Chromosome painting reveals specific patterns of chromosome occurrence in mitomycin C- and diethylstilboestrol-induced micronuclei

Cultures of human blood lymphocytes from three subjects were incubated with the clastogen mitomycin C (MMC, 500 ng/ml) and the aneugen diethylstilboestrol (DES, 80 microM) 23 h before harvesting, to induce formation of micronuclei (MN) and numerical and structural alterations in metaphase chromosomes. We used fluorescence in situ hybridization (FISH) with painting probes for all human chromosomes to determine which chromosomes had contributed material to the induced MN. MMC treatment induced an approximately 18-fold increase in MN and led to a significant increase in hypodiploidy and structural chromosome aberrations in metaphase preparations. Undercondensation of pericentromeric heterochromatin of chromosomes 9 and 1 occurred in 20-75% of metaphases and FISH disclosed an abundance of material from these chromosomes in induced MN (62-69% from chromosome 9 and 7-12% from chromosome 1). DES treatment of lymphocytes induced a seven-fold increase in MN frequency and four-fold increase in the frequency of numerical aberrations; structural aberrations were not significantly increased. FISH analysis showed that material from all chromosomes was present in DES-induced MN, with material from chromosome 1 present in 16% of MN and material from each other chromosomes being present in 2-10% of MN. Material from chromosomes 14, 19 and 21 was significantly more frequent material from chromosome Y significantly less frequent in DES-treated cells than in controls. The findings of the MMC studies indicate that the heterochromatin block of chromosome 9 is a specific target for MMC-induced undercondensation, which induces a preferential occurrence of chromosome 9 material in MN. DES, in contrast, does not trigger heterochromatin decondensation and fails to induce such a significant appearance of material of particular chromosomes in MN.     

Spontaneous and spindle poison-induced micronuclei and chromosome non-disjunction in cytokinesis-blocked lymphocytes from two age groups of women

Fluorescence in situ hybridization (FISH) was used to evaluate spontaneous and aneuploidogen-induced micronucleus frequencies and non-disjunction of chromosomes X and 8 in cultured binucleated lymphocytes of women of two age groups. Demecolcine and vincristine were used as model aneuploidogens to induce micronuclei and chromosome malsegregation. Four of the women were aged 22-26 (mean 24.3) years and four 47-50 (mean 49.0) years. Pancentromeric FISH was applied to micronuclei to identify chromosomes and double-color centromeric FISH, performed in binucleates of two young and two older women, was used to assess the involvement of chromosomes X and 8 in micronuclei and non-disjunction. It was confirmed that age increases micronucleus frequency. Micronuclei containing whole chromosomes predominated in older females. Age also enhanced micronuclei containing acentric chromosome fragments. The inclusion of chromosomes X and 8 in micronuclei was enhanced by age and chromosome X was generally overrepresented. Non-disjunction of chromosomes X and 8 also increased with age, chromosome X being the more sensitive. Treatment of lymphocytes with vincristine and demecolcine increased micronucleus frequency and malsegregation of chromosomes X and 8 in both age groups. Comparison of the estimated frequencies of micronucleation and non-disjunction for all human chromosomes showed that non-disjunction is the main type of chromosome malsegregation.     

Induction of chromosomal aberrations by the rhodium(III) complex cis-[Rh(biq)(2)Cl(2)]Cl in cultured human lymphocytes

The genotoxicity of the rhodium(III) complex cis-[Rh(biq)(2)Cl(2)]Cl (complex R) in cultured human lymphocytes was studied using the chromosome aberrations (CAs) assay. Lymphocyte cultures were initiated from two adult healthy non-smoking male volunteers and were exposed to the complex for a duration of 3 or 20 h prior to cell collection. The reduction in mitotic indices (MI) and the induction of CAs represented the toxic and clastogenic effects of the different treatments, respectively. Complex R proved to be an intermediate toxic clastogen with a MI(50) of 1.0 microg/ml and a minimum positive dose (MPD) of 0.1 microg/ml. Like bleomycin, complex R exerted its clastogenic effects without the need for metabolic activation and induced CAs of all types in lymphocytes treated in the G(2) and late S phases and, therefore, can be considered a radiomimetic. In addition, it induced more total CAs of chromatid-type than of chromosome-type. The reduction in the frequencies of CAs following the 20 h treatment as compared with those induced following the 3 h treatments indicated that human lymphocytes in the presence of complex R can partially tolerate the lesions involved in CA production. Based on the biological effects of complex R and the similarities between its functional group and that of bleomycin, possible mechanisms for complex R genotoxicity are discussed.     

Noscapine hydrochloride-induced numerical aberrations in cultured human lymphocytes: a comparison of FISH detection methods and multiple end-points

The cytokinesis block in vitro micronucleus (MN) assay in combination with CREST staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows mechanistic information on the induction of numerical chromosomal aberrations to be obtained through a rapid and simple microscopic analysis. These techniques can now be used to investigate relationships between the induction of chromosomal loss, non-disjunction and polyploidy by aneuploidy-inducing agents. In the present study, we treated 72 h cultured lymphocytes for the last 24 h of culture with various concentrations of the cough medicine noscapine hydrochloride (NOS) (3.9-120 micro g/ml) in the presence of either cytochalasin B (CYB) (3 micro g/ml) or 5-bromo-2'-deoxyuridine (BrdU) (1 micro M). Using the CREST staining modified MN assay in the CYB-treated cultures, we detected significant increases in CREST-positive but not CREST-negative MN in both binucleated and, to a lesser extent, mononucleated cells, demonstrating the ability of this compound to induce chromosomal loss. In addition, using FISH with chromosome 1- and 9-specific classical satellite probes, a significant induction of chromosomal non-disjunction in the binucleated lymphocytes and polyploidy in the mononucleated lymphocytes was seen, indicating that polyploidy induced by NOS may occur without progression through a normal anaphase and/or telophase. In the BrdU-treated cultures, a dose-dependent induction of hypodiploidy, hyperdiploidy and polyploidy was observed using FISH with a chromosome 9-specific alpha-satellite probe in the labeled cells. By comparison, in the unlabeled non-cycling cells, only a slight increase in hyperdiploidy/polyploidy but not hypodiploidy was seen. A comparison of the effects seen at different concentrations shows that at the lower effective concentrations, all three types of numerical aberrations, chromosomal loss, non-disjunction and polyploidy, contributed to the numerical aberrations seen, whereas at the highest concentration tested, polyploidy was the predominant alteration. These studies indicate that FISH in combination with CYB or BrdU immunfluorescent staining can be sensitive tools for the identification of aneuploidy-inducing agents.     

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.     

Confirmation of the chromosome damaging effects of lamivudine in in vitro human peripheral blood lymphocytes

The aim of this study was to investigate genotoxic effects of lamivudine (an analogue of cytidine) using in vitro sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus (MN) tests in human peripheral lymphocytes. The cells were treated with 75, 100, 125, and 150 microg/ml concentrations of lamivudine (roughly 30-60 times higher than plasma levels achieved in patients receiving this drug) for two (24- and 48-hr) treatment periods. Lamivudine induced SCEs at the highest concentration (150 microg/ml) in the 24-hr treatment, and at 125 and 150 microg/ml in the 48-hr treatment, when compared to the solvent control. During both treatment periods, structural chromosome aberrations were significantly increased at 100, 125, and 150 microg/ml lamivudine concentrations. However, the increases of SCEs (22%) and CAs (50%) were weak. In addition, lamivudine reduced both the proliferation index (PI) and the mitotic index (MI) significantly at all concentrations for the two treatment periods. The MI was reduced by lamivudine in a dose-dependent manner during both the 24- and 48-hr treatment periods. In contrast, the PI was reduced by lamivudine only during the 48-hr treatment period. A weak but significant increase in MN formation was observed following lamivudine treatment at 100, 125, and 150 microg/ml for 48 hr, but no significant increase in micronuclei were observed following 24-hr treatment. In conclusion, lamivudine has a weak genotoxic effect at elevated doses on human peripheral lymphocytes.     

Does formaldehyde induce aneuploidy?

Formaldehyde (FA) was tested for a potential aneugenic activity in mammalian cells. We employed tests to discriminate between aneugenic and clastogenic effects in accordance with international guidelines for genotoxicity testing. The cytokinesis-block micronucleus test (CBMNT) in combination with fluorescence in situ hybridisation (FISH) with a pan-centromeric probe was performed with cultured human lymphocytes and the human A549 lung cell line. FA induced micronuclei (MN) in binuclear cells of both cell types under standard in vitro test conditions following the OECD guideline 487. FISH analysis revealed that the vast majority of induced MN were centromere negative, thus indicating a clastogenic effect. A similar result was obtained for MN induced by γ-irradiation, whereas the typical aneugens colcemid (COL) and vincristine (VCR) predominantly induced centromere-positive MN. Furthermore, COL and VCR clearly enhanced the MN frequency in mononuclear lymphocytes in the CBMNT, whereas such an effect was not observed for γ-irradiation and FA. In experiments with the Chinese hamster V79 cell line, the aneugens COL and VCR clearly increased the frequency of tetraploid second division metaphases, whereas FA did not cause such an effect. Altogether, our results confirm the clastogenicity of FA in cultured mammalian cells but exclude a significant aneugenic activity.     

Cytogenetic effects of permethrin in cultured human lymphocytes.

The pyrethroid insecticide permethrin was tested for its ability to induce sister chromatid exchanges (SCE), micronuclei (MN) and structural chromosome aberrations (CA) in cultured human peripheral blood lymphocytes. Permethrin was tested in the range of 5-500 micrograms/ml in the absence and in the presence of a rat liver activation system (S9 mix). Small elevations in the SCE frequencies were found and even though statistically significant may have no biological meaning, the more so since there was no dose-effect relationship. Permethrin induced both MN and CA when it was evaluated in the absence of a metabolic activation system. Nevertheless, it cannot be said that S9 mix suppressed the activity in itself. The effect of permethrin seemed to be time of exposure dependent. Permethrin could be characterized as a S-phase independent agent with greater potential for inducing chromosomal damage than sister chromatid exchanges.     

17-alpha-ethinylestradiol and norgestrel in combination induce micronucleus increases and aneuploidy in human lymphocyte and fibroblast cultures

Oral contraceptives are highly efficient and easily administered drugs; however, it must not be forgotten that they are composed of chemical substances which can be classified as potential carcinogens. Testing of a substance for genotoxicity represents a reliable approach both to evaluate the genetic hazard and to obtain information on its possible tumorigenic (cancerogenic) properties. The present study was undertaken to evaluate through carefully planned and controlled investigations the in vitro cytogenetic effects of oral contraceptives (ethynilestradiol and norgestrel mixed in the proportion 1:5) using three different concentrations, with two different durations of treatment (48 and 72 h), on two types of human cells (lymphocytes and fibroblasts) and a series of short-term test procedures: sister chromatid exchange (SCE), micronucleus test (MN), and chromosome aberrations (CA). In addition, the FISH procedure and in vitro anaphase and metaphase preparation analyses were performed. In contrast to CA and SCE frequencies, the frequency of MN in treated blood lymphocytes showed higher values by comparison with the controls, although the difference was statistically significant only for the lowest concentration (P = 0. 016). When using pancentromeric alphoid probes, the FISH procedure gave positive signals in more than 85% of micronuclei, clearly indicating that MN may contain whole chromosomes rather than acentric fragments. Unlike the lymphocytes, the fibroblasts showed dose-dependent effects, although those treated with the highest hormone concentrations showed an increased number of highly damaged cells (cytoplasmatic vacuolization, nuclear fragmentation, etc.), a decreased number of anaphase cells, a large number of which were abnormal, and a reduction of mitotic index. In conclusion, our data confirm that hormones do not induce structural chromosome aberrations in lymphocytes and indicate that ethynilestradiol and norgestrel have an aneugenic effect on fibroblast and lymphocyte cultures; FISH analysis on micronuclei from lymphocyte cultures and anaphase preparations from fibroblast cultures support this hypothesis. Teratogenesis Carcinog. Mutagen. 20:147-159, 2000.     

X chromosome territories in human female lymphocytes.

We developed an improved bromodeoxyuridine (BrdU)-DNA assay procedure for flow cytometric analysis. Which makes possible cytochemical investigation against cells after BrdU-DNA assay, such as immunochemical staining or in situ hybridization. Using this method, we performed fluorescence in situ hybridization (FISH) technique using DNA probes that recognize whole X chromosome of human lymphocytes in peripheral blood (resting cells) and cultured cells stimulated by phytohemagglutinin (PHA) (proliferative cells). Cultured cells were fractionated precisely into each stage of cell cycle by cell sorter after flow cytometric analysis. Following FISH procedure, the cells were examined for the volumes of two X chromosome territories in the nuclei using the optical section images on a confocal laser scanning microscope (CLSM). And we analyzed the variation in the volume ratio (R) of two X chromosome territories at different stages in cell cycle. Our data indicated that there were no significant difference between the volume of two X chromosome territories in female human lymphocytes at resting (G0) stage. Furthermore, they also showed no significant alternation throughout the cell cycle. These results may challenge the traditional concepts for inactivated chromosome in two points. First, the inactivated X chromosome is more decondensed throughout the cell cycle than previously thought. Second, although the inactivated X chromosome appears more decondensed during its self replication it dose not bring a great change in the volume of its territory.     

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.     

Evaluation and characterization of micronuclei induced by the antitumour agent ASE [3beta-hydroxy-13alpha-amino-13, 17-seco-5alpha-androstan-17-oic-13, 17-lactam-p-bis(2-chloroethyl)amino phenylacetate] in human lymphocyte cultures

3beta - Hydroxy - 13alpha - amino - 13, 17 - seco - 5alpha - androstan - 17 -oic-13,17-lactam-p-bis(2-chloroethyl)amino phenylacetate (ASE) is a homo-aza-steroidal ester of p-bis(2-chloroethyl) amino phenyl acetic acid and has been shown to display antineoplastic, mutagenic and genotoxic activity. In the present study an effort has been made to evaluate the ability of ASE to induce micronuclei (MN) in human lymphocytes treated in vitro using the cytokinesis-block assay. Lympocytes were treated with different concentrations of ASE (0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 20 microg/ml) at two different cell culture times, 21 and 41 h after culture initiation. ASE treatment lasted until cell harvest, for 51 and 31 h, respectively. Two types of cultures were used, whole blood and isolated lymphocyte cultures. The content of induced MN was identified by FISH analysis, using an alpha-satellite DNA probe, in binucleate cells. Our results suggest that ASE is capable of increasing MN frequencies in human lymphocytes under both culture conditions. This increase is related to the concentration in a linear dose-dependent manner and is also dependent on the duration of treatment. FISH analysis has shown that the induced MN resulted mainly from breakage events. Additionally, a weak aneugenic effect was found at the higher concentrations in whole blood cultures as well as in isolated lymphocyte cultures. Cytotoxic effects of ASE were observed under both cell culture conditions with a linear dose-dependent relationship according to CBPI evaluation and were more pronounced in isolated lymphocyte cultures.     

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.     

Influence of donor age on vinblastine-induced chromosome malsegregation in cultured peripheral lymphocytes.

The incidence of spontaneous aneuploidy in human somatic and germ cells is known to be positively associated with aging. However, the influence of age on the individual susceptibility to chemically induced chromosome malsegregation has not been elucidated. In this study the spindle poison vinblastine (VBL) was used as a model compound to assess the influence of donor age on chemically induced chromosome malsegregation in cultured lymphocytes. Blood cultures from 20 female donors belonging to two different age groups (10 <30 years and 10 >50 years) were treated with VBL (7.5 ng/ml) from 43 h after mitogen stimulation until harvest at 60 h, i.e. during the time interval corresponding to G2/M. In order to block cytokinesis, cytochalasin B (6 microg/ml) was added to cultures at 44 h. For each donor the incidence of micronuclei, polyploidy and malsegregation (non-disjunction and loss) of chromosomes X and 8 was determined using fluorescence in situ hybridization with chromosome-specific centromeric probes. Both background incidence of micronuclei and spontaneous chromosome X non-disjunction were significantly elevated in older donors. Individual responses to VBL treatment showed wide interindividual variability, which was not significantly associated with the age of the donor. In both age classes chromosome X was more susceptible than chromosome 8 to both spontaneous and VBL-induced malsegregation. These results indicate that donor age has a limited influence on the aneugenic effects exerted by VBL in peripheral lymphocytes in vitro. Other factors have to be considered to account for the large interindividual variation in sensitivity to VBL challenge observed in this work.     

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.     

The in vivo or ex vivo origin of micronuclei measured in human biomonitoring studies

The micronucleus test (MNT) is a well-established assay in genotoxicity testing and human biomonitoring. The cytokinesis-block micronucleus test (CBMNT) is the preferred method for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. However, it is unclear to what extent mutagen exposure either leads to the formation of MN already in vivo or to the formation of MN ex vivo during cell culture as a consequence of persisting DNA damage. MN that were already induced in vivo can be determined by scoring MN in mononuclear lymphocytes 24 h after the start of the lymphocyte culture (i.e. in lymphocytes that did not divide yet). Results obtained for cancer patients after chemotherapy suggest that mutagen exposure in vivo mainly leads to the formation of MN during ex vivo proliferation of lymphocytes as a consequence of mis-repair of persistent damage. If these results also apply to other kinds of mutagen exposure, increased MN frequencies in the CBMNT can only be expected for exposures leading to a sufficient amount of damage that persists during ex vivo lymphocyte culture. For a better understanding of the origin of increased MN frequencies and the correct interpretation of results obtained with the CBMNT, further research is recommended: MN in mononuclear lymphocytes should be additionally scored 24 h after the start of the cultures, comparative investigation with the CBMNT and the MNT with reticulocytes should be performed and the kinetics of MN formation in lymphocyte cultures and the repair capacity of lymphocytes for different kinds of DNA damage should be characterised.     

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.     

Direct and indirect non-disjunction in the origin of trisomy in cultured human lymphocytes

The aim of the present work was to investigate the processes involved in the origin of trisomic karyotypes, i.e. co-migration of sister chromatids (mitotic non-disjunction, MND) and recovery of micronuclei (MN) originating from lagging chromosomes/chromatids at anaphase (mitotic indirect non-disjunction, MIND), and to evaluate their relative contribution to aneuploidy in human lymphocytes mitotically activated in vitro. Therefore, phytohaemagglutinin-stimulated human lymphocytes from one donor were treated with 10 and 25 nM colchicine and analysed through two cell cycles by means of both molecular (FISH with centromeric DNA probes specific for chromosomes 7 and 11) and classical cytogenetic techniques. The following events were analysed: (i) chromosome/chromatid loss (a MN-generating event) in M(1) bipolar ana-telophases; (ii) MN recovery in M(2+) prophases; (iii) non-disjunction and loss of chromosomes 7 and 11 by FISH analysis in cytochalasin B-induced binucleate cells; (iv) spontaneous frequency of trisomic cells by chromosome counting and FISH analysis in M(1) c-metaphases; (v) induced frequency of trisomic cells by chromosome counting and FISH analysis in M(2) c-metaphases. Our results indicate that MND plays a major role compared with MIND in the origin of trisomic karyotypes, being approximately 4- to 5-fold higher in colchicine-treated cells. Moreover, remarkable reductions in the observed frequencies of trisomic cells were recorded in comparison with the expected ones, with an observed/expected frequency ratio of trisomic M(2) c-metaphases ranging between 1/3 and 1/6.     

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

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

Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies

Formaldehyde (FA) was tested for its genotoxicity in human blood cultures. We treated blood samples at the start of the culture to follow FA-induced DNA damage (DNA-protein crosslinks, DPX), its repair and its genetic consequences in form of sister chromatid exchanges (SCE) and micronuclei (MN). Our results clearly indicate that DPX (determined by the comet assay) are induced at FA concentrations of > or =25 microM. DPX induced by FA concentrations up to 100 microM are completely removed before lymphocytes start to replicate. SCE are induced at concentrations >100 microM parallel to the induction of cytotoxicity (measured as reduction of the replication index). MN were not induced by FA concentrations up to 250 microM (the highest analyzable concentration) added at the start of the blood cultures in the cytokinesis-block micronucleus (CBMN) test. FA-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented division of damaged cells. MN were only significantly induced in human blood when proliferating cells were exposed to FA during the last cell cycle before preparation. Several human biomonitoring studies reported increased frequencies of SCE and MN in lymphocytes of subjects exposed to FA. Our results characterize the genotoxic potential of FA in cultured lymphocytes and lead to the conclusion that cytogenetic effects of FA are very unlikely to occur in blood cultures of FA-exposed subjects.