Mutagenesis vol. 19 no. 2 pp. 129-135,
March 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
In vitro DNA damage by arsenic compounds in a human lymphoblastoid cell line (TK6) assessed by the alkaline Comet assay
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain and 1European Commission, Institute for Health and Consumer Protection, ECVAM Unit, Joint Research Centre, 21020 Ispra, Italy
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Abstract |
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Arsenic is classified as a carcinogen for humans, but as a possible genotoxic agent. Thus, taking into account the controversial data about how arsenic compounds are able to induce genetic damage, we investigated the possible genotoxic activity of different arsenic compounds in the TK6 human lymphoblastoid cell line using the alkaline Comet assay. Eight different inorganic and organic arsenical compounds have been selected as follows: three inorganic (sodium arsenite, sodium arsenate and sodium hexafluorarsenate) and five organic (monomethylarsonic and dimethylarsinic acids, arsenobetaine, tetramethylarsonium iodide and tetraphenylarsonium chloride). According to their toxicity and genotoxicity, the highest concentration tested was 10 mM, and the duration of the treatments was 30 min or 3 h. The results indicate that some compounds belonging to both the organic and inorganic species were able to induce significant increases in the tail moment, the parameter used to determine genotoxicity. Thus, the inorganic compounds sodium arsenite and sodium arsenate (but not sodium hexafluoroarsenate) were genotoxic, while among the organoarsenic species tested only tetramethylarsonium iodide and tetraphenylarsonium chloride compounds (but not monomethylarsonic, dimethylarsinic acids and arsenobetaine) induced significant increases in the tail moment. Nevertheless, genotoxic induction was generally only observed at the highest doses tested.
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Introduction |
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Arsenic is an important toxic agent from both natural and anthropogenic sources. It is widely found in water, soil and air. Arsenic occurs naturally in the drinking water of millions of people at higher concentrations than those considered safe, which is an important health concern in certain areas. Thus, based on strong epidemiological evidence, arsenic has been considered by the International Agency for Research on Cancer (IARC, 1987
) as a human carcinogen and chronic exposure has been associated with different types of cancer in skin, lung, bladder and liver (Abernathy et al., 1999). Nevertheless, it is still unclear how arsenic causes cancer and what type of arsenic compound(s) is (are) most likely to be associated with carcinogenicity.
This metalloid exists in different oxidative states and in several inorganic and organic forms. The most known inorganic arsenic forms are arsenate and arsenite, which mammals are able to methylate to monomethylarsonic and dimethylarsinic acids, although with different efficiencies between species. In addition to the inorganic forms present in water, seafood also contains high levels of arsenic, thus, its consumption can suppose an additional intake of arsenic in the organic form, mainly arsenobetaine, arsenocholine and arsenosugars (Cullen and Reimer, 1989).
Several studies have been carried out to determine the genotoxic potential of arsenic compounds (Basu et al., 2001). In vitro and in vivo studies have shown the genotoxicity of inorganic arsenic. It increases the frequency of micronuclei, chromosome aberrations and sister chromatid exchanges in both animals and humans, but it does not induce point mutations (Gradecka et al., 2001). In this context, no general agreement has been reached about its mechanism of action. Thus, in bacteria arsenic shows rare mutagenic effects, while in in vitro and in vivo studies on DNA damage induction it seems that arsenic acts indirectly by inhibiting DNA repair. On the other hand, all the results obtained in cytogenetic studies show that arsenic is a clear clastogenic agent.
To gain further insight into the genotoxic effects of arsenic, here we report the results obtained in a wide study on the genotoxicity of eight different arsenic compounds, comprising inorganic and organic forms, using the Comet test as assay system. Since most of the studies on the genotoxicity of arsenic compounds have been carried out with sodium arsenite and with the two most frequent methylated forms, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), we consider that the selected compounds, containing a wide range of both organic and inorganic arsenic forms, give us a complete view under the same experimental conditions of the genotoxic properties of such compounds. It must be emphasized that some of these compounds have never been evaluated for genotoxicity before.
The single cell gel electrophoresis (SCGE) or Comet assay has been considered a rapid, simple and sensitive technique for measuring DNA damage (Fairbairn et al., 1995). Thus, in in vitro studies the Comet assay has been shown to be a very sensitive method for detecting the genetic damage induced by different genotoxic agents such as radiation (Tice et al., 1990), herbicides (Ribas et al., 1995) and heavy metals (Hartmann and Speit, 1994), as well as for examining DNA repair under a variety of experimental conditions (Fairbairn et al., 1995). With this assay, effects such as DNA single-strand breaks, incomplete excision repair sites and alkali-labile sites can be easily analyzed from the DNA that migrates out of the cell nucleus.
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Materials and methods |
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Cell culture
The TK6 human lymphoblastoid cell line was used. This is a standard cell line with a stable karyotype often used in genotoxicity studies. The TK6 cells were maintained in suspension culture in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and 2.5 U/ml amphotericin B. The cultures were incubated at 37°C in a 5% CO2 incubator at 95% humidity. Asynchronous cultures in the exponential phase of growth were used in all experiments.
Chemicals
Sodium metaarsenite (NaAsO2), sodium hydrogenarsenate heptahydrate (Na2HAsO4.7H2O) and arsenobetaine (C5H11AsO2) were purchased from Fluka, Sigma-Aldrich (Milano, Italy); sodium hexafluoroarsenate (NaAsF6) was from Alfa Aesar (Cologno Monzese, Milano, Italy); MMA (CH3H2AsO), DMA [(CH3)2HAsO2] and tetramethylarsonium iodide [(CH3)4AsI] were purchased from Trichemical Laboratory (Osaka, Japan); tetraphenylarsonium chloride hydrate [(C6H5)4AsCl.H2O] was from Aldrich (Milano, Italy). RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin, amphotericin B (AMPH), sodium pyruvate and low melting point (LMA) and normal melting point (NMA) agarose were purchased from Gibco BRL, Life Technologies (Paisley, UK); phosphate-buffered saline (PBS), 30% (w/w) hydrogen peroxide, EDTA and the stains used (ethidium bromide (EtBr), 0.4% trypan blue solution and fluorescein diacetate (FDA)) were purchased from Sigma Chemical Co. (St Louis, MO); sodium chloride and dimethylsulfoxide (DMSO) were from Panreac Química SA (Barcelona, Spain); N-laurosylsarcosine sodium and Triton X-100 were from Fluka Chemical AG (Buchs, Switzerland); sodium hydroxide, and absolute ethanol were from Carlo Erba Reagenti (Milano, Italy); Tris buffer was purchased from US Biochemical (Cleveland, OH).
Purity of As compounds
All arsenic compounds tested were analyzed for different arsenic species impurities by high pressure liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICPMS). The organic purity of the organoarsenic species was determined by protonic nuclear magnetic resonance (1H NMR) at the Bioindustry Park, Ivrea, Italy. The three inorganic arsenic compounds were analyzed for elemental impurities by ICPMS or radiochemical separation neutron activation analysis (Farina, 2003). The results of the analysis (not shown) indicate that the arsenic compounds tested had a suitable degree of purity to avoid possible artifacts due to the presence of inorganic/organic impurities.
Treatments
Cell cultures were centrifuged and the pellet was resuspended in RPMI 1640 medium (106 cells in 1 ml). Each arsenic compound was dissolved in bidistilled water except for tetraphenylarsonium chloride, which was dissolved in DMSO. All concentrations were prepared immediately prior to incubation. Aliquots of 10 µl of each metal salt solution, at the different concentrations tested, were added to the cultures for 30 min/3 h at 37°C. An aliquot of 10 µl H2O2 (150 or 2000 µM) was included as a positive control and 10 µl of metal vehicle (DMSO or bidistilled H2O) was used as a negative control. After incubation, cells were washed with RPMI 1640 medium and resuspended in PBS for the Comet assay. Cell viability was evaluated as soon as possible, with a mix of FDA and EtBr (Strauss, 1991). Two hundred cells were scored for each treatment. More than 70% of the cells were viable at the highest dose under all culture conditions, which agrees with the usual testing conditions used in the Comet assay (Henderson et al., 1998).
Comet assay
The Comet assay was performed as previously described (Singh et al., 1988) with minor modifications. The cell samples (
40 000 cells in 20 µl) were carefully resuspended in 75 µl of 0.5% LMA, layered onto microscope slides precoated with 150 µl of 0.5% NMA (dried for 10 min at 65°C) and spread with a coverslip. After solidification (for 10 min at 4°C) and removal of the coverslip, 75 µl of 0.5% LMA was again added to the slides, covered and kept for 15–20 min at 4°C. Then the coverslips were removed and the slides were immersed in cold fresh lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100 and 1% laurosylsarcosinate, pH 10) for 2 h at 4°C in a dark chamber. To avoid the occurrence of additional DNA damage, the following steps were performed under dim light. Afterwards, the slides were placed for 40 min in a horizontal gel electrophoresis tank filled with cold electrophoretic buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13.5) to allow DNA unwinding. Electrophoresis was performed in the same buffer for 20 min at 0.73 V/cm and 300 mA. Unwinding and electrophoresis were done in an ice bath. After electrophoresis, the slides were neutralized twice for 5 min with 0.4 M Tris (pH 7.5) and fixed with 3 ml of absolute ethanol for 3 min. The slides were stained with 50 µl of EtBr (0.4 µg/ml) just before analysis. Finally, the images were examined at 400x magnification with a Komet 3.1 Image Analysis System (Kinetic Imaging Ltd, Liverpool, UK) fitted with an Olympus BX50 fluorescence microscope equipped with a 480–550 nm wide band excitation filter and a 590 nm barrier filter. One hundred randomly selected cells (50 cells from each of the two replicate slides) were analyzed per sample. The Olive tail moment, the percentage of DNA in the tail and tail length were used as measures of DNA damage and computed using Komet version 3.1 software.
Statistical analysis
The statistical significance of the results was determined by a general linear model. Homogeneity of variances between dose levels was determined using Levene’s test. Independent tests using Dunnett’s correction for multiple test adjustment were performed to compare each level of dose to the negative control when the overall F-test was significant. A result was considered statistically significant at P < 0.05. All analyses were performed using SAS proc MIXED v8.0 (SAS Institute, Cary, NC).
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Results |
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First of all, the purity of the tested compounds was determined. Elemental analysis of the arsenic salts used showed that the 38 elements (impurities) determined were in concentrations ranging from 13 to <0.5 ng/g. Analysis of seven chemical species of arsenic in individual arsenic compounds showed contamination levels <1%, the highest being inorganic trivalent arsenic in sodium arsenate and arsenobetaine (0.8%). NMR analysis indicated the presence of organic impurities in the organoarsenic compounds tested in the range 0.2–0.7%.
Figure 1 shows the results obtained after treatment of the TK6 cell line with five concentrations of sodium arsenite (from 0.001 to 10 mM), in treatments lasting 30 min. A clear positive dose-dependent relationship was observed when the genetic damage was expressed as the Olive tail moment. This was observed in two independent experiments, 0.1 mM being the lowest concentration that induced significant levels of DNA damage. Positive controls, run in parallel with hydrogen peroxide (0.15 mM), induced significant tail moment values. Since after the 30 min treatments sodium arsenite acted as a clear genotoxic agent, a longer treatment (3 h) was not carried out.
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Sodium arsenate was unable to induce DNA damage after a short (30 min) treatment with concentrations up to 10 mM. Thus, treatments lasting for 3 h were also conducted. In this case the highest concentration assayed (10 mM) was clearly genotoxic in the two independent experiments conducted, nevertheless, at a concentration of 1 mM only one of the experiments showed a significant increase in the tail moment (Figure 2).
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The other inorganic pentavalent salt tested (sodium hexafluoroarsenate) was not genotoxic in either of the two studies carried out (Figure 3).
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Figure 4 shows the genotoxic effect of MMA after short (30 min) and long treatments (3 h). As can be seen, neither of the experiments conducted under the two treatment conditions led to a significant increase in the tail moment of the treated TK6 cells. Similar negative results were also obtained with the dimethylated form of arsenic (Figure 5). Even though DMA treatments lasting for 3 h produced a significant increase in one replicate at the highest concentration tested (10 mM), no increase were observed when two new replicates were carried out. Thus, it appears that neither of the two normal metabolites of arsenic are able to induce DNA damage, as measured by the Comet test.
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Arsenobetaine, another organic form of arsenic evaluated in this study, only showed a significant response at the lowest dose tested (0.001 mM) in the treatment lasting for 30 min. Nevertheless, neither of the other experimental conditions of treatment revealed DNA damage induction (Figure 6). As a consequence, we consider the positive result to be marginal. Thus, arsenobetaine does not seem to act as a genotoxic agent under the experimental conditions reported.
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Tetramethylarsonium iodide treatments lasting for 30 min were unable to induce any increase in the levels of DNA damage. Nevertheless, when the treatment period was longer (3 h), clear and significant increases in the tail moment were observed in the two independent experiments, although only at the highest concentration (10 mM) assayed and without a direct dose–response tendency (Figure 7).
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All the compounds referred to above give >70% cell viability at the highest dose evaluated (10 mM) and for all the experimental conditions. However, the last compound tested, tetraphenylarsonium chloride, did show a higher toxicity and, as a consequence, the highest concentration tested in the experiment lasting for 30 min was 3 mM. This compound gave clear and significant increases in the values assessing the induction of DNA damage (Figure 8). Thus, in one experiment all the concentrations show significant increases in tail moment, although in the other experiment only the two highest concentrations were able to induce significant increases. Therefore, tetraphenylarsonium chloride was not tested with treatments lasting for 3 h and it was considered to be genotoxic.
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All the results obtained in the present study with regards to the toxicity and the genotoxicity of the eight arsenic compounds evaluated in the Comet test using TK6 cells are summarized in Table I.
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Discussion |
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Although it is generally accepted that arsenic is a genotoxic carcinogen in humans, it remains unclear how arsenic causes genetic damage and what type of arsenic compounds are most likely associated with such genotoxic activity. Our results obtained using the Comet test indicate that, in both inorganic and organic forms of arsenic, some compounds are able to induce DNA damage. Nevertheless, taking into account the levels of induced damage, the positive compounds seem to be weak genotoxic agents.
Concerning the significance of the results obtained it should be noted that, from the analysis carried out to detect the presence of possible impurities, our results indicate that the amounts of impurities in the culture medium are too low to induce any detectable toxic or genotoxic effects under our experimental conditions. Thus, the arsenic compounds used were considered sufficiently pure to exclude possible artefacts due to these inorganic/organic impurities.
Whilst inorganic arsenic compounds have been epidemiologically proven to be carcinogenic to humans, their carcinogenicity in animal experiments has been problematical (Wang et al., 2002), although recently its transplacental carcinogenicity has been shown in pregnant female mice exposed to sodium arsenite via their drinking water (Waalkes et al., 2003). These compounds are not usually genotoxic in bacteria, do not induce point mutations in mammalian cells (Basu et al., 2001), and are unable to induce mitotic recombination in Drosophila (Rizki et al., 2002); nevertheless, they are generally effective in inducing cytogenetic damage. In our study, sodium arsenite was clearly genotoxic in the Comet assay, which would confirm the results previously obtained by other authors using the same assay system (Hartmann and Speit, 1996; Ho et al., 2000; Mourón et al., 2001; Sordo et al., 2001). It has not been proven that inorganic arsenic reacts directly with DNA, but it has been suggested that arsenite induces oxidative damage causing single- and double-strand breaks (Hei et al., 1998), although oxidative stress is only one of the possible mechanisms involved in arsenite genotoxicity. Thus, arsenite treatment can modify cellular functions by changing the phosphorylation profiles of cellular proteins (Huang et al., 1995) and inhibiting DNA repair enzymes (Lynn et al., 1997; Yager and Wiencke, 1997), two mechanisms which could be related. Thus, it has recently been shown that arsenite treatment induces phosphorylation of the Mre11 protein (involved in ataxia telangiectasia-like disorder) which, although it does not affect formation of the Rad50–NBS1–Mre11 repair complex, it affects formation of Rad50–NBS1–Mre11 nuclear foci upon DNA damage (Yuan et al., 2002).
In our study the inorganic pentavalent form of arsenic was also able to induce DNA breakage as measured in the Comet test, although to a lesser extent than the trivalent form and only when the treatment lasted for 3 h. This is generally observed when evaluating the few works carried out with arsenate assaying for different genetic end-points. Thus, arsenite always acts as a more potent genotoxic agent than arsenate (Wan et al., 1982; Ramos-Morales and Rodríguez-Arnaiz, 1995). Scarce information exists as to how arsenate acts, although it is considered that the observed effects may be the result of arsenite produced by arsenate reduction.
Hexafluoroarsenate was the third inorganic arsenic compound tested and no genotoxic effects were detected under any of the conditions assayed. No information regarding the possible genotoxicity of this compound has been found in the literature, nevertheless, we have included this compound as a useful tool for the understanding of mechanisms of arsenic toxicity. This choice was based on the hypothesis that stability of the AsF6– complex, due to the strong electronegativity of F, means the As atom is unavailable for normal metabolic activity.
The metabolism of arsenic in mammals has been extensively studied, indicating that inorganic arsenic compounds are methylated to MMA and DMA. This has been considered as a detoxification process, because the methylated metabolites, in comparison with inorganic arsenic, are less toxic and more easily excreted in urine (Vather, 1988). Nevertheless, recent studies suggest that methylated arsenic species, mainly in the trivalent state, may be more toxic than the inorganic forms (Styblo et al., 2000). In addition, although reports of effective cancer induction in animals by inorganic arsenic are rare, DMA has proved to be tumorigenic in the most common strains of mice and rats (Wang et al., 2002). However, there is controversy about these results, mainly due to the very high doses of arsenic compounds used in such experiments.
In our study both MMA and DMA were unable to significantly increase the level of DNA damage under the experimental conditions used. With DMA, and using the Comet test, a previous study (Mourón et al., 2001) also reported a lack of effect and showed that treatment reduced the level of DNA damage to below the control values. Based on these data, the authors supposed that DMA might act on DNA by inducing crosslinks. In another study carried out with human lymphocytes and using the Comet assay, both MMA and DMA induced significant increases in DNA single-strand breaks, but with long treatments (24 h) and with a large variability between donors (Sordo et al., 2001). On the other hand, other authors have shown that DMA causes DNA single-strand breaks in a human pulmonary cell culture system (Tezuka et al., 1993). In this case DNA damage was measured in an alkaline elution method, where breaks resulted from inhibition of repair polymerization using inhibitors such as aphidicolin and hydroxyurea, nevertheless, with this method no effects were observed with MMA (Yamanaka et al., 1997). The same group has been unable to demonstrate in vivo genotoxicity of DMA in MutaTM Mouse (Noda et al., 2002). This apparent contradictory genotoxic activity has been explained by supposing that induced lesions, mainly oxidative damage, are not particularly mutagenic because of efficient repair. Nevertheless, the organic arsenic compounds have been shown to induce cytogenetic damage in mammalian cells (Basu et al., 2001; Hughes, 2002), although they required much higher doses than the inorganic arsenic compounds. In this context, the recent study by Mass et al. (2001) is interesting because it shows that the trivalent methylated arsenicals are directly genotoxic, as proven by nicking supercoiled 
X174 DNA, and also induce clear effects in the Comet assay, in contrast to the lack of effects of arsenite, arsenate and the pentavalent forms of MMA and DMA. Recent studies with the trivalent forms of MMA and DMA suggest oxidative stress as a possible mechanism of action of arsenic carcinogenesis (Kitchin and Ahmad, 2003). This has been confirmed by Schwerdtle et al. (2003) in cultured human cells and in isolated PM2 DNA, showing that the induction of high levels of oxidative damage takes place at very low physio logically relevant doses. Thus, biomethylation of inorganic arsenic may be involved in inorganic arsenic-induced genotoxicity/carcinogenicity.
Arsenobetaine is mainly present in seafood and it is ingested upon consumption of crab, lobster, shrimp and fish, although it is rapidly excreted into urine unchanged (Le et al., 1993) and, for this reason, it has been considered as not harmful. Nevertheless, as arsenosugars from seaweeds (also present in seafood) have been shown to increase DMA levels in urine (Ma and Le, 1998), there are some doubts about the safety of arsenobetaine. A recent study has been conducted to investigate this point by carrying out a follow-up of people before and after refraining from eating seafood for 3 days (Hsueh et al., 2002). The results indicate that intake of seafood does not modify the proportions of organic and inorganic arsenicals, reinforcing the proposal that DMA is not formed from organic arsenic compounds in seafood. In vitro studies on the cytotoxicity and carcinogenic potential of arsenobetaine in the BALB/3T3 cell line support this hypothesis, the chemical form of arsenobetaine remaining unchanged (Sabbioni et al., 1993).
Only two reports have been found on the genotoxicity of arsenobetaine. In bacteria, no genotoxicity was detected in the Salmonella test with and without S9 mix metabolic activation, and similar results were obtained in a forward mutation test for the HGPRT gene in V79 Chinese hamster cells (Jongen et al., 1985). This lack of effect would agree with our results, although it must be remembered that arsenic compounds are generally weak inducers of such genotoxic effects. Nevertheless, in a study on the cytogenetic potential of arsenobetaine in mammalian cell cultures, a slight increase was observed in the frequency of chromosomal aberrations, but this included chromatid gaps, which for many authors are not chromosomal aberrations. This study also shows that arsenobetaine does not induce sister chromatid exchanges (Kaise et al., 1998). All these results, including our data, seem to support the view that arsenobetaine is not genotoxic.
Although MMA and DMA are the predominant metabolites of inorganic arsenic in humans, trimethylarsine oxide can also be found in urine, but at very low concentrations. This trimethylated form is not genotoxic in cultured human fibroblasts when looking for chromosomal damage (Oya-Ohta et al., 1996). In addition, another trimethylated form, alkylaminotrimethyl arsonic acid, was also not genotoxic in mouse fibroblast cells (Kaise et al., 1998). In this context, our positive results with the tetramethylarsonium iodide and tetraphenylarsonium chloride are surprising. No data have been found in the literature on the possible genotoxic role(s) of these compounds. The reasons for testing these two compounds are that the first is a product of degradation of cooked seafood containing arsenobetaine, while the second is an organoarsenic compound considered as a possible carcinogen (Lenga, 1988).
Summing up all the results obtained in this work we can conclude that some organic and inorganic forms of arsenic may induce DNA damage. It must be stressed that although the most common methylated forms are not detected as genotoxic, the tetramethyl form evaluated here clearly induces DNA damage in the human lymphoblastoid cell line used.
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This work was supported by a UAB–JRC (Ispra) contract (15469-1999-11-F1ED ISP ES) and seed grants from the Spanish Ministry of Education and Culture (PM98-0179) and the UE (CT99-01142).
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2To whom correspondence should be addressed. Email: ricard.marcos{at}uab.es
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References |
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