte cultures of three male donors. The frequency of MNwas much higher in multinucleate cells (mean 100–204 MNper 1000 cells) than in binucleate cells (mean 8.2–21.0MN per 1000 cells), tetranucleate cells containing more MN thantrinucleate cells. The presence of whole chromosomes in theMN was studied in two separate experiments by immunofluorescenceusing antikinetochore (CREST) serum and by a centromeric alphoidDNA oligomer probe (in situ hybridization, ISH). In the tri-and tetra-nucleate cells produced by Cyt-B, MN were clearlymore often kinetochorepositive (K+) (mean 82–86%) andcentromere-positive (C+) (mean 73–83%) than in mononucleatecells of cultures containing no Cyt-B (mean 63% for CREST and50% for ISH), indicating that most of the excess MN in the multinucleatecells were due to whole chromosomes. The binucleate lymphocyteshad about as high prevalence of K+ MN (mean 79–84%) asthe tri- and tetra-nucleate cells, despite their low MN count.Also in the ISH analysis, the majority of MN in binucleate cellswere positively stained (mean 58–62%). If it is assumedthat the extra labelled MN are due to Cyt-B, the present findingssuggest that Cyt-B could be responsible for {small tilde}45–57%(CREST data) or {small tilde}17–23% (ISH data) of MN inbinucleate cells. The presence of whole chromosomes in the majorityof human lymphocyte MN is problematic when the assay is usedfor biomonitoring of exposure to clastogens, where the usuallysmall effects expected may be masked by the high background.The identification of kinetochores or centromeres in MN mighthelp in dealing with this problem.     

Detection of the centromere in micronuclei by fluorescence in situ hybridization: its application to the human lymphocyte micronucleus assay after treatment with four suspected aneugens

The human lymphocyte micronucleus (MN) test combined with fluorescencein situ hybridization (FISH) of a centrom-eric probe is considereda useful screening assay to distinguish between clastogenicand aneugenic agents. Four suspected aneuploidy-inducing chemicals,acetaldehyde (AA), diethylstilbestrol (DES), diethylstilbestroldipropionate (DESdp) and griseofulvin (GF), have been evaluatedwith the assay. All compounds induced a significant increaseof MN at all doses tested. After the application of the FISHtechnique with a pancentromeric DNA sequence, DES, DESdp andGF showed a statistically significant increase in the percentageof positive signals compared with the control culture. GF inducedthe highest percentage of centromere-positive MN observed todate (>90% on average). AA did not show a significant differencein the percentage of centromere-positive MN. The results indicatethat in human lymphocytes DES, DESdp and GF act primarily asaneugens, while AA seems capable of causing both chromosomebreakage and aneuploidy. ¹To whom correspondence should be addressed     

In vitro and in vivo evaluation of the antihypertensive drug atenolol in cultured human lymphocytes: effects of long-term therapy

The genotoxicity of atenolol, a beta-blocker antihypertensive drug, both in vitro and in vivo, was cytogenetically tested for its ability to induce sister chromatid exchange (SCE) and micronuclei (MN) in cultured peripheral lymphocytes. Also, fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out, on the one hand, on four patients under antihypertensive treatment with atenolol and, on the other hand, on four matched control individuals taking an oral dose of atenolol. The in vitro study was performed on the control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma. When a comparison was made, the frequency of SCE did not show significant differences in any case. A statistically significant increase in the frequency of MN was detected in patients but not in control individuals either in vitro or in vivo. FISH analysis revealed statistically significant differences between patients and control individuals without the drug with respect to the frequency of centromeric signals in MN. Taking all these observations together, our data suggest that chronic exposure to atenolol resulted mainly in the induction of chromosome loss, so an aneugenic activity could be predicted. Different sensitivity to the compound was observed among control individuals. Nevertheless, all of them responded to the presence of atenolol in the same way in both assays. Interindividual variability was also reported. The intervariability seen in patients suggested an adaptive response to the chemical after long-term therapy.     

Evaluation of the cytogenetic damage induced by the antihypertensive drug nimodipine in human lymphocytes.

The aim of this work was a study of the genotoxic potential of chronic long-term therapy with the antihypertensive drug nimodipine by measures of sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral human lymphocytes of patients with long-term exposure to this drug. Peripheral human lymphocytes of control individuals exposed in vitro to nimodipine were also studied to assess the effect of the drug itself. Fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out on five patients under antihypertensive treatment with nimodipine. The in vitro study was performed on five control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma (controls/medium). The in vivo study showed no genotoxic effects of long-term therapy with nimodipine because the frequencies of SCE and MN in exposed patients did not show significant differences as compared with control individuals. A statistically significant increase in the frequency of MN was detected in controls/medium as compared with control individuals without the drug. FISH analysis revealed statistically significant differences with respect to the frequency of centromeric signals in nimodipine-induced MN in vitro. With regard to the in vivo results, chronic long-term therapy with nimodipine is not associated with increased genotoxicity. The differing results in vivo and in vitro could be due to extensive metabolism of nimodipine, indicating that the cytogenetic effect observed was due to the drug itself rather than its metabolites or to an adaptive response to nimodipine in vivo.     

Micronuclei assay and FISH analysis in human lymphocytes treated with six metal salts

The capability of some metal compounds for inducing micronuclei (MN) in human lymphocytes was studied. In this investigation, Al (III), Cd (II), Hg (II), Sb (V), Te (VI), and Tl (I) salts were considered. The FISH (fluorescence in situ hybridization) technique with a centromeric probe was coupled with the MN assay in binucleated cells in order to detect both centromere-positive MN (C+ MN) due to malsegregation phenomena and centromere-negative MN (C- MN) due to chromosome breakage. The blood of two young nonsmoking male donors was employed for all experiments. In both donors, all the tested metal compounds, with the exception of Tl(2)SO(4), showed a statistically significant increase of MN compared to controls, at least at one dose. FISH analysis revealed an increase in the fraction of C+ MN for Al, Cd, and Hg compounds, and of C- MN for the Sb salt; however, this was not a statistically significant increase. A different efficiency was observed for the different metal compounds, in particular, KSbO(3) and CH(3)HgCl, which were highly genotoxic, whereas the others showed minimal effects.     

The centromere as a target for the induction of chromosome damage in resting and proliferating mammalian cells: assessment of mitomycin C-induced genetic damage at kinetochores and centromeres by a micronucleus test in mouse splenocytes

The cytokinesis-block micronucleus assay (MN) on murine splenocyteswas used for the estimation of chromosome damage in a restingcell population in vivo that can be induced to proliferate invitro. Mitomycin C at different doses (10^–8, 6x10^–8,10^–7, 6x10^–7 and 10^–6 M) was used to inducecytogenetic damage in resting and cycling splenocytes. Antikinetochoreantibodies (CREST) and two-colour fluorescence in situ hybridization(FISH) with minor and major satellite DNA were applied. Theseapproaches allowed the detailed characterization of the mechanismsby which MN originates, since it was possible to identify breaksinduced in pericentric heterochromatin (resulting in MN containingthe major but not the minor satellite DNA) or detachment/disruptionof kinetochore (resulting in different frequencies of MN containingkinetochore or both probes). Based on the evidence that restingand cycling mouse splenocytes are characterized by differentspatial distribution of centromeric regions, the hypothesiswas tested that the damage induced by mutagens at centromeresis influenced by the phase of the cell cycle in which the cellsare treated. Data presented here show that resting and cyclingsplenocytes are both sensitive to mitomycin C action, and indicatethat this compound has an aneugenic potential, besides its strongclastogenic activity. In particular, results obtained afterCREST and FISH characterization of MN differed when cells weretreated during proliferation, suggesting a disruption/detachmentof kine-tochores induced by mitomycin C at this cell stage.Furthermore, under the same treatment condition the proportionof MN containing the major satellite DNA only was greater thanexpected on the basis of random breakage at this site. Treatmentof resting cells produced aneugenic damage, but without evidenceof disruption/detachment of kinetochores or preferential breakageat the centromere. These results indicate that the amount andtype of chromosome damage induced by mitomycin C in mouse splenocytesdiffer in relation to the proliferative status of treated cells. ³To whom correspondence should be addressed