CREST Staining Research Articles

Di(2-ethylexyl) phthalate and chromosomal damage: Insight on aneugenicity from the cytochalasin-block micronucleus assay

Bis(2-ethylhexyl) phthalate is the most abundant phthalate used as plasticizer to soften plastics and polymers included in medical devices. Human and environmental exposure may occur because DEHP is not chemically bound to plastics and can easily leach out of the materials. This phthalate is classified as reproductive toxicant and possible carcinogen to humans. The genotoxic potential has still to be clarified, but there are indications suggesting that DEHP may have aneugenic effects. To further investigate DEHP genotoxicity, the cytochalasin-block micronucleus assay was applied and combined with the CREST staining to characterise micronucleus content and gain insights on its genotoxic mode of action. Chromosomal damage was also analysed in metaphase and ana-telophase cells and the morphology of the mitotic spindle was investigated to evaluate the possible involvement of this cellular apparatus as a target of DEHP.Our findings indicated that DEHP induced a statistically significant increase in the frequency of micronuclei as well as in the frequency of CREST-positive micronuclei. Consistently, disturbance of chromosome segregation and induction of numerical chromosome changes were observed together with changes in spindle morphology, formation of multipolar spindles and alteration of the microtubule network. Experiments performed without metabolic activation demonstrated a direct action of DEHP on chromosome segregation not mediated by its metabolites.In conclusion, there is consistent evidence for an aneugenic activity of DEHP. A thresholded genotoxic activity was identified for DEHP, disclosing possible implications for risk assessment.

Dietary exposure to mercury and its relation to cytogenetic instability in populations from “La Mojana” region, northern Colombia

Fish consumption and chronic exposure to low doses of mercury (Hg) seems to activate several molecular mechanisms leading to carcinogenic and/or teratogenic processes. However, Hg genotoxic effects on humans are not completely described. In the present study, we assessed cytogenetic damage in isolated human peripheral lymphocytes using the cytokinesis-block micronucleus cytome assay (CBMN-Cyt), micronucleus formation with anti-kinetochore antibody (CREST staining), levels of total Hg in hair (T-Hg), fish consumption, and estimated Hg dose. The study comprised 39 non-exposed, and 73 residents from La Mojana region, an area with a well-documented Hg contamination. Data showed a significant increase in micronuclei (MNBN), nucleoplasmic bridges (NPB), and necrotic and apoptotic cell frequencies in residents of “La Mojana.” The overall mean T-Hg level in hair for exposed residents was 1.12 ± 0.94 mg kg−1 and 0.15 ± 0.05 in individuals from the reference area. Approximately 40% of analyzed individuals showed T-Hg levels that exceeded US Environmental Protection Agency (USEPA) reference dose. Increased T-Hg levels in hair were related to increased MNBN frequencies and high fish consumption. Other cellular markers, such as necrotic and apoptotic cell frequencies, were also correlated with high fish intake and T-Hg contents. Results of the CREST staining demonstrated that in vivo exposure to Hg induces genetic instability by chromosome fragment loss (clastogenic). Additionally, a high average intake of some fish species, particularly with carnivorous habits like Caquetaia kraussii, Hoplias malabaricus, and Sorubin cuspicaudus, seems to increase MNBN frequencies significantly.

A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements.

Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood

Mice heterozygous for a p53 null mutation develop tumours induced by genotoxic carcinogens with a shorter latency than wild type mice and have been proposed as an alternate animal model for carcinogenicity testing. Some literature data suggest that p53+/− mice might also be more sensitive to the short-term effects of genotoxic agents and manifest a haploinsufficiency phenotype that could contribute to the higher tumour susceptibility. We have compared the induction of micronuclei in bone marrow and blood of p53+/− and p53+/+ isogenic mice after treatment with a single or multiple doses of melphalan (MLP), a crosslinking genotoxic carcinogen. We have also characterized the mechanism of micronucleus induction with CREST staining of kinetochore proteins to distinguish between chromosome break- and chromosome loss-induced micronuclei. Significant increases of micronucleated bone marrow polychromatic erythrocytes and blood reticulocytes were induced under all MLP exposure conditions. The frequency of micronucleated blood erythrocytes increased linearly with duration of exposure. Micronuclei were essentially a consequence of chromosome break events. After a single MLP dose, a significant reduction of the frequency of polychromatic erythrocytes in bone marrow of p53+/+ animals suggested the induction of cytotoxicity/cell cycle delay. This effect was not observed in p53+/− mice. We believe this finding to provide some evidence of a haploinsufficiency phenotype in the modulation of cell cycle/apoptotic pathways mediated by the p53 protein. In bone marrow of wild type mice, an increased effect of multiple MLP doses was detected over that of a single administration, whereas, in p53+/− mice, no differential effect was found of different exposure durations. Possibly, the probability of micronucleus formation increased under chronic exposure because of increased cell division in response to peripheral anemia and a reduction of p53 protein level had a small effect on cell cycle modulation and on such indirect mechanism of micronucleus induction. However, pairwise comparisons between the frequencies of cells with micronuclei in wild type and p53+/− mice under all exposure conditions did not show statistically significant differences, suggesting that the observed effects of p53 haploinsufficiency were weak and temporary and a higher/faster induction of irreversible chromosome damage could not account for the increased susceptibility of p53+/− mice to MLP-induced tumours.

A role for the Rab6A′ GTPase in the inactivation of the Mad2-spindle checkpoint

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A', regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A' function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150(Glued), a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A' likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A', through its interaction with p150(Glued) and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.

Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-dioxane in the bone marrow and liver of young CD-1 mice

1,4-Dioxane, a widely used industrial chemical and rodent hepatocarcinogen, has produced mixed, largely negative results in the mouse erythrocyte micronucleus assay. In contrast, a recent report has indicated that 1,4-dioxane induces micronuclei in mouse hepatocytes following in vivo treatment. The objective of this study was to confirm these earlier results and identify the origin of the induced micronuclei. Following an initial range-finding study, mice were administered 1,4-dioxane by gavage at doses ranging from 1500 to 3500 mg/kg. The test animals were also implanted with BrdU-releasing osmotic pumps to allow cell proliferation to be measured in the liver and to increase the sensitivity of the hepatocyte assay. Upon sacrifice, the frequency of micronuclei in the bone marrow erythrocytes and in the proliferating BrdU-labeled hepatocytes was determined. Significant dose-related increases in micronuclei were seen in both the liver and the bone-marrow with significant increases being detected at all the tested doses in the bone marrow and at the 2500 and 3500 mg/kg doses in the liver. Using CREST staining or pancentromeric FISH to determine the origin of the induced micronuclei, it was determined that 80–90% of the micronuclei in both tissues originated from chromosomal breakage. Small increases in centromere-containing micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as well as in the ratio of bone marrow PCE:NCE were also observed. Based on these results, we conclude that at high doses: (i) dioxane exerts genotoxic effects in both the mouse bone marrow and liver; (ii) the induced micronuclei are formed primarily from chromosomal breakage; and (iii) dioxane can interfere with cell proliferation in both the liver and bone marrow.

Catalytic inhibitors of topoisomerase II are DNA-damaging agents: induction of chromosomal damage by merbarone and ICRF-187.

Merbarone is a catalytic inhibitor of topoisomerase II (topo II) that has been proposed to act primarily by blocking topo II-mediated DNA cleavage without stabilizing DNA-topo II-cleavable complexes. In this study merbarone was used as a model compound to investigate the genotoxic effects of catalytic inhibitors of topo II. The clastogenic properties of merbarone were evaluated using in vitro and in vivo micronucleus (MN) assays combined with CREST staining. For the in vitro MN assay, ICRF-187, a different type of catalytic inhibitor, and etoposide, a topo II poison, were used for comparison. Treatment of TK6 cells with all three of these drugs resulted in highly significant dose-related increases in kinetochore-lacking MN and, to a lesser extent, kinetochore-containing MN. In addition, a good correlation between p53 accumulation and MN formation was seen in the drug-treated cells. A mouse MN assay was performed to confirm that similar DNA-damaging effects would occur in vivo. Bone marrow smears from merbarone-treated B6C3F1 mice showed a dose-related increase in micronucleated polychromatic erythrocytes with a mean of 26 MN per 1000 cells being seen at the 60 mg/kg dose. Almost all MN lacked a kinetochore signal, indicating that merbarone was predominantly clastogenic under these conditions in vivo. The present study clearly shows that merbarone is genotoxic both in vitro and in vivo, and demonstrates the inaccuracy of earlier statements that merbarone and other catalytic inhibitors block the enzymatic activity of topo II without damaging DNA.

Micronuclei, CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality

Purpose : To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. Materials and methods : Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/mum and 27.6 keV/mum protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. Results : MN yield and the frequency of BN cells carrying multiple MN (2) were significantly higher after exposure to 27.6 keV/mum protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/mum protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6keV/mum protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3Gy). Conclusions : Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.

CREST staining of micronuclei from free-living rodents to detect environmental contamination in situ.

In this work immunofluorescent antikinetochore (CREST) staining was used to analyse bone marrow micronuclei (MN) from free-living animals belonging to four different rodent species. Yellow-necked mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus) were trapped in the Czech Republic, Algerian mice (Mus spretus) in Spain and house mice (Mus musculus domesticus) in Italy. Animals were collected in areas displaying low or high environmental pollution in order to investigate the sensitivity of CREST analysis on bone marrow MN as a biomarker of environmental stress in situ. Differences in total MN frequencies between animals collected in control or contaminated areas were statistically significant for two species, whereas the differences in CREST+ MN were statistically significant for three species. Interestingly, the percentages of CREST+ MN in animals collected in the control areas were very low (3. 2-8.7%), suggesting that activities inducing alterations in the distribution of chromosomes are very rare in natural conditions. The increased frequencies of CREST+ MN observed in areas with high environmental impact indicate that activities producing loss of chromosomes at mitosis may be characteristic of anthropogenic environments such as industrial settlements around petrochemical factories. Our data suggest that the analysis of CREST+ MN may represent a sensitive end-point for the detection of environmental contamination by genotoxic xenobiotics, offering the advantage of providing information on the mechanism of action of environmental contaminants.

Induction of micronuclei in bone marrow by two pesticides and their differentiation with CREST staining: an in vivo study in mice

Two pesticides, organophosphate phosphamidon (PHO) and organochlorine dieldrin (DED) were assayed by the mouse bone marrow micronucleus test, to ascertain whether they showed genotoxic activity in vivo. Two doses, sub-lethal (PHO=3 mg/kg b.wt.; DED=60 mg/kg b.wt.) and lethal (PHO=5 mg/kg b.wt.; DED=90 mg/kg b.wt.), of each substance were administered intraperitoneally to 9–10-week old CBA male mice, in acute and repeated exposure. The sub-lethal dose was also administered at two different times and twice at 24-h intervals. Both PHO and DED proved able to induce a dose-dependent increase of micronucleated polychromatic erythrocytes (PCE). The two pesticides also showed a different detoxification time. Furthermore, the CREST staining with antikinetochore antibodies allowed us to conclude that the two chemicals are clastogens.

Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors at various endpoints in vitro

The mammalian lignans enterolactone (ENL) and enterodiol (END) are formed by intestinal bacteria from the plant lignans matairesinol (MAT) and secoisolariciresinol (SEC), respectively, which are ingested with different types of food. ENL and END are weak estrogens. According to epidemiological and biochemical studies, lignans may act as anticarcinogens, but little is known about their genotoxic potential. We have therefore investigated the effects of ENL, END, MAT and SEC on cell-free microtubule assembly and at the following genetic endpoints in cultured male Chinese hamster V79 cells: disruption of the cytoplasmic microtubule complex, induction of mitotic arrest, induction of micronuclei and their characterization by CREST staining, and mutagenicity at the HPRT gene locus. The lignans were tested at concentrations of 200 μM in the cell-free system and 100 μM in cultured cells, which represents the limit of solubility in each assay. The established aneuploidogen diethylstilbestrol and the clastogen 4-nitroquinoline- N-oxide were used as positive reference compounds. As none of the four lignans had any activity at the endpoints studied, we conclude that ENL, END, MAT and SEC are devoid of aneuploidogenic and clastogenic potential under the experimental conditions used in this study.

Aneuploidogenic and clastogenic potential of the mycotoxins citrinin and patulin.

The aneuploidogenic and clastogenic potentials of the mycotoxins citrinin (CIT) and patulin (PAT) were studied by determining inhibition of microtubule assembly under cell-free conditions and by measuring induction of mitotic arrest and micronuclei in cultured Chinese hamster V79 cells. Both CIT and PAT inhibited cell-free microtubule polymerization in a concentration-dependent manner. PAT, but not CIT, bound covalently to reactive thiol groups of microtubule proteins. At concentrations without gross cytotoxicity, mitotic arrest and CREST-positive micronuclei, i.e. micronuclei containing whole chromosomes/chromatids, were induced by CIT and PAT in V79 cells. The time course of micronucleus induction and positive CREST staining indicate the aneuploidogenic potential of CIT and PAT. CREST-negative micronuclei, i.e. micronuclei containing acentric chromosomal fragments, were induced by PAT but not by CIT, implying a clastogenic potential of PAT. The aneuploidogenic and clastogenic potential of PAT and the aneuploidogenic potential of CIT may well contribute to the putative carcinogenicity of these mycotoxins in long-term animal studies.

Immunofluorescence analysis of diazepam-induced mitotic apparatus anomalies and chromosome loss in Chinese hamster cells.

To study the mechanisms leading to diazepam (DZ)-induced chromosome loss, we evaluated the effect of the drug on the distribution of cytoplasmic and mitotic apparatus proteins using specific antibodies. The use of antibodies directed against dynein and kinetochores (CREST staining) suggested that chromosomes arranged in monopolar spindles were interacting with short fibers originating from the monopole. Interestingly, nearly 50% of DZ-induced monopolar mitoses showed a punctate staining of centrosomes when evaluated with an anti-gamma-tubulin antibody. The extent of phosphorylation of mitotic proteins was not affected by drug treatment, as shown by staining the cells with an antibody against mitotic phosphorylated proteins (MPM-2). After recovery of DZ, nearly 20% of anaphases were abnormal and mainly consisted of multipolar anaphases and lagging chromosomes; this was consistent with a high frequency of kinetochore-containing micronuclei as evaluated by CREST antibody staining in cells that had divided only once after drug removal, i.e. binucleate cells obtained by cytochalasin-B treatment. Our data confirmed that DZ is a powerful inducer of chromosome loss in cultured rodent cells. Moreover, our results indicate that DZ interfered with the correct assembly of centrosomes.

Measurement and characterization of micronuclei in cultured primary lung cells of mice following inhalation exposure to benzene.

The genotoxic effects of benzene in lung cells of mice exposed to single acute doses by inhalation have been estimated by cytogenetic analysis of micronuclei in primary cultures of lung fibroblasts. Mice were nose-only exposed to 1000 p.p.m. for 30 or 60 min or to 3500 p.p.m. for 30 min and sacrificed 24 h after the end of exposure. Lung fibroblasts were cultured attached to coverslips for 72 h, the last 48 h in the presence of 0.75 microgram/ml cytochalasin B. Micronuclei were scored in binucleate cells. The mechanism(s) of micronucleus induction was characterized by immunofluorescent staining of kinetochore proteins (CREST staining), which allowed micronuclei due to chromosome loss (kinetochore-positive) to be distinguished from those produced by chromosome breakage (kinetochore-negative). Three- and 4-fold statistically significant increases in total micronucleus frequencies were observed in all benzene-exposed mice with respect to unexposed controls. The effect was neither concentration nor time dependent. This is compatible with a plateau dose-effect relationship for the effects on bone marrow, which is explained by saturation of metabolism. Both chromosome loss and chromosome breakage appear to contribute to micronucleus formation, suggesting that in addition to chromosome rearrangements, aneuploidy may be a relevant early genotoxic event associated with benzene carcinogenicity. Under the same treatment conditions no micronucleus induction could be shown in spleen lymphocytes, suggesting that with very short benzene exposures cells at the first contact site with local metabolizing capacity have a higher probability of genetic alterations potentially leading to neoplasia.

The centromeres of the Indian muntjac: Evidence for the existence of multiple centromeres?

Unlike the centromeres of other species, the ‘compound’ centromeres of the Indian muntjac span over exceptionally extended regions (Brinkley et al., 1984). We extend this concept and show that some of these centromeres are divisible into several chromomeres in which the light staining regions alternate with the dark staining C-band positive segments. Unlike the centromeres of other species where the centromere replicates as one unit, the replication of the sub-units constituting the centromere of the X-chromosome in the muntjac occurs at different times as at least three independent segments. The CREST staining of the centromere regions of even the smallest (Y 2) chromosome is interrupted by non-staining segments. Electron microscopy shows similar interruptions in the continuity of the trilamellar kinetochore. Sister chromatid exchanges occur in the region of the centromeres and chromatid breaks within the centromere region occur in the non-fluorescent segments. We interpret these data to suggest that the centromere regions of the Indian muntjac are made up of independent multiple centromeres interrupted by non-centromeric chromatin. Relevance of these parameters in mutagenesis is briefly discussed.

Current cytogenetic methods for detecting exposure and effects of mutagens and carcinogens.

Most mutagens and genotoxic carcinogens are efficient inducers of chromosomal alterations in exposed cells. Two important classes of aberrations, namely structural and numerical, are recognized and both types of aberrations are associated with congenital abnormalities and neoplasia in humans. These alterations can be easily detected and quantified in human peripheral blood lymphocytes. Conventional staining techniques can be used to detect these aberrations; this technique was used to estimate absorbed dose in the case of a radiation accident in Goiania, Brazil. A recently introduced fluorescent in situ hybridization technique (FISH) using DNA probes has increased the sensitivity and ease of detecting chromosome aberrations, especially stable chromosome aberrations. This technique allows, to some extent, the estimation of absorbed radiation dose from past exposures. Numerical aberrations can be directly estimated in metaphases by counting the number of FISH-painted chromosomes. Micronuclei are formed by lagging chromosome fragments or whole chromosomes during the anaphase stage of cell division. The nature of micronuclei as to whether they possess a centromere can be determined either by CREST staining (calcinosis, Raynoud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) or FISH with centromere-specific DNA probes. In several carcinogen-exposed populations, such as heavy smokers or people exposed to arsenic, aneuploidy appears to be more common than structural aberrations. In victims of radiation accidents, aneuploidy (hyperploidy) has been found to be common in addition to structural aberrations.

Preferential sensitivity of acrocentric chromosomes to the aneugenic effect of colchicine.

In order to evaluate the predisposition to the aneuploidy-inducing agent colchicine (Col) on lymphocytes from trisomic 21 patients compared with their parents and with a control group of subjects without trisomic children, we performed the micronucleus (MN) assay associated with C-banding, CREST staining, and nucleolar organizing region (NOR)-banding. According to our results Col behaves as an aneugenic agent independently of the population studied for CREST and C-banding. The Col-induced MN exhibited a clear majority (> 80%) of positive NOR-MN, meaning that they contain a NOR region transcriptionally active or inactive. The same data were observed in trisomic 21 individuals, their parents, and the control group, without significant differences between them. These results seem to suggest a preferential effect of the aneugen Col on acrocentric chromosomes in all of the three groups studied.

Effects of 5-azacytidine on the centromeric region of human fibroblasts studied by CREST staining and in situ hybridization on cytokinesis-blocked cells.

Several interphase methodologies have been applied in this study to investigate whether chemically induced undercondensation of the pericentromeric region of human fibroblast chromosomes promotes structural or numerical aberrations at the cell cycles following treatment. To achieve this aim, the effects of the hypomethylating agent 5-azacytidine (5- azaC) were studied on cytokinesis-blocked binucleated fibroblasts. This approach allowed the distribution of whole chromosomes or chromosome fragments in the daughter nuclei and micronuclei of treated cells to be followed, since the daughter nuclei of a single mitosis are maintained in one cytoplasm by treatment with the actin inhibitor cytochalasin B. Antikinetochore staining and in situ hybridization with an alpha-satellite probe capable of detecting all human centromeres on 5-azaC-induced micronuclei in binucleated fibroblasts indicated that the predominant effect of the chemical is to induce micronuclei lacking centromeres, suggestive of induced chromosome aberrations. Double in situ hybridization with alphoid and classical satellite DNA probes specific for chromosome 1 on binucleated fibroblasts was used to discriminate induced aneuploidy from breakage effects in the target area of hybridization. The results showed that undercondensation of the pericentromeric heterochromatin of human fibroblast chromosomes by treatment with 5-azaC produces structural chromosome aberrations involving the classical satellite DNA, whereas there was no evidence that the chemical induced chromosomal aneuploidy.

Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was studied for the induction of micronuclei (MN). We have carried out the MN assay in bone marrow polychromatic erythrocytes in mice, in cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN assay in vitro was performed in the presence and in the absence of S9. To further extend the study, an antikinetochore antibody (CREST staining) was used to distinguish MN containing whole chromosomes (kinetochore positive) from those containing acentric fragments (kinetochore negative). When tested in vivo quercetin failed to induce micronuclei, a result which is in agreement with other published reports. When tested in vitro in V79 cells quercetin clearly induces micronuclei in the absence of S9 and also in the presence of S9 for the highest dose used. When tested in vitro in human lymphocytes quercetin shows a significant induction of micronuclei in the absence and in the presence of S9. The presence of s9 compared to its absence is not significant for any of the systems used. Both in the presence and absence of S9, quercetin appears to behave as a clastogenic agent in human lymphocytes inducing a significant majority of kinetochore-negative MN.

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 and in situ hybridization (FISH) with centromeric DNA probes are able to distinguish between micronuclei (MN) containing lagging chromosomes or acentric fragments. Different frequencies of signal-positive MN induced by mitomycin C (MMC) were obtained by Miller et al. (Mutagenesis, 6, 297-302, 1991) between CREST labelling and FISH with the mouse major-gamma-satellite DNA probe (major probe). Both modes of identifying the presence of an entire chromosome in a MN are theoretically prone to misclassification. Breaks induced in pericentric heterochromatin can produce fragment-containing MN with a major signal. Alternatively, alterations of kinetochore proteins can produce CREST-negative MN containing lagging chromosomes. To improve the reliability of MN differentiation two additional DNA probes, the mouse minor satellite DNA probe (minor probe) and the telomere repeat (5'-TTAGGG-3')7, have been used and double labelling has been employed with minor/major and minor/telomere probes. At 1 mg/kg of MMC the labelling frequencies of MN with CREST and the minor probe were identical (18.5 and 19%, respectively) and the major probe showed a higher labelling rate (30.5%). Using double-labelling the difference between minor and major probe responses was confirmed (17 and 31.5%, respectively). At 5 mg/kg of MMC, CREST labelling gave the lowest (6%), the minor probe gave intermediate (10 and 11.5% after single- and double-labelling, respectively) and the major probe gave the highest signal frequencies (16.5 and 15% after single- and double-labelling, respectively). The CREST and the minor signal frequencies were not significantly different at either dose of MMC whereas the minor and the major signal frequencies were significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)