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Carcinogenesis Advance Access originally published online on June 15, 2006
Carcinogenesis 2006 27(12):2511-2518; doi:10.1093/carcin/bgl102
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Aneuploidy-inducing capacity of two widely used pesticides

Marta Mattiuzzo, Mario Fiore, Ruggero Ricordy and Francesca Degrassi*

Institute of Molecular Biology and Pathology, CNR, Department of Genetics and Molecular Biology University ‘La Sapienza’, 00185 Rome, Italy

*To whom correspondence should be addressed at: Institute of Molecular Biology and Pathology, CNR, Department of Genetics and Molecular Biology, University ‘La Sapienza’, Via degli Apuli, 4 00185 Rome, Italy. Tel: +39064457527; Email: f.degrassi{at}caspur.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aneuploidy-inducing activity of alachlor and dichlorvos, two pesticides representing an important source of human exposure to potential carcinogens, has been evaluated in a cytokinesis block micronucleus assay combined with anti-kinetochore (CREST) staining to detect chromosome loss and in situ hybridization with chromosome-specific centromeric probes for the analysis of non-disjunction. Cytofluorimetric analysis to assess potential interference of the chemicals with cell cycle progression and TUNEL assay to detect apoptosis were also performed. The results obtained show that both environmental compounds induced significant and dose-related increases of total micronuclei (MN) and CREST-positive MN as compared with the concurrent solvent control. The chemicals were also capable of promoting chromosome non-disjunction. However, the two pesticides differed in their mode of action: alachlor induced both chromosomal aberrations and aneuploidy, while the genotoxic activity of dichlorvos was only related to aneuploidy induction. Cytofluorimetric analyses showed that dichlorvos caused a marked accumulation of cells in the G2/M phase of cell cycle and indicate a potential for this chemical to interfere with mitosis. Furthermore, dichlorvos induced CREST-positive MN at a concentration lower than the one producing apoptosis, suggesting that dichlorvos-induced aneuploid cells may persist in the growing cell population.

Abbreviations: CHO assay, Chinese hamster ovary cell assay; EPA, Environmental Protection Agency; FISH, fluorescent in situ hybridization; FITC, fluorescein isothiocyanate; MN, micronuclei


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Aneuploidy is the condition of a cell with whole chromosome gains or losses. The ubiquitous nature of aneuploidy in most malignant tumours and in many early stage carcinomas suggests that this condition is strongly associated with the tumorigenic process (1,2). However, the relative contribution of abnormal chromosome numbers in the development of cancer as compared with gene mutations or structural chromosome aberrations is still a matter of debate (3–5). Errors in chromosome segregation may lead to chromosome missegregation and chromosome instability, i.e. the occurrence of aneuploidy at an increased rate in a cell population (6,7). It is now becoming clear that defects in cellular structures controlling the mitotic process could have a crucial role in the origin of chromosome instability and in the aetiology and progression of cancer (8). Thus, a crucial issue in carcinogenic risk prevention is the identification of environmental compounds able to interfere with mitotic structures and control mechanisms which regulate chromosome segregation, since exposure to these agents may promote aneuploidy, chromosome instability and cancer (9). This is particularly true for environmental carcinogens that lack evidence of mutagenic activity or that produce inconclusive results in bacterial and mammalian mutagenicity assays.

In the present work we investigated the aneuploidy-inducing activity of two widely used pesticides which represent an important source of human exposure to potential harmful chemicals among agriculture workers. The compounds analysed in the work are the herbicide alachlor, classified as probable human carcinogen by Environmental Protection Agency (EPA) (10), and the insecticide dichlorvos, a possible carcinogen to humans (11). Alachlor [2-chloro-2',6'-diethyl-N-(methoxymethyl) acetanilide] is a pre- and post-emergence herbicide, mainly used for field application in the production of corn, soybeans and peanuts. According to EPA estimates, alachlor annual usage was 3150–4500 tonnes in 1999, which ranks this chemical among the 25 most used herbicides in the USA (12). The compound has been found to be carcinogenic in different tissues of rats (13). More recently, a possible association between alachlor application and lymphohematopoietic cancers among agriculture workers has been demonstrated (14). Mutagenicity studies were generally negative in bacterial systems and a negative finding was published in the HPRT/CHO assay (Chinese hamster ovary cell assay)(15). However, alachlor was positive in inducing mutation and recombination in Drosophila melanogaster assays (15). Induction of chromosomal aberrations and sister chromatid exchanges (SCE) was observed in cultured mammalian cells (15) but results for micronuclei (MN) induction were equivocal, suggesting either a clastogenic (16) or an aneugenic action (17) by an in vitro exposure of human lymphocytes to the chemical.

Dichlorvos (2, 2-dichlorovinyl dimethyl phosphate) is a common organophosphate insecticide with anti-cholinesterase activity. It was estimated that ~450 tonnes of dichlorvos was used in the USA in the year 1989 (11). It is applied on crops (mainly tobacco), to control parasites in livestock, and against flies and mosquitoes inside the house (11). Moreover, human exposure to dichlorvos is also expected with the medical use of trichlorfon, a drug employed in human medicine for Alzheimer’s disease treatment (18). The drug spontaneously metabolizes to dichlorvos (11). Based on experimental carcinogenicity data, IARC classified dichlorvos as a probable human carcinogen (11). More recently, this regulatory assessment has been questioned (19,20) and industrial epidemiology studies of workers manufacturing or handling dichlorvos have been strongly requested (21). In vitro mutagenicity has been observed in several non-mammalian model systems (reviewed in ref. 19) but its in vivo mutagenic activity has been confirmed only in the liver of {lambda}LacZ transgenic mice (22). Induction of chromosomal aberrations and MN in vivo have been reported in Syrian hamster and rat but other authors did not observe MN induction after dermal application in mouse (reviewed in ref. 19). Therefore, data concerning dichlorvos genotoxicity are still inconclusive.

Thus, both pesticides are potential human carcinogens in wide use among agriculture workers. However, their ability to induce gene mutations or chromosomal aberrations in genotoxicity assays is still unclear. Therefore, we have decided to investigate whether or not these two environmental compounds exert their carcinogenic action through aneuploidy by analysing their influence on chromosome segregation in human cells.

The aneuploidy-inducing capacity of the two pesticides has been assessed in a human lymphoblastoid cell line known to maintain high levels of mixed oxigenase activity (23). To assess the performance of the test system we employed the well-known aneuploidy-inducing taxol as the positive control (24,25). This chemotherapeutic drug interferes with chromosome segregation by stabilizing microtubules in mitotic cells thereby producing non-functional mitotic spindles (26). The cytokinesis block micronucleus assay is nowadays widely used to assess chromosome damage (27). The assay has been implemented by including the anti-kinetochore (CREST) staining to distinguish the MN induced by structural chromosome damage from the MN originated from lagging chromosomes at anaphase (28). Chromosome missegregation, i.e. non-disjunctional events within the main daughter nuclei of a binucleated cell, can also be identified using chromosome-specific centromeric probes in fluorescence in situ experiments (28). Both approaches have been applied to study the aneuploidy-inducing capacity of the pesticides and to investigate their action mechanisms on mitosis. To better evaluate the biological action of these substances on cultured human cells, induction of apoptosis and alterations in cell cycle progression following treatment with the two pesticides have been also investigated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture and chemicals
Human lymphoblastoid AHH-1 cells were maintained in RMPI 1640 medium containing GlutaMAX (Gibco-BRL, Paisley, UK), 10% foetal bovine serum (FBS; Cambrex Bio Science Verviers, Belgium), 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were grown at 37°C in 5% CO2. In these conditions the average generation time is 16 h. For each experiment, dichlorvos (Fluka Riedel-de Haën, Schweiz) and alachlor (Fluka) stock solutions were freshly prepared at a concentration of 10 µg/ml and 20 mg/ml in dimethylsulfoxide (DMSO), respectively. 10 µM (8.5 µg/ml) taxol (Paclitaxel, SIGMA-Aldrich, St Louis, MO) and 1.5 mg/ml cytochalasin B (SIGMA) stock solutions were prepared in DMSO and stored at –20°C.

Treatment schedule
For each experimental point, 300 000 cells/ml were seeded in 25 cm2 flasks. The next day cultures received DMSO alone (control) or different amounts of the test chemicals dissolved in DMSO. DMSO concentration in control cultures never exceed 0.5% (v/v). Cytochalasin B (3 µg/ml) was also added to each culture to inhibit cytokinesis and produce binucleated cells. Samples were harvested 18 h from the beginning of the treatment. Cell cycle analysis and TUNEL assay were carried out on cultures treated with the same schedule except for cytochalasin B addition.

Trypan blue assay
An aliquot of 90 µl of cell suspension was incubated with 10 µl of 0.4% Trypan blue solution (SIGMA) for 10 min. Cells were then dropped on cleaned microscopic slides and the number of cells incorporating the dye (Trypan blue-positive) was counted under phase contrast. Two hundred cells were scored for each concentration in three independent experiments.

MN assay in binucleated cells
Cells were washed in phosphate-buffered saline (PBS) without calcium and magnesium (PBS2–: 140 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4 and 1.5 mM KH2PO4) and cytocentrifuged on polylysine-coated coverslips. Coverslips were incubated 2 min at room temperature in 75 mM KCl and then fixed in 90% methanol. After blocking in 20% goat serum for 1 h, AHH-1 cells were incubated with anti-kinetochore antibody (Antibodies Incorporated, Davies, CA) for 2 h at room temperature, washed in PBS2– + 0.1% Tween-20 and again incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-human antibody (ICN Biomedicals, Costa Mesa, CA) for 45 min. DNA was counterstained with 4'-6-Diamidino-2-phenylindole (DAPI, SIGMA) and coverslips were sealed in an antifade solution (Vector Laboratories, Burlingame, CA). All preparations were examined under an Olympus Vanox microscope equipped with a x100 (1.35 NA) oil immersion objective. A minimum of 500 binucleated cells were scored on immunostained slides to evaluate the distribution of mono-, bi- and more than binucleated cells using DAPI fluorescence. On the same slides binucleated cells with well-preserved cytoplasm were scored to evaluate the frequencies of total MN and CREST-positive and CREST-negative MN. MN were located using DAPI fluorescence and the presence of a CREST signal was analysed under blue violet illumination. Necrotic or apoptotic cells were not included in the scoring of binucleated cells, according to criteria proposed by Fenech (27). For each experimental point 1000 binucleated cells were analysed, in three independent experiments. Statistical significance of differences between control and treated samples was calculated using the {chi}2-test. The data from the same groups from the three experiments were pooled together since a {chi}2-test for homogeneity showed no significant variations between control values from different experiments (29).

Fluorescent in situ hybridization (FISH) with centromeric chromosome-specific probes in binucleated cells
At the end of an 18 h treatment, cells were cytocentrifuged on coverslips, fixed in a 3:1 methanol:acetic acid mixture, air dried and stored at –20°C. Before in situ hybridization, coverslips were fixed again in 3:1 methanol:acetic acid for 1 h at room temperature, heated for 2 h at 65°C, treated 5 min at 37°C with 50 µg/ml pepsin (SIGMA) in 0.01 M HCl, fixed in 1% formaldehyde and then dehydrated in an ethanol series. FITC-labelled chromosome 7 alphoid probe and rhodamine-labelled chromosome 11 alphoid probe (QBIOgene, Irvine, CA) were pre-heated 10 min at 96°C. An aliquot of 10 µl of {alpha}-satellite mixed probes was applied on a coverslip and denaturation took place for 2 min at 71°C. Hybridization was performed overnight at 37°C and the final post-hybridization washings were carried out in 0.1x SSC at 60°C. The slides were then sealed in a DAPI-containing antifade solution (Vector). Slides were examined under an Olympus Vanox fluorescence microscope. DAPI fluorescence was used to locate binucleated cells and hybridization signals were analysed using the appropriate filters. A total of 1000 binucleated cells were observed for each experimental point. Statistical significance of differences between control and treated samples was calculated using the {chi}2-test.

Cell cycle analysis
DNA content and bromodeoxyuridine (BrdU) incorporation were determined by flow cytometry on a FACStar Plus apparatus (Becton&Dickinson, San Diego, CA). Briefly, 30 min before harvesting, 45 µM BrdU was added to the culture medium. Cells were then fixed in a 1:1 methanol: PBS2– mixture and DNA was denaturated in 3 N HCl for 45 min. After neutralizing in 0.1 M Na2B4O, cells were incubated with a mouse monoclonal anti-BrdU antibody (Becton&Dickinson) for 1 h at room temperature, washed in PBS2– + 0.5% Tween-20 and then incubated for 45 min with Alexa Fluor 488-conjugated anti mouse antibody (Molecular Probes, Eugene, OR). After extensive washing, cells were resuspended in PBS2– containing 20 µg/ml propidium iodide and analysed for their DNA content (red fluorescence) and BrdU incorporation (green fluorescence). Ten thousand events were collected for each sample. Biparametric dot plots of DNA and BrdU content were obtained using WinMDI software.

Evaluation of apoptosis by TUNEL assay
Test chemical treated cells were cytocentrifuged onto polylysine-coated coverslips and briefly rinsed in PBS2–. Cells were fixed 5 min at room temperature with cold methanol and washed in PBS2–. Specimens were then incubated for 1 h at 37°C in a humidified chamber with TUNEL (terminal transferase dUTP nick end labelling) reaction mixture (Roche Applied Science, Germany), washed three times in PBS2– and then counterstained with DAPI solution. Label solution was used as the negative control. Fluorescence images were acquired with a CCD camera (Diagnostic Instruments, Serling Heights, MI). Images were processed using AdobePhotoshop 7.0 program. For each experimental point at least 1000 cells were analysed for TUNEL reaction in three independent experiments. The data presented are the mean ± SE of 3000–4000 cells. Statistical significance of differences between control and treated samples was calculated using the {chi}2-test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Alachlor and dichlorvos exert both cytotoxic and cytostatic effects
To identify an adequate range of doses for the analysis of MN we first investigated cytotoxicity and the growth inhibitory effects of the pesticides and the reference chemical, as suggested by the international working group on MN assay (30). To this aim, the frequencies of Trypan blue-positive cells and the incidence of binucleated cells have been analysed after an exposure lasting approximately one cell cycle in the human lymphoblastoid AHH-1 cell line. As shown in Table I, taxol did not show evidence of induced cytotoxicity within the tested dose range (2.5–10 nM). With respect to dichlorvos, a weak but statistically significant increase in Trypan blue-positive cells was observed only at the two highest concentrations. On the contrary, exposure to alachlor (5–40 µg/ml) of AHH-1 cells decreased cell viability in a dose-dependent fashion, accounting for up to 60% of Trypan blue-positive cells at the highest concentration (Table I). A dose-related decrease in the frequencies of binucleated cells with a parallel increase of mononucleated cells was produced by all three chemicals suggesting that during the 18 h exposure time, fewer cells completed a cell cycle in the presence of the cytokinesis inhibitor cytochalasin B (Table I). The highest taxol concentration decreased the binucleated frequency to ~50% of the respective control value and an even stronger cytostatic effect was found for the two pesticides. The highest concentration of alachlor and dichlorvos decreased the binucleated frequencies to ~32 and 34% of their control value, respectively. These values exceeded the 50–60% decrease in binucleated cell frequency suggested as an upper limit for MN testing (30). The highest concentration was nevertheless included in our procedure to investigate the action mechanisms of the two pesticides in a wide range of doses.


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  		Table I Frequencies of Trypan blue-positive cells and percentage of mononucleated (1N), binucleated (2N) and polinucleated (>2N) cells in cytochalasin B-treated cultures

 
MN and CREST-positive MN are induced by both pesticides
Production of chromosomal and/or aneugenic damage by the pesticides was investigated using the MN assay with anti-kinetochore staining in binucleated cells. The aneugenic compound taxol was very effective in producing MN, which were essentially CREST-positive MN (Table II and Figure 1) in accordance with the mechanism of action of this anti-cancer drug. The increase in CREST-positive MN was statistically significant (P < 0.001) for all tested doses. Consistently, CREST-negative MN were not increased over the control value after taxol treatment (Figure 1). In alachlor-treated cells, frequencies of micronucleated binucleated cells (P < 0.05), MN (P < 0.01) and CREST-positive MN (P < 0.05) were also significantly increased from the lower tested concentration (5 ug/ml, Table II). As shown in Figure 1, both CREST-negative and CREST-positive MN were induced by alachlor, with CREST-positive MN accounting for approximately two-thirds of induced MN at all tested concentrations. These results suggest that more than one mechanism may be involved in the genotoxic activity of the pesticide, producing both clastogenic and aneugenic damage. Exposure of AHH-1 cells to dichlorvos produced statistically significant increases in micronucleated cells and total MN from the dose of 20 ng/ml (P < 0.001); CREST-positive MN were also significantly increased by the same concentration (P < 0.001). Similar to taxol, induction of CREST-positive MN was responsible for the observed increase in MN, and CREST-negative MN were not induced by dichlorvos. This strongly suggests interference of chromosome segregation by the compound.


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  		Table II Frequencies of micronucleated binucleated cells (MNBNC), MN, CREST-positive MN (CRESTpos MN) and CREST-negative MN (CRESTneg MN) in binucleated AHH-1 cells

 

Figure 1
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  		Fig. 1 Induction of micronuclei (black triangles), CREST-positive micronuclei (black circles) and CREST-negative micronuclei (black squares) following treatment with taxol, alachlor or dichlorvos of AHH-1 cells. The graphs shown are the mean ± SE of three independent experiments.

 
Non-disjunction frequencies are higher in alachlor- or dichlorvos-treated cells
Beside chromosome loss, another error in chromosome segregation is the migration of both copies of a chromosome in one nucleus of a binucleated cell, i.e. non-disjunction. This type of segregation error was analysed in test chemical treated cells by counting the numbers of hybridization signals for alphoid centromeric probes specific to chromosome 7 and 11 on each nucleus of a binucleated cell. The results of this analysis are reported in Table III. As expected, a significant induction of chromosomes 7 and 11 non-disjunction was found in taxol-treated samples (P < 0.05 and < 0.001 for 5 and 10 nM, respectively). Some increases in chromosome missegregation were observed also after alachlor exposure. Mitotic non-disjunction frequencies for both chromosomes increased up to a maximum at an exposure dose of 10 µg/ml and a reduction in non-disjunction frequencies was observed at higher doses, possibly indicating toxicity effects. The {chi}2-test showed a significant difference (P < 0.05) between treated and control samples only with a dose of 10 µg/ml. Frequencies of non-disjunction in dichlorvos-treated samples were constantly higher than the respective control, starting with exposures of at least 10 ng/ml dose. However, differences were not statistically significant at the {chi}2-test.


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  		Table III Frequencies of cells showing chromosome non-disjunction (NDBN cells) for chromosomes 7 and 11 per 1000 binucleated cells as detected by FISH analysis with centromeric probes

 
Distinct changes in cell cycle progression are observed after alachlor or dichlorvos exposure
To investigate cell cycle progression after pesticides treatment, cytofluorimetric analysis of DNA content and BrdU incorporation was carried out to evaluate accumulation in specific cell cycle phases and investigate DNA replication activity by immunostaining with an anti-BrdU antibody (Figure 2A). Cell cycle profiles of taxol-treated samples showed an increase in the G1 fraction up to 10 nM (Figure 2B, taxol). At the maximum tested dose (20 nM) a clear mitotic block was produced with an accumulation of the vast majority of the cells in the G2/M fraction at the expense of G1 and S phases (Figure 2B, taxol). This is in agreement with the microtubule stabilizing properties of this chemotherapeutic agent (26). Treatment with alachlor produced a small but dose-dependent increase in the G1 population with a concomitant decrease in the S phase fraction, suggesting impairment of the G1/S transition after treatment with this chemical (Figure 2B, alachlor). Exposure of AHH-1 cells to dichlorvos resulted in a dose-dependent decrease in the fraction of S phase cells. Consistently, at the two highest concentrations the cell cycle profile was profoundly altered with the vast majority of the cells in the G1 or G2/M phase of the cell cycle (Figure 2B, dichlorvos). This indicates that the insecticide affects cell cycle progression at different stages.


Figure 2
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  		Fig. 2 Cell cycle distribution of AHH-1 cells grown for 18 h in the presence of different concentrations of taxol, alachlor or dichlorvos. Figure 2A: Bivariate flow cytometric analysis of DNA content (x axis) and BrdU incorporation (y axis). G1 cells: 2C DNA content and no BrdU incorporation; S-phase cells: intermediate DNA content and BrdU incorporation; G2/M cells: 4C DNA content and no BrdU incorporation. Figure 2B: the graphs report the percentage of cells in the different stages of the cell cycle.

 
Apoptosis is not involved in the aneugenic effects of the pesticides
To characterize the contribution of apoptosis to the observed induction of MN, we examined apoptosis-induced DNA fragmentation by in situ immunofluorescence detection of free 3'-OH DNA strand breaks using the terminal transferase-based TUNEL assay in cells grown with or without the different chemicals for about one cell cycle. Figure 3A shows that the FITC-conjugated TUNEL reaction identified AHH-1 cells that could be morphologically recognized as apoptotic by their nuclear fragmentation (arrow) and also cells that did not yet display apoptotic bodies (arrowhead). The histogram in Figure 3B shows that the frequency of TUNEL-positive cells was statistically increased over the control value starting from 5 nM taxol, a concentration that induced a high level of CREST-positive MN and hence aneuploid cells. This suggests that induction of apoptosis may eliminate aneuploid cells from the growing population (31). However, low levels of aneuploidy do not seem to promote apoptosis, as in the case of the 2.5 nM taxol concentration. In dichlorvos-treated samples, a significant induction of apoptosis was observed at 40 ng/ml, whereas the increase in CREST-positive MN was statistically significant at the dose of 20 ng/ml. Induction of apoptosis in alachlor-treated cells was statistically significant only at 20 µg/ml, a concentration that produced >30% dead cells in the Trypan blue assay. Thus, induction of apoptosis must not be responsible for the high cytotoxicity observed by Trypan blue exclusion assay in alachlor-treated cells or for MN induction in dichlorvos-treated samples.


Figure 3
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  		Fig. 3 Induction of apoptosis in AHH-1 cells grown for 18h in the presence of different concentrations of taxol, alachlor or dichlorvos. Figure 3A: Fluorescence microphotographs of TUNEL-stained AHH-1 cells. DNA is visualized by DAPI fluorescence (DAPI) and apoptotic nuclei are recognizable by their intense FITC fluorescence after terminal deoxynucleotidyl transferase (TdT) reaction (TUNEL reaction). Arrow points to an apoptotic cell with nuclear fragmentation; arrowhead points to an apoptotic cell without apoptotic bodies. The graphs shown in Figure 3B are the mean ± SE of at least two different experiments. 40 µg/ml alachlor was not tested for apoptosis, since it produced massive necrosis (> 60% Trypan blue positive cells). *P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Aneuploidy induction has been reported in cultured mammalian cells or in entire animals after treatment with several established human carcinogens, including asbestos, cadmium, diethylstilbestrol or after exposure to other chemicals, mainly microtubule-interacting chemicals, for which evidence of carcinogenicity in humans is still inadequate (32). Furthermore, aneuploidy occurs in the very early stages of cellular transformation in hamster cells treated with genotoxic and non-genotoxic carcinogens (33,34). This suggests that chemically induced aneuploidy may be extremely relevant for environmental carcinogenesis.

The present study shows that induction of aneuploidy, as detected by CREST-positive MN and in situ hybridization with centromeric probes in binucleated human cells, is an important feature of the genotoxic action of alachlor and dichlorvos, two potential human carcinogens. However, the two pesticides differ in their mode of action, in that alachlor promotes the formation of both chromosomal aberrations and aneuploidy while the genotoxic activity of dichlorvos may be only related to aneuploidy.

In the case of alachlor a consistent fraction of MN lack the centromeric staining, indicating that besides numerical aberrations structural chromosomal aberrations are also produced following treatment with this herbicide. A significant induction of MN lacking anti-kinetochore staining or centromeric FISH was previously observed in isolated human lymphocytes (16), whereas a more recent paper suggested the presence of aneuploidy (17). The present results show that alachlor acts both as an aneuploidy-inducing and a clastogenic agent in our experimental conditions, which indicates that the lymphoblast cell line used in our study may maintain some metabolic function(s) producing chemical species promoting aneuploidy (23). It is noteworthy that a clear induction of CREST- positive MN is observed only at concentrations significantly affecting cell viability in alachlor-treated samples. This suggests that general cell toxicity is an important effect exerted by this chemical on cultured mammalian cells.

The biological action of dichlorvos appears to be mainly connected to its ability to induce numerical chromosome damage, as shown by the induction of CREST-positive MN. The insecticide induces CREST-positive MN at a concentration lower than the ones significantly inducing apoptosis or alterations in cell cycle progression, excluding the contribution of general cell toxicity or apoptosis to its capacity to promote aneuploidy. These results are in agreement with several reports that investigated the aneuploidy-inducing capacity of trichlorfon, which spontaneously metabolizes to dichlorvos at neutral pH (11). Induction of chromosome loss and non-disjunction has been observed in lymphoblastoid cells after trichlorfon exposure (35) and has been linked to its capacity to interfere with mitotic spindle assembly and block cells in mitosis (36). Furthermore, trichlorfon has been found to promote aneuploidy in male and female meiotic cells in rodents (36–38) by interfering with spindle formation (39). The hypothesis that dichlorvos interferes with mitosis is supported by the present cytofluorimetric data showing a marked accumulation of treated cells in the G2/M phase of the cell cycle, similar to what was observed after treatment with the spindle poison taxol. Preliminary data from our lab also show that dichlorvos blocks cells in mitosis, possibly by interfering with mitotic spindle assembly (data not shown). Studies of dichlorvos-induced neurotoxicity have suggested a potential mechanism for its effects on mitosis. Enhanced phosphorylation of neuronal microtubules and microtubule-associated protein 2 (MAP2) due to an increased activity of microtubule-associated kinases has been observed in rat brain after dichlorvos treatment, which suggests an action of this chemical on tubulin metabolism (40). Investigations dealing with the influence of dichlorvos on mitosis and microtubule assembly in mammalian cells will help to elucidate the mechanism(s) promoting aneuploidy after this pesticide.

In contrast to taxol, a clear accumulation of cells in the G1 phase is also observed after dichlorvos treatment in cytofluorimetric experiments. This indicates that G1/S checkpoint is activated in dichlorvos-treated cells by a mechanism that may be potentially different from its effects on mitosis. It can be hypothesized that the observed G1/S checkpoint may be related to its interference with the activity of kinases modulating the transition from the G1 to the S phase of the cell cycle, due to the potential ability of the insecticide to dissociate and release phosphate moieties.

In conclusion, induction of chromosome missegregration at mitosis represents an important property of the two pesticides under study. This feature may be particularly relevant in relation to cancer as we observed that low levels of aneuploidy following dichlorvos or taxol treatment do not trigger apoptosis. This suggests that induced aneuploid cells could persist in the growing cell population.


   Acknowledgments
 
The authors thank P. Mosesso for critical reading of the manuscript. This work was partially supported by the grant PMS/DML/19/UO6 from the Italian Ministry of Health to F. Degrassi.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 20, 2005; revised May 26, 2006; accepted May 27, 2006.


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