Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Jul 9.
Published in final edited form as: Toxicol Mech Methods. 2013 Feb 6;23(6):389–395. doi: 10.3109/15376516.2012.762570

Oxidative DNA damage after acute exposure to arsenite and monomethylarsonous acid in biomethylation-deficient human cells

Ruben Orihuela 1, Chikara Kojima 1, Erik J Tokar 1, Rachel J Person 1, Yuanyuan Xu 1, Wei Qu 1, Michael P Waalkes 1
PMCID: PMC12239147  NIHMSID: NIHMS2088185  PMID: 23301828

Abstract

The carcinogen inorganic arsenic (iAs) undergoes biomethylation (BMT) in some cells. The methylated metabolite, monomethylarsonous (MMA3+), may cause oxidative DNA damage (ODD). With chronic iAs exposure, BMT-competent cells show ODD while BMT-deficient do not. To further define these events, we studied ODD produced by acute iAs or MMA3+ in the BMT-deficient human prostate cell line, RWPE-1. ODD, measured by the immuno-spin trapping method, was assessed after exposure to iAs or MMA3+ alone, with the arsenic BMT inhibitor selenite or after glutathione (GSH) depletion. The expression of oxidative stress-related genes (HO-1, SOD-1, SOD-2, Nrf2 and Keap-1) was also assessed. Exposure to iAs at 24 h (0–20 μM), stimulated ODD only at levels above the LC50 of a 48 h exposure (17 μM). If iAs induced ODD, it also activated oxidative stress-related genes. Selenium did not alter iAs-induced ODD. MMA3+ at 24 h (0–0.5 μM) caused ODD at levels below the LC50 of a 48 h exposure (1.5 μM), which were greatly increased by GSH depletion but not selenite. MMA3+ induced ODD at levels not activating oxidant stress response genes. Overall, iAs induced ODD in BMT-deficient cells only at toxic levels. MMA3+ caused ODD at non-toxic levels, independently of cellular BMT capacity and in a fashion not requiring further BMT.

Keywords: Arsenic, immuno-spin trapping, reactive oxygen species

Introduction

Arsenic is a multisite carcinogen commonly found in drinking water (IARC, 2012; ROC, 2011). Exposure to this metalloid is recognized as a worldwide environmental health issue (IARC, 2012). Chronic exposure to inorganic arsenic (iAs) has been linked to lung, skin and urinary bladder cancer and is possibly associated with liver, kidney and prostate cancer (IARC, 2012; ROC, 2011).

iAs can be enzymatically methylated in some cells via arsenic (+3 oxidation state) methyltransferase (AS3MT) that uses S-adenosyl-L-methionine as the methyl-group donor (Hayakawa et al., 2005; Thomas et al., 2007). Arsenic biomethylation (BMT) was initially considered to be a detoxicating process because of the low toxicity of the ultimate product, dimethylarsinic acid (DMA5+) compared with that of the inorganic arsenicals (Sakurai et al., 2002). However, it is now clear that intermediate trivalent methylated arsenicals, such as methylarsonous acid (MMA3+), are up to 20 times more cytotoxic than iAs (Styblo et al., 2000). For this reason, the concept has emerged that arsenic BMT is a pathway to toxic activation because it produces toxic trivalent methylated arsenical intermediates (Gomez et al., 2005; Kojima et al., 2005, 2009; Sakurai et al., 2006). Indeed, we have demonstrated MMA3+ to be carcinogenic in mice after in utero exposure (Tokar et al., 2012). Several researchers have proposed that MMA3+ is the potential ultimate carcinogenic form of arsenic (Hirano et al., 2004; Kitchin, 2001) and it will induce malignant transformation in vitro (Bredfeldt et al., 2006). If the BMT of iAs is blocked with selenite, acquisition of a malignant phenotype in vitro is blocked (Kojima et al., 2009). However, the molecular mechanisms by which arsenicals in any form cause cancer are not fully understood.

Arsenic can stimulate production of reactive oxygen species (ROS) in target cells (Barchowsky et al., 1999; Shi et al., 2004) and ROS can attack key biomolecules, distort cell signaling and induce apoptosis disrupting physiological function (Tseng, 2004). This could contribute to its carcinogenic potential, as for instance, an oxidative attack on a macromolecule such as DNA has oncogenic implications. Indeed, arsenic exposure has also been associated with oxidative DNA damage (ODD). Multiple studies have shown that inorganic or methylated arsenicals increase the generation of oxidatively damaged DNA bases, such as 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxo-dG) (De Vizcaya-Ruiz et al., 2009; Eblin et al., 2006; Gomez et al., 2005; Jomova & Valko, 2011; Kessel et al., 2002). However, the formation of artifactual 8-oxo-dG during DNA isolation and preparation has been raised as a general concern for production of high background levels of 8-oxo-dG, detracting from methods using its assessment for measuring DNA radical formation and prompting a search for new methods (Ramirez et al., 2006, 2007). Recently, the immuno-spin trapping (IST) method for measuring ODD was developed (Ramirez et al., 2006, 2007). In this method, DNA radicals are trapped in situ by the spin-trap agent DMPO, which converts DNA radicals to stable DNA-nitrone adducts in place before DNA isolation, thus preventing the formation of isolation artifacts (Ramirez et al., 2006, 2007). This greatly facilitates accurate ODD analysis by providing a low background (Ramirez et al., 2006, 2007), and has been applied to ODD formed with arsenicals in cells during chronic exposure (Kojima et al., 2009).

In previous work, we exposed the established iAs BMT-deficient human prostate cell line (RWPE-1) as well as the BMT-competent liver cell line (TRL1215) to chronic low levels of iAs until malignant transformation occurred (Kojima et al., 2009). The liver and prostate are both potential targets of arsenic carcinogenesis in humans (IARC, 2012). The BMT-capable liver cells showed increases in ODD gradually starting at about 5 weeks of iAs exposure and peaking at 18 weeks, the approximate point of malignant transformation (Kojima et al., 2009). On the other hand, the BMT-deficient prostate cells never showed increases in ODD even when treated with iAs for 30 weeks, the approximate point of acquisition of malignant phenotype (Kojima et al., 2009). Thus, ODD may be a key event in initiating the carcinogenic process with iAs in cells where iAs undergoes BMT. However, iAs-induced ODD is not obligatory in all target cells of arsenic carcinogenesis for transformation.

Glutathione (GSH) is one of the most important antioxidant molecules maintaining cellular redox status (Valko et al., 2007), and helps protect against the cytolethality of arsenicals by scavenging the ROS (Sakurai et al., 2004). Furthermore, GSH depletion clearly enhances arsenic toxicity in various model systems (Hirano et al., 2004; Kojima et al., 2005; Roychowdhury et al., 2003). It is also evident that GSH is involved with the transport of iAs, which is effluxed as a tri-GSH conjugate by ABCC1 (Leslie et al., 2004). Thus, GSH plays a major role in arsenic toxicology.

Many studies have shown that acute DNA damage can be induced by iAs, although the ability to biomethylate arsenicals and the survivability at the levels of iAs used are often not realistically considered as factors in this outcome (Chai et al., 2007; Pi et al., 2005; Ruiz-Ramos et al., 2009). Such factor would have direct impact on the relevance at any acute findings to carcinogenic mechanisms. In the present study, we test the hypothesis that ODD can occur in BMT-deficient cells with iAs but only at levels that will kill most of the cells. Further, we test the proximate BMT metabolite, MMA3+, as a primary factor in arsenical-induced ODD. The effects of GSH depletion and inhibition of any minimal residual BMT with selenite was assessed. Activation of oxidant stress response of genes during this process was also studied.

Methods

Chemicals and reagents

L-Buthionine-sulfoximine (BSO), sodium m-arsenite (NaAsO2) and sodium selenite (Na2SeO3) were obtained from Sigma-Aldrich (St. Louis, MO). DMPO was purchased from Alexis Biochemicals (San Diego, CA). MMA3+ was a gracious gift from Dr William R. Cullen, Department of Chemistry, University of British Columbia.

Cells and culture conditions

The RWPE-1 cell line is a normal human prostate epithelial cell line that is known from prior work to very poorly methylate iAs (Kojima et al., 2009). Cells were cultured in keratinocyte serum-free medium (Gibco/Invitrogen, Grand Island, NY) containing 50 μg/mL bovine pituitary extract (Gibco/Invitrogen) and 5 ng/mL epidermal growth factor (Gibco/Invitrogen) and 1% antibiotic/antimycotic mixture (100 U/mL penicillin and 100 μg/mL streptomycin). Cells were grown at 37 ◦C in a humidified atmosphere of 5% CO2 and 95% air.

Cell viability

Cells were exposed to various concentrations of iAs or MMA3+ for 48 h. Cell viability was assessed using the trypan blue dye exclusion method as previously described (Tokar et al., 2010). Cytolethality is expressed as lethal concentration in 50% of the cells (LC50) and is derived using a wide range of concentrations from the linear portion of three or more separate viability curves per treatment group to enable generation of descriptive statistics (mean +/− SD). LC50 were determined at a later time point (48 h) than ODD (24 h), so that ODD could be measured in cells that could potentially subsequently die from their arsenical exposures. The impact of selenite and BSO treatments on the arsenical LC50 values were determined. LC50 values are listed at appropriate points in the text.

Determination of ODD

Cells were grown in media containing iAs (0, 5, 10 and 20 μM) or MMA3+ (0, 0.25, 0.5 and 1 μM) for 24 h in the presence or absence of BSO (50 μM) to deplete GSH or selenite (1 μM) to block iAs BMT prior to ODD measurement. DNA was purified by standard procedures and ODD was measured using the IST method (Ramirez et al., 2006, 2007) as applied to cells (Kojima et al., 2009). ODD is measured as DNA-nitrone adducts using DMPO in this assay (Ramirez et al., 2006, 2007) and adjusted to control set to as 100%. The primary antibody was anti-DMPO polyclonal serum (1:10 000; Cayman Chemicals, Ann Arbor, MI) and the secondary antibody was goat anti-rabbit igG conjugated to horseradish peroxidase (1:10 000; Pierce, Rockford, IL). Data represent nine separate determinations per concentration.

Gene expression

Gene expression at the transcript level was quantified by RT-PCR analysis by the method described by Woods et al. (2009). Briefly, total RNA was extracted from cells using TRIzol agent (Invitrogen, Carlsbad, CA), followed by purification with RNeasy columns (Qiagen, Valencia, CA). RNA was reverse transcribed with MuLV reverse transcriptase and oligo-dT primers. Forward and reverse primers were designed with ABgene Primer Express software (Foster City, CA, USA) as follows: nuclear factor (erythroid-derived 2)-like 2 (Nrf2; 5′-CCATGCCTTCTTCCACGAA-3′ and 5′-AGGGC CCATGGATTTCAGTT-3′); heme oxygenase-1 (HO-1; 5′-ATCATGGCTTGGCCTACATTG-3′ and 5′-CACGGATG TGCACCTCCTT-3′); superoxide dismutase 1 (SOD1; 5′-TGT ACCACTGCAGGACCTCATT-3′ and 5′-GGTCTCCAACAT GCCTCTCTTC-3′); superoxide dismutase 2 (SOD2; 5′-ACATCAACGCGCAGATCATG-3′ and 5′ TCGGTGACG TTCAGGTTGTTC-3′); Kelch-like ECH-associated protein 1 (Keap1; 5′-CCTCTGGCCGGGTAATAGG-3′ and 5′-CCCCT CCCAGGTATCCAAGA-3′); β-actin (5′-TCCTCCTGAGCG CAAGTACTCT-3′ and 5′-GCTCAGTAACAGTCCGCCTAG AA-3′); glyceraldehyde 3-phosphate dehydrogenase (GADPH, 5′-CCTCCCGCTTCGCTCTCT-3′ and 5′-CTGGCGACGCA AAAGAAGA-3′). The SYBR green PCR master mix (Applied Biosystems, Cheshire) was used for RT-PCR analysis. The cycle time values of the selected genes were first normalized with β-actin and GADPH of the same sample, and then the relative expression was calculated and expressed as percent control with control set to 100%.

Statistical analysis

The data are expressed as the mean ±SD and represent three or more independent determinations. Statistical significance was determined by Student’s t-test or analysis of variance followed by Dunnett’s multiple comparison test, as appropriate. All tests were two-sided and a p value ≤0.05 was considered to indicate statistical significance.

Results

Acute cytolethality of iAs and MMA3+ in BMT-deficient cells

BMT-deficient prostate cells were exposed to iAs or MMA3+ and cytolethality was assessed at 48 h. The LC50 for iAs in BMT-deficient cells was 17.1 ± 1.9 μM compared to 1.5 ± 0.3 μM for MMA3+. Thus, the methylated metabolite was much more cytotoxic.

ODD induced by acute iAs or MMA3+ exposure

The cells were acutely exposed to iAs and ODD was assessed at 24 h (Figure 1), allowing for the examination of ODD in cells that would potentially die at a later point. After acute exposure to iAs, there was no evidence of ODD until exposure levels (20 μM, Figure 1a) exceeded the 48 h LC50 concentration (17.1 μM). Thus, for ODD to occur in these BMT-deficient cells the concentration of iAs had to be subsequently lethal to the majority of cells. Acute exposure to MMA3+ (24 h) induced significant increases in ODD in these BMT-deficient cells (Figure 1b) at concentrations (0.25 and 0.5 mM) well below the LC50 of a 48 h exposure (1.5 μM). The increase in ODD was modest but concentration related.

Figure 1.

Figure 1.

Open in a new tab

ODD in BMT-deficient cells. The cells were exposed to (a) 0, 5.0, 10.0 or 20 μM iAs or (b) 0, 0.25 or 0.5 μM MMA3+ for 24 h. ODD is measured as DNA-nitrone adducts in this assay and adjusted to controls set to 100%. For experimental details see “Methods” section. Results are expressed as the mean ±SD (n = 9). *Significant difference (p<0.05) in comparison to matched control (vehicle).

Effect of BMT inhibition or GSH depletion on ODD

Selenium effectively inhibits iAs BMT (Walton et al., 2003). Indeed, in previous work, we found that sodium selenite (1 μM) blocked ODD and subsequent malignant transformation induced by chronic exposure to low concentrations of iAs (0.5 μM) in BMT-competent liver cells (Kojima et al., 2009). Thus, we studied the effect of selenite co-exposure with iAs in these BMT-deficient cells in order to be sure any possible BMT was blocked. The addition of selenite had no effect on iAs-induced ODD which still occurred only at levels of iAs (20 μM) in excess of the LC50 (Figure 2a). Addition of selenite did not significantly alter iAs-induced LC50 at 48 h of exposure (15.0 ± 2.0 mM).

Figure 2.

Figure 2.

Open in a new tab

ODD in BMT-deficient cells after acute iAs exposure with selenite or GSH depletion. The cells were exposed to 0, 5.0, 10.0 or 20 μM As in the presence of (a) sodium selenite or (b) GSH depletion for 24 h. ODD is measured as DNA-nitrone adducts in this assay and adjusted to controls set to 100%. For experimental details see “Methods” section. Results are expressed as the mean ±SD (n = 9). *Significant difference (p<0.05) in comparison to control (vehicle).

The effect of GSH depletion was also studied. iAs-treated GSH-depleted cells showed ODD increases at 10 μM iAs but the levels were not significantly different from iAs alone (Figure 2b). Thus, this could be viewed as GSH depletion enhancing iAs-induced ODD to a certain extent. However, GSH depletion did decrease iAs LC50 to 2.1 ± 1.2 mM at 48 h so levels required for ODD were still in excess of the LC50.

The addition of selenite had no effect on MMA3+-induced ODD in these BMT-deficient cells (Figure 3a). This, together with the BMT-deficiency of the cells in question, clearly indicates that BMT is not needed for MMA3+ to be active in inducing ODD. Selenite addition did not alter the LC50 of MMA3+ (1.4 ±0.6 μM) compared to MMA3+ alone (1.5 ±0.3 μM). For MMA3+ exposure in GSH depleted BMT-deficient cells there was a clear increase in ODD (Figure 3b), which was MMA3+ concentration related. The LC50 of MMA3+ at 48 h was decreased by GSH depletion to 0.4 ±0.2 μM, a concentration still higher than the minimum able to generate ODD (i.e. 0.25 μM) in non-GSH depleted cells.

Figure 3.

Figure 3.

Open in a new tab

ODD in BMT-deficient cells after acute MMA3+exposure with selenite or GSH depletion. The cells were exposed to 0, 0.25 or 0.5 μM MMA3+ in (a) the presence of sodium selenite or (b) GSH depletion for 24 h. ODD is measured as DNA-nitrone adducts in this assay and adjusted to controls set to 100%. For experimental details see “Methods” section. Results are expressed as the mean ±SD (n = 9). *Significant difference (p<0.05) in comparison to control (vehicle). †Significant difference in comparison to arsenic-treated cells.

Expression of genes related to oxidative stress response

Arsenical exposure can cause oxidative stress, resulting in generation of ROS and stimulation of the oxidative response, events clearly relevant to ODD. Thus, we examined the expression of various genes linked to oxidative stress response (SOD-1, SOD-2, Keap-1, Nrf2 and HO-1), after acute exposure to iAs or MMA3+ at a sub-toxic levels that might indicate oxidant stress below the threshold for ODD production.

When the prostate cells were exposed to a non-cytotoxic concentration of iAs (10 μM), HO-1 expression was markedly upregulated (Figure 4a). GSH depletion caused even higher expression, consistent with ODD data (Figure 2b). On the other hand, exposure to 0.25 μM MMA3+ had no effect on HO-1 expression itself, but when combined with cellular GSH depletion there were large increases in HO-1 expression (Figure 4b). With regard to SOD-1, SOD-2, Keap-1 and Nrf2, in general, iAs (10 μM) caused significant increases in expression while GSH depletion did not cause further increases. MMA3+ exposure alone (0.25 μM) did not induce this battery of oxidant response genes, but when MMA3+ was combined with GSH depletion significant increases occurred in SOD-1, SOD-2, Keap-1 and Nrf2 expression (Figure 5).

Figure 4.

Figure 4.

Open in a new tab

Expression of HO-1 after iAs or MMA3+ exposure with or without GSH depletion to (a) 10 μM iAs or (b) 0.25 μM MMA3+ with or with GSH depletion for 24 h. Expression was quantified by RT-PCR with control set at 100%. For experimental details see “Methods” section. Note broken y-axis in (a) and (b). Results are expressed as the mean ±SD (n = 3). *Significant difference (p<0.05) in comparison to control (vehicle). †Significant difference in comparison to matched arsenical-treated cells.

Figure 5.

Figure 5.

Open in a new tab

Expression of antioxidant response genes after iAs or MMA3+ exposure with or without GSH depletion (a) 10 μM iAs or (b) 0.25 μM MMA3+ or with GSH depletion for 24 h. Expression was quantified by RT-PCR with control set to 100%. For experimental details see “Methods” section. Results are expressed as the mean ± SD (n = 3). *Significant difference (p<0.05) in comparison to control (vehicle). †Significant difference in comparison to matched arsenical-treated cells.

Discussion

In a prior work, the BMT-deficient prostate cell line used in this study was chronically exposed for up to 30 weeks to iAs and showed no evidence of ODD, even though it acquired a malignant phenotype (Kojima et al., 2009). When liver cells, which are adept at iAs BMT, were likewise chronically exposed to iAs they showed a gradual build-up of ODD to the point of malignant transformation, which occurred approximately 40% more rapidly than in the prostate cells (Kojima et al., 2009). These data provide compelling evidence that during chronic exposure, iAs BMT is obligatory for ODD to occur but this DNA damage is not always required for iAs-induced malignant transformation (Kojima et al., 2009). More recently, we found BMT-deficient human keratinocyte cells can also undergo malignant transformation with chronic iAs exposure in about 30 weeks (Sun et al., 2011). Again, these cells showed no increase in ODD during the process of iAs induced malignant transformation (Sun et al., 2011). Thus, a pattern seems to emerge where ODD is linked to BMT capacity and makes transformation occur more rapidly, but ODD is not absolutely required for malignant conversion. Similar to these findings, in the present study, acute iAs exposure of BMT-deficient prostate cells did not induce ODD unless the metalloid exceeded cytolethal levels. In these BMT-deficient cells, ODD was produced by iAs exposure only at levels which exceeded the LC50, making the relevance of this event dubious to the carcinogenic process. Again this observation infers that iAs-induced ODD formation in cells that show a survival rate relevant to cancer would be dependent on the BMT-ability of particular cells (Kojima et al., 2009; Sun et al., 2011). These factors should be considered in analyses of studies showing ODD with iAs.

The effect of MMA3+ on ODD in BMT-deficient cells is also illuminating. Even if ODD is not obligatory to malignantly transformation with all iAs-exposed cells, it appears to hasten the process (Kojima et al., 2009; Sun et al., 2011). In the present study, MMA3+ exposure caused ODD at levels that were well below those inducing cytolethality. For this reason, a comparative study between the malignant transformation of the BMT-deficient prostate cell line with iAs and MMA3+ should be considered as a priority, and is now on-going in our laboratory.

iAs has been shown to stimulate ROS production and can cause the inhibition of important antioxidant enzymes (Chouchane & Snow, 2001; Petrick et al., 2001; Samikkannu et al., 2003). Cells have developed different defense mechanisms against ROS, such as small non-enzymatic antioxidants molecules such as GSH or various antioxidant stress response genes. Interestingly, in the present work, although iAs exposure provoked an increase in various antioxidant response genes, such as SOD-2, HO-1, Keap-1 and Nrf2, this did not occur concurrently with the formation of ODD. Indeed, iAs ODD was only detected after reaching cytolethal concentrations apparently when the cellular defenses had been overwhelmed. On the other hand, several antioxidant response genes were upregulated after exposure to non-cytotoxic concentrations of iAs perhaps indicating that this response was adequate to defend against negative effect of non-lethal ROS.

In contrast, acute MMA3+ exposure did cause ODD formation in these cells at relatively low, non-toxic concentrations. Even though trivalent methylated arsenicals appear able to produce ROS more efficiently than iAs (Barchowsky et al., 1999; Gomez et al., 2005), these concentrations of MMA3+ did not increase the response of the antioxidant stress genes, suggesting differences in the capacity for cellular defense against MMA3+. Differences in the ROS formation and response between iAs and MMA3+ at the cellular level have been previously described (Eblin et al., 2006, 2008). Using a BMT-deficient human urothelial cell line (UROtsa) it has been proposed that iAs and MMA3+ may generate different ROS species and activate different antioxidant defense responses by distinct mechanisms (Eblin et al., 2006). Nonetheless, the exact linkage between antioxidant response gene activation, ODD and exposure to iAs or MMA3+ needs to be clarified.

Another mechanism by which the cell can protect against arsenical toxicity is to efflux the metalloid via conjugation with GSH by the enzyme glutathione S-transferase (GST) (Hirano et al., 2004; Kobayashi & Hirano, 2008; Lee et al., 1989). Increasing cellular GSH levels and GST levels or activity provide tolerance to arsenic (Lee et al., 1989). In our study, GSH depletion increased the cytolethality of iAs and MMA3+, confirming that GSH plays an important role protecting the cells against the toxic effects of arsenical exposure. Overall, these results agree with prior work that indicates that GSH likely may scavenge the ROS produced by iAs and MMA3+ and helps efflux arsenicals (Leslie et al., 2004), and that GSH depletion markedly increases arsenical-induced cytolethality (Hirano et al., 2004; Kojima et al., 2005). Results from this study also add the growing body of literature that finds trivalent methylated arsenicals more toxic than iAs (Hughes, 2002; Petrick et al., 2000, 2001; Shaw et al., 2003; Styblo et al., 2000), although the mechanism is not well understood. It is known that MMA3+ is one of the most genotoxic and toxic of all iAs metabolites, and even though it is an intermediate product of iAs metabolism, MMA3+ is believed by some to be responsible for most of the toxic effects of iAs (Valenzuela et al., 2005), which presumably would include carcinogenesis, as exemplified by tumor formation in mice (Tokar et al., 2012) and cellular malignant transformation in vitro (Bredfeldt et al., 2006).

Even though the prostate cells studied here are BMT deficient, in this work we used sodium selenite which is effective at blocking iAs methylation (Kojima et al., 2009; Walton et al., 2003). Acute exposure to iAs with selenite co-exposure did not alter the cytolethality or the ODD induction from iAs alone, fortifying the concept that only at cytolethal levels was iAs able to produce ODD in these BMT-deficient cells. MMA3+ and selenite co-exposure did not alter the methylated intermediates’ ability to induce ODD at sub-cytolethal levels. The fact that selenite did not alter ODD induction by MMA3+ suggests that MMA3+ alone can cause ODD without any additional BMT being necessary.

Conclusion

In conclusion, the ability of acute iAs and MMA3+ exposure to produce ODD in iAs BMT-deficient cells was studied. Previously, we found that after chronic exposure to iAs, BMT is required for ODD to occur and also accelerates malignant transformation (Kojima et al., 2009). In this study, we have demonstrated that ODD formation after acute iAs exposure in BMT-deficient cells can occur, but only after exposure to concentrations of iAs that exceed the LC50, making relevance to carcinogenesis dubious. Thus, the relevance of finding iAs-induced ODD in acute studies in cells that do not methylate iAs may be of questionable value and should be critically evaluated. MMA3+ was able to cause ODD in BMT-deficient cells at non-cytolethal levels suggesting it can cause ODD and with no further methylation. Overall, this study points out that acute in vitro studies with arsenic-induced DNA damage needs to be considered in the light of cell survival to be relevant to the carcinogenic process.

Acknowledgements

The authors thank Drs Nigel Walker, John Bucher, Chris McPherson and Mathilde Triquigneaux for critical review of this manuscript. The authors also thank Matthew W. Bell for aid with the graphics.

Footnotes

Declaration of interest

The authors report no conflicts of interest.

This article may be the work product of an employee or a group of employees of the NIEHS, NIH; however, the statements contained herein do not necessarily represent the statements, opinions or conclusions of the NIEHS, NIH or the US Government. The content of this publication does not necessarily reflect the views or the policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

References

  1. Barchowsky A, Klei LR, Dudek EJ, et al. (1999). Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite. Free Radic Biol Med 27:1405–12. [DOI] [PubMed] [Google Scholar]
  2. Bredfeldt TG, Jagadish B, Eblin KE, et al. (2006). Monomethylarsonous acid induces transformation of human bladder cells. Toxicol Appl Pharmacol 216:69–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chai CY, Huang YC, Hung WC, et al. (2007). Arsenic salt-induced DNA damage and expression of mutant p53 and COX-2 protein in SV-40 immortalized human uroepithelial cells. Mutagenesis 22:403–8. [DOI] [PubMed] [Google Scholar]
  4. Chouchane S, Snow ET. (2001). In vitro effect of arsenical compounds on glutathione-related enzymes. Chem Res Toxicol 14:517–22. [DOI] [PubMed] [Google Scholar]
  5. De Vizcaya-Ruiz A, Barbier O, Ruiz-Ramos R, Cebrian ME. (2009). Biomarkers of oxidative stress and damage in human populations exposed to arsenic. Mutat Res 674:85–92. [DOI] [PubMed] [Google Scholar]
  6. Eblin KE, Bredfeldt TG, Gandolfi AJ. (2008). Immortalized human urothelial cells as a model of arsenic-induced bladder cancer. Toxicology 248:67–76. [DOI] [PubMed] [Google Scholar]
  7. Eblin KE, Bowen ME, Cromey DW, et al. (2006). Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture. Toxicol Appl Pharmacol 217:7–14. [DOI] [PubMed] [Google Scholar]
  8. Gomez SE, del Razo LM, Munoz Sanchez JL. (2005). Induction of DNA damage by free radicals generated either by organic or inorganic arsenic (AsIII, MMAIII, and DMAIII) in cultures of B and T lymphocytes. Biol Trace Elem Res 108:115–26. [DOI] [PubMed] [Google Scholar]
  9. Hayakawa T, Kobayashi Y, Cui X, Hirano SA. (2005). New metabolic pathway of arsenite: arsenite-glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Arch Toxicol 79:183–91. [DOI] [PubMed] [Google Scholar]
  10. Hirano S, Kobayashi Y, Cui X, et al. (2004). The accumulation and toxicity of methylated arsenicals in endothelial cells: important roles of thiol compounds. Toxicol Appl Pharmacol 198:458–67. [DOI] [PubMed] [Google Scholar]
  11. Hughes MF. (2002). Arsenic toxicity and potential mechanisms of action. Toxicol Lett 133:1–16. [DOI] [PubMed] [Google Scholar]
  12. IARC (2012). International Agency for Research on Cancer: a review of human carcinogens-part C: arsenic, metals, fibres and dust. Vol. 100C. Lyon: IARC Press, 41–96. [Google Scholar]
  13. Jomova K, Valko M. (2011). Advances in metal-induced oxidative stress and human disease. Toxicology 283:65–87. [DOI] [PubMed] [Google Scholar]
  14. Kessel M, Xian Liu S, Xu A, et al. (2002). Arsenic induces oxidative DNA damage in mammalian cells. Mol Cell Biochem 234/235:301–8. [PubMed] [Google Scholar]
  15. Kitchin KT. (2001). Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol 172:249–61. [DOI] [PubMed] [Google Scholar]
  16. Kobayashi Y, Hirano S. (2008). Effects of endogenous hydrogen peroxide and glutathione on the stability of arsenic metabolites in rat bile. Toxicol Appl Pharmacol 232:33–40. [DOI] [PubMed] [Google Scholar]
  17. Kojima C, Ramirez DC, Tokar EJ, et al. (2009). Requirement of arsenic biomethylation for oxidative DNA damage. J Natl Cancer Inst 101:1670–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kojima C, Sakurai T, Waalkes MP, Himeno S. (2005). Cytolethality of glutathione conjugates with monomethylarsenic or dimethylarsenic compounds. Biol Pharm Bull 28:1827–32. [DOI] [PubMed] [Google Scholar]
  19. Lee TC, Wei ML, Chang WJ, et al. (1989). Elevation of glutathione levels and glutathione S-transferase activity in arsenic resistant Chinese hamster ovary cells. In Vitro Cell Dev Biol 25:442–4. [DOI] [PubMed] [Google Scholar]
  20. Leslie EM, Haimeur A, Waalkes MP. (2004). Arsenic transport by the human multidrug resistance protein 1 (MRP1/abcc1). Evidence that a tri-glutathione conjugate is required. J Biol Chem 279:32700–8. [DOI] [PubMed] [Google Scholar]
  21. Petrick JS, Ayala-Fierro F, Cullen WR, et al. (2000). Monomethylarsonous acid [MMA (III)] is more toxic than arsenite in Change human hepatocytes. Toxicol Appl Pharmacol 163:203–7. [DOI] [PubMed] [Google Scholar]
  22. Petrick JS, Jagadish B, Mash EA, Aposhian HV. (2001). Monomethylarsonous acid (MMA(III)) and arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate dehydrogenase. Chem Res Toxicol 14:651–6. [DOI] [PubMed] [Google Scholar]
  23. Pi J, He Y, Bortner C, et al. (2005). Low level, long-term inorganic arsenite exposure causes generalized resistance to apoptosis in cultured human keratinocytes: potential role in skin co-carcinogenesis. Int J Cancer 116:20–6. [DOI] [PubMed] [Google Scholar]
  24. Ramirez DC, Gomez-Mejiba SE, Mason RP. (2006). Immuno-spin trapping of DNA radicals. Nat Methods 3:123–7. [DOI] [PubMed] [Google Scholar]
  25. Ramirez DC, Gomez-Mejiba SE, Mason RP. (2007). Immuno-spin trapping analyses of DNA radicals. Nat Protoc 2:512–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. ROC (2011). Arsenic and inorganic arsenic compounds. Report on Carcinogens (ROC). 12th ed. Research Triangle Park (NC): US Department of Health and Human Services, Public Health Services, National Toxicology Program, 50–4. [Google Scholar]
  27. Roychowdhury S, Wolf G, Keilhoff G, Horn TFW. (2003). Cytosolic and mitochondrial glutathione in microglial cells are differently affected by oxidative/nitrosative stress. Nitric Oxide 8:39–47. [DOI] [PubMed] [Google Scholar]
  28. Ruiz-Ramos R, Lopez-Carrillo L, Rios-Perez AD, et al. (2009). Sodium arsenite induces ROS generation, DNA oxidative damage, HO-1 and c-Myc proteins, NF-kappaB activation and cell proliferation in human breast cancer MCF-7 cells. Mutat Res 674:109–15. [DOI] [PubMed] [Google Scholar]
  29. Sakurai T, Kojima C, Kobayashi Y, et al. (2006). Toxicity of a trivalent organic arsenic compound, dimethylarsinous glutathione in a rat liver cell line (TRL 1215). Br J Pharmacol 149:888–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sakurai T, Kojima C, Ochiai M, et al. (2004). Cellular glutathione prevents cytolethality of monomethylarsonic acid. Toxicol Appl Pharmacol 195:129–41. [DOI] [PubMed] [Google Scholar]
  31. Sakurai T, Qu W, Sakurai MH, Waalkes MP. (2002). A major human arsenic metabolite, dimethylarsinic acid, requires reduced glutathione to induce apoptosis. Chem Res Toxicol 15:629–37. [DOI] [PubMed] [Google Scholar]
  32. Samikkannu T, Chen CH, Yih LH, et al. (2003). Reactive oxygen species are involved in arsenic trioxide inhibition of pyruvate dehydrogenase activity. Chem Res Toxicol 16:409–14. [DOI] [PubMed] [Google Scholar]
  33. Shaw JR, Glaholt SP, Greenberg NS, et al. (2003). Acute toxicology of arsenic to Daphnia pulex: influence of organic functional groups and oxidation state. Environ Toxicol Chem 26:1532–7. [DOI] [PubMed] [Google Scholar]
  34. Shi H, Shi X, Liu KJ. (2004). Oxidative stress and apoptosis in metal ion-induced carcinogenesis. Free Radic Biol Med 37:582–93. [DOI] [PubMed] [Google Scholar]
  35. Styblo M, Del Razo LM, Vega L, et al. (2000). Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rats and human cells. Arch Toxicol 74:289–99. [DOI] [PubMed] [Google Scholar]
  36. Sun Y, Kojima C, Chignell CF, et al. (2011). Arsenic predisposes skin keratinocytes to UV-induced oxidative DNA damage yet enhances their survival. Toxicol Appl Pharmacol 255:242–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thomas DJ, Li J, Waters SB, et al. (2007). Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals. Exp Biol Med 232:3–13. [PMC free article] [PubMed] [Google Scholar]
  38. Tokar EJ, Diwan BA, Thomas DJ, Waalkes MP. (2012). Tumors and proliferative lesions in adult offspring after maternal exposure to methylarsonous acid during gestation in CD1 mice. Arch Toxicol 86:975–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tokar EJ, Qu W, Liu J, et al. (2010). Arsenic-specific stem cell selection during malignant transformation. J Natl Cancer Inst 102:638–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tseng CH. (2004). The potential biological mechanisms of arsenic-induced diabetes mellitus. Toxicol Appl Pharmacol 197:67–83. [DOI] [PubMed] [Google Scholar]
  41. Valenzuela OL, Cruz-Gonzalez BL, Garcia-Vargas GG, et al. (2005). Urinary trivalent methylated arsenic species in a population chronically exposed to inorganic arsenic. Environ Health Perspect 113:250–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Valko M, Leibfritz D, Moncol J, et al. (2007). Free Radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84. [DOI] [PubMed] [Google Scholar]
  43. Walton FS, Waters SB, Jolley SL, et al. (2003). Selenium compounds modulate the activity of recombinant rat AsIII-methyltransferase and the methylation of arsenite by rat and human hepatocytes. Chem Res Toxicol 16:261–5. [DOI] [PubMed] [Google Scholar]
  44. Woods CG, Fu J, Xue P, et al. (2009). Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages. Toxicol Appl Pharmacol 238:27–36. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES