Centromere-negative Micronuclei Research Articles

In vivo micronuclei in uncultured T-lymphocytes of male railroad transit workers and referents

In the biomonitoring of human genotoxic effects, micronuclei (MN) usually are scored in phytohaemagglutinin-stimulated cultured lymphocytes. MN also can be examined in uncultured lymphocytes, which facilitates the analysis of genotoxic damage incurred in vivo. Characterization of MN in cultured lymphocytes by fluorescence in situ hybridization (FISH) has shown a clear over-representation of the X and Y chromosomes in the MN of males. However, it is not known if this phenomenon also occurs in vivo. The purpose of the present study was to assess the frequency and composition of MN formed in vivo from immunomagnetically isolated uncultured T-lymphocytes of men. To evaluate the possible effects of genotoxic exposure on in vivo MN, we examined 17 railroad workers occupationally exposed to complex chemical mixtures and 14 referents, all nonsmokers. The results showed similar total frequencies of micronucleated cells among the exposed workers and the referents. When the MN were characterized by FISH, there were no significant differences between the exposed and referents with regards to the frequency of centromere-positive or centromere-negative MN. Centromeric label was observed in 69% of all MN, indicating that most of the MN contained whole chromosomes (or chromatids). 80% of the centromere-positive MN harbored autosomes, 12% Y chromosomes, and 8% X chromosomes. The occurrence of the Y- and X-chromosomes in MN was, respectively, 5.5- and 3.8-times greater than would be expected assuming an equal contribution by all chromosomes. Thus, sex chromosomes appear to be over-represented in lymphocyte MN of men in vivo, confirming previous results obtained in vitro.

A combined analysis of XRCC1, XRCC3, GSTM1 and GSTT1 polymorphisms and centromere content of micronuclei in welders

The aims of the present study were to assess clastogenic and aneugenic properties of welding fumes using fluorescent in situ hybridization (FISH) with a human pancentromeric DNA probe. The involvement of genetic polymorphisms in DNA repair genes (p.Arg399Gln of XRCC1 and p.Thr241Met of XRCC3) and in detoxification genes (GSTM1 and GSTT1) on the centromere content of micronuclei (MN) was also evaluated. This study included 27 male welders working without any collective protection device and a control group (n = 30). The welders showed significantly higher levels of chromosome/genome damage compared to the controls. The frequencies of MN and centromere-positive MN (C+MN) per 1,000 binucleated cells were significantly higher in the exposed group than in the control group (7.1 per thousand +/- 3.7 versus 4.9 per thousand +/- 1.8; P = 0.012 and 3.5 per thousand +/- 1.8 versus 2.4 per thousand +/- 1.2; P = 0.018, respectively, Mann-Whitney U-test). The centromere-negative MN (C-MN) frequency was higher in the exposed subjects than in the controls (3.6 per thousand +/- 3.4 versus 2.5 per thousand +/- 1.4), but the Mann-Whitney U-test did not yield a significant result. In the total population, the GSTM1 and GSTT1 polymorphisms significantly affected the frequencies of C-MN and C+MN defined by FISH. GSTM1 positive subjects showed an increased C-MN frequency and GSTT1 null subjects showed an elevated C+MN frequency. When GSTM1 and GSTT1 genotypes were included in multiple regression analysis, the effect of the occupational exposure could better be demonstrated; both C+MN and C-MN were significantly increased in the welders. Our results suggest that the combined analysis of genetic polymorphisms and centromeres in MN may improve the sensitivity of the micronucleus assay in detecting genotoxic effects.

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.

Cytogenetic biomonitoring of a floriculturist population in Italy: micronucleus analysis by fluorescence in situ hybridization (FISH) with an all-chromosome centromeric probe

Flower production in greenhouses associated with a heavy use of pesticides is very wide-spread in the western part of the Ligurian region (Italy). The formation of micronuclei in peripheral blood lymphocytes is a valuable cytogenetic biomarker in human populations occupationally exposed to genotoxic compounds. In the present study we investigated the micronucleus frequency in peripheral blood lymphocytes of 52 floriculturists and 24 control subjects by use of the cytokinesis-block methodology associated with fluorescence in situ hybridization with a pan-centromeric probe that allowed to distinguish centromere-positive (C+) and centromere-negative (C−) micronuclei. The comparison between floriculturists and controls did not reveal any statistically significant difference in micronucleus frequency, although an increase was observed with increasing pesticide use, number of genotoxic pesticides used and duration of exposure. An increase in C+ as well as in C− micronuclei and in the percentage of C+ micronuclei with respect to the total number of micronuclei was detected in floriculturists, suggesting a higher contribution of C+ micronuclei in the total number scored. The percentage C+ micronuclei was not related to the duration of exposure or to the number of genotoxic pesticides used, but a higher percentage (66.52% versus 63.78%) was observed in a subgroup of subjects using benzimidazolic compounds, compared with the floriculturist population exposed to a complex pesticide mixture not including benzimidazolics. These results suggest a potential human hazard associated with the exposure to this class of aneuploidy-inducing carcinogens.

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.

Cytogenetic monitoring of industrial radiographers using the micronucleus assay

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years±11.2 , in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv±49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls ( 10.7‰±5.2 versus 6.6‰±3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C−MN) in exposed subjects than in controls ( 8.5‰±4.9 versus 2.2‰±1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C−MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.

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.

Micronucleus incidence and their chromosomal origin related to therapy in acute lymphoblastic leukemia (ALL) patients: Detection by micronucleus and FISH techniques

Micronucleus assay and dual color-fluorescence in situ hybridization (DC-FISH), using centromere-specific and whole chromosome-specific painting probes, are considered a useful screening test to determine the incidence of micronucleus, their origin and contents. The patients with acute lymphoblastic leukemia (ALL), who had undergone chemotherapy, were analysed before and after treatment with vincristine, methotrexate, daunomycin, prednisone, and asparaginase. The incidence of micronuclei after the antileukemic agent treatment was significantly higher than before the treatment. Application of DC-FISH using a combination of whole chromosome-specific painting probes and the same chromosome-specific alpha-satellite centromeric probe showed that there were no significant differences in the micronucleus incidence for any specific chromosome (chromosomes 7, 8, 11, 17, X, and Y). There were no significant differences between the incidence of centromere-positive micronuclei and the incidence of centromere-negative micronucleus. We concluded that antileukemic agents induced the somatic genetic damage but this damage is not related to any specific chromosome studied.

Detection and characterization of micronuclei in a murine liver epithelial cell line, by application of the in vitro cytokinesis block MN assay and PRINS.

The cytokinesis block micronucleus assay was applied to murine cell line C6, derived from fetal liver, after an optimal protocol had been designed. Micronucleus frequencies were assayed after exposure to three concentrations of colcemid or diepoxybutane. Two-colour primed in situ DNA synthesis (PRINS) was applied to simultaneously label telomeric and centromeric (minor satellite DNA) sequences. Both chemicals induced a highly significant increase in MN and the effect was dose dependent. Diepoxybutane did not appear to significantly increase the frequency of centromere-positive micronuclei. Colcemid, as expected, induced high frequencies of centromere-positive micronuclei at all concentrations tested; in addition a significant increase in centromere-negative micronuclei was observed at 10(-5) M. Many centromere-positive micronuclei carried three or four telomeres, thus indicating that a duplicated (non-disjoined) chromosome with two chromatids was contained in the micronucleus. This observation leads to the conclusion that micronuclei deriving from missegregation could be due to errors occurring before the onset of anaphase. The results obtained on C6 cells are in good agreement with those obtained on other cell systems, indicating that this cell line can be considered for in vitro aneuploidy evaluation.

A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay

A cytogenetic study was performed in 215 nuclear power plant workers occupationally exposed to radiation using the micronucleus-centromere assay for peripheral blood lymphocytes. As control population served administrative staff with yearly doses below 1 mSv. The increase of the micronucleus frequency with age, observed in the non-smoking control population, is mainly due to an enhanced number of centromere-positive micronuclei, pointing to an increased chromosome loss. No differences in the number of micronuclei, centromere-positive and centromere-negative micronuclei between smokers and non-smokers are observed. An analysis of the micronucleus data vs. the dose accumulated over the 10 years preceding the venepuncture shows no significant clastogenic or aneuploidogenic effects of the exposure in the studied population which is representative for workers in the nuclear industry at present. According to the linear fits to our data an increase of the micronucleus frequency pro rata 0.5 per 1000 binucleated cells per year, related to the centromere-negative micronuclei, may be expected for workers with the maximal tolerable dose of 20 mSv/year.

Delayed cytotoxic and genotoxic effects in a human cell line following X-irradiation

Background: In order to clarify the relationship between delayed reproductive death and radiation-induced genomic instability, the colony-forming efficiency of surviving, irradiated human squamous carcinoma cells and centromere positive as well as centromere negative micronuclei in surviving progeny were examined. Materials and methods: Colony-forming ability and micronucleus (MN) frequency in binucleated cells 24h after the addition of cytochalasin B during 2 weeks of post-irradiation growth were determined in a squamous cell carcinoma cell line (SCL-II) of human origin. In addition, centromeres in micronuclei were detected using FISH. Results: In the human epithelial cell line used for these experiments, delayed reproductive death was pronounced and persisted for at least 2 weeks after irradiation. Although there is evidence for an increased rate of centromere positive micronuclei, but not of centromere negative micronuclei, arising during the first week of post-irradiation proliferation, this decreases later while the rate of delayed reproductive death remains elevated. Conclusion: In the studied cell line, the observed delayed reproductive death is not closely related to the investigated criteria of radiation-induced genomic instability. This casts doubt on the common assumption that delayed reproductive death is a direct manifestation of radiation-induced genomic instability.

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.

A study of the aneugenic activity of trichlorfon detected by centromere-specific probes in human lymphoblastoid cell lines

The potential of the pesticide trichlorfon to induce mitotic aneuploidy has been investigated in genetically engineered human lymphoblastoid cell lines. Trichlorfon failed to induce micronuclei in the AHH-1 and MCL-5 cell lines when treated in media at normal cell culture pH (pH 7.3). Under a treatment pH of 5.5, trichlorfon exposures resulted in the induction of both chromosome loss and chromosome non-disjunction as measured by fluorescence in situ hybridisation (FISH) using a pan-centromeric probe for all human centromeres and centromere probes specific for chromosomes 2, 7 and 18. At treatment concentrations greater than 20 μg/ml trichlorfon also induced structural chromosome damage resulting in the production of centromere negative micronuclei.

Analysis of radiation-induced micronuclei in two-cell human-hamster embryos using telomeric and centromeric FISH probes.

Simultaneous, fluorescent in situ hybridization using a centromeric human alpha satellite DNA probe and a telomeric DNA probe was used to analyze the chromosome content of micronuclei induced in two-cell human-hamster embryos by in vitro gamma-ray irradiation of human spermatozoa. In unirradiated samples, about 26% of micronuclei were centromere positive, indicating that both structural chromosome aberrations and numerical changes are involved in the spontaneous production of micronuclei. After exposure of spermatozoa to radiation, a significant increase in the number of micronuclei was found. About 77% of induced micronuclei contained only telomeric signals suggesting that they originated from acentric fragments. However, both centromere-positive and centromere-negative micronuclei increased with radiation dose. These results are consistent with the well known clastogenic effect of ionizing radiation and with its weak aneugenic effect.

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 gamma irradiation and by Vinblastine sulfate. In a cytokinesis-block micro-nucleus 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 alpha 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.

Immunofluorescent and confocal laser cytometric analyses of centromeres in V79 cells

Previously, a modified anticentromere antibody (ACA) technique was established in the V79 Chinese hamster lung cells to simulataneously analyze chromosome damage and aneuploidy induced by various agents. Using this method, cyclophosphamide (CP) was evaluated further in the presence and absence of S9 activation for micronucleus/aneuploidy induction. The specific binding nature of ACA to the centromeric region was also analyzed using a confocal scanning laser cytometry. The results indicated that CP was primarily a clastogen and S9 activation was required for its activity. Vinblastine, the positive control for aneuploidy, produced predominantly centromere containing micronuclei and the addition of S9 was not required for its activity. X-radiation, the positive control for clastogenicity, predominantly produced centromere negative micronuclei confirming its clastogenicity. An evaluation of centromeric region under the standard fluorescence microscope indicated that ACA generally binds to most centromeric regions in a cell. However, by confocal imaging it was found that ACA binds to the central core proteins of the centromere region and not to the peripheral proteins.

Centromere analysis of micronuclei induced by 2-aminoanthraquinone in cultured mouse splenocytes using both a gamma-satellite DNA probe and anti-kinetochore antibody.

We have tested 2-aminoanthraquinone (2-AAQ) as a potential aneugen in a cytokinesis-blocked mouse splenocyte micronucleus (MN) assay. Binucleated cells (BNC) were evaluated for MN, and the MN were further probed with two indicators of centromere presence: an anti-kinetochore autoantibody and a DNA probe for the mouse gamma-satellite locus. A dose-dependent increase in the frequency of BNC with MN was observed. At the highest 2-AAQ concentration (10 micrograms/ml), the frequency of BNC containing MN was increased greater than 10-fold over background. Both centromere-positive and centromere-negative MN were significantly increased. At least 62% of MN at all 2-AAQ doses were positive for the gamma-satellite DNA probe, while 30-53% were labeled with the antikinetochore antibody. In contrast with the 2-AAQ results, after treatment with the aneugen demecolcine (positive control), greater than 80% of MN labelled positive with both probes. This discordance in the results with the two probes after 2-AAQ exposure suggests that the mode of action of this chemical may be as an aneugen by disruption of the kinetochore proteins, as a clastogen with a preferential cleavage site at or near the gamma-satellite locus, or both. Our results also suggest that the use of either of these probes individually may not be an adequate measure of centromere presence. Nevertheless, positive results for both markers provides strong evidence that 2-AAQ is aneugenic.