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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Toxicol Appl Pharmacol. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2783778
NIHMSID: NIHMS145527

Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

Abstract

Malignant transformation was demonstrated in UROtsa cells following 52 wk exposure to 50 nM monomethylarsonous acid (MMAIII); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMAIII [URO-MSC(+)] and after subsequent removal of MMAIII [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMAIII, URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12 wk exposure; in particular, a significant increase in DNA single strand breaks at 12 wk exposure consistently elevated through 52 wk. The persistence of DNA damage in URO-MSC cells was assessed after a 2 wk removal of MMAIII. URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36 wk MMAIII exposure, suggesting the presence of MMAIII is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMAIII results in: the induction of DNA damage that remains elevated upon removal of MMAIII; increased levels of ROS that play a role in MMAIII induced-DNA damage; and decreased PARP activity in the presence of MMAIII.

Keywords: Arsenic, Monomethylarsonous acid, Bladder cancer, UROtsa, DNA damage

1. Introduction

Numerous research efforts are focused on determining the critical role of the arsenic metabolite, monomethylarsonous acid (MMAIII), in the induction of bladder carcinogenesis. Multiple mechanisms have been implicated in the MMAIII-induced transformation of the human bladder urothelial cell model, UROtsa, following chronic, low-level exposure. Acute exposure to MMAIII have been shown to generate reactive oxygen species (ROS) resulting in oxidative stress and perturbations of cell signaling pathways involved in tumorigenesis (Eblin et al., 2006, 2007). However, the ability of chronic, low-level MMAIII to induce DNA damage and alter DNA repair activity has not been extensively studied. The research herein was carried out to determine the ability of chronic, low-level MMAIII to induce DNA damage, generate elevated levels of ROS implicated in damaging DNA, evaluate the specific type of DNA damage occurring, and investigate the effect of MMAIII in the alteration of DNA repair activity. Of particular importance was the assessment of these parameters in the presence and absence of MMAIII following chronic, low-level exposures. The ability of MMAIII exposure to permanently alter normal cell function after the subsequent removal of an environmentally relevant exposure, exemplifies the carcinogenic potential of the biomethylated metabolite, MMAIII.

Inorganic arsenic is a known human carcinogen associated with pleiotropic toxicities including cancers of the skin, lung, liver, and bladder, in addition to non-carcinogenic health effects (Chen et al., 1988; Brown and Ross, 2002; Marshall et al., 2007). Sources of inorganic arsenic exposure include air, water, and food from both natural and anthropogenic sources (Ramirez-Solis et al., 2004). Following exposure, inorganic arsenic undergoes a series of biotransformation processes alternating between reduction of pentavalent arsenic to trivalent arsenic and sequential methylation steps resulting in monomethylarsenic acid (MMAIII,V) and dimethylarsenic acid (DMAIII,V) metabolites (Aposhian, 1997; Healy et al., 1998; Vahter, 2002). Of particular interest in these studies, the toxicity of the trivalent metabolite, MMAIII, is 20 times greater than that of arsenite (AsIII) (Styblo et al., 2002; Bredfeldt et al., 2006). The level of MMAIII used in this study [50 nM MMAIII or approximately 3.75 ppb] represents an environmentally relevant exposure level (the current EPA inorganic arsenic standard for drinking water is set at 10 ppb), and a concentration which falls within the range of MMAIII detected in the urine of humans exposed to inorganic arsenic in their water supply (Aposhian et al., 2000; Mandal et al., 2001, 2004). Despite the classification of arsenic as a human carcinogen, the exact mechanisms of arsenic carcinogenicity are not well understood due, in part, to the multitude of complex factors attributed to playing a role in the induction of carcinogenesis.

Numerous mechanisms have been implicated in arsenical-induced malignant transformation. Exposure to MMAIII has been linked to the alteration of signaling events including processes of the MAPK, NFkB, JAK-STAT, AP-1, and WNT signaling pathways (Ludwig et al., 1998; Simeonova et al., 2001; Ahlborn et al., 2008; Tun-Kyi et al., 2008; Jensen et al., 2009). Other mechanisms include sulfhydryl depletion, altered DNA repair, the formation of DNA adducts and/or damage as a result of oxidative stress, and the alteration of DNA methylation and histone modifications in promoter regions (National Research Council, 2001; Bredfeldt et al., 2004; Jensen et al., 2008, 2009). In particular, chronic exposure to low-level MMAIII in UROtsa cells has been shown to produce oxidative stress as a result of elevated ROS. ROS have been demonstrated to play a role in altered signaling pathways involved in proliferation and anchorage-independent growth; all of which contribute to the MMAIII-induced malignant transformation of UROtsa cells (Eblin et al., 2007, 2008, and 2009).

Several lines of evidence have shown that inorganic and organic arsenicals can produce ROS and free radicals such as superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (·OH), singlet oxygen (1O2), and lipid peroxyl radical, LOO. (Shi et al., 2004a, 2004b; Eblin et al., 2006; 2008). The generation of ROS can cause structural alterations in DNA, affect cytoplasmic and nuclear signal transduction pathways, and modulate activity of proteins involved in cell proliferation, differentiation, and apoptosis (Wiseman and Halliwell, 1996; Leonard et al., 2004; Beyersmann and Hartwig, 2008). With respect to genotoxicity, the application of radical scavengers has revealed the involvement of arsenical-induced ROS in the induction of DNA damage in arsenite exposed rats (Ramanathan et al., 2003). ROS may have a significant role in the incidence of DNA damage following arsenical exposure, providing evidence against a direct genotoxic mechanism of action involved in arsenical-induced cancer. Previous research has demonstrated that ROS can act as intermediates in the DNA damaging activity in supercoiled phiX174 DNA and DNA in peripheral human lymphocytes following acute, low-level MMAIII exposure (Nesnow et al., 2002; Schoen et al., 2004). However, the ability of the biomethylated metabolite, MMAIII, to induce a genotoxic insult following chronic, low-level exposure in a human bladder model has not been adequately examined.

The ability of trivalent arsenicals to attach to zinc binding structures prevalent in DNA repair proteins may provide a target for arsenical exposure, resulting in the alteration/inhibition of critical DNA repair enzymes involved in maintaining genomic integrity (Kitchin et al., 2008). DNA damage in the form of single strand breaks is repaired through the base excision repair process. A critical repair enzyme involved in base excision repair is poly (ADP-ribose) polymerase (PARP) (Caldecott, 2007). The capacity of chronic, low-level MMAIII exposure to decrease activity of such repair enzymes in the presence of a genotoxic insult, such as ROS, could be a potential mechanism in arsenical-induced carcinogenesis.

The primary route of arsenic elimination is urinary excretion; therefore, the bladder is a key target organ of toxicity as a result of exposure to both inorganic arsenic and multiple biomethylated metabolites (Tapio and Grosche, 2006). The immortalized, non-tumorigenic human urothelial cell line, UROtsa, and the MMAIII-induced malignant transformed variant, URO-MSC, were used as in vitro models to study the molecular mechanisms leading to the development of MMAIII-induced malignant transformation (Petzoldt et al., 1995; Sens et al., 2004; Bredfeldt et al., 2006). URO-MSC cells in the presence of MMAIII [URO-MSC(+)] were used to investigate the generation of ROS, the induction of DNA damage, and the alteration of the DNA repair enzyme, PARP, following exposure to chronic, biologically relevant concentrations of MMAIII. The persistence of DNA damage and the alteration of DNA repair activity was also studied in URO-MSC cells after the 2 wk withdrawal of MMAIII [URO-MSC(-)] to examine the biological alterations in URO-MSC(-) cells following previous chronic MMAIII exposure. Analysis in the absence of MMAIII was necessary to ensure that the experimentation focused on the persistent arsenical-induced alteration of biological processes within the UROtsa cell line, and not the chemical effect caused by the presence of MMAIII.

2. Materials and Methods

2.1 Reagents

Dulbecco's Modified Eagle Medium, fetal bovine serum, antibiotic-antimycotic, and 1X trypsin-EDTA (0.25%) were acquired from Gibco Invitrogen Corporation (Carlsbad, CA). Diiodomethylarsine (MMAIII iodide, CH3AsI2) was prepared by the Synthetic Chemistry Facility Core (Southwest Environmental Health Sciences Center, Tucson, AZ) using the method of Millar et al., 1960. Etoposide was obtained from Trevigen, Inc. (Gaithersburg, MD). Water used in studies was distilled and de-ionized.

2.2 Dosing solutions

Preparation of dosing solution and procedures were derived from Bredfeldt et al., 2006. Pure MMAIII was stored in ampules at 4 °C. Fresh stock solutions of 25 mM MMAIII were made and diluted to a final concentration of 5 μM prior to dosing (1 to 100 dilution) to obtain a final concentration of 50 nM MMAIII. All dosing solutions were sterile filtered with a 0.2 μm acrodisc and stored in sealed, sterile tubes at 4 °C that were opened only for dosing in a sterile cell culture hood. As previously reported by Gong et al., 2001, MMAIII solutions in distilled, de-ionized water were stable for approximately 4 months at 4 °C with no degradation observed when monitored using HPLC-ICP MS.

2.3 Cell culture

UROtsa cells were a generous gift from Drs. Donald and Maryann Sens (University of North Dakota). URO-MSC cells were created in our laboratory as reported by Bredfeldt et al., 2006. Cell culture conditions were derived from those previously described by Rossi et al., 2001 and Bredfeldt et al., 2004. UROtsa cells were cultured in a growth medium of DMEM containing 5% v/v FBS and 1% antibiotic–antimycotic. Culture medium was changed every 2 days. Cultured cells were incubated in an atmosphere that was 5% CO2:95% air at 37 °C. Confluent cells were removed from plates with trypsin–EDTA (0.25%) and subcultured at a ratio of 1:3. All UROtsa and URO-MSC cells used in this study tested negative for the presence of mycoplasma contamination. MMAIII-treated cells were continuously cultured in a medium enriched with 50 nM MMAIII and dosed every other day to ensure continuous presence of MMAIII. Parallel cultures of UROtsa cells were maintained in MMAIII-free medium and served as passage-matched controls. Experimentation was carried out with MMAIII in the culture medium to investigate cellular alterations in the presence of MMAIII; and after withdrawal of MMAIII from culture medium to examine biological changes in UROtsa cells altered after removal of MMAIII. UROtsa cells chronically exposed to low-level MMAIII were evaluated in the presence of MMAIII at the time of experimentation, and after a 2 wk removal of MMAIII following previous chronic, low-level exposure (Fig. 1). Persistence of MMAIII in cell culture conditions was determined by exposing UROtsa cells to 50 nM MMAIII and then measuring the presence of the various arsenic species in both cell lysates and media 1-4 days after initial MMAIII exposure using HPLC/ICP-MS (Bredfeldt et al., 2004). Results demonstrated MMAIII was below the limit of detection (0.5 ppb) after 3 days. The 2 wk removal of MMAIII and cell culture splitting upon cell confluency prior to experimentation was to ensure that MMAIII was no longer present within URO-MSC(-) cells. Previous studies by Bredfeldt et al., 2004 evaluated the ability of UROtsa to metabolize AsIII to MMAIII, MMAV, and DMAV. The most abundant metabolite produced in those studies was MMAIII. UROtsa cells treated for 24 h with 50 nM MMAIII did not metabolize MMAIII to more toxic products, as detected by HPLC/ICP-MS. Demonstrating that MMAIII is the chemical responsible for the observed effects as >90% of the dose remained in the form of MMAIII. DMAIII was not detected in these same UROtsa cells treated with 50 nM MMAIII.

Figure 1
Summary of experimental design. Non-tumorigenic UROtsa cells were chronically exposed to 50 nM MMAIII for 52 wk resulting in the malignantly transformed URO-MSC cell line. Cells were exposed to MMAIII for 12, 24, 36 and 52 wk to examine the progressive ...

2.4 Alkaline comet assay

The comet assay was performed using a modified protocol of Trevigen Inc. (Gaithersburg, MD) to measure DNA strand breaks. URO-MSC cells at 12, 24, 36, and 52 wk in the presence of MMAIII [URO-MSC12(+), URO-MSC24(+), URO-MSC36(+), and URO-MSC52(+)] and after a 2 wk removal of MMAIII [URO-MSC12(-), URO-MSC24(-), URO-MSC36(-), and URO-MSC52(-)] were utilized and cultured in 100 × 20 mm tissue culture dishes (BD Falcon, Franklin Lakes, NJ) at 5 × 105 cells per plate and allowed to reach 90% confluency. Adherent cells were removed via trypsin-EDTA (0.25%), quenched with 5% FBS/Media, and centrifuged at 400 rpm for 5 min. Cells were resuspended in 1X PBS (Ca2+ and Mg2+ free) and counted via trypan blue exclusion assay (Bredfeldt et al., 2006). Cells were washed in ice cold 1X PBS (Ca2+ and Mg2+ free) and resuspended at 1 × 105 cell/ml in ice cold 1X PBS (Ca2+ and Mg2+ free). Cells (1 × 105) were combined with a low melting point agarose at a ratio of 1 to 10, allowed to solidify onto a comet slide, and placed in prechilled lysis solution overnight. Comet slide was placed in alkali solution (1 h) followed by horizontal electrophoresis. After electrophoresis, slides were rinsed in distilled H2O and immersed in 70% ethanol for 5 min and allowed to dry. Prior to fluorescence microscopy, samples were stained with SYBR green from Trevigen Inc. (Gaithersburg, MD).

2.5 Image analysis

Comet images were viewed via epifluorescence microscopy at excitation and emissions of 494/521 nm. The images were captured using an Olympus IMT-2 inverted fluorescence microscope, using a 20× objective. The camera used was a Hamamatsu ORCA-100 CCD camera (chip resolution 1280 × 1024) in bin2 mode, with the actual image resolution set at 640 × 512 using the SimplePCI version 6.5 (Hamamatsu) instrument control software. At least 75 randomly selected cells were analyzed per n-value. An image analysis system based on the Matlab computer program (Mathworks, Natick, MA) developed by Dr. Joceline Lega (Department of Mathematics, University of Arizona) was used to analyze individual cells to determine mean comet profiles, assess mean comet tail length, and average comet moment.

2.6 Spectrofluorometric analysis of endogenous ROS

URO-MSC cells exposed to 50 nM MMAIII for 12, 24, and 52 wk in the presence of MMAIII or following removal of MMAIII (2 wk) were grown to confluency, trypsinized, and counted. Cells (200 cells/μl) were added to Eppendorf tubes (1.5 ml), washed with PBS, and loaded with 25 μM CM-H2DCFDA (Molecular Probes/Invitrogen, Carlsbad, CA) for 30 min at 37 °C. Cells were washed and re-suspended in PBS. Cell suspensions were added to a 96-well plate and fluorescence emission measured at 530 nm on a SpectraMax Gemini XS multi-well plate reader (Molecular Devices Co., Sunnyvale, CA) with excitation filter set at 485 nm. Temperature was maintained at 37 °C and readings were performed every minute for a total of 1 h (Wang and Joseph, 1999).

2.7 Effect of ROS scavengers on DNA damage in URO-MSC cells

UROtsa cells exposed 50 nM MMAIII for 12 or 52 wk followed by removal of MMAIII for 2 wk [URO-MSC12(-)/URO-MSC52(-)] were treated with antioxidants as previously described (Eblin et al., 2009): potassium iodide (KI; 5 mM), superoxide dismutase (SOD; 100 units/ml), or catalase (CAT; 200 units/ml) every 2 days. After a 14 day incubation of URO-MSC12(-)/URO-MSC52(-) cells with the various antioxidants, cells were removed from culture flasks via trypsinization and counted via trypan blue exclusion assay. DNA damage was detected using the alkaline comet assay. A minimum of 75 randomly selected cells were analyzed per n-value, n=4, for each condition with protocol performed similar to methods section 2.3 and section 2.4 as previously described.

2.8 Analysis of Single Stranded DNA damage via flow cytometry

A monoclonal antibody, F7-26, from Cell Technology Inc. (Mountain View, CA) was used to detect single stranded DNA in UROtsa cells chronically exposed to MMAIII for 12, 24, 36, and 52 wk in the presence of MMAIII and after the 2 wk removal of MMAIII exposure. Cell samples were cultured in 75 cm2 tissue culture flasks from Sarstedt (Newton, NC) until confluence was reached. Cells were removed via trypsin-EDTA (0.25%), and resuspended in serum enriched media. Cell count was performed using the trypan blue exclusion assay. URO-MSC(+) and URO-MSC(-) cells: 12, 24, 36, 52 wk MMAIII exposure and UROtsa control (n=4) cells (1 × 106) were transferred into individual 5 ml polystyrene round bottom tubes (BD Falcon, Franklin Lakes, NJ) and fixed using 1 ml fixative solution (6:1 methanol/PBS solution) for 48 h. Samples were denatured using 1× denaturing buffer, formamide (Sigma Co., St. Louis, MO), for 10 min at 80 °C. Samples were then blocked and stained using a mouse anti-ssDNA (primary) antibody and anti-mouse IgM FITC labeled (secondary) antibody (Cell Technology, Mountain View, CA). After subsequent staining and washes, cells (1 × 106) were placed in sterile PBS. Samples were analyzed at the Arizona Cancer Center Cytometry Core Facility using a FACScan cytometer (BD Biosciences, San Jose CA) equipped with a 15 mW Argon laser. Cells were analyzed via excitation/emission wavelengths at 488/530 nm. The protocol for the detection of ssDNA using the mouse anti-ssDNA monoclonal antibody was adapted from Frankfurt et al., 1996 and Cell Technology Inc. (Mountain View, CA).

2.9 Poly (ADP-ribose) polymerase (PARP) Activity Assay

PARP activity in chronic MMAIII exposed UROtsa cells was determined using a semi-quantitative ELISA in a 96 well format. Relative PARP activity was determined through detection of poly (ADP-ribose) polymer, PAR, which was immobilized onto histone proteins on the bottom of the 96-well plates. URO-MSC(+) and URO-MSC(-) cells at 12, 24, 36, and 52 wk MMAIII exposure were seeded at 5 × 103 cells/200 μl media and allowed to grow overnight in a 96-well flat-bottom plate. Adherent cells were lysed with cell extraction buffer (100 μl/well) and protein levels quantified via the bicinchoninic acid assay. Lysates were incubated with PARP substrate cocktail containing NAD+, which allows for the generation of PAR based on relative PARP activity within the samples. An anti-PAR monoclonal antibody (50 μl), a goat anti-mouse IgG-HRP conjugate (50 μl), and horseradish peroxidase substrate were used to generate a colorimetric signal. 50 μl of TACS-Sapphire was added to the samples and allowed to incubate for 10 min; reaction was stopped by adding 50 μl of phosphoric acid. Calculated absorbance was correlated with relative PARP activity. Samples were read using a VERSAmax plate reader (Molecular Devices Co., Sunnyvale, CA) at an absorbance of 450 nm. The protocol for the detection of PARP activity was adapted from Trevigen Inc. (Gaithersburg, MD).

2.10 Statistics

Graphs were generated in Microsoft Office Excel (Microsoft Corp., Redmond, WA). Comet assay data analyzed included an n=4 for each experimental value with a minimum of 75 cells assessed per each n-value. Detection of ROS via spectrofluorometric analysis included an n=3 for each experimental value. Detection of ssDNA damage via flow cytometry was expressed as the average of four experiments. PARP activity was expressed as the average of three experiments. Error bars within each column represent the standard error of the mean (± SEM). Statistical analysis utilized and p-value cutoff for each experiment is noted in corresponding figure legends. Statistical significance was determined using the Student's t-test for comparison of samples, ANOVA followed by Bonferroni's multiple comparison test, or Cruzick's non-parametric trend analysis which is an extension of the Wilcoxon rank-sum test using Stata10 (College Station, TX).

3. Results

3.1 DNA damage assessed through the alkaline comet assay

The majority of studies examining DNA damage as a result of exposure to inorganic or trivalent methylated arsenic species have focused on the ability of these arsenicals to induce DNA strand breaks following acute exposure (Liu et al., 2000; Schwerdtle et al., 2003; Chai et al., 2007); however, our model focuses on the biological changes taking place within the non-tumorigenic, urothelial cell line, UROtsa, during chronic, low-level exposure to the biomethylated metabolite, MMAIII. The URO-MSC cell lines were used to examine the extent of DNA damage in the presence of MMAIII through utilization of the alkaline comet assay. The DNA damaging agent, etoposide, was utilized as a positive control. Etoposide, a topoisomerase II inhibitor, was utilized as a positive control due to the ability to induce DNA single- and double- strand breaks (Sinha et al., 1988; Van Maanen et al., 1988). The average comet tail moment is defined as the product of the tail length and the fraction of total DNA in the tail. The mean comet moment of URO-MSC cells in the presence of MMAIII is first elevated at 12 wk exposure [URO-MSC12(+)] and remains consistently elevated through 52 wk exposure [URO-MSC52(+)] (Fig. 2).

Figure 2
Comparison of the average comet moment in URO-MSC cells in the presence/absence of MMAIII following chronic exposure. Graph depicts changes in comet moment between MMAIII exposed URO-MSC cells and untreated UROtsa control (n=4), each n value ≥ ...

To determine if the changes in DNA damage were related to the presence of MMAIII, or whether the changes were associated with the transformation of UROtsa cells, the alkaline comet assay was performed on URO-MSC cells after MMAIII was removed from culture conditions for 2 wk. The average comet moment of URO-MSC(-) cells decreased in contrast to URO-MSC(+) cells in the presence of MMAIII with the same length of exposure; however, the DNA damage in URO-MSC cells removed from MMAIII increased in a time-dependent manner following previous exposure to MMAIII (Fig. 2). These data demonstrate that in the presence of MMAIII DNA damage levels in URO-MSC(+) cells remain elevated and sustained throughout chronic exposure. In addition, although DNA damage levels in URO-MSC(-) cells decrease compared to URO-MSC(+) cells, the disparity in DNA damage levels between the two decrease as the length of exposure to MMAIII increases.

3.2 Role of ROS in DNA damage following chronic, low-level exposure to MMAIII

At environmentally relevant concentrations arsenite and MMAIII can generate DNA damaging ROS (Eblin et al., 2006). Previous studies by Eblin et al., 2006, 2008 have focused on the generation of ROS species following acute exposures in the presence of MMAIII; however, the elevation of ROS in URO-MSC cells following chronic, low-level MMAIII exposure has not been analyzed. To establish ROS as a key factor involved in generating DNA damage seen in URO-MSC cells it was necessary to determine whether URO-MSC(+)/URO-MSC(-) cells have elevated levels of ROS. In the presence of MMAIII, URO-MSC(+) cells have significantly elevated levels of ROS at 12 wk exposure [URO-MSC12(+)] which remain elevated through 52 wk in the presence of MMAIII (Fig. 3). URO-MSC(-) cells exposed to MMAIII for 12, 24, and 52 wk followed by cell culture growth for a period of 2 wk in the absence of MMAIII were assessed for the production of endogenous ROS. URO-MSC12(-), URO-MSC24(-), and URO-MSC52(-) cells have elevated levels of endogenous ROS in a time-dependent manner when compared to untreated UROtsa control, despite removal of MMAIII following previous chronic exposure (Fig. 3).

Figure 3
Detection of ROS generation following chronic MMAIII exposure in URO-MSC(+) and URO-MSC(-) cells. Spectrofluorometric analysis of ROS in URO-MSC12(+), URO-MSC24(+), and URO-MSC52(+) demonstrate a constitutive increase in ROS levels beginning at 12 wk ...

To demonstrate that ROS may be responsible for the increase in DNA damage seen in URO-MSC cells chronically exposed to MMAIII, it was necessary to determine whether ROS scavengers would lead to a decrease in DNA damage in URO-MSC cells cultured in the absence of MMAIII (2 wk). Because URO-MSC12(-) cells demonstrated the initial increase in DNA damage and elevation in ROS levels following chronic MMAIII exposure, these cells were chosen to demonstrate the role of ROS in generating DNA damage. To further exemplify the involvement of ROS in damaging DNA, the malignantly transformed URO-MSC52(-) cells demonstrating the greatest levels of DNA damage were also incubated with ROS scavengers. URO-MSC12(-)/URO-MSC52(-) cells were incubated with the ROS scavengers: KI (5 mM), SOD (100 units/ml), and CAT (200 units/ml) every 2 days for a 14 day period. URO-MSC12(-)/URO-MSC52(-) cells showed a significant decrease in DNA damage as measured using the alkaline comet assay following incubation with SOD, CAT, or KI (Fig. 4a, 4b). These data show that DNA damage following MMAIII exposure is partially dependent on the generation of ROS. Interestingly, previous studies have indicated that superoxide, O2-, is the primary ROS species produced by arsenite exposure. Superoxide formation in biological systems has the potential to result in the production of H2O2, 1O2, and ˙OH, all of which have significant roles in the generation of oxidative stress, perturbations in signaling pathways, and DNA damage (Wiseman and Halliwell, 1996; Buetler et al., 2004; Leonard et al., 2004; Shi et al., 2004a, 2004b; Beyersmann and Hartwig, 2008). Incubation of URO-MSC cells with SOD, KI, or CAT significantly reduced the levels of DNA damage, supporting the role of arsenical-induced O2-, ·OH, and H2O2 in producing a genotoxic insult.

Figure 4
a.) Decrease in DNA damage in URO-MSC12(-) cells incubated with ROS scavengers following chronic, low-level exposure to MMAIII. b.) Decrease in DNA damage in URO-MSC52(-) cells incubated with ROS scavengers following chronic, low-level exposure to MMA ...

3.3 Analysis of DNA single strand breaks

To verify the extent of single strand DNA (ssDNA) breaks, flow cytometry was conducted using a mouse primary antibody directed against ssDNA breaks. Results demonstrate a statistically significant increase in single stranded DNA damage in URO-MSC12(+), URO-MSC24(+), URO-MSC36(+), and URO-MSC52(+) cells compared to UROtsa control, consistent with the level of damage seen after treatment with the positive control, etoposide (Fig. 5). By 12 wk of exposure to MMAIII, a 3-fold increase in ssDNA was present, and the increase was maintained throughout continuous exposure to MMAIII. URO-MSC12(-), URO-MSC24(-), URO-MSC36(-), and URO-MSC52(-) cells out of MMAIII demonstrate a time-dependent increase in ssDNA damage (Fig. 5). The ssDNA damage closely resembles the results seen when MMAIII-induced DNA damage was measured using the alkaline comet assay (Fig. 2). Taken together, these data demonstrate that not only does significant damage to DNA occur in the presence of low-level MMAIII, but even upon removal of chronic, low-level MMAIII exposure URO-MSC(-) cells maintain an increase in DNA damage. The DNA damage levels in URO-MSC(-) cells remain elevated relative to untreated UROtsa levels after the removal of the MMAIII exposure and increase in a time-dependent manner. As the length of exposure to MMAIII increases in URO-MSC cells, the difference in the levels of ssDNA damage between URO-MSC(+) cells and URO-MSC(-) cells decreases.

Figure 5
Comparison of ssDNA damage in the presence/absence of MMAIII following chronic, low-level exposure to MMAIII in URO-MSC cells. (*) Marks statistically significant difference (p ≤ 0.05) between URO-MSC(+) and URO-MSC(-) determined using Student's ...

3.4 PARP repair activity following chronic MMAIII exposure

Inactivation of DNA repair enzymes via trivalent arsenic species is believed to occur through an interaction with potential molecular targets containing zinc finger domains (Hartwig et al., 2003a; 2003b). One particular DNA repair enzyme of interest involved in the repair of single stranded DNA damage is poly (ADP-ribose) polymerase, PARP. In the presence of MMAIII, PARP activity in URO-MSC(+) cells increased with exposure; however, levels are only significantly increased above UROtsa control levels in URO-MSC36(+), and URO-MSC52(+) cells (Fig. 6). PARP activity levels in URO-MSC(+) cells are comparable to levels of PARP in UROtsa cells incubated with etoposide, which was utilized as a positive control. In regards to DNA repair activity in the absence of MMAIII, relative PARP activity significantly increases in a time-dependent manner in cultured URO-MSC24(-), URO-MSC36(-), and URO-MSC52(-) cells. The results demonstrate that in the presence of MMAIII, URO-MSC24(+), URO-MSC36(+), and URO-MSC52(+) cells have a general decrease in PARP activity compared to URO-MSC(-) cells devoid of MMAIII; yet, significant difference is only reached in URO-MSC52(+) cells compared to URO-MSC52(-) cells.

Figure 6
Comparison of relative PARP activity in the presence/absence of MMAIII following chronic, low-level exposure to MMAIII in URO-MSC cells. Graph depicts changes in PARP activity between MMAIII exposed URO-MSC cells and untreated UROtsa control. (*) Marks ...

4. Discussion

Inorganic arsenicals do not react with DNA directly to cause DNA damage, nor are they significantly mutagenic in most classical mutagenesis assays at low concentrations (Hei et al., 1998; Klein et al., 2007). However, the arsenic species, AsIII and MMAIII, have been noted to play an indirect role in DNA damage via the generation of ROS (Hei et al., 1998; Schwerdtle et al., 2003; Shi et al., 2004a, 2004b; Eblin et al., 2006; Klein et al., 2007). The induction of chromosomal aberrations, DNA strand breaks, and oxidative DNA base damage have been noted following low-level arsenic exposure in cultured mammalian cell lines (Schoen et al., 2004).

To determine whether chronic exposure to 50 nM MMAIII results in an increase in DNA damage, the extent of DNA damage in UROtsa cells in the presence of MMAIII after 12, 24, 36, and 52 wk exposure was analyzed via alkaline comet assay. DNA damage was significantly increased in UROtsa cells after 12 wk exposure [URO-MSC12(+)]. This increase in DNA damage in the presence of MMAIII remained statistically elevated and at comparable levels through 52 wk exposure [URO-MSC24(+), URO-MSC36(+), and URO-MSC52(+)] (Fig. 2). To examine the biological implications of chronic, low-level MMAIII on the ability to generate DNA damage in the absence of MMAIII, the alkaline comet assay was carried out in URO-MSC cells after a 2 wk removal of MMAIII. The DNA damage levels were significantly decreased compared to URO-MSC(+) cells at the time of experimentation; however, these levels remain significantly elevated above control and increased with prolonged exposure to MMAIII. The linear increase in DNA damage demonstrated in URO-MSC(-) cells coincide with growing evidence of malignant transformation within UROtsa cells following chronic, low-level exposure to MMAIII. Bredfeldt et al., 2006 demonstrated that URO-MSC cells obtain characteristics of anchorage independent growth after 24 wk MMAIII exposure and tumor growth in immunocompromised mice after 52 wk MMAIII exposure. The combination of the DNA damage data in the presence and absence of MMAIII exposure strengthens the possibility of an indirect genotoxic insult as a result of arsenical-induced ROS.

Chronic MMAIII exposure can alter cell signaling events, which can lead to an upregulation of endogenous ROS as a result of secondary ROS generation in URO-MSC cells no longer exposed to MMAIII. Although experimentation has been shown to produce elevated levels of ROS in the presence of MMAIII, the secondary ROS generation in the absence of MMAIII has the potential to regenerate elevated levels of ROS resulting in subsequent DNA damage (Eblin et al., 2006). Previous studies, have demonstrated the inactivation of ROS with antioxidants in URO-MSC cells results in a decrease in colony formation in soft agar, suggesting that activated Ras and NADPH oxidase could play a role in producing secondary ROS. Eblin et al. 2009, has also established the importance of constitutive activation of the MAPK pathway and subsequent ROS generation leading to the upregulation of Ras in URO-MSC cells following chronic MMAIII exposure. Activation of Ras through ROS can act through a positive feedback mechanism, resulting in increased ROS production (Serù et al., 2004). This is important as it establishes a possible mechanism for maintaining elevated endogenous ROS following chronic MMAIII exposure, which may contribute to the induction of DNA damage, even after the removal of MMAIII.

Chronic, low-level exposure to MMAIII results in elevated levels of ROS. Results indicate that in the presence of MMAIII, URO-MSC(+) cells have significantly elevated levels of ROS at 12 wk exposure [URO-MSC12(+)] which remain elevated through 52 wk in the presence of MMAIII [URO-MSC52(+)]. URO-MSC(-) cells have elevated levels of endogenous ROS, which increase in a time-dependent manner when compared to untreated UROtsa cells despite removal of MMAIII following previous chronic exposure (Fig. 3). Following acute exposure Eblin et al., 2006 demonstrated that 50 nM MMAIII in UROtsa cells results in the generation of ROS; furthermore, pretreatment of these cells with superoxide dismutase or catalase ameliorate the increase in ROS. The production of ROS in UROtsa cells following chronic MMAIII exposure has the potential to induce malignant transformation. Previous investigations have demonstrated that acute exposure to MMAIII results in increased ROS and numerous alterations of growth signaling pathways including the activation of signaling cascades involving stress-related cellular proliferation (Eblin et al., 2006, 2007; Wang et al., 2007). Furthermore, evidence of the involvement of ROS as a potential player involved in cellular hyperproliferation and anchorage independent growth, two common characteristics of malignant transformation, have been established (Eblin et al., 2009).

Support for the importance of ROS in the induction of DNA damage following chronic, low-level MMAIII exposure comes from the partial decrease in DNA damage in URO-MSC12(-) and URO-MSC52(-) cells incubated with ROS scavengers. Incubation of URO-MSC12(-)/URO-MSC52(-) cells with the ROS scavengers SOD, CAT, and KI each resulted in a significant decrease in the level of DNA damage produced (Fig. 4a, 4b). Previous studies have indicated that superoxide, O2-, is the primary ROS species produced by AsIII exposure (Buetler et al., 2004; Shi et al., 2004). Due to the ability of O2- to form other free radicals (H2O2 and ·OH) involved in damaging DNA, blocking O2- with SOD could potentially decrease the ability of downstream free radicals to generate further DNA damage. These results establish the significance of ROS in the induction of DNA damage demonstrated in URO-MSC cells.

As the generation of DNA damage in URO-MSC cells was shown to be dependent on the MMAIII-induced ROS, it was important to determine the type of DNA damage being generated in order to investigate the importance of DNA repair enzymes particular to the type of DNA damage present. Utilizing a monoclonal antibody generated against ssDNA, URO-MSC(+) cells had a similar trend in DNA damage as URO-MSC12(+), URO-MSC24(+), URO-MSC36(+), and URO-MSC52(+) that were previously demonstrated using the comet assay (Fig. 5). Single stranded DNA damage was first elevated at 12 wk MMAIII exposure and consistently elevated through 52 wk. In the absence of MMAIII there was a decrease in ssDNA damage, but with prolonged previous, chronic exposure to MMAIII the difference between ssDNA damage levels between URO-MSC(+) and URO-MSC(-) cells decreased. In the absence of MMAIII, URO-MSC12(-), URO-MSC24(-), URO-MSC36(-), and URO-MSC52(-) resulted in a time-dependent increase in ssDNA damage (Fig. 5) similar to the results demonstrated using the comet assay (Fig. 2).

Arsenical exposure not only contributes to the generation of ROS involved in DNA damage, but has also been shown to inhibit DNA repair mechanisms specific to ROS-induced damage (Snow et al., 1999). As the role of MMAIII-induced generation of ROS and subsequent involvement in DNA damage has been investigated, it was important to determine if the presence/absence of MMAIII resulted in altered repair activity of PARP, a key repair enzyme in DNA single strand break repair. In URO-MSC(+) cells, PARP activity only slightly increased following chronic exposure to 50 nM MMAIII. PARP activity levels were significantly elevated above control UROtsa levels after 36 wk exposure; however, these levels remained decreased when compared to URO-MSC(-) cells removed from MMAIII exposure. URO-MSC cells following a 2 wk removal of MMAIII had a time-dependent increase in PARP activity. The time-dependent increase in PARP activity in URO-MSC(-) cells may be elevated as a result of the elevated levels of DNA damage. PARP activity levels in URO-MSC(-) cells may be elevated to account for the increase in DNA damage. These data demonstrate that in the presence of MMAIII, PARP activity levels are reduced, which could explain the vast elevation of DNA damage; however, after removal of MMAIII exposure, PARP activity levels elevate allowing for subsequent initiation of DNA repair activity. The increase in PARP activity may explain the subsequent decrease in DNA damage levels seen in URO-MSC(-) cells devoid of MMAIII when compared to URO-MSC(+) cells.

In summary, exposure to 50 nM MMAIII results in an increase in ROS following chronic exposure. Of great significance, the concentrations of MMAIII used in this study represent an environmentally relevant level of exposure. In addition, ROS upregulation was determined to play a role in the induction of DNA damage; in particular, the ability to generate ssDNA damage. The DNA damage demonstrated in URO-MSC12(-)/URO-MSC52(-) can be decreased through treatment with the ROS scavengers SOD, KI, and CAT which block O2-, ·OH, and break down H2O2, respectively. These data demonstrate that chronic, low-level MMAIII-induced ROS have the capacity to cause genotoxic insult. Following chronic, low-level MMAIII exposure in URO-MSC cells, DNA damage in the presence of MMAIII is significantly elevated after 12 wk and remains elevated through 52 wk. Upon removal of MMAIII for 2 wk, DNA damage levels in URO-MSC cells remain elevated and increase in a time-dependent manner when compared to control UROtsa despite the absence of MMAIII. In 2004, Yamauchi et al. demonstrated that the length of inorganic arsenic exposure dictates the ability of individuals to recover from oxidative DNA damage. In the URO-MSC(-) cells following previous chronic, low-level exposure DNA damage levels remain elevated, possibly due to an increase in the length of time necessary to recover. The decreased levels of PARP activity in the presence of MMAIII may also explain the significantly elevated levels of DNA damage first occurring at 12 wk in the presence of 50 nM MMAIII. When MMAIII is removed for 2 wk, a time-dependent increase in PARP activity may account for the decrease in DNA damage levels in URO-MSC(-) cells out of MMAIII exposure. Taken together, the increased DNA damage as a result of MMAIII-induced ROS in concert with an overall decrease in repair capacity in the presence of MMAIII may play a key role in generating an indirect genotoxic insult and an overall contribution in the progression of arsenic-induced bladder carcinogenesis.

Acknowledgments

The authors would like to thank Drs. Donald and Mary Ann Sens and Dr. Scott Garret for the UROtsa cells and assistance with culturing conditions, Dr. Joceline Lega (Department of Mathematics, University of Arizona) for the comet analysis software, Doug Cromey (SWEHS Imaging Core Facility) for his help with image analysis, and the Synthetic Chemistry Facility Core (Southwest Environmental Health Sciences Center, Tucson, AZ) for the Diiodomethylarsine (MMAIII). The analyses for the biotransformation and persistence of MMAIII were performed by the Analytical Section of the Hazard Identification Core from our NIEHS-supported Superfund Basic Research Program Grant (ES 04940). We wish to acknowledge Michael Kopplin for performing the metal analyses. The research herein was made possible by the NIEHS Superfund Basic Research Program (ES 04940) and the Southwest Environmental Health Sciences Center (ES 06694). SMW is funded by an NIEHS Training Grant (ES 07091).

Footnotes

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