Induction of DNA strand breaks, DNA-protein crosslinks and sister chromatid exchanges by arsenite in a human lung cell line

Toxicology in Vitro 20 (2006) 279–285 www.elsevier.com/locate/toxinvit

Induction of DNA strand breaks, DNA-protein crosslinks and sister chromatid exchanges by arsenite in a human lung cell line Silvana Andrea Mourón, Claudia Alejandra Grillo, Fernando Noel Dulout, Carlos Daniel Golijow ¤ Centro de Investigaciones en Genética Básica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Calle 60 y 118 s/n, 1900, La Plata, Argentina Received 21 July 2005; accepted 21 July 2005 Available online 6 September 2005

Abstract Based on in vitro studies, several modes of action for arsenic have been suggested, although the mechanisms responsible for arsenic carcinogenesis have not been well established. In our previous study a dose-dependent increment in DNA migration was detected at low doses of sodium arsenite, but at higher dose levels a reduction in the migration was observed, suggesting the induction of DNA adducts. In order to conWrm this hypothesis we performed the experiments considering other parameters and modiWcations of the standard alkaline comet assay. Additionally, the induction of sister chromatid exchanges was evaluated. The present study showed the induction by sodium arsenite of single strand breaks and DNA-protein adducts assessed by comet assay as well as of sister chromatid exchanges in the human lung Wbroblast cell line MRC-5. The standard alkaline comet assay also revealed, at the highest arsenic concentration tested, a reduction in all the considered parameters in relation to untreated cells and the other doses. On the other hand, the incubation with proteinase K induced a dose-dependent increment in DNA migration as a consequence of the release of proteins joined to the DNA. Thus, sodium arsenite was able to induce both DNA-strand breaks and protein-DNA adducts in arsenic exposed MRC-5 cells, depending on the concentrations of arsenic salts tested. © 2005 Elsevier Ltd. All rights reserved. Keywords: Sodium arsenite; Comet assay; SCE

1. Introduction Arsenic is a metalloid found in water, soil and air from natural and anthropogenic sources and all human populations are exposed to it in one form or another. For the general population, the major sources of exposure are via food or drinking water, and also, from industrial emissions. Inorganic arsenic (iAs) is classiWed as a known human carcinogen, associated with increased risk for cancer of the lung, skin, urinary bladder, kidneys and liver, as well as hyperkeratosis, pigmentation

*

Corresponding author. Tel./fax: +54 221 421 1799. E-mail address: [email protected] (C.D. Golijow).

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changes and adverse eVects on the circulatory and nervous system (IARC, 1987; NRC, 1999; IPCS/WHO, 2001). However, the carcinogenic eVects of inorganic arsenicals in animal models could not be conclusively demonstrated (Kitchin, 2001; T.S. Wang et al., 2002; J. Wang et al., 2002). Inorganic arsenic can be ingested as either arsenite (iAs III) or arsenate (iAs V) depending on the pH of the environment. Although iAs V is less toxic, it is reduced biologically to iAs III before methylation. There are marked diVerences in arsenic toxicity among mammalian species, and man seems to be more sensitive than most experimental animals. Among human, ethnicity is associated with diVerences in arsenic toxicity, probably due to genetic polymorphisms operating in both methylation process as

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well as in arsenic derivatives tolerance (Vahter, 1999, 2000; Thomas et al., 2001). Based on in vitro studies, several modes of action for arsenic have been suggested, although the mechanisms responsible for arsenic carcinogenesis have not been well established (for review Basu et al., 2001; Gebel, 2001; Kitchin, 2001; Pott et al., 2001; Hughes, 2002; Rossman, 2003). Our previous study showed a signiWcant dosedependent increment in the extent of DNA migration and the percentage of tailed cells with 2.5 M and 5 M of sodium arsenite. But, cells treated with 10 M of this salt presented lower increments than the other doses (Mourón et al., 2001). This reduction in DNA migration could be reXecting the induction of DNA-protein crosslinks (DPC) at higher dose levels, a fact reported by other authors at diVerent concentrations (SchaumlöVel and Gebel, 1998; Gebel et al., 1998; Ramirez et al., 2000). In order to conWrm this hypothesis we performed the experiments considering other parameters and modiWcations of the standard alkaline comet assay. In addition, the eVects obtained in the comet assay were compared to another cytotoxic and genotoxic indicator such as sister chromatid exchanges (SCEs) induction to better understand the mechanisms of action of trivalent inorganic arsenic.

2. Materials and methods 2.1. Cell culture and treatments The human lung Wbroblast cell line MRC-5 was used for the experiments. Cells were grown as monolayers in Minimal Essential Medium (GIBCO BRL, Los Angeles, California, USA), supplemented with 10% inactivated fetal calf serum, 50 IU/ml of penicillin and 50 g/ml of streptomycin sulphate at 37 °C in a 5% CO2 atmosphere. Cells were treated with 2.5 M, 5 M and 10 M of sodium arsenite (NaAsO2) (Sigma, St. Louis, Missouri, USA) according to previously reported data concerning this salt (Lin and Tseng, 1992; Radha and Natarajan, 1998; Sciandrello et al., 2002). A control group (untreated cells) was incorporated in each assay. All experiments were performed twice in independent trials to assess reproducibility. 2.2. Sister chromatid exchanges assay Before each experience, cells were grown until conXuence in order to obtain cells in the G0/G1 phase due to the cessation of growth by contact inhibition. Then, cells were grown in culture medium added with 10 g/ml of 5⬘-bromo-2⬘-deoxyuridine (BrdU) (Sigma) in complete darkness for 38 h. Twelve hours after the initiation of the culture, the cells were treated with sodium arsenite for the Wnal 26 h. Colchicine (0.1 g/ml Wnal concentration)

was added 2 h prior to the harvest of the cultures. The SCE assay was performed according to Perry and WolV (1974) as we previously described (Mourón et al., 2004). SCEs were scored in 50 cells per treatment and experience (N D 100 cells). Proliferation of cells was evaluated in more than 100 cells, determining the proportion of Wrst (M1), second (M2) and third or more (M3) mitotic divisions. The proliferation index (PI) was calculated according to the formula PI D (M1 + 2M2 + 3M3)/total cells. 2.3. Comet assay Subcultures for experiments were set up the day before treatment. Approximately, 2 £ 105 cells at logarithmic growth phase were treated with sodium arsenite for 2 h. Cell viability was determined immediately after treatment using the trypan blue stain exclusion method. The standard alkaline comet assay was performed according to the method of Singh et al. (1988) with some small modiWcation (Tice, 1995) as we previously described (Mourón et al., 2001). BrieXy, after agarose solidiWcation, the slides were immersed overnight at 4 °C in freshly lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10) containing 1% Triton X-100 and 10% dimethylsulfoxide, added just before use. After lysis, the slides were placed on an horizontal gel electrophoresis unit Wlled with fresh electrophoretic buVer (300 mM NaOH, 1 mM Na2EDTA, pH > 13) and left for 20 min for DNA unwinding and then electrophoresed for 30 min at 1.25 V/cm (300 mA). These measures were performed at 4 °C under dim light. After electrophoresis, the slides were washed with neutralizing buVer (0.4 M Tris, pH 7.5) and the cells were stained with SYBR Green I (Molecular Probes, Eugene, Oregon, USA) at the recommended dilution. Individual cells were visualized at 400£ magniWcation on an Olympus BX40 Xuorescent microscope (equipped with a 515–560 nm excitation Wlter), connected through a Sony 3CCD-IRIS Color Video Camera. The image for each individual cell was acquired immediately after opening the microscope shutter to the computer monitor, employing the Image Pro Plus 3.0 program (Media Cybernetics, Silver Spring, Madison, USA). Pictures of 75 randomly selected cells from each slide were analyzed. The following comet parameters were evaluated: length migration (Mig), tail moment (TM) and percentage of tail (%T). These parameters were calculated using the Image Pro Plus 3.0 software. 2.4. Detection of DNA crosslinks To verify the possibility of sodium arsenite to induce DNA-protein crosslinks, cells were incubated with proteinase K according to the method of Woqniak and Blasiak (2002). BrieXy, after cell lysis the slides were washed

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three times (5 min, 4 °C) in TE buVer containing 10 mM Tris, pH 10, 1 mM EDTA and drained. Aliquots of 100 l of TE buVer (as control) or 100 l of proteinase K in buVer (Invitrogen—Life Technologies), at 1 mg/ml were applied to the agarose containing cells and incubated for 2 h at 37 °C in a moist chamber. Further steps were done as described above. 2.5. Statistical analysis Statistical evaluation was done with SPSS 11.0.1 software (SPSS Inc., LEAD Technologies, Chicago, Illinois, USA). In the SCE test a total of 100 metaphases, when it was possible, were analyzed for each treatment and the mean and standard error were calculated. The eVect of chemical treatment on frequency of SCEs was analyzed using the non-parametric Kruskal–Wallis one-way ANOVA test and the Mann–Whitney test. In the comet assay a total of 150 cells were evaluated for each treatment. The median and interquartile range was calculated for the comet parameters for each treatment. Also, the Kruskal–Wallis test was used in order to analyze total diVerences between groups for length migration, tail moment and percentage of tail parameters. To evaluate diVerences between each sample pair the Mann–Whitney test was employed.

3. Results 3.1. SCE assay Table 1 exhibits the SCEs frequencies detected in MRC-5 cells treated with diVerent doses of sodium arse-

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nite and its untreated control. The cell growth was considerably diminished at 5 M of NaAsO2, where only 35 metaphases could be counted, and none second division metaphase could be found with 10 M of sodium arsenite. Cytotoxic eVect of arsenic salt was also reXected by the proliferation index that decreased at 2.5 M concentration with respect to untreated cells as a consequence of an increment of M1 cells and a relative drop of M2 cells (Table 1). SCEs frequencies were signiWcantly increased in treated cells in relation to controls (H D 81.9, p < 0.001). The Mann–Whitney test demonstrated signiWcant diVerences only between each dose and the control group (p < 0.001). 3.2. Comet assay The treatment with sodium arsenite produced an increment in all the considered parameters in relation to untreated cells assessed by the standard alkaline comet assay (Table 2). The percentage of viable cells did not demonstrate cytotoxic eVects with the employed doses. When migration, tail moment and % tail were considered the Kruskal–Wallis test showed highly signiWcant diVerences between groups (p < 0.001). Mann–Whitney test revealed signiWcant diVerences between the control group and cells treated with 2.5 M (p < 0.005) and 5 M of sodium arsenite (p < 0.001). However, no diVerences were found between untreated cells and those treated with 10 M of NaAsO2. As we previously reported (Mourón et al., 2001), with this treatment a reduction in all the comet parameters analyzed was observed. These results might be indicating a complex way for arsenite carcinogenesis. In this sense the induction of

Table 1 Frequencies of sister chromatid exchanges (SCEs) in MRC-5 cells treated with sodium arsenite NaAsO2

N

SCEs (§SE)

M1

M2

M3

PI

0 M 2.5 M 5 M 10 M

100 92 35 0

3.24 (0.14) 5.23 (0.18)¤¤¤ 6.2 (0.41)¤¤¤ –

36 87 100 –

184 155 40 –

0 0 4 –

1.84 1.64 1.33 –

Proliferation index (PI) was calculated in more than 100 cells, determining the proportion of Wrst (M1), second (M2) and third or more (M3) mitotic divisions. N D number of metaphases analyzed. ¤¤¤ Denotes signiWcant diVerences in relation to control group (p > 0.001).

Table 2 Response of sodium arsenite on human lung Wbroblasts assessed by the standard alkaline comet assay NaAsO2

Miga

TMa

% Ta

%V

0 M 2.5 M 5 M 10 M

0 (0–72) 45 (0–160)** 66.5 (16–178)*** 29.5 (0–197)

0 (0–6.3) 2.6 (0–27.2)** 3.8 (1–18.4)*** 2 (0–13.3)

0 (0–7.9) 4.2 (0–29.7)** 6.1 (0.8–21)*** 3.3 (0–15.8)

91.2 89.3 93 89.1

The number of cells in each treatment was 150; Mig D tail length; TM D tail moment; % V D percentage of viable cells. SigniWcant diVerences in relation to control group is denoted by an asterisk (*): **p < 0.005; ***p < 0.001. a Expressed as median and interquartile range.

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Table 3 Comet parameters of MRC-5 cells incubated with sodium arsenite and post-treated with proteinase K and control group (only with TE) NaAsO2

PI

Miga

TMa

% Ta

%V

0 M

TE

0 (0–61)

0 (0–8)

0 (0–9.8)

92.7

*

*

**

PK

0 (0–107)

0 (0–20.6)

0 (0–22.8)

TE

32.5 (0–82)

2.8 (0–9.6)

4.4 (0–12.9)

**

**

**

PK

57 (0–113)

6.3 (0–20.4)

10.1 (0–22.3)

5 M

TE PK

35.5 (0–123) 56 (0–156)

2.9 (0–15.6) 6.8 (0–26.2)

5.3 (0–17.7) 9.4 (0–23.7)

90.6

10 M

TE

93.1

2.5 M

PK

25 (0–93)

2.3 (0–12.4)

4 (0–18)

***

***

***

117 (42–209)

13.6 (3.8–30.8)

14.6 (6.1–29.7)

92.4

The number of cells in each treatment was 150; PI D post-incubation with TE or PK-TE; Mig D tail length; TM D tail moment; % T D percentage of DNA in tail; % V D percentage of viable cells. * p < 0.01; **p < 0.005; ***p < 0.001: denote signiWcant diVerences between proteinase K treatment and the control group (TE). a Expressed as median and interquartile range.

DNA adducts could be happening at the higher concentration tested. So that, a modiWcation of the comet assay was performed to verify the possible induction of protein-DNA adducts. The control assay (only with TE buVer) also showed a slight decrease in DNA migration in cells treated with 10 M in relation to the other doses, reXected in all the analyzed parameters (Table 3). The Kruskal–Wallis test showed signiWcant diVerences between groups (p < 0.001). The Mann–Whitney test revealed signiWcant diVerences between the control group and each dose (p < 0.01). On the other hand, the incubation with proteinase K induced the release of proteins joined to the DNA and consequently a dose-dependent increment in DNA migration was observed (Table 3). The Kruskal–Wallis test showed signiWcant diVerences between groups (p < 0.001). The Mann–Whitney test revealed signiWcant diVerences: (i) between the control group and doses of 2.5 M (p < 0.05), 5 M (p < 0.01) and 10 M (p < 0.001); (ii) between cells treated with 2.5 M and those treated with 10 M (p < 0.001); (iii) dose 5 M respected to dose 10 M (p < 0.01). Comparison of proteinase K treated slides and its control showed signiWcant diVerences in most of the treatments and parameters (Table 3). Thus, sodium arsenite was able to induce both DNAstrand breaks and protein-DNA adducts in As exposed MRC-5 cells, depending on the concentrations of arsenic salts tested.

4. Discussion The present study showed that sodium arsenite can induce single strand breaks and DNA-protein adducts assessed by comet assay as well as sister chromatid exchanges in a human Wbroblast cell line.

A signiWcantly increased SCE response has been observed when the human Wbroblast cells were exposed to sodium arsenite (p < 0.001). However, the cell growth was highly diminished at 5 M of NaAsO2, appearing to be no cell growth at all at 10 M. The toxicity was also demonstrated in the proliferative index values, which decrease by increasing sodium arsenite concentrations. These results were concordant with other studies that used human lymphocytes (Jha et al., 1992; Hartmann and Speit, 1994; Lerda, 1994; Kochhar et al., 1996; Rasmussen and Menzel, 1997; Mahata et al., 2004a). In contrast, other in vitro studies did not show cytotoxic eVects with these concentrations of sodium arsenite salts (Lin and Tseng, 1992; Radha and Natarajan, 1998; Sciandrello et al., 2002; Mahata et al., 2004b). Contrarily, Dulout et al. (1996) studying a native Andean population from Northwestern Argentina exposed to arsenic in drinking water, demonstrated the induction of micronuclei and aneuploidy, but not of SCEs or clastogenic eVects in peripheral blood lymphocytes. These heterogeneous results obtained with human lymphocytes could be revealing variability among individuals in their sensitivity to SCE induction by arsenic, also observed by Rasmussen and Menzel (1997). Also, Gebel et al. (1997) did not Wnd an increment of sister chromatid exchanges exposing in vitro human lymphocytes to sodium arsenate (iAs V). On the other hand, when cells were exposed to As2O3 (iAs III) they found similar results, where a signiWcant increment in SCEs and cytotoxic eVects at 5 M were observed. Moreover, the use of diVerent cell types for in vitro studies revealed the ability of sodium arsenite to induce micronuclei, endoreduplication, aneugenic and clastogenic eVects (Lee et al., 1985; Vega et al., 1995; Huang et al., 1995; Mäki-Paakkanen et al., 1998; Nilsson et al., 1999; Basu et al., 2001).

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When the standard alkaline comet assay was performed, signiWcant increments in all the considered parameters were observed at 2.5 M and 5 M of sodium arsenite in relation to the untreated cells. However, no diVerences were found between the control group and those cells treated with 10 M of NaAsO2. As we previously reported (Mourón et al., 2001), with this treatment a reduction in all the comet parameters analyzed was observed. These results might be indicating the induction of DNA adducts at higher concentrations. So that, a modiWcation of the comet assay was performed to verify the possible induction of protein-DNA adducts. The control assay (only with TE buVer) also showed a slight decrease in DNA migration in cells treated with 10 M in relation to the other doses. However, the incubation with proteinase K induced the release of proteins joined to the DNA and consequently a dose-dependent increment in DNA migration was observed. Therefore, this study demonstrated the induction of both DNA-strand breaks and protein-DNA adducts by sodium arsenite assessed by two variants of the comet assay. The response of MRC-5 cells would be depending on the concentrations of arsenic salts used. The results presented in the literature in relation to the induction of DNA-strand breaks by sodium arsenite were somewhat contradictory. On one side, Hartmann and Speit (1994) demonstrated an increment on the length of DNA migration in human blood cells but at higher concentrations (10¡4–10¡3 M). Likewise, Guillamet et al. (2004) demonstrated a signiWcant dose-dependent increment in the tail moment after the treatment of the TK6 cell line with 0.1–10 mM of sodium arsenite. However, at the lowest concentration of 0.001 mM a slight reduction in the comet parameter was observed. Probably, these results could be reXecting the generation of DNA crosslinks at the lower concentrations tested, but the induction of DNA-strand breaks at higher and probably cytotoxic concentrations. Other authors using human Wbroblasts observed the induction of DNAstrand breaks at 5 M of sodium arsenite (Yih and Lee, 2000). Evidently each cell line responds in a similar way after the exposure to sodium arsenite, with the induction of DNA adducts and DNA-strand breaks, but with diVerences in concentration susceptibilities. On the other side, iAs III was shown to induce strand breaks at the low concentration of 0.01 M, but the induction of DPC with doses up to 1 M led to a tail moment decrease. At higher doses the tail moment again increased, presumably because of cytotoxicity. When proteinase K was added to the comet assay, a continuously increasing tail moment was found, indicative of disruption of DNA-protein crosslinks generated by iAs III (SchaumlöVel and Gebel, 1998; Gebel et al., 1998). Additionally, other authors demonstrated the induction of DNA-strand breaks with arsenite above

283

0.25 M in two human leukemia cells and above 10 M in Chinese hamster ovary cells, assessed by standard comet assay. Nevertheless, digestion with E. coli formamidopyrimidine-DNA glycolylase, which is known to catalyze the excision of oxidized bases, and proteinase K, which is eVective in releasing DNA-strand breaks from DNA-protein crosslinks, increased the level of DNA-strand breakage. These results suggested the simultaneous participation of oxidative DNA damage and the induction of adducts and DNA-strand scissions, a fact also reported in other works (Wang et al., 2001; T.S. Wang et al., 2002; J. Wang et al., 2002; Bau et al., 2002). Induction of DNA-protein crosslinks by arsenite assessed by an adduct precipitation method has also been reported in a human hepatic cell line. The proportion of DPC correlated linearly with intracellular concentration of trivalent iAs, indicating that they represent the eVect of the intracellular dose of As III (Ramirez et al., 2000). Recently, oxidative stress and inhibition of the repair system have been postulated as other modes for arsenic carcinogenesis (Nilsson et al., 1999; Hartwig et al., 1997; Liu et al., 2001; Schewerdtle et al., 2003; Shi et al., 2004). Also, it may aVect DNA methylation (Zhao et al., 1997; Goering et al., 1999; Zhong and Mass, 2001) and could enhance the expression of certain oncogenes (Trouba et al., 2000; Vega et al., 2001; Simeonova et al., 2002). However, arsenic does not appear to be a point mutagen in standard assays (Rossman et al., 1980; Lee et al., 1985; Wiencke et al., 1997). Although arsenic is a well-established human carcinogen, the mechanisms for cancer induction remain unclear. Carcinogenesis by arsenic is undoubtedly a complex process, suggesting that it does not Wt the model of a classical initiator or tumor promoter. So, arsenic seems to have the properties of both types of agents. Further studies will be required to provide more deWnitive data concerning the mechanisms involved in arsenic carcinogenesis.

Acknowledgment Dr. Silvana Mourón, Fellowship from Consejo Nacional de Investigaciones CientíWcas y Técnicas (CONICET), Argentina.

References Basu, A., Mahata, J., Gupta, S., Giri, A., 2001. Genetic toxicology of a paradoxical human carcinogen, arsenic: a review. Mutation Research 488, 171–194. Bau, T., Wang, T., Chung, C., Wang, A., Jan, K., 2002. Oxidative DNA adducts and DNA-protein cross-links are the major DNA lesions induced by arsenite. Environmental Health Perspectives 110 (suppl. 5), 753–756.

284

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Dulout, F., Grillo, C., Seoane, A., Maderna, R., Nilsson, R., Vahter, M., Darroudi, F., Natarajan, A., 1996. Chromosomal aberrations in peripheral lymphocytes from native Andean women and children from Northwestern Argentina exposed to arsenic in drinking water. Mutation Research 370, 151–158. Gebel, T., 2001. Genotoxicity of arsenical compounds. International Journal of Hygiene and Environmental Health 203, 249– 262. Gebel, T., Christensen, S., Dunkelberg, H., 1997. Comparative and environmental genotoxicity of antimony and arsenic. Anticancer Research 17, 2603–2608. Gebel, T., Birkenkamp, P., Luthin, S., Dunkelberg, H., 1998. Arsenic(III) but not antimony (III) induces DNA-protein crosslinks. Anticancer Research 18, 4253–4258. Goering, P., Aposhian, H., Mass, M., Cebrián, M., Beck, B., Waalkes, M., 1999. The enigma of arsenic carcinogenesis: role of metabolism. Toxicological Sciences 49, 5–14. Guillamet, E., Creus, A., Ponti, J., Sabbioni, E., Fortaner, S., Marcos, R., 2004. In vitro DNA damage by arsenic compounds in a human lymphoblastoid cell line (TK6) assessed by the alkaline comet assay. Mutagenesis 19 (2), 129–135. Hartmann, A., Speit, G., 1994. Comparative investigations of the genotoxic eVects of metals in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test. Environmental and Molecular Mutagenesis 23, 299–305. Hartwig, A., GröblinghoV, U., Beyersmann, D., Natarajan, A., Filon, R., Mullenders, L., 1997. Interaction of arsenic(III) with nucleotide excision repair in susceptibility to environmental carcinogenesis. Environmental and Health Perspectives 104 (Suppl. 3), 547–551. Huang, R., Ho, I., Yih, L., Lee, T., 1995. Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human Wbroblasts. Environmental and Molecular Mutagenesis 25, 188–196. Hughes, M., 2002. Arsenic toxicity and potential mechanism of action. Toxicology Letters 133, 1–16. IARC, 1987. Arsenic and arsenic compounds. In: IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans—Overall Evaluations of Carcinogenicity: an Update of IARC Monographs (Suppl. 7). International Agency for Research on Cancer, Lyon, pp. 100–106. IPCS/WHO, 2001. Arsenic and arsenic compounds. In: Environmental Health Criteria 224, second ed. International Program on Chemical Safety, World Health Organization, Geneva. Jha, A., Noditi, M., Nilsson, R., Natarajan, A., 1992. Genotoxic eVects of sodium arsenite on human cells. Mutation Research 284, 215– 221. Kitchin, K., 2001. Recent advances in arsenic carcinogenesis: modes of action, animal model systems and methylated arsenic metabolites. Toxicology and Applied Pharmacology 172, 249–261. Kochhar, T.S., Howard, W., HoVman, S., Brammer-Carleton, L., 1996. EVect of trivalent and pentavalent arsenic in causing chromosome alterations in cultured Chinese hamster ovary (CHO) cells. Toxicology Letters 84 (1), 37–42. Lee, T., Huang, R., Jan, K., 1985. Sodium arsenite enhances the citotoxicity, clastogenicity and 6-thiogaunine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells. Mutation Research 148, 83–89. Lerda, D., 1994. Sister-chromatid exchange (SCE) among individuals chronically exposed to arsenic in drinking water. Mutation Research 312, 111–120. Lin, J., Tseng, S., 1992. Chromosomal aberrations and sister-chromatid exchanges induced by N-nitroso-2-acetylaminoXuorene and their modiWcations by arsenite and selenite in Chinese hamster ovary cells. Mutation Research 265 (2), 203–210. Liu, S., Athar, M., Lippai, I., Waldren, C., Hei, T., 2001. Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. PNAS 98 (4), 1643–1648.

Mahata, J., Chaki, M., Ghosh, P., Das, L.K., Baidya, K., Ray, K., Natarajan, A.T., Giri, A.K., 2004a. Chromosomal aberrations in arsenic-exposed human populations: a review with special reference to a comprehensive study in West Bengal, India. Cytogenetics and Genome Research 104 (1–4), 359–364. Mahata, J., Chaki, M., Ghosh, P., Das, L.K., Baidya, K., Ray, K., Natarajan, A.T., Giri, A.K., 2004b. EVect of sodium arsenite on peripheral lymphocytes in vitro: individual susceptibility among a population exposed to arsenic through the drinking water. Mutagenesis 19 (3), 223–229. Mäki-Paakkanen, J., Kurttio, P., Paldy, A., Pekkanen, J., 1998. Association between the clastogenic eVect in peripheral lymphocytes and human exposure to arsenic through drinking water. Environmental and Molecular Mutagenesis 32, 301–313. Mourón, S., Golijow, C., Dulout, F., 2001. DNA damage by cadmium and arsenic salts assessed by the single cell gel electrophoresis assay. Mutation Research 498, 47–55. Mourón, S., Grillo, C., Golijow, C., Dulout, F., 2004. A comparative investigation of DNA strand breaks, sister chromatid exchanges and k-ras gene mutations induced by cadmium salts in cultured human cells. Mutation Research 568/2, 221–231. Nilsson, R., Natarajan, A., Hartwig, A., Dulout, F., De la Rosa, M., Vahter, M., 1999. Clastogenic eVects and inXuence of inorganic arsenic on DNA-repair in mammalian systems. In: Sarkar, B. (Ed.), Metals and Genetics. Plenum Publ Corp., New York, pp. 21–40. NRC, 1999. Risk Assessment of Arsenic in Drinking Water. Subcommittee on Arsenic in Drinking Water. National Research Council, National Academy Press, Washington, 263. Perry, P., WolV, S., 1974. New Giemsa method for the diVerential staining of sister chromatids. Nature 251, 156–158. Pott, W., Benjamín, S., Yang, R., 2001. Pharmacokinetics, metabolism and carcinogenicity of arsenic. Reviews of Environmental Contamination and Toxicology 169, 165–214. Radha, S., Natarajan, A., 1998. Sodium arsenite-induced chromosomal aberrations in the Xq arm of Chinese hamster cell lines. Mutagenesis 13 (3), 229–234. Ramirez, P., Del Razo, L., Gutierrez-Ruiz, M., Gonsebatt, M., 2000. Arsenite induces DNA-protein crosslinks and cytokeratin expression in the WRL-68 human hepatic cell line. Carcinogenesis 21 (4), 701–706. Rasmussen, R., Menzel, D., 1997. Variation in arsenic-induced sister chromatid exchange in human lymphocytes and lymphoblastoid cell lines. Mutation Research 386, 299–306. Rossman, T., 2003. Mechanism of arsenic carcinogenesis: an integrated approach. Mutation Research 533, 37–65. Rossman, T., Stone, D., Molina, M., Troll, W., 1980. Absence of arsenite mutagenicity in E. coli and Chinese hamster cells. Environmental Mutagenesis 2, 371–379. SchaumlöVel, N., Gebel, T., 1998. Heterogeneity of the DNA damage provoked by antimony and arsenic. Mutagenesis 13 (3), 281–286. Schewerdtle, T., Walter, I., Mackiw, I., Hartwig, A., 2003. Induction of oxidative DNA damage by arsenite and its trivalent and pentavalent methylated metabolites in cultured human cells and isolated DNA. Carcinogenesis 24 (5), 967–974. Sciandrello, G., Barbaro, R., Caradonna, F., Barbata, G., 2002. Early induction of genetic instability and apoptosis by arsenic in cultured Chinese hamster cells. Mutagenesis 17 (2), 99–103. Shi, H., Shi, X., Liu, K., 2004. Oxidative mechanism of arsenic toxicity and carcinogenesis. Molecular and Cell Biochemistry 255, 67–78. Simeonova, P., Wang, S., Hulderman, T., Luster, M., 2002. c-Src dependent activation of the epidermal growth factor receptor and mitogen-activated protein kinase pathway by arsenic. Journal of Biological Chemistry 277 (4), 2945–2950. Singh, N., McCoy, M., Tice, R., Schneider, E., 1988. A simple technique for quantiWcation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191.

S.A. Mourón et al. / Toxicology in Vitro 20 (2006) 279–285 Thomas, D., Styblo, M., Lin, S., 2001. The cellular metabolism and systemic toxicity of arsenic. Toxicology and Applied Pharmacology 176, 127–144. Tice, R., 1995. The single cell gel/comet assay: a microgel electrophoretic technique for the detection of DNA damage and repair in individual cells. In: Phillips, D., Venitt, S. (Eds.), Environmental Mutagenesis. Bios ScientiWc Publishers, Oxford, pp. 315–339. Trouba, K., Wauson, E., Vorce, R., 2000. Sodium arsenite-induced dysregulation of proteins involved in proliferative signaling. Toxicology and Applied Pharmacology 164, 161–170. Vahter, M., 1999. Methylation of inorganic arsenic in diVerent mammalian species and population groups. Science Progress 82, 69–88. Vahter, M., 2000. Genetic polymorphism in the biotransformation of inorganic arsenic and its role in toxicity. Toxicology Letters 112– 113, 209–217. Vega, L., Gonsebatt, M., Ostrosky-Wegman, P., 1995. Aneugenic eVect of sodium arsenite on human lymphocytes in vitro: an individual susceptibility eVect detected. Mutation Research 334, 365–373. Vega, L., Styblo, M., Patterson, R., Cullen, W., Wang, C., Germolec, D., 2001. DiVerential eVects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes. Toxicology and Applied Pharmacology 172, 225–232. Wang, T.S., Chung, C.H., Wang, A.S., Bau, D.T., Samikkannu, T., Jan, K.Y., Cheng, Y.M., Lee, T.C., 2002. Endonuclease III, formamidopyrimidine-DNA glycosilase and proteinase K additively enhance

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arsenic-induced DNA strand breaks in human cells. Chemical Research in Toxicology 15, 1254–1258. Wang, J., Qi, L., Moore, M., Ng, J., 2002. A review of animal models for the study of arsenic carcinogenesis. Toxicology Letters 133, 17–31. Wang, T., Hsu, T., Chung, C., Wang, A., Bau, D., Jan, K., 2001. Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells. Free Radical Biology and Medicine 31 (3), 321–330. Wiencke, J., Yager, J., Varkonyi, A., Hultner, M., Lutze, L., 1997. Study of arsenic mutagenesis using the plasmid shuttle vector pZ189 propagated in DNA repair proWcient human cells. Mutation Research 386, 335–344. Woqniak, K., Blasiak, J., 2002. Free radicals-mediated induction of oxidized DNA bases and DNA-protein cross-links by nickel chloride. Mutation Research 514 (1–2), 233–243. Yih, L., Lee, T., 2000. Arsenite induces p53 accumulation through an ATM-dependent pathway in human Wbroblasts. Cancer Research 60, 6346–6352. Zhao, C., Young, M., Diwan, B., Coogan, T., Waalkes, M., 1997. Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proceedings of the National Academy of Sciences USA 94, 10907–10912. Zhong, C., Mass, M., 2001. Both hypomethylation and hypermethylation of DNA associated with arsenite exposure in cultures of human cells identiWed by methylation-sensitive arbitrarily-primed PCR. Toxicology Letters 122 (3), 223–234.