* Institute of Hygiene and Occupational Medicine, University Hospital, Hufelandstrasse 55, 45122 Essen, Germany; Institute of Environmental Analysis, University of Duisburg-Essen, Universitätsstrasse 3-5, 45141 Essen, Germany; and
Industrial Toxicology Research Centre, 226 001 Lucknow, India
1 To whom correspondence should be addressed at Universität Essen, Universitätsklinikum, Institut für Hygiene und Arbeitsmedizin, Hufelandstraße 55, 45147 Essen, Germany. Fax: 0201/723 4546. E-mail: elke.dopp{at}uni-essen.de.
Received March 17, 2005; accepted May 7, 2005
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ABSTRACT |
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Key Words: DNA damage; arsenic; dimethylarsinous acid; monomethylarsonous acid; cellular uptake; electroporation; cytotoxicity; micronuclei.
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INTRODUCTION |
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Microorganisms take up Asi(V) in the form of arsenate via phosphate transporters (Bun-ya et al., 1996; Rosen, 2002
; Yompakdee et al., 1996
) and it can be assumed that arsenate is taken up similarly in mammals, although this has not been demonstrated. In contrast, glycerol channels appear to play a key role in the cellular uptake of Asi(III), e.g., the glycerol channel Fps1p has been shown to mediate the uptake of both Asi(III) and Sbi(III) in the yeast Saccharomyces cerevisiae, and the mammalian aquaglyceroporins AQP7 and AQP9 have been reported to catalyse uptake of Asi(III) and Sbi(III) (Liu et al., 2002
). Asi(V) is reduced to Asi(III) by arsenate reductases (Ji and Silver, 1992
; Oden et al., 1994
). Asi(III) is extruded from cells or sequestered in intracellular compartments, either as free arsenite or as a conjugate with GSH or other thiols (Ghosh et al., 1999
; Kala et al., 2000
). In addition, arsenic can be methylated (Aposhian, 1997
), although this process may increase arsenic toxicity rather than contributing toward detoxification since certain metabolic intermediates are more toxic than the inorganic arsenic (Styblo et al., 2000
).
Dopp et al. (2004) have demonstrated that only a relatively small percentage (about 12%) of the initial Asi loading is taken up by CHO-9 cells and can be detected in cell free extract. The highest arsenic uptake was detectable at relatively low concentrations (500 nM Asi(III): 1.2%, 1 µM Asi(V): 1.6%) and the relative uptake decreased with increasing arsenic concentrations in the external medium. The authors concluded that a defense mechanism exists involving arsenic extrusion and that uptake at higher arsenic concentrations is inhibited. Trivalent methylarsenic species were more membrane-permeable than the pentavalent forms; up to 10% of the initial trivalent methylarsenic acid dose was taken up. The order of cellular uptake for the arsenic compounds in the trivalent state was DMA(III)>MMA(III)>Asi(III), and for pentavalent arsenic compounds Asi(V)>MMA(V)>DMA(V)>TMAO (Dopp et al., 2004
). The higher uptake of the trivalent methylated arsenicals may be responsible for the reported greater genotoxic effects of these compounds (e.g., Mass et al., 2001
; Schwerdtle et al., 2003
; Yamanaka et al., 2004
).
In the present study, we compare the uptake capabilities of fibroblasts (CHO-9 cells) used for genotoxicity studies with those of hepatoma cells (Hep G2). These cells were chosen because the liver is the primary site of arsenic metabolism within the body and skin, along with the bladder, is a target organ for arsenic carcinogenicity. The cellular uptake of the arsenic compounds was forced by electroporation to test the hypothesis that arsenic-induced genotoxic effects are related to the intracellular arsenic concentration and dependent upon uptake. Also, we investigated whether the arsenic compounds are bound to membranes or whether they are present in the cytosol. For these investigations we removed the cell membranes by osmotic lysis and subsequent centrifugation before the intracellular arsenic concentrations were measured by ICP-MS.
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MATERIALS AND METHODS |
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Hep G2 cells (ATCC, HB 8065) were maintained at 37°C and 5% CO2 in Minimum Essential Medium (MEM) with Earle's BSS and sodium-bicarbonate (CC PRO, Germany) supplemented with 10% heat-inactivated FCS (Gibco), non-essential amino acids (0.1 mM), sodium-pyruvate (1 mM), and 100 IU/ml penicillin/streptomycin (CC PRO, Germany).
Reagents.
Sodium arsenite (AsNaO2) and sodium arsenate (AsHNa2O4·7H20) were purchased from Fluka (Seelze, Germany) and Sigma (Taufkirchen, Germany), respectively. Dimethylarsinic acid (Me2AsOOH) was obtained from Strem (Kehl, Germany), and monomethylarsonic acid (MeAsO(OH)2) as well as trimethylarsine oxide (Me3AsO) were from Tri-Chemical Laboratories Inc. (Yamanashi, Japan). Monomethyldiiodoarsine (MeAsI2) and dimethyliodoarsine (Me2AsI) were purchased from Argus Chemicals (Vernio, Italy). Preparation of dilute solutions of these iodide precursors results in the formation of the corresponding acids, monomethylarsonous acid (MeAs(OH)2) and dimethylarsinous acid (Me2AsOH) (Gong et al., 2001; Millar et al., 1960
). All chemicals were of analytical grade or of the highest quality obtainable. Solutions of arsenicals were prepared in sterile McCoys 5A, Ham's F12K, or MEM medium, respectively, and stored at 20°C until used. The stability of the arsenicals in the culture medium was tested by HPLC-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) using the method of Le et al. (2000)
. Due to the high volatility and sensitivity to oxidation of dimethylarsinous acid, solutions of this arsenical were always prepared immediately before each experiment and were discarded if not used within a two-day period. All other arsenicals tested, including the trivalent compounds sodium arsenite and monomethylarsonous acid, were found to be stable over a minimum two month period. All arsenicals used in the cytotoxicity, genotoxicity, uptake, and electroporation experiments are listed in Table 1.
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Cytotoxicity test.
CHO-9 and Hep G2 cells were treated with the arsenicals at different concentrations for 24 h, and all experiments were performed in duplicate. Cell viability was evaluated immediately after exposure. Treated and untreated cells were harvested by trypsin treatment (Sigma). Cell counting was performed following trypan blue staining. The cell suspension was mixed with an equivalent volume of 0.4% trypan blue solution (Sigma) and subsequently evaluated under the light microscope. Cell viability is expressed as percentage of surviving cells compared to the total number of cells. Significance was tested by using the Student's t-test.
Cytokinesis blocked micronucleus assay (CBMN) and Nuclear division index (NDI).
For MN analysis, 2 x 105 CHO-9 cells were seeded in each well of Quadriperm-dishes (Viva-Science, Sartorius, Göttingen, Germany) and cultured overnight. Then the arsenic compounds were applied for 24 h at different concentrations (0.1 µM to 7.5 mM). Subsequently, the cells were washed twice with PBS and incubated for an additional period of 24 h with cytochalasin B (3 µg/ml) to block cytokinesis (recovery time). Cells were fixed by treating with Carnoy's solution (methanol:acetic acid, 3:1) for 5 min and stained with May-Grünwald and 6% Giemsa solution. The frequency of MN formation was expressed as percentage of binucleated (bn) cells with MN. 2000 bn cells were evaluated in each case, and all experiments were performed at least in duplicate.
Using the cytokinesis-blocked MN assay, the extent and progression of nuclear division could be assessed by analysing the NDI. After exposure of cells to non-cytotoxic concentrations of the arsenic compounds for 24 h, cells were incubated with cytochalasin B (3 µg/ml) for additional periods of 14 h, 28 h, or 35 h, respectively. The frequency of mononucleated (MI), binucleated (MII), and multinucleated (MIII + MIV) cells after cytochalasin-B treatment was determined. A minimum of 1000 cells/slide was analyzed.
Electroporation.
Electroporation according to Eppendorf Soft Pulse technology was carried out in hypoosmolar buffer (250 mOsmol/kg), in which the cells absorb water shortly before the pulse and then swell up as a result of the treatment. A number of effects, including a decreased optimal pulse voltage, ensure that the plasma membrane can be permeated more easily. The tolerance of CHO-9 cells to hypoosmolar conditions was tested in preliminary experiments. The survival rate of the cells was >90%.
Electroporation and micronucleus assay.
A suspension of cells (0.5 x 106) was prepared in medium for electroporation (basal medium + 0.5 % FCS) and centrifuged. The tested arsenic species were dissolved in hypoosmotic buffer (concentration: 500 µM) and added to the cells to obtain a final volume of 800 µl. The cells were transferred to electroporation cuvettes with a 4 mm gap. The hypoosmotic exposure lasted 30 min, and electroporation was performed at 530 V for 40 µs. The cell suspension was allowed to stand for a maximum of 10 min at room temperature and was then centrifuged (190 x g, 5 min). The cell pellet was washed twice in fresh culture medium and resuspended in 5 ml fresh medium containing cytochalasin B (3 µg/ml). Following an incubation period of 24 h (recovery time), the CBMN-assay was carried out. The experiments were set up as follows: (a) unexposed control, (b) unexposed control and electroporation, (c) exposed with arsenicals, and (d) exposed with arsenicals and electroporation. All experiments were performed in triplicate.
Cellular uptake of arsenic.
To assess the membrane permeability of the arsenic compounds under normal and forced conditions, CHO-9 cells were incubated with the arsenic compounds at different concentrations (0.1 µM10 mM) for an exposure time of 1 h and 24 h. Since the results obtained from experiments with exposure periods of 1 h and 24 h did not differ significantly, all uptake experiments were performed over a period of 1 h. For comparison of arsenic uptake in different cell lines, 8.8 x 106 CHO or 3.3 x 106 Hep G2 cells were exposed to the different arsenic species. Following incubation, cells were washed with PBS and subsequently resuspended in 10 ml fresh culture medium. After cell counting, the cell suspension was centrifuged for 5 min at 190 x g and the pellet was resuspended in 10 ml distilled water to lyse the cells (for at least 30 min). The absence of intact cells was confirmed by microscopic examination. From this cell solution two kinds of samples were prepared: (a) whole-cell extract with membranes and proteins present, and (b) cell-free (membrane removed) extract, obtained by osmotic lysis of the whole-cell extract with subsequent centrifugation (1700 x g, 15 min) to remove the membranes. The samples were stored at 20°C until ICP-MS analysis.
For electroporation approximately 1 x 106 CHO-9 cells were prepared in basal medium + 0.5% FCS and centrifuged. Solutions of each arsenic species (5, 50, 500 µM) were prepared in hypoosmotic buffer and added to the cells to achieve a final volume of 800 µl. The cells were transferred to electroporation cuvettes with a 4 mm gap. Samples were treated with electrical impulses of 530 V for 40 µs and all experiments were performed in triplicate. The cell suspension was allowed to stand for 10 min at room temperature and then centrifuged (5 min, 190 x g) and washed twice in fresh culture medium. Whole-cell and cell-free extract samples were prepared as described above.
Analysis of intracellular arsenic concentration.
Total arsenic concentrations in the cell extracts were determined by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7500a, Agilent Technologies, Germany). The ICP-MS was operated at 1260 W rf-power, with argon flows of 15 l min1 (plasma gas), 0.98 l min1 (carrier gas), and 0.9 l min1 (auxiliary gas). Solutions (up to 1 in 100 dilutions) were delivered at 0.3 ml/min to a Babington nebulizer and routed through a double-pass Scott-type spray chamber maintained at 2°C. The signals 75As (1000 ms), 77Cl (500 ms), and 115In (1000 ms) were monitored. Apart from the signal obtained at m/z 75 for the analyte arsenic and from that at m/z 115 for the internal standard indium, the signal at m/z 77 was monitored in order to control chloride interference. Quantitation was performed by external calibration with an arsenate standard solution and validated by analyzing CRM SERO B2.
Statistics.
The chi-square-test was used for the statistical analysis of the results of the micronucleus assay and the two-tailed Student's t-test for evaluating the data from the cytotoxicity test and the NDI.
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RESULTS |
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The cellular uptake of the arsenic species by CHO-9 cells increased after treatment of cells using electroporation. The increased cellular uptake under electroporative conditions was significant for Asi(III) (p < 0.001), Asi(V) (p < 0.001), MMA(III) (p < 0.01), MMA(V) (p < 0.05), and DMA(V) (p < 0.05) (Fig. 1).
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Micronucleus Induction after Electroporation
The induction of micronucleated cells (MN) by the pentavalent organoarsenic species MMA(V), DMA(V), and TMAO was assessed in CHO-9 cells after electroporation. These compounds were chosen for electroporation experiments because they were negative in the MN assay under normal exposure conditions (Figs. 57). No significant differences in MN induction were observed between the unexposed control and the unexposed control after electroporation. Exposure of CHO-9 cells to 500 µM MMA(V), DMA(V), and TMAO for 30 min did not induce a significant increase of MN formation. After forced uptake of the arsenic compounds via electroporation, the number of induced MN was significantly increased (p < 0.05, Fig. 8).
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DISCUSSION |
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The pentavalent methylated arsenic species are negatively charged at physiological pH (MMA (V) pKa1 2.6, pKa2 8.2, DMA(V) pKa 6.3, TMAO pKa 3.6) and were poorly taken up by both cell lines (0% to a maximum of 2%). No significant genomic damage was observed for MMA(V), DMA(V), or TMAO(V). After forced uptake of these species by electroporation the number of MN increased significantly in CHO-9 cells; intracellular concentrations of MMA(V), DMA(V), and TMAO(V) were also higher following electroporation. These data demonstrate that higher internal amounts of pentavalent organoarsenic species within the cell cause a higher degree of DNA damage and indicate that resistance to MMA(V), DMA(V), and TMAO(V) is controlled at the uptake level.
The reduction of the pentavalent arsenic species to the trivalent analogue, as described by the Challenger mechanism (Challenger, 1945), is a metabolic process catalysed by MMA(V) reductase/human glutathione-S-transferase-omega (hGSTO-1) (Aposhian et al., 2004
), hence the possibility of the observed genotoxic effects being caused by the trivalent metabolites rather than by the pentavalent substrate itself must be considered. Preliminary data from HPLC-ICP-MS analysis of the membrane-removed cell-free extracts from MMA(V), DMA(V), and TMAO(V) incubations did not reveal the presence of trivalent arsenic species (data not shown); it remains to be established, however, as to whether this remains the case over a 24 h incubation period.
We have shown that most of the arsenic species (except DMA(III) and Asi(III) in CHO-9 cells) are not bound to cellular membranes, but are able to enter the cytosol (no significant difference in arsenic concentration in whole-cell extract and membrane-removed cell extract). The differences between DMA(III) as well as Asi(III) concentrations in whole-cell extract and in membrane-removed cell extract may be explained by the presence of a specific exporter for these species. Active efflux of Asi(III) has been reported in bacteria, yeast, and mammalian cells (Kimura et al., 2005; Rosen, 2002
), and Shiobara et al. (2001)
have discussed the possibility of DMA(III) efflux from the liver. Furthermore, the detection of DMA(III) in urine has also been reported (Aposhian et al., 2000
; Mandal et al., 2001
). The apparent lack of Asi(III) export in Hep G2 cells may be explained by considering the high arsenic biotransformation capacity of hepatocytes. Efficient biotransformation of inorganic arsenic to methylarsenic species would remove the need for the extrusion of Asi(III) as a significant resistance mechanism.
Asi(III), Asi(V), MMA(III), and DMA(III) were shown to induce genomic damage in CHO-9 cells after an incubation period of 24 h. In contrast, the pentavalent organoarsenic species MMA(V), DMA(V), and TMAO(V) did not induce genotoxic effects up to a concentration of 7 mM. DMA(III) was most reactive of all arsenicals tested in inducing micronuclei (>10-fold increase compared to control). Two mechanisms could be responsible for the observed genotoxic effects of the trivalent arsenicals: (1) induction of oxidative damage causing single- and/or double-strand breaks (Hei et al., 1998) and (2) modification of cellular functions by changing the phosphorylation profiles of cellular proteins (Huang et al., 1995
) and inhibition of DNA repair enzymes (Hartwig et al., 2003
; Lynn et al., 1997
; Yager and Wiencke, 1997
).
The dimethylated trivalent arsenic forms were more effective in expressing DNA damage than the monomethylated trivalent forms, possibly due to the involvement of such reactive oxygen species (ROS) as dimethylarsenic peroxy-radicals [(CH3)2AsOO] (Kenyon and Hughes, 2001; Noda et al., 2002
; Yamanaka et al., 2001
). DMA(III) has been shown to form ROS which cause DNA damage in vivo (Bernstam and Nriagu, 2000
; Liu et al., 2001
; Lynn et al., 1998
; Mandal et al., 2001
; Wu et al., 2001
).
Kessel et al. (2002) showed that arsenite is a potent chromosomal mutagen and that oxyradicals are involved in the mutagenic process. As a result of ROS production by arsenic DNA strand breaks (Okayasu et al., 2003
), oxidative DNA damage (Bau et al., 2002
; Hartwig et al., 1997
; Schwerdtle et al., 2003
) and the formation of micronuclei can occur (Basu et al., 2004
; Bernstam and Nriagu, 2000
; Kato et al., 2003
). Arsenic can either enhance or reduce nitric oxide (NO) production, depending on the type of cell, the arsenic species, and the concentration of the tested arsenical. Suppression of NO production has been shown to reduce arsenite-induced oxidative DNA damage and micronucleus formation (Gurr et al., 1998
, 2003
). An elevated resistance to As-induced genotoxicity in Hep G2 cells was reported by Gebel et al. (2002)
. The authors did not find elevated numbers of induced micronuclei in Hep G2 cells after arsenic exposure.
A cell-cycle delay in arsenite-treated human leukaemia cells was reported by McCabe et al. (2000). Kligerman et al. (2003)
also observed a retardation of the cell-cycle after treatment of human lymphocytes with Asi(III) and (V), MMA(III) and (V), and DMA(III) and (V) for 24 h. In our study, a recovery period was instituted, i.e., CHO-9 cells were treated with the different arsenicals for 24 h and after the treatment period, cells were incubated for additional 14 h to 35 h with cytochalasin B (recovery period). Hence, in contrast to the results of McCabe et al. (2000)
and Kligerman et al. (2003)
we detected no significant cell-cycle delay after exposing CHO-9 cells to Asi(III) and (V), MMA(III) and (V), DMA(V), and TMAO. The NDI was significantly modified only after incubation of cells with DMA(III) and a short recovery period of 14 h. Taken together, these data indicate that arsenic compounds are able to temporarily delay the cell cycle, but that this delay is reversible. These results are in agreement with data of previous studies published by Dopp et al. (2004)
where CHO-9 cells were exposed to organoarsenicals for periods of 1 h. After an extended exposure time of 24 h (and a recovery period of 14 h) in the present study, the DMA(III)-induced cell-cycle delay was still detectable.
In conclusion, the presented results reveal that the uptake capabilities of arsenic compounds are dependent upon both the cell type and the arsenic species: We noted a resistance to intracellular accumulation of arsenic, either due to increased resistance at the uptake level or an enhanced efflux mechanism by Hep G2 cells in comparison to CHO-9 cells. We have shown that the poorly membrane-permeable pentavalent organoarsenic compounds MMA(V), DMA(V), and TMAO(V) are able to induce DNA damage, but only under forced uptake conditions. The induced genotoxic effects were directly correlated to the increased intracellular arsenic concentrations. These data reveal that cellular resistance to MMA(V), DMA(V), and TMAO(V) occurs at the uptake and not intracellular level. We found that Asi(V), MMA(III), MMA(V), DMA(V), and TMAO were present in the cytosol and not externally bound to cell membranes for either fibroblasts or heptaocytes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Aposhian, H. V., Gurzau, E. S., Le, X. R., Gurau, A., Healy, S. M., Lu, X., Ma, M., Yip, L., Zakharyan, R. A., Mariorino, R. M., Dart, R. C., Tircus, M. G., Gonzalez-Ramirez, D., Morgam, D. L., Avram, D., and Aposhian, M. M. (2000). Occurrence of monomethylarsonous acid urine of humans exposed to inorganic arsenic. Chem. Res. Toxicol. 13, 693697.[CrossRef][ISI][Medline]
Aposhian, H. V., Zakharyan, R. A., Avram, M. D., Sampayo-Reyes, A., and Wollenberg, M. L. (2004). A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species. Toxicol. Appl. Pharmacol. 198, 327335.[CrossRef][ISI][Medline]
Bau, D. T., Wang, T. S., Chung, C. H., Wang, A. S., Wang, A. S., and Jan, K. Y. (2002). Oxidative DNA adducts and DNA-protein cross-links are the major DNA lesions induced by arsenite. Environ. Health Perspect. 110, 753756.[ISI][Medline]
Basu, A., Ghosh, P., Das, J. K., Banerjee, A., Ray, K., and Giri, A. K. (2004). 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. 13, 820827.
Bernstam, L., and Nriagu, J. (2000). Molecular aspects of arsenic stress. J. Toxicol. Environ. Health B. Crit. Rev. 3, 293322.[CrossRef][ISI][Medline]
Bhattacharjee, H., Gosh, M., Mukhopadhyay, R., and Rosen, B. P. ( 1999). Arsenic defence mechanisms. In Transport of Molecules Across Microbial Membranes (J. K. Broome-Smith, S. Baumberg, C. J. Sterling, and F. B. Ward, Eds.), Vol. 58, pp. 5879. Society for General Microbiology, Leeds.
Bun-ya, M., Shikata, K., Nakade, S., Yompakdee, C., Harashima, S., and Oshima, Y. (1996). Two new genes, PHO86 and PHO87, involved in inorganic phosphate uptake in Saccharomyces cerevisiae. Curr. Genet. 29, 344351.[CrossRef][ISI][Medline]
Challenger, F. (1945). Biological methylation. Chemical Reviews 36, 315361.[CrossRef][ISI]
Dopp, E., Hartmann, L. M., Florea, A. M., von Recklinghausen, U., Pieper, R., Shokouhi, B., Rettenmeier, A. W., Hirner, A. V., and Obe, G. (2004). Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells. Toxicol. Appl. Pharmacol. 201, 156165.[CrossRef][ISI][Medline]
Drobná, Z., Waters, S. B., Walton, F. S., LeCluyse, E. L., Thomas, D. J., and Styblo, M. (2004). Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes. Toxicol. Appl. Pharmacol. 201, 166177.[CrossRef][ISI][Medline]
Gebel, T. W., Leister, M., Schumann, W., and Hirsch-Ernst, K. (2002). Low-level self tolerance to arsenite in human Hep G2 cells is associated with a depressed induction of micronuclei. Mutat. Res. 514, 245255.[ISI][Medline]
Goering, P. L., Aposhian, H. V., Mass, M. J., Cebrian, M., Beck, B. D., and Waalkes, M. P. (1999). The enigma of arsenic carcinogenesis: Role of metabolism. Toxicol. Sci. 49, 514.[Abstract]
Ghosh, M., Shen, J., and Rosen, B. P. (1999). Pathways of As(III) detoxification in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 96, 50015006.
Gong, Z., Lu, X., Culler, W. R., Le, X. C. (2001). Unstable trivalent arsonic metabolites, monomethylarsonous acid and dimethylarsinous acid. J. Anal. Atom Spectrom 16, 14091413.[CrossRef][ISI]
Gradecka, D., Palus, J., and Wasowicz, W. (2001). Selected mechanisms of genotoxic effects of inorganic arsenic compounds. Int. J. Occup. Med. Environ. Health 14, 317328.[Medline]
Gurr, J. R., Liu, F., Lynn, S., and Jan, K. Y. (1998). Calcium-dependent nitric oxide production is involved in arsenite-induced micronuclei. Mutat. Res. 416, 137148.[ISI][Medline]
Gurr, J. R., Yih, L. H., Samikkannu, T., Bau, D. T., Lin, S. Y., and Jan, K. Y. (2003). Nitric oxide production by arsenite. Mutat. Res. 533, 173182.[Medline]
Hartwig, A., Groblinghoff, U. D., Beyersmann, D., Natarajan, A. T., Filon, R., and Mullenders, L. H. (1997). Interaction of arsenic(III) with nucleotide excision repair in UV-irradiated human fibroblasts. Carcinogenesis 18, 399405.[Abstract]
Hartwig, A., Blessing, H., Schwerdtle, T., and Walter, I. (2003). Modulation of DNA repair processes by arsenic and selenium compounds. Toxicology 193, 161169.[CrossRef][ISI][Medline]
Hei, T. K., Liu, S. X., and Waldren, C. (1998). Mutagenicity of arsenic in mammalian cells: Role of reactive oxygen species. Proc. Natl. Acad. Sci. U.S.A. 95, 81038107.
Huang, R. N., Ho, I. C., Yih, L. H., and Lee, T. C. (1995). Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts. Environ. Mol. Mutagen. 25, 188196.[ISI][Medline]
Ji, G., and Silver, S. (1992). Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258. Proc. Natl. Acad. Sci. U.S.A. 89, 94749478.
Kala, S. V., Neely, M. W., Kala, G., Prater, C. I., Atwood, D. W., Rice, J. S., and Liebe, M. W. (2000). The MRP2/cMOAT transporter and arsenic-glutathione formation are required for biliary excretion of arsenic. J. Biol. Chem. 275, 3340433408.
Kato, K., Yamanaka, K., Hasegawa, A., and Okada, S. (2003). Active arsenic species produced by GSH-dependent reduction of dimethylarsinic acid cause micronuclei formation in peripheral reticulocytes of mice. Mutat. Res. 539, 5563.[ISI][Medline]
Kenyon, E. M., and Hughes, M. F. ( 2001). A concise review of the toxicity and carcinogenicity of dimethylarsinic acid. Toxicology 160, 227236.[CrossRef][ISI][Medline]
Kessel, M., Liu, S. X., Xu, A., Santella, R., and Hei, T. K. (2002). Arsenic induces oxidative DNA damage in mammalian cells. Mol. Cell. Biochem. 234, 301308.[CrossRef][ISI]
Kimura, A., Ishida, Y., Wada, T., Yokoyama, H., Mukaida, N., and Kondo, T. (2005). MRP-1 expression levels determine strain specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice. Toxicol. Appl. Pharmacol. 203, 5361.[CrossRef][ISI][Medline]
Kligerman, A. D., Doerr, C. L., Tennant, A. H., Harrington-Brock, K., Allen, J. W., Winkfield, E., Poorman-Allen, P., Kundu, B., Funasaka, K., Roop, B. C., Mass, M. J., and DeMarini, D. M. (2003). Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: Induction of chromosomal mutations but not gene mutations. Environ. Mol. Mutagen. 42, 192205.[CrossRef][ISI][Medline]
Le, X. C., Lu, X., Ma, M., Cullen, W. R., Aposhian, H. V., and Zheng, B. (2000). Speciation of key arsenic metabolic intermediates in human urine. Anal. Chem. 72, 51725177.[CrossRef][ISI][Medline]
Liu, S. X., Athar, M., Lippai, I., Waldren, C., and Hei, T. K. (2001). Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proc. Natl. Acad. Sci. U.S.A. 98, 16431648.
Liu, Z., Shen, J., Carbrey, J. M., Mukhopadhyay, R., Agre, P., and Rosen, B. P. (2002). Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9. Proc. Natl. Acad. Sci. U.S.A. 99, 60536058.
Lynn, S., Shiung, J. N., Gurr, J. R., and Jan, K. Y. ( 1998). Arsenite stimulates poly(ADP-ribosylation) by generation of nitric oxide. Free Radic. Biol. Med. 24, 442449.[CrossRef][ISI][Medline]
Lynn, S., Lai, H. T., Gurr, J. R., and Jan, K. Y. (1997). Arsenite retards DNA break rejoining by inhibiting DNA ligation. Mutagenesis 12, 352358.
Mandal, B. K., Ogra, Y., and Suzuki, K. T. (2001). Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal, India. Chem. Res. Toxicol. 14, 371378.[CrossRef][ISI][Medline]
Mass, M. J., Tennant, A., Roop, B. C., Cullen, W. R., Styblo, M., Thomas, D. J., and Kligerman, A. D. (2001). Methylated trivalent arsenic species are genotoxic. Chem. Res. Toxicol. 14, 355361.[CrossRef][ISI][Medline]
McCabe, M. J., Jr., Singh, K. P., Reddy, S. A., Chelladurai, B., Pounds, J. G., Reiners, J. J., Jr., and States, J. C. (2000). Sensitivity of myelomonocytic leukemia cells to arsenite-induced cell cycle disruption, apoptosis, and enhanced differentiation is dependent on the inter-relationship between arsenic concentration, duration of treatment, and cell cycle phase. J. Pharmacol. Exp. Ther. 295, 724733.
Millar, I. T., Heaney, H., Heinecky, D. M., Fernelius, C. (1960). Methyldiiodoarsine, in Rochow EG (ad). Inorganic Synthesis 6, 113115.
Noda, Y., Suzuki, T., Kohara, A., Hasegawa, A., Yotsuyanagi, T., Hayashi, M., Sofuni, T., Yamanaka, K., and Okada, S. (2002). In vivo genotoxicity evaluation of dimethylarsinic acid in MutaMouse. Mutat. Res. 513, 205212.[ISI][Medline]
Oden, K. L., Gladysheva, T. B., and Rosen, B. P. (1994). Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione. Mol. Microbiol. 12, 301306.[ISI][Medline]
Okayasu, R., Takahashi, S., Sato, H., Kubota, Y., Scolavino, S., and Bedford, J. S. (2003). Induction of DNA double strand breaks by arsenite: Comparative studies with DNA breaks induced by X-rays. DNA Repair 2, 309314.[CrossRef][ISI][Medline]
Rosen, B. P. (2002). Transport and detoxification systems for transition metals, heavy metals and metalloids in eukaryotic and prokaryotic microbes. Comparative Biochem. Physiol. A Molec. Integ. Physiol. 133, 689693.[CrossRef][ISI]
Rossman, T. G., Uddin, A. N., and Burns, F. J. (2004). Evidence that arsenite acts as a cocarcinogen in skin cancer. Toxicol. Appl. Pharmacol. 198, 394404.[CrossRef][ISI][Medline]
Schwerdtle, T., Walter, I., Mackiw, I., and 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, 967974.
Shiobara, Y., Ogra, Y., and Suzuki, K. T. (2001). Animal species difference in the uptake of dimethylarsinous acid (DMA(III)) by red blood cells. Chem. Res. Toxicol. 14, 14461452.[CrossRef][ISI][Medline]
Smith, A. H., Hopenhayn-Rich, C., Bates, M. N., Goeden, H. M., Hertz-Picciotto, I., Duggan, H. M., Wood, R., Kosnett, M. J., and Smith, M. T. (1992). Cancer risks from arsenic in drinking water. Environ. Health Perspect. 97, 259267.[ISI][Medline]
Styblo, M., Del Razo, L. M., Vega, L., Germolec, D. R., LeCluyse, E. L., Hamil, G. A., Reed, W., Wang, C., Cullen, W. R., and Thomas, D. J. (2000). Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch. Toxicol. 74, 289299.[CrossRef][ISI][Medline]
Tchounwou, P. B., Wilson, B., and Ishaque, A. (1999). Important considerations in the development of public health advisories for arsenic and arsenic-containing compounds in drinking water. Rev. Environ. Health 14, 211229.[Medline]
Wu, M. M., Chiou, H. Y., Wang, T. W., Hsueh, Y. M., Wang, I. H., Chen, C. J., and Lee, T. C. (2001). Association of blood arsenic levels with increased reactive oxidants and decreased antioxidant capacity in a human population of northeastern Taiwan. Environ. Health Perspect. 109, 10111017.[ISI][Medline]
Yager, J. W., and Wiencke, J. K. (1997). Inhibition of poly(ADP-ribose) polymerase by arsenite. Mutat. Res. 386, 345351.[CrossRef][ISI][Medline]
Yamanaka, K., Takabayashi, F., Mizoi, M., An, Y., Hasegawa, A., and Okada, S. (2001). Oral exposure of dimethylarsinic acid, a main metabolite of inorganic arsenics, in mice leads to an increase in 8-oxo-2'-deoxyguanosine level, specifically in the target organs for arsenic carcinogenesis. Biochem. Biophys. Res. Commun. 287, 6670.[CrossRef][ISI][Medline]
Yamanaka, K., Kato, K., Mizoi, M., An, Y., Takabayashi, F., Nakano, M., Hoshino, M., and Okada, S. (2004). The role of active arsenic species produced by metabolic reduction of dimethylarsinic acid in genotoxicity and tumorigenesis. Toxicol. Appl. Pharmacol. 198, 385393.[CrossRef][ISI][Medline]
Yompakdee, C., Bun-ya, M., Shikata, K., Ogawa, N., Harashima, S., and Oshima, Y. (1996). A putative new membrane protein, Pho86p, in the inorganic phosphate uptake system of Saccharomyces cerevisiae. Gene 171, 4147.[CrossRef][ISI][Medline]
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