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.     

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.     

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.     

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.     

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.     

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.     

Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes.

The human lymphocyte micronucleus (MN) assay is relatively insensitive to genotoxic agents that predominantly induce excision-repairable lesions such as adducts and abasic sites. In this study we have explored the possibility of using cytosine arabinoside (ARA) to convert excision-repairable DNA lesions to micronuclei (MN) within one cell cycle. The system consisted of human lymphocytes as target cells, the cytokinesis-block (CB) method for identifying cells that had completed one nuclear division only, and X-rays, methylnitrosourea (MNU), and ultraviolet light (UV) as mutagens. With each mutagen we have observed significant increments in induced MN in the cultures that had also been treated with ARA during G1. The slope of the dose-response curves for induction of MN was increased by a factor of approximately 1.8 for X-rays and 10.3 for UV and significant MNU induction of MN was only achieved in the cultures treated with ARA. Furthermore, a 24-hr gap between mutagen exposure and the start of the assay did not abolish the increased sensitivity in the cultures treated with ARA. These observations suggested that the combined ARA and cytokinesis-block micronucleus (CBMN) method may enhance the detection of exposure to genotoxic agents that predominantly induce excision-repairable lesions.     

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.     

Detection of chromosome loss and gain induced by griseofulvin, estramustine, and vanadate in binucleated lymphocytes using FISH analysis.

Cytochalasin B-blocked binucleated human lymphocytes from a healthy male donor were used to detect micronucleus induction and other aneuploidy events (chromosome loss and gain) after treatment with griseofulvin (GF), estramustine (EM), and sodium orthovanadate (Na(3)VO(4)). A two-color FISH was performed by using centromeric probes for chromosome 2 (FITC labeled) and the X chromosome (TRITC labeled) to measure chromosome loss and gain events in binucleated cells. GF induced mainly aneuploid binucleates involving the X chromosome, but this was not associated with preferential loss of one of the two chromosomes. EM preferentially induced aneuploidy of chromosome 2, and Na(3)VO(4) of the X chromosome. Our results indicate that chromosome malsegregation events (chromosome loss and/or gain) are probably not randomly induced, suggesting that different mechanisms leading to aneuploidy may be either chromosome-dependent or compound- and dose- related.     

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.     

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.     

Enhancement of genetic instability in human B cells by Epstein-Barr virus latent infection

The level of genetic instability, as assessed by micronucleus (MN) formation, was higher in Epstein-Barr virus (EBV)-converted B-cell lines with one copy of the EBV genome integrated in each cell than in the parental, EBV-negative, B lymphoma cells. MN induced by EBV latency, as analysed by in situ hybridization, contained mainly centromeric regions, indicating that the presence of EBV affects the segregation of entire chromosomes. The instability was inhibited by treatment with antioxidants. Flow cytometric analysis indicated that there was a higher basal level of peroxides in EBV(+) cells. Direct oxidative stress caused by hydrogen peroxide (which is known to be both apoptogenic and mutagenic) enhanced the number of MN only in an EBV-converted clone. These cells were also resistant to apoptosis, as expected, suggesting that in the parental EBV cells apoptosis may efficiently eliminate cells with genetic damage. These results show for the first time a direct involvement of EBV in the induction of genetic instability, suggesting that it could contribute to tumour progression.     

Evaluation of the genotoxic effects of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay

The present work reports on the genotoxicity of the boron neutron capture (BNC) reaction in human metastatic melanoma cells (A2058) assessed by the cytokinesis block micronucleus assay (CBMN) using p-borono-L-phenylalanine (BPA) as the boron delivery agent. Different concentrations of BPA (0.48, 1.2 and 2.4 mM) and different fluences of thermal neutrons were studied. Substantial genotoxic potential of alpha and lithium particles generated inside or near the malignant cell by the BNC reaction was observed in a dose-response manner as measured by the frequency of micronucleated binucleated melanoma cells and by the number of micronuclei (MN) per binucleated cell. The distribution of the number of MN per micronucleated binucleated cell was also studied. The BNC reaction clearly modifies this distribution, increasing the frequency of micronucleated cells with 2 and, especially, > or =3 MN and conversely decreasing the frequency of micronucleated cells with 1 MN. A decrease in cell proliferation was also observed which correlated with MN formation. A discrete genotoxic and anti-proliferative contribution from both thermal neutron irradiation and BPA was observed and should be considered secondary. Additionally, V79 Chinese hamster cells (chromosomal aberrations assay) and human lymphocytes (CBMN assay) incubated with different concentrations of BPA alone did not show any evidence of genotoxicity. The presented results reinforce the usefulness of the CBMN assay as an alternative method for assessment of the deleterious effects induced by high LET radiation produced by the BNC reaction in human melanoma cells.     

The genotoxic effect of potassium metabisulfite using chromosome aberration, sister chromatid exchange, micronucleus tests in human lymphocytes and chromosome aberration test in bone marrow cells of rats

Potassium metabisulfite (PMB) is used as an antimicrobial substance in many kinds of foods. In the present study, the effects of PMB on chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) formation in human lymphocytes and as well as its effect on CAs in bone marrow cells of rats were investigated. The human lymphocytes were treated with 25, 50, 100, and 200 microg/ml of PMB for 24 and 48 hr. PMB was also intraperitoneally (ip) injected to the rats as a single dose of 150, 300, and 600 mg/kg body weight (b.w.) for 12 and 24 hr before sacrifice. PMB induced abnormalities such as structural and numerical (total) CAs, SCEs, and MN formations in a dose dependent manner in the lymphocytes of the 24- and 48-hr treatment periods. In addition, PMB showed a cytotoxic effect by decreasing the replication index (RI), mitotic index (MI) and nuclear division index (NDI) in a dose dependent manner in human lymphocytes. The compound induced CA as well and decreased the MI in bone marrow cells of rats. It might be concluded that PMB had a high genotoxic and cytotoxic risk.     

An evaluation of micronucleus induction in bone marrow and in hepatocytes isolated from collagenase perfused liver or from formalin-fixed liver using four-week-old rats treated with known clastogens

The bone marrow (BM) micronucleus (MN) test is a sensitive assay for identifying clastogens. However, some clastogenic compounds and metabolites may never reach the BM. The liver has been suggested as an alternative tissue to BM but adult rat liver has a low mitotic index that increases the difficulty of evaluating hepatocytes (HEP) for MN induction. Chemical mitogens and partial hepatectomy have been used to increase HEP proliferation to improve the sensitivity for detection of clastogenic compounds, but these practices raise concerns for the evaluation of drug candidates. The use of 4-wk-old rats provides an alternative to mitogenic stimulation because livers from these animals have ∼5.4% of their HEP in S-phase. HEP were isolated by collagenase perfusion, or from formalin-fixed tissue, from 4-wk-old treated rats. Six compounds were evaluated for the incidence of MN in HEP that were isolated by both methods. The results for MN induction by these compounds were similar for the two methods and confirmed that formalin-fixed tissue is an acceptable source of cells for evaluating MN induction in HEP. BM polychromatic erythrocytes (PCE) also were harvested at the end of the live phase for each study and then evaluated for the incidence of MN. Diethylnitrosamine and 2-nitrofluorene induced MN in HEP but had no effect in PCE. 2-Acetylaminofluorene, cyclophosphamide and 7, 12-dimethylbenz[a]anthracene did not induce MN in HEP but were positive in PCE. The direct-acting clastogen, mitomycin C, was positive in both HEP and PCE. These results indicate that this modified liver micronucleus test, using 4-wk-old rats, offers an alternative to existing methods that use mitogens or partial hepatectomy to stimulate cell replication. Analysis of MN from formalin-fixed tissue provides additional flexibility by allowing the investigator to assess MN induction at a later time. Environ. Mol. Mutagen. 29:379–385, 1997 © 1997 Wiley-Liss, Inc.     

Benzene metabolite, 1,2,4-benzenetriol,induces micronuclei and oxidative DNA damage in human lymphocytes and HL60 cells

The triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), is readily oxidized to its corresponding quinone via a semiquinone radical. During this process, active oxygen species are formed that may damage DNA and other cellular macromolecules. The ability of BT to induce micronuclei (MN) and oxidative DNA damage has been investigated in both human lymphocytes and HL60 cells. An antikinetochore antibody based micronucleus assay was used to distinguish MN containing kinetochores and potentially entire chromosomes (kinetochore-positive, K⁺) from those containing acentric chromosome fragments (kinetochore-negative, K^?). BT increased the frequency of MN formation twofold in lymphocytes and eightfold in HL60 cells with the MN being 62% and 82% K⁺, respectively. A linear dose-related increase in total MN, mainly in K⁺-MN, was observed in both HL60 cells and lymphocytes. Addition of copper ions (Cu²⁺) potentiated the effect of BT on MN induction threefold in HL60 cells and altered the pattern of MN formation from predominantly K⁺ to K^?. BT also increased the level of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), a marker of active oxygen-induced DNA damage. Cu²⁺ again enhanced this effect. Thus, BT has the potential to cause both numerical and structural chromosomal changes in human cells. Further, it may cause point mutations indirectly by generating oxygen radicals. BT may therefore play an important role in benzene-induced leukemia. © 1993 Wiley-Liss, Inc.     

Aneugen-induced micronuclei (MN) in human lymphocytes may be discerned using image analysis techniques when cell-cycle stage is taken into account

We show that for the in vitro cytochalasin-B human lymphocyte micronucleus (MN) test, the quantification of the DNA content of MN and the difference in DNA content between the two macronuclei in the binucleate cells without MN, as measured by image analysis, gives a first estimation of the aneugenic potential of a test compound. Cultures of isolated human lymphocytes were exposed either to γ-rays as a clastogen or to carbendazim (MBC) as an an-eugen. The lymphocytes were stained with Feulgen stain and the MN were analyzed for DNA content with a Magiscan 2A image analyzer. The mean DNA content of MN induced by MBC were statistically higher than γ-irradiation-induced MN. It was demonstrated that in culture the lymphocytes, as well as the MN, are in different stages of the cell cycle, but this will not affect the discriminating power of the MN DNA content when only G1 cells are considered, or when DNA content of the MN is expressed relative to the total genome. The identification of Gl and G2 cell populations from image analysis data was performed by extrapolation of DNA content data from Gl- and G2-sorted lymphocytes with a FacStar plus flow sorter. It was demonstrated that in MBC-treated cells the DNA rearrangement between the macronuclei in binucle-ates without MN was on the average higher than in γ-irradiated and untreated cells, which points to aneugenic effects of MBC without the formation of MN. In contrast to DNA content measurements, the area of the MN is not a reliable measure for discriminating clastogens from aneugens. © 1995 Wiley-Liss, Inc.     

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 γ-irradiation and by Vinblastine sulfate. In a cytokinesis-block micronucleus 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 α 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. © 1996 Wiley-Liss, Inc.     

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

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

Effect of sulforaphane on micronucleus induction in cultured human lymphocytes by four different mutagens

Isothiocyanates (ITCs) are commonly found in cruciferous vegetables. A variety of biological activities have been ascribed to ITCs, such as inhibition of cytochrome P450 enzymes and induction of phase II enzymes in animal models. ITCs are also able to block cell-cycle progression and induce apoptosis in human cancer cells in vitro. In this study, we evaluated the ability of the ITC sulforaphane to protect cultured human lymphocytes from micronucleus (MN) induction by four different mutagens: ethyl methanesulfonate (EMS), vincristrine (VIN), H(2)O(2) and mitomycin C (MMC). To understand the mechanisms of action of sulforaphane, the cultures were treated with the compound before, during and after treatment with the mutagens; in addition, the cultures were evaluated for the induction of apoptosis. Up to 10 microM, sulforaphane was non-genotoxic by itself, while 30 microM sulforaphane reduced the replicative index of the cells by more than 60%. Moreover, 1-10 microM sulforaphane reduced the MN frequency induced by EMS, VIN, H(2)O(2) and MMC in at least one of the treatment protocols; it had no effect on H(2)O(2)-MN induction in the post-treatment protocol, and it increased MN induction by MMC in the pre-treatment protocol. Apoptosis was produced in the cultures treated with sulforaphane alone. The fraction of apoptotic cells was increased after co- or post-treatment with sulforaphane and EMS and MMC, suggesting that sulforaphane-mediated apoptosis may remove highly damaged cells induced by these agents. Other mechanisms are involved in the anti-genotoxic activity of sulforaphane against VIN and H(2)O(2). Taken together, our findings indicate that under certain conditions sulforaphane possesses anti-genotoxic activity in vitro and that further studies are warranted to characterize this property in vivo.