Mutagenesis Advance Access originally published online on March 15, 2007
Mutagenesis 2007 22(4):255-261; doi:10.1093/mutage/gem010
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Genotoxicity induced by arsenic compounds in peripheral human lymphocytes analysed by cytokinesis-block micronucleus assay
1Department of Human and Environmental Sciences, University of Pisa, Via S. Giuseppe 22, 56126 Pisa, Italy 2Department of Neurosciences, University of Pisa, Via Roma 67, 56126 Pisa, Italy 3European Commission, Institute for Health and Consumer Protection, ECVAM Unit, JRC-Ispra, TP 580, Via E. Fermi 1, 21020 Ispra, VA, Italy
This work focuses on the analysis of genotoxic effects on human peripheral lymphocytes exposed in vitro to different arsenic (As) compounds by means of the cytokinesis-block micronucleus assay. The study was carried out by challenging peripheral human lymphocytes with six As compounds in trivalent or pentavalent forms such as arsenite (AsIII) and arsenate (AsV) and organoarsenic species such as monomethylarsonous acid (MMAsIII), monomethylarsonic acid (MMAsV), dimethylarsinic acid (DMAsV) and trymethylarsine oxide (TMAOV). For AsIII and AsV at concentrations of 4 and 32 µM, respectively, an increase of micronuclei (MN) frequency was found. MMAsIII and MMAsV induced a statistically significant increase of MN frequency at the dose of 2 and 500 µM, respectively. For DMAsV, no significant increase of MN was observed, although a decrease of the nuclear division index (NDI) was evident, indicating a cytotoxic effect. The genotoxic mechanism of action of MMAsIII was further evaluated by means of fluorescence in situ hybridization analysis. Due to a higher percentage of centromere-positive MN, MMAsIII showed a clear aneuploidogenic property. Finally, for TMAOV no genotoxicity was observed up to 1 mM. These results show how speciation is important in determining the genotoxic and cytotoxic effects of As compounds in human peripheral lymphocytes and support the emerging hypothesis that the induction of aneuploidy could be a mechanism by which As exerts its carcinogenic properties.
 |
Introduction |
|---|
 Top Introduction Materials and methodsResultsDiscussionReferences |
|---|
Significant exposure to arsenic (As) occurs through both anthropogenic and natural sources. Occupational exposure to arsenic is common in the smelting industry as by-products of ores containing lead, zinc and nickel and is increasing in the microelectronics industry. Populations are exposed also through the commercial use of arsenic compounds in products such as pesticides, herbicides and paints, and through the burning of fossil fuels. The human exposure to As-contaminated water can induce the onset and progression of pathologies like anaemia, neuropathies, hyperpigmentation, skin irritation and lung, dermal and bladder cancer. For this last reason, As is now considered by International Agency for Research on Cancer to be a Class I carcinogen (1
). As a consequence of these toxic effects on human health, many countries have adopted standard rules to limit the amount of arsenic in drinking water, leading the United States National Research Council to recommend to the Environmental Protection Agency the value of 10 µg/litre of As. Moreover, industrial pollution can contribute to the increase of As in the environment, so that inorganic arsenic is still nowadays considered of primary importance by both toxicologists and ecotoxicologists (2,3).
Following ingestion, inorganic arsenic, both in the trivalent and in the pentavalent form, is metabolized by successive oxidation, methylation and reduction steps leading to organoarsenic metabolites. Cullen and Remer (4) described the biomethylation of arsenic as follows:
 |
In mammalian cells, arsenic methylation was suggested to be an enzymatic process involving the action of specific arsenic methyltransferases to add a methyl group to form monomethylated (MMAs) and then dimethylated (DMAs) species either in pentavalent (MMAsV, DMAsV) or in trivalent (MMAsIII, DMAsIII) chemical form (5). However, recent findings by Hayakawa et al. (6) suggest that these methylation processes are not direct mechanisms (e.g. AsIII 
MMAsIII), but rather they are activated via the formation of glutathione (GSH) complexes (arsenic triglutathione or monomethylarsonic diglutathione) by Cyt19 arsenic methyltransferase. A fraction of As in the body can be eliminated in urine, whereas the rest can be trapped mainly in tissues rich in sulfhydryl groups like derma, hairs and nails (7). The primary metabolic compound found in urine, and usually used as a biomarker of exposure, is DMAsV. Latest data reveal that also another metabolite, trimethylarsine oxide (TMAOV), can be found, in minimal quantity, in urine (5) but it is not yet known if the presence of this compound is due to a methylation mechanism or is triggered by the accumulation through the diet (e.g. some types of fish and crustaceans) of arsenic in an organic form (8).
Arsenic genotoxicity has been analysed extensively in a wide range of in vivo (9–12) and in vitro (5,13–15) studies and the overall conclusion is that there is a clear induction of genotoxic effects, including an increase in micronucleus (MN) frequency and a decrease in the proliferation index that reflects its toxic potential. Nesnow et al. (16) demonstrated the induction of damage on the 
X174 phage DNA by arsenate, MMAsV, MMAsIII, DMAsV and DMAsIII. However, it was subsequently observed that only MMAsIII and DMAsIII induced a double nick in the phage DNA (17). The results obtained by Schwerdtle et al. (17), by analysing DNA damage and evaluating the frequencies of DNA strand breaks recognized by the Fpg enzyme in PM2 isolated DNA and in HeLa S3 cells, showed that MMAsIII and DMAsIII were clearly more powerful in the induction of DNA breaks also in comparison with inorganic arsenic salts. Kligerman et al. (18) were able to demonstrate that in human lymphocytes, the speciation of As compounds induces increases in cytotoxic and genotoxic effects going from inorganic to organic substances and, moreover, from the pentavalent to the trivalent form. In fact, considering several biomarkers like chromosome aberrations (CA), sister chromatid exchanges (SCE) and DNA fragmentation, the following scheme in terms of increased toxicity was presented: AsV, MMAsV, DMAsV, AsIII, MMAsIII and DMAsIII (18). Authors also tested the mutagenicity of arsenicals in mouse lymphoma cells, in Salmonella and by prophage induction in Escherichia coli, concluding that the trivalent methylated arsenicals were powerful clastogens and DNA-damaging agents, but not point mutagens or SCE inducers; moreover, they observed that several of the arsenicals were also spindle poisons, suggesting a role for these forms in inducing aneuploidy (18). Recent findings on the molecular mechanisms associated to toxic effects of As have revealed that, also if in different way, it is able to interfere with oxidative stress, diminishing DNA repair capacity, altering DNA methylation patterns, cell proliferation and apoptosis (5). Concerning arsenic genotoxicity, two different mechanisms have been proposed: the interference with the DNA repair processes and the induction of oxidative stress. According to the first mechanism, trivalent As would produce DNA damage by the inhibition of the repair enzymes (19). It seems in fact that As can inhibit the excision phase of DNA repair processes at a concentration of 
2.5 µM (19). Moreover, arsenate inhibits the DNA ligase through the inactivation of the essential thiolic groups (19). The second mechanism, that could justify the As genotoxicity, seems to be due to its capacity to activate the reactive oxygen species in the redox reactions (20). More recently, Kligerman et al. (21) investigated the spindle inhibitory properties of six arsenicals on human lymphoblasts. In their study, these authors observed that the trivalent methylated arsenicals are the more potent aneuploidy-inducing forms of arsenic, whereas none of the tested pentavalent arsenicals gave significant results. Moreover, MMAIII was the most potent of the arsenicals tested (21). In conclusion, the authors showed that trivalent methylated arsenicals not only possess DNA-damaging and clastogenic capabilities (17,18) but are also able to induce aneuploidy (21).
The aim of this study was to evaluate the genotoxic effects of inorganic and organic arsenic compounds in human peripheral lymphocytes by the MN test in order to better characterize the genotoxicity related to the chemical characteristics of the substances. Moreover, to better discriminate between the clastogenic and aneuploidogenic properties of MMAsIII, we evaluated the presence of positive (C+ MN) or negative (C– MN) MN by applying the fluorescence in situ hybridization (FISH) technique with an All Human Centromere Probe.
|
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chemicals
The following compounds were tested: sodium arsenite (AsIII) (CAS No. 7784-46-5), sodium arsenate (AsV) (CAS No. 7631-89-2), monomethylarsonous acid (MMAsIII) (CAS No. 25400-23-1), monomethylarsonic acid (MMAsV) (CAS No. 124-58-3), dimethylarsinic acid (DMAsV) (CAS No. 75-60-5) and trimethylarsine oxide (TMAOV) (CAS No. 4964-14-1). All the compounds were freshly dissolved in MilliQ water at 25°C at the stock concentrations of 10–2 or 10–3 M. Aliquots of the concentrated solutions were added to culture medium (not exceeding 1% v/v) in order to obtain the final concentrations to be tested in the experiments. Then, the resulting solutions were equilibrated for 2 h at 25°C before incubation with cells. Under these conditions, compounds were stable and no transformation or change of the oxidation state occurred as determined by high performance liquid chromatography (HPLC) inductively coupled plasma mass spectrometry (ICP-MS). Mitomycin C was used as a positive clastogenic control at the concentration of 170 ng/ml in sterile distilled water. Griseofulvin was used as an aneuploidogenic control at the concentration of 43 µM. Before the treatment, the solutions were sterile filtered using a 0.2-µm mini-filter (Minisart, Sartorius, Italy).
Donors and blood collection
To assess the genotoxic effects of As compounds, the study was performed on human peripheral lymphocytes of three healthy male volunteers. All subjects were informed of the scope of the study and gave their written consent that was submitted and approved by the local Ethics Committee. Approximately 4–6 ml of blood was drawn by venipuncture in Li-heparin vials (Emoplast, Vacutest, Lot No. 1734) according to standard procedure. The donors were chosen according to the following criteria: young age (their mean age was 31.66 ± 2.51 years), non-smokers, without pharmacological treatments for at least 3 weeks before donation and without any radiological examination performed within the previous 3 months. Donors were excluded from the study if any chronic illness, genetic or oxidative stress-related pathologies, such as obesity, diabetes mellitus or cancer, were reported in the pedigree (22).
Cytokinesis-block micronucleus assay
The cytokinesis-block micronucleus (CBMN) assay was performed according to the procedure described by Migliore et al. (23). Briefly, blood was drawn from the volunteers and whole blood was used to prepare two paired independent lymphocyte cultures for each subject. Standard medium was prepared with RPMI 1640 (Gibco BRL, Italy) supplemented with 10% foetal bovine serum (Gibco BRL), 1.5% phytohaemagglutinin (Gibco BRL) and 1% penicillin–streptomycin (Gibco BRL). After 24 h, the cells were exposed to concentrations of As compounds ranging from 0.5 to 4 µM for AsIII, 4 to 32 µM for AsV, 0.01 to 2 µM for MMAsIII, 50 to 1000 µM for MMAsV, 50 to 250 µM for DMAsV and 100 to 1000 µM for TMAOV. Cytochalasin B (6 µg/ml) was added at 44 h to block the cytokinesis process, and lymphocyte cultures were harvested after 72 h (24). Cells were then treated with an hypotonic solution (0.075 M KCl) to lyse erythrocytes for 3 min, prefixed in 3:5 methanol:acetic acid, washed once with methanol and subsequently fixed twice with a 7:1 methanol:acetic acid fixative solution. Finally, the cell solution was dropped onto cold glass slides. The staining procedure was performed by immersing the air-dried slides in a 2% Giemsa solution. Two thousand binucleated cells for each experimental point were examined blindly, following the scoring criteria adopted by the HUman MicroNucleus Project (25). We evaluated the binucleated micronucleated lymphocytes (BNMN) frequency as the number of binucleated lymphocytes containing one or more MN per 1000 binucleated cells. Moreover, 500 lymphocytes were scored to evaluate the percentage of binucleated cells and the nuclear division index (NDI) was calculated according to the following formula:
 |
Fluorescence in situ hybridization
Slides for FISH analysis were prepared as described in the previous paragraph until the fixation procedure. In fact, slides for FISH analysis were prepared by using a 3:1 ratio of methanol:acetic acid fixative solution. After the fixing procedure, the slides were allowed to dry for at least 1 h. Slides for probing were pre-treated by an incubation period of 30 min at 37°C in 2x standard saline citrate (SSC), pH 7.0 (Sigma Aldrich, Italy), with 0.5% of Igepal (Sigma Aldrich) followed by a 70, 80 and 95% ethanol dehydration steps for 2 min. Probe and slides denaturation was performed as follows: All Human Centromere probe (Cat. No. PAHC0001-R, Resnova, Italy) was denaturated for 5 min at 95°C and placed on ice until use. Slides were instead denatured for 2 min in 70% formamide, 2x SSC. Slides were then dehydrated as already described above. The hybridization procedure was obtained by placing the slides with 15 µl of probe, covered with a coverslip, overnight at 37°C in a humidified chamber (50% formamide, 2x SSC). The post-hybridization washing steps were performed as follows: 5 min at 65°C in 1x washing buffer (0.5x SSC, 0.1% sodium dodecyl sulphate) and 5 min in 1x washing buffer (4x SSC, 0.1% Tween 20) at room temperature. Finally, the slides were dehydrated in 70, 80 and 95% ethanol for 2 min. To visualize the fluorescence, the slides were counterstained by applying 25 µl of 4',6-diamidino-2-phenylindole/antifade (0.5 µg/ml). For the statistical analysis, 50 MN for positive or negative centromeres were scored for each treatment considered.
Statistical analyses
Statistical analyses, where reported, were performed by the general linear model (GLM) and the differences were considered statistically significant at P <0.05 when the evaluation of the treatment was analysed. For each donor, the increase in the BNMN frequency was evaluated and tested for statistical significance using Fisher's exact test.
|
Results |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
For all the As compounds tested (AsIII, AsV, MMAsIII, MMAsV, DMAsV and TMAOV), the dose range used was derived from preliminary experiments in which it was possible to observe the concentration range where the substances were exerting their toxic effects (data not shown). When compounds with cytotoxic activity have been used, the highest analysed concentration chosen was that with a reduction of cell viability (evaluated by means of the NDI) of
50%.
Results obtained with the MN test for all the donors exposed to inorganic trivalent As demonstrated a statistically significant increase in the frequency of BNMN (
) as analysed by GLM (Table 1). When the effect of the treatment was considered for each donor, the statistical significance, performed by the Fisher's exact test, was homogenous in the group, starting from the concentration of 2 µM (P < 0.01). In order to better characterize the effects of AsIII, the NDI was also evaluated (Table 1).
|
After exposure to inorganic pentavalent As, the analysis of BNMN
shows, starting from 16 µM, concentration-dependent increases (Table 2) in MN in lymphocytes. The mean of the BNMN donor considered indicates that statistical significance (P < 0.05) was reached at the two highest doses tested (16 and 32 µM). The treatment with this compound for each donor shows a statistically significant increase at 16 and 32 µM (P < 0.01 and P < 0.001) for two donors and at 8, 16 and 32 µM (P < 0.01 and P < 0.001) for the third.
|
The analysis of BNMN frequency after exposure of lymphocytes to MMAsIII at concentrations ranging from 0.01 to 2 µM is presented in Table 3. This As compound induces a significant increase in the percentage of BNMN at the highest concentrations tested. Moreover, for a better understanding of the genotoxic properties of MMAsIII we evaluated the presence of positive (C+ MN) or negative (C– MN) MN by applying the FISH technique with an All Human Centromere Probe. The experiment performed at the highest concentration tested (2 µM) leads to a statistical increase of BNMN, showing that the effect is clearly aneuploidogenic since we found, by analysing 50 BNMN,
80% of C+ MN (Figure 1). In the FISH experiment, AsIII, as an inorganic control, was also analysed. The results demonstrate that the genotoxic effect of this compound can be considered both clastogenic and aneuploidogenic (50% of C+ and C–) (Figure 1).
|
|
Concerning the exposure of lymphocytes to MMAsV (Table 4) (concentrations tested from 50 µM to 1 mM), the genotoxic effect was higher if compared with those of the previous tested compounds. The concentration-dependent increase of the BNMN
was observed starting from 100 µM (Table 4). Although some values are missing for donors 2 and 3 (Table 4) and the first statistically significant values are highly variable, 100 µM for donor 1 and 3 and 500 µM for donor 2, the final appearance of the data indicates clear genotoxic effects.
|
Tables 5 and 6 report the corresponding data related to the exposure of lymphocytes to DMAsV and TMAOV. Although an increased toxicity is demonstrated for DMAsV (Table 5) by the clear decrease in the NDI (Table 5) for all the donors analysed, no statistical significance in the BNMN frequency was observed, apart from donor 2 (P < 0.05) at 250 µM DMAsV. TMAOV, tested in a range of concentrations from 100 to 1000 µM (Table 6), did not show any mutagenic activity.
|
|
|
Discussion |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study, the genotoxic effects induced by two inorganic (trivalent and pentavalent) chemical compounds of As as well as by four organoarsenic species (MMAsV, MMAsIII, DMAsV and TMAOV) in human peripheral lymphocytes have been analysed. The genotoxic effects of inorganic arsenic compounds have a wide range of documented literature data (26
,18). Like our data, Kligerman et al. (18) were able to show that methylated trivalent species, as well as some of the other species, are able to induce genotoxic effects in human lymphocytes analysed by means of CA, SCE, aberrant cells, replicative index and mitotic index (18). Considering the genotoxic capacity of As compounds, our data, compared to those of Kligerman et al. (18), focus the attention on a specific toxic biomarker like MN by analysing the modulation of the frequency of binucleated micronucleated human peripheral lymphocytes. This latter biomarker is of fundamental importance to better understand the molecular mechanisms involved in the toxic effects since it is able to show also the real interference with the mitotic apparatus. Numerous studies, in fact, have demonstrated that these As compounds can immediately interact with DNA causing CA, SCE and MN in many cell systems. Sodium arsenite, starting from a concentration of 0.5 µM, has been shown to cause a significant induction of MN in human lymphocytes in vitro (27). Moreover, a significant induction of CA, SCE and MN in chinese hamster overy (CHO) cells from 10 µM was observed (28). Despite this wide range of data, genotoxicity studies on human peripheral lymphocytes are rare. In order to verify the genotoxic effects of different arsenic compounds, we have used the CBMN assay (24) on human peripheral lymphocytes. This simple, sensible and reproducible methodology represents a valid screening system to investigate the genotoxic capacity of chemicals. Arsenic compounds have been analysed in lymphocytes from a population of three male non-smokers and healthy donors, with a mean age of 32 years old. In fact, many studies have demonstrated that the MN frequency is an endpoint highly variable due to factors such as gender, females show a higher MN frequency than the one observed in males (29,30), and age, in both sexes the frequency has been demonstrated to be considerably higher in individuals >40 years of age (29,31). We used different donors to take into account the possible interindividual variability in the treatment with different compounds, since genotypic variations regarding the arsenic metabolism could interfere with the susceptibility to the toxic, genotoxic and possibly carcinogenic effects of the elements. In fact, the presence of variations in the intraindividual elimination of MMAs with urine has been interpreted as first evidence of the existence of genetic polymorphisms in metabolic capacity (32,33), and several observations suggest that human metabolism of arsenic compounds varies among different individuals based on several factors, including genes involved in the biotransformation of arsenic (34,35). Genotypic variation may not only affect the metabolism of arsenic but also modify the individual susceptibility to arsenic-induced diseases, including cancer (36).
From the present study, it emerged that a significant increase in MN frequency was induced above the concentration of 2 µM. Sodium arsenate is known to induce a significant increase in CA and MN in mouse lymphoid cells (37). Our results are in complete agreement with literature data on these inorganic arsenic salts since, in CHO cells, at least a one order of magnitude increase in the genotoxic effects of arsenite, compared to arsenate, has been reported (38–40). Concerning the NDI (Tables 1 and 2) together with an increase on the BNMN, we observed a concentration-dependent decrease of the NDI, a clear indication of a cytotoxic effect induced by the treatment with both compounds.
Concerning the data on the organic compounds, like MMAs and DMAs, screened in this study, recent results on the cytotoxic capacity of these substances (15) indicate that the hypothesis of the methylation process as a detoxification pathway should be reconsidered. In this scenario, we have focussed our attention on the genotoxic properties of MMAsIII, MMAsV and DMAsV. The trivalent methylated arsenicals are species that have the capability to interact with cytosolic proteins and that, under those circumstances, can produce some of the toxic effects. Moreover, these compounds have been considered potent cytotoxins in primary culture of rat hepatocytes and in this cell model, they have been shown to be significantly more cytotoxic compared with inorganic compounds, like arsenite and arsenate (41,42). By using a different endpoint, like the primary DNA damage, evaluated by the comet assay on human isolated lymphocytes, Mass et al. (43) demonstrated that the MMAsIII and DMAsIII are more potent in inducing DNA strand breaks in respect to arsenite. Moreover, Kligerman et al. (18) evaluating CA on human peripheral lymphocytes showed that MMAsIII and DMAsIII are the most potent As clastogenic forms. In the present study, we have obtained the following scale of genotoxicity, evaluated by the BNMN frequency: MMAsIII > MMAsV > DMAsV (Tables 3–5). Moreover, in the case of MMAsIII, this shows the highest toxic effect in our study, and FISH analysis reveals that the induction of MN is due to an aneuploidogenic mechanism, since 80% of the MN analysed contained centromeres. This observation is in agreement with the results of a recent study by Kligerman et al. (21) aimed at evaluating the spindle inhibitory properties of six arsenicals (NaAsIII, NaAsV, MMAsIII, MMAsV, DMAsIII and DMAsV), and demonstrating that arsenic metabolites can interfere with cell division via tubulin disruption, with MMAsIII being the most potent one tested. In summary, Kligerman et al. (21) have suggested that exposure to arsenic can lead to aneuploidy, with the trivalent methylated arsenicals being the more potent aneuploidy-inducing forms of arsenic. Here, we have provided direct evidence that MMAsIII exerts its genotoxic effect acting mainly as an aneuploidogenic compound. We did not evaluate DMAsIII in the present study; however, its genotoxic mechanism of action requires further clarification; in fact, Ochi et al. (44,45) showed that dimethylarsinic acid causes mitotic delay, multipolar spindles and multipolar divisions, centrosome abnormality and CA in the Chinese hamster V79 mitotic cells. Both DMAsIII and MMAsIII were shown to interfere with tubulin polymerization in human lymphoblasts (21). However, whereas MMAsIII caused a significant concentration-related increase of the mitotic index, DMAsIII effects on the mitotic index were quite variable and inconsistent, suggesting the need for further studies.
An increased cytotoxicity was observed for the three compounds MMAsIII, MMAsV and DMAsV (Tables 3–5); however, the apparent higher cytotoxic effect of DMAsV compared with the other compounds could be due to the fact that we used much more higher concentrations than those used for the other compounds (100-fold higher than those of MMAsIII). Ochi et al. (45,46) observed that at equitoxic levels, the cytotoxic effect of DMAsIII was about three orders of magnitude more potent than DMAsV on Chinese hamster V79 cells. They suggested that some of the cytotoxic effects of DMAsV could be attributed to the action of DMAsIII, generated from DMAsV in an intracellular reducing environment. Their hypothesis was supported by evidence that the depletion of cellular GSH did not influence the cytotoxic effects of DMAsIII, whereas it enhanced the cytotoxicity of DMAsV (45,46), suggesting that at least in their experimental system the interaction between GSH and reactive materials generated from DMAsV was critical for the cytotoxicity of DMAsV. Interestingly, DMAsV was the most potent mitotic inhibitor of the three pentavalent arsenicals examined by Kligerman et al. (21) on human lymphoblasts; however, it had no significant effect on tubulin polymerization. Based on this evidence, and considering that we used a concentration of DMAsV almost 100-fold higher than that of MMAsIII, we can speculate that part of the cytotoxic effect of DMAsV observed in the present study could be due to the intracellular formation of a small amount of DMAsIII, which has been observed to be much more powerful than DMAsV (45). In our study, no toxic effect for TMAOV was observed suggesting, since this compound was found together with the TMAOIII, as a metabolite in urine, that it could be a detoxification product.
Arsenic has been associated with a wide range of cancers, including cancer of lung, kidney, liver, skin and urinary bladder. Several mechanisms have been proposed to explain the carcinogenic properties of As, including arsenic-induced oxidative stress, perturbation of important intracellular pathways involved in cell proliferation, differentiation and apoptosis, DNA damage, followed by cell cycle arrest, a direct or indirect inhibition of DNA repair enzymes by arsenic, the perturbation of DNA methylation and induced genomic instability (36).
Li and Broome (47) observed that arsenic was able to induce apoptosis in myeloid leukaemia cells by inhibiting tubulin polymerization, leading to a mitotic arrest, and suggested the possibility to design arsenic-based antimitotic agents to be used in cancer therapy. Recent data (21) provide evidence that some species of arsenicals, mainly DMAsIII and MMAsIII, interfere with tubulin polymerization. The present results provide further evidence that speciation plays an important role in determining the cytotoxic and genotoxic effects of As compounds. Moreover, we provide evidence that MMAsIII shows an aneuploidogenic mechanism of genotoxicity, bringing into highlight the need for further studies to better understand the molecular mechanisms of action of arsenic compounds for a better comprehension of arsenic-induced carcinogenicity and for the development of arsenic-based cancer therapies.
|
Acknowledgments |
|---|
We thank Dr Cristina Balia, Dr Federico Tarchi and Dr Elena Pardini for their valuable contribution to some experimental phases of the work. This work has been supported by the 15469-1999-11F1ED ISP ES project.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +39050836223; Fax: +39050551290; Email: l.migliore{at}geog.unipi.it
|
References |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
1. International Agency for Research on Cancer (IARC). Some drinking-water disinfectants and contaminants, including arsenic. In: IARC Monographs on the Evaluation of the Carcinogenic Risk to Humans, Vol. 8 (2004) Lyon, France: IARC.
2. Sabbioni E, Fischbach M, Pozzi G, Pietra R, Gallorini M, Piette JL. Cellular retention, toxicity and carcinogenic potential of seafood arsenic. I. Lack of cytotoxicity and transforming activity of arsenobetaine in the BALB/3T3 cell line. Carcinogenesis (1991) 12:1287–1291.
3. Sakurai T, Kaise T, Ochi T, Saitoh T, Matsubara C. Study of in vitro cytotoxicity of a water soluble organic arsenic compound, arsenosugar, in seaweed. Toxicology (1997) 122:205–212.[CrossRef][ISI][Medline]
4. Cullen WR, Remer KJ. Arsenic speciation in the environment. Chem. Rev. (1989) 89:713–764.[CrossRef][ISI]
5. Kitchin TK. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. (2001) 172:249–261.[CrossRef][ISI][Medline]
6. Hayakawa T, Kobayashi Y, Cui X, Hirano S. A new metabolic pathway of arsenite: arsenic-glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Arch. Toxicol. (2005) 79:183–191.[CrossRef][ISI][Medline]
7. Mandal BK, Ogra Y, Anzai K, Suzuki TK. Speciation of arsenic in biological samples. Toxicol. Appl. Pharmacol. (2004) 198:307–318.[CrossRef][ISI][Medline]
8. Yoshida T, Yamamuchi H, Fan Sun G. Chronic health effects in people exposed to arsenic via the drinking water: dose-response relationships in review. Toxicol. Appl. Pharmacol. (2004) 198:243–252.[CrossRef][ISI][Medline]
9. Basu P, Ghosh JK, Das A, Banerjee K, Ray AK, Giri W. Micronuclei as biomarkers of carcinogen exposure in populations exposed to arsenic through drinking water in West Bengal, India: a comparative study in three cell types. Cancer Epidemiol. Biomarkers Prev. (2004) 13:820–827.
10. Wegner R, Radon K, Heinrich-Ramm R, Seemann B, Riess A, Koops F, Poschadel B, Szadkowski D. Biomonitoring results and cytogenetic markers among harbour workers with potential exposure to river silt aerosols. Occup. Environ. Med. (2004) 61:247–253.
11. Chen CJ, Hsu LI, Wang CH, Shih WL, Hsu YH, Tseng MP, Lin YC, Chou WL, Chen CY, Lee CY, Wang LH, Cheng YC, Chen CL, Chen SY, Wang YH, Hsueh YM, Chiou HY, Wu MM. Biomarkers of exposure, effect, and susceptibility of arsenic-induced health hazards in Taiwan. Toxicol Appl Pharmacol. (2005) 206:198–206.[CrossRef][ISI][Medline]
12. Martinez V, Creus A, Venegas W, Arroyo A, Beck JP, Gebel TW, Surralles J, Marcos R. Micronuclei assessment in buccal cells of people environmentally exposed to arsenic in northern Chile. Toxicol. Lett. (2005) 155:319–327.[CrossRef][ISI][Medline]
13. Hughes MF. Arsenic toxicity and potential mechanisms of action. Toxicol. Lett. (2002) 133:1–16.[CrossRef][ISI][Medline]
14. Ramirez T, Garcia-Montalvo V, Wise C, Cea-Olivares R, Poirier LA, Herrera LA. S-adenosyl-L-methionine is able to reverse micronucleus formation induced by sodium arsenite and other cytoskeleton disrupting agents in cultured human cells. Mutat. Res. (2003) 528:61–74.[ISI][Medline]
15. Dopp E, Hartmann LM, Florea AM, von Recklinghausen U, Pieper R, Shokouhi B, Rettenmeier AW, Hirner AV, Obe G. Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells. Toxicol. Appl. Pharmacol. (2004) 201:156–165.[CrossRef][ISI][Medline]
16. Nesnow S, Roop BC, Lambert G, Kadiiska M, Mason RP, Cullen WP, Mass MJ. DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species. Chem. Res. Toxicol. (2002) 15:1627–1634.[CrossRef][ISI][Medline]
17. Schwerdtle T, Walter L, Mackiw I, Hartwig A. Induction of oxidative DNA damage by arsenite and its trivalent and pentavalent methylated metabolites in cultured human cells and isolated DNA. Carcinogenesis (2003) 24:967–974.
18. Kligerman AD, Doerr CL, Tennant AH, et al. Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: induction of chromosomal mutations but not gene mutations. Environ. Mol. Mutagen. (2003) 42:192–205.[CrossRef][ISI][Medline]
19. Hartwig A, Blessing H, Schwerdtle T, Walter I. Modulation of DNA repair processes by arsenic and selenium compounds. Toxicology (2003) 193:161–169.[CrossRef][ISI][Medline]
20. Hei TK, Filipic M. Role of oxidative damage in the genotoxicity of arsenic. Free Radic. Biol. Med. (2004) 37:574–581.[CrossRef][ISI][Medline]
21. Kligerman AD, Doerr CL, Tennant AH. Oxidation and methylation status determine the effects of arsenic on the mitotic apparatus. Mol. Cell. Biochem. (2005) 279:113–121.[CrossRef][ISI][Medline]
22. Drobna Z, Waters B, Walton FS, LeCluyse EL, Thomas DJ, Styblo M. Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes. Toxicol. Appl. Pharmacol. (2004) 201:166–177.[CrossRef][ISI][Medline]
23. Migliore L, Frenzilli G, Nesti C, Fortaner S, Sabbioni E. Cytogenetic and oxidative damage induced in human lymphocytes by platinum, rhodium and palladium compounds. Mutagenesis (2002) 17:411–417.
24. Fenech M. The in vitro micronucleus technique. Mutat. Res. (2000) 455:81–95.[ISI][Medline]
25. Bonassi S, Fenech M, Lando C, et al. Human micronucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei. Environ. Mol. Mutagen. (2001) 37:31–45.[CrossRef][ISI][Medline]
26. Gebel TW. Genotoxicity of arsenical compounds. Int. J. Hyg. Environ. Health (2001) 203:249–262.[CrossRef][ISI][Medline]
27. Schaumloffel N, Gebel T. Heterogeneity of the DNA damage provoked by antimony and arsenic. Mutagenesis (1998) 13:281–286.
28. Gurr JR, Liu F, Lynn S, Jan KJ. Calcium-dependent nitric oxide production is involved in arsenite-induced micronuclei. Mutat. Res. (1998) 416:137–148.[ISI][Medline]
29. Bonassi S, Bolognesi C, Abbondandolo A, et al. Influence of sex on cytogenetic end points: evidence from a large human sample and review of the literature. Cancer Epidemiol. Biomarkers Prev. (1995) 4:671–679.[Abstract]
30. Nordic Study Group. A Nordic data base on somatic chromosome damage in humans. Nordic Study Group on the Health Risk of Chromosome Damage. Mutat. Res. (1990) 241:325–337.[CrossRef][ISI][Medline]
31. Ishikawa H, Tian Y, Yamauchi T. Influence of gender, age and lifestyle factors on micronuclei frequency in healthy Japanese populations. J. Occup. Health (2003) 45:179–181.[CrossRef][ISI][Medline]
32. Vahter M. Genetic polymorphism in the biotransformation of inorganic arsenic and its role in toxicity. Toxicol. Lett. (2000) 112–113.
33. Wang TS, Chung CH, Wang AS, Bau DT, Samikkannu T, Jan KY, Chang YM, Lee TC. Endobuclease III, formamidopyrimidine-DAN glycosilase, and proteinase K additively enhance arsenic-induced DNA strand breaks in human cells. Chem. Res. Toxicol. (2000) 15:1254–1258.[CrossRef]
34. Chiou HY, Hsueh YU, Hsieh LL, et al. Arsenic methylation capacity, body retention, and null genotypes of glutathione S-transferase M1 and M2 among current arsenic-exposed residents of Taiwan. Mutat. Res. (1997) 386:197–207.[CrossRef][ISI][Medline]
35. Marnell LL, Garcia-Vargas GG, Chowdhury UK, et al. Polymorphisms in the human monomethylarsonic acid (MMA V) reductase/hGSTO1 gene and changes in urinary arsenic profiles. Chem. Res. Toxicol. (2003) 16:1507–1513.[CrossRef][ISI][Medline]
36. Tapio S, Grosche B. Arsenic in the aetiology of cancer. Mutat. Res. (2006) 612:215–246.[CrossRef][ISI][Medline]
37. Moore MM, Harrington-Brock K, Doerr CL. Relative genotoxic potency of arsenic and its methylated metabolites. Mutat. Res. (1997) 386:279–290.[CrossRef][ISI][Medline]
38. Li JH, Rossman TG. Inhibition of DNA ligase activity by arsenite: a possible mechanism of its comutagenesis. Mol. Toxicol. (1989) 2:1–9.[Medline]
39. Tabakova S, Hunter ES III, Gladen BC. Developmental toxicity of inorganic arsenic in whole embryo culture: oxidation state, dose, time, and gestational age dependence. Toxicol. Appl. Pharmacol. (1996) 138:298–307.[CrossRef][ISI][Medline]
40. World Health Organization (WHO). Environmental Health Criteria 224: Arsenic and Arsenic Compounds. (2001) Geneva: International Programme on Chemical Safety (IPCS), WHO.
41. Styblo M, Del Razo ML, LeCluyse EL, Hamilton GA, Wang C, Cullen WR, Thomas DJ. Metabolism of arsenic in primary cultures of human and rat hepatocytes. Chem. Res. Toxicol. (1999) 12:560–565.[CrossRef][ISI][Medline]
42. Styblo M, Del Razo LM, Vega L, Germolec DR, LeCluyse EL, Hamilton GA, Wang C, Cullen WR, Thomas DJ. Comparative to toxicity of trivalent and pentavalent inorganic and methylated arsenicals in human cells. Arch. Toxicol. (2000) 74:289–299.[CrossRef][ISI][Medline]
43. Mass MJ, Tennant BC, Roop WR, Cullen M, Styblo DJ, Thomas AD, Kligerman AD. Methylated trivalent arsenic species are genotoxic. Chem. Res. Toxicol. (2001) 14:355–361.[CrossRef][ISI][Medline]
44. Ochi T, Nakajima F, Nasui M. Distribution of gamma-tubulin in multipolar spindles and multinucleated cells induced by dimethylarsinic acid, a methylated derivative of inorganic arsenics, in Chinese hamster V79 cells. Toxicology (1999) 136:79–88.[CrossRef][ISI][Medline]
45. Ochi T, Suzuki T, Isono H, Schlagenhaufen C, Goessler W, Tsutsui T. Induction of structural and numerical changes of chromosome, centrosome abnormality, multipolar spindles and multipolar division in cultured Chinese hamster V79 cells by exposure to a trivalent dimethylarsenic compound. Mutat. Res. (2003) 530:59–71.[ISI][Medline]
46. Ochi T, Meguro S, Namikoshi M, Oya-Ohta Y, Kaise T. Dimethylarsinic acid causes inhibition of microtubule assembly and inhibition of calcium-sensitive disassembly of microtubules via interaction with glutathione. Appl. Organomet. Chem. (2002) 16:432–436.[CrossRef]
47. Li YM, Broome JD. Arsenic targets tubulins to induce apoptosis in myeloid leukemia cells. Cancer Res. (1999) 59:776–780.
Received on July 4, 2006; revised on January 12, 2007; accepted on January 31, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Vahidnia, G.B. van der Voet, and F.A. de Wolff Arsenic neurotoxicity A review Human and Experimental Toxicology, October 1, 2007; 26(10): 823 - 832. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||