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Keywords:

  • arsenicals;
  • mode of action;
  • reactive oxygen species;
  • DNA damage;
  • Comet assay

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

Although it is widely known that arsenic-contaminated drinking water causes many diseases, arsenic's exact mode of action (MOA) is not fully understood. Induction of oxidative stress has been proposed as an important key event in the toxic MOA of arsenic. The authors' studies are centered on identifying a reactive species involved in the genotoxicity of arsenic using a catalase (CAT) knockout mouse model that is impaired in its ability to breakdown hydrogen peroxide (H2O2). The authors assessed the induction of DNA damage using the Comet assay following exposure of mouse Cat+/+ and Cat/ primary splenic lymphocytes to monomethylarsonous acid (MMAIII) to identify the potential role of H2O2 in mediating cellular effects of this metalloid. The results showed that the Cat/ lymphocytes are more susceptible to MMAIII than the Cat+/+ lymphocytes by a small (1.5-fold) but statistically significant difference. CAT activity assays demonstrated that liver tissue has approximately three times more CAT activity than lymphocytes. Therefore, Comet assays were performed on primary Cat+/+, Cat+/, and Cat/ hepatocytes to determine if the Cat/ cells were more susceptible to MMAIII than lymphocytes. The results showed that the Cat/ hepatocytes exhibit higher levels of DNA strand breakage than the Cat+/+ (approximately fivefold) and Cat+/ (approximately twofold) hepatocytes exposed to MMAIII. Electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trap agent detected the generation of ·OH via MMAIII when H2O2 was present. These experiments suggest that CAT is involved in protecting cells against the genotoxic effects of the ·OH generated by MMAIII. Environ. Mol. Mutagen. 54:317–326, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

Human ingestion of arsenic-contaminated drinking water can lead to cancer of the bladder [Pou et al., 2011], liver [Wadhwa et al., 2011], and lung [IARC, 2012] and noncancerous diseases such as cardiovascular disease [Chen et al., 2011], neuropathy [Hafeman et al., 2005], and skin lesions [Argos et al., 2011], as reviewed by Jomova et al. [2011]. Arsenic has also been shown to be a transplacental carcinogen with adult mice developing cancer after in utero exposure to sodium arsenite [Waalkes et al., 2007] and monomethylarsonous acid (MMAIII) [Tokar et al., 2012]. Exposure of the Webber prostate epithelial (WPE)-stem human prostate epithelial stem/progenitor cell line to sodium arsenite and subsequent transplantation into nude male mice leads to the development of aggressive tumors [Tokar et al., 2010a,b].

Even though a plethora of research has been done to clarify the mode of action (MOA) of arsenic, no single pathway has been identified as the definitive cause for its toxicity, and it has been suggested that several MOAs may be acting in concert depending on the cancer or the disease being studied. The suggested MOAs include inhibition of DNA damage repair pathway via interactions of arsenicals with enzymatic sulfhydryl groups in DNA repair enzymes [Kitchin and Wallace, 2008], epigenetic alterations [Zhou et al., 2008] of essential genes such as WNT5A, which is involved in development and differentiation [Jensen et al., 2009], cytotoxicity followed by proliferative regeneration [Cohen et al., 2007], and genetic damage leading to chromosomal changes [Kligerman et al., 2003; Mahata et al., 2003].

We have been investigating the genotoxic aspects of arsenic's MOA. Mass et al. [2001] showed that the methylated forms of arsenic are highly genotoxic, and these might be the ultimate genotoxic forms of arsenic [Kligerman et al., 2003]. These active arsenicals are poor point mutagens but are potent clastogens that can lead to changes in chromosome structure [Kligerman et al., 2010] and can also interfere with the spindle, leading to changes in chromosome number [Kligerman et al., 2005]. Although the exact genotoxic MOA of the methylated forms of arsenic has not been determined, it has been suggested that after arsenic exposure, the production of reactive oxygen species (ROS) such as superoxide (O2·), hydrogen peroxide (H2O2), and the hydroxyl radical (·OH) can interact with many cellular physiological pathways [Liu et al., 2001]. Wang et al. [2001] have suggested that arsenic-induced DNA strand breaks are a result of the excision of oxidative DNA adducts and DNA–protein crosslinks. Oxidative damage could also lead to frank chromosome breakage, depending on the location of the lesions and their persistence during the cell cycle as well as the repair capacity of the cells in which the lesions occur [Kligerman et al., 2010].

It is known that chemically-induced DNA damage via the production of ROS and direct interactions with DNA can lead to carcinogenesis [Klaunig et al., 2011], as has been previously shown in UROtsa cells [Wnek et al., 2011]. ·OH is one of the ROS that may be involved in arsenic's genotoxic MOA through the production of O2· [Hei et al., 1998; Liu et al., 2001; Nesnow et al., 2002]. This species is produced by the one-electron reduction of oxygen and can decompose spontaneously or be broken down by the enzyme superoxide dismutase (SOD) to produce H2O2. This in turn can be enzymatically decomposed by catalase (CAT) to form H2O and O2 (Fig. 1). Studies in our laboratory have shown that a deficiency in Sod1 increases the levels of DNA damage in primary mouse splenic lymphocyte cells exposed to methylated arsenicals [Tennant and Kligerman, 2011]. These results suggest that O2· is involved in the DNA damage-inducing ROS produced after exposure to arsenic. Because ·OH is produced downstream of O2·, we hypothesized that ·OH might be involved in the genotoxic pathway of MMAIII. To test this hypothesis, we exposed CAT homozygous knockout (Cat−/−) primary splenic lymphocytes and hepatocytes to MMAIII and compared the genotoxic potential of this arsenic metabolite via the Comet assay to similarly exposed wild-type (Cat+/+) and heterozygous (Cat+/−) primary splenic lymphocytes and hepatocytes. In addition, we investigated if CAT deficiency induces susceptibility to the toxic effects of arsenic. We then used electron spin resonance to identify the radical(s) produced by MMAIII.

image

Figure 1. Pathway depicting the formation of different forms of reactive oxygen species, such as superoxide (O2·), hydrogen peroxide (H2O2), and the hydroxyl radical (·OH), formed via aerobic respiration and their degradation by antioxidants such as superoxide dismutase (SOD) and catalase (CAT).

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MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

Chemicals

MMAIII (89%) was obtained from Dr. Kirk Kitchin (U.S. Environmental Protection Agency, Research Triangle Park, NC). This compound forms MMAIII in aqueous solution. Methyl methanesulfonate (MMS; CAS# 66-27-3), CAT (EC 1.11.1.6, Lot # 125H7050 for CAT assay experiments; CAT from Aspergillus niger for electron spin resonance experiments), and ethylene glycol tetraacetic acid (EGTA) were purchased from Sigma-Aldrich (St. Louis, MO). Roswell Park Memorial Institute (RPMI) Medium 1640, Williams medium E, fetal bovine serum (FBS), 1× Hank's balanced salt solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin–streptomycin mix, Gluta-MAX®, 0.4% trypan blue stain, and Sybr Gold® were bought from Invitrogen™ (Carlsbad, CA). Collagenase (Lot # 49M11443) was purchased from Worthington Biochemical (Lakewood, NJ). Sodium hydroxide and H2O2 were obtained from Fisher Scientific (Pittsburgh, PA). Ketamine (Ketaset) and xylazine (AnaSed®) were procured from Butler Schein Animal Health (Dublin, OH). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Dojindo Molecular Technologies (Rockville, MD). SOD from bovine kidney was purchased from Calzyme Laboratories (San Luis Obispo, CA). Chelex-100 resin was bought from Bio-Rad Laboratories (Hercules, CA).

Animals

CAT heterozygous (Cat+/) mice were obtained from Dr. Ye-Shih Ho [Ho et al., 2004], rederived by Charles River Laboratory (Wilmington, MA) and bred to produce homozygous wild-type (Cat+/+), heterozygous (Cat+/−), and homozygous knockout (Cat−/−) offspring. Mice were housed in an animal care facility at the U.S. Environmental Protection Agency (Research Triangle Park, NC) with a 12-hr light/dark cycle and given food and water ad libitum. Animal care, euthanasia, aseptic removal of spleens, and liver perfusions were performed according to established protocols approved by the Institutional Animal Care and Use Committee of the National Health and Environmental Effects Research Laboratory.

Genotyping

Genotyping was performed as previously described [Tennant and Kligerman, 2011] with modifications. Three- to 5-month-old male mice were used in the experiments. To determine the Cat genotype, mouse tissue was obtained via ear punch biopsy and incubated at 55°C in a lysing solution containing 10 mM Tris pH 8.0, 2.5 mM MgCl2, 100 mM KCl, 100 mAU ml−1 proteinase K (Invitrogen™), and 0.45% Tween 20 for 3.5 hr. Samples were heated to 95°C for 15 min. A PCR was performed to amplify DNA fragments using 5 µl of the lysed tissue, 12.5 µl of a reaction mixture containing HotStarTaq® Plus Master Mix (Qiagen, Valencia, CA), and 5 µl of a primer mix containing 1 µM neomycin primer (5′-TTGGCGGCGAATGGGCTGAC), 2 µM CAT forward primer (5′-GGGACTTCTGGAGTCTTCGTCCC), and 1 µM CAT reverse primer (5′-GCCTGGAGAACAGGCTGTGCC). Reaction mixtures were heated to 95°C for 5 min, followed by 35 cycles of heating at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec. The reaction was finished with a 7-min elongation step at 72°C. The wild-type genome does not harbor the neomycin cassette sequence; therefore, it will only produce a 429-bp fragment, whereas the knockout genome will produce a 650-bp fragment; a heterozygous genome will yield both fragments. The amplified DNA fragments were separated on a 2% agarose gel with banding patterns indicating genotype.

Preparation of Primary Splenic Lymphocytes

Spleens were prepared as previously described [Tennant and Kligerman, 2011], with some modifications. The spleens were removed from male mice, placed in sterile RPMI 1640 medium, and rinsed two times in PBS followed by mincing in RPMI 1640. The minced suspension was then transferred to a 15-ml polypropylene tube. Next, 5 ml of Lympholyte® (Cedarlane Labs, Burlington, ON, Canada) was added to the homogenate. The homogenate was centrifuged for 20 min at 1,300g. The middle layer was collected and decanted into a new 15-ml tube and rinsed in 10 ml PBS and centrifuged for 10 min at 500g. The supernatant was discarded, and the pelleted cells were resuspended in PBS and centrifuged at 500g for 5 min. The supernatant was discarded, and the pelleted cells were resuspended in 2 ml RPMI 1640 supplemented with 15% FBS. Cell viability was measured using 0.4% trypan blue stain.

Preparation of Primary Hepatocytes via a Two-Step Liver Perfusion

The two-step liver perfusion was performed as previously described [Li et al., 2010], with some modifications. All solutions used were placed in a water bath set to 43°C so that the temperature of the liquid coming out of the catheter and into the vena cava would be ∼37°C. An intraperitoneal injection of 0.1 ml/10 g of body weight of 100 mg ml−1 ketamine and 20 mg ml−1 xylazine mixed in PBS was administered to each mouse. A 22 G × 1 catheter was inserted into the vena cava. The syringe was removed, and a solution of Hank's Balanced Salt Solution (HBSS) supplemented with 0.5 mM EGTA was pumped into the mouse through the vena cava for 5 min at a rate of 5 ml min−1. The hepatic vein was then cut before the liver expanded due to the pressure exerted by the EGTA being perfused. The liver was then perfused with Williams Medium E supplemented with 0.01 M HEPES and 0.004 N NaOH (WMI) and 60 U ml−1 of collagenase at a rate of 5 ml min−1 for 8–10 min. The catheter was removed from the vena cava, the liver excised, and the gall bladder removed carefully. The liver was then placed in a plastic Petri dish containing WMI at room temperature. The liver was clamped using hemostatic forceps, and using a surgical blade, several incisions were made on the lobes of the liver. The capsule was pulled using curved-end forceps, and the liver was agitated to release hepatocytes. The homogenate was then aspirated using a 10-ml wide-bore borosilicate pipette and poured through sterile gauze into a 50-ml polypropylene tube. The hepatocytes were centrifuged at 50g for 1 min. The pellet was then washed twice with cold WMI supplemented with 10% FBS, 100 U penicillin and 100 µg streptomycin mix, 0.01 M HEPES, 0.004 N NaOH, and 1× GlutaMAX™, resuspended in 10 ml of the same medium, and kept cold. Cell viability was measured using 0.4% trypan blue stain.

Exposures with H2O2 and MMAIII

All test chemicals were dissolved in filter-sterilized dH2O, which was also used as a negative control for DNA damage.

Splenic Lymphocytes

Exposures were conducted by adding 100 µl of each of the dilutions of stock H2O2 to yield final concentrations of 10, 20, 30, and 40 µM to individual 900-µl cell suspensions (7.5 × 105 cells per milliliter) and incubated on ice for 15 min in a 5-ml plastic tube. Cells were exposed to MMAIII at a concentration of 10 and 20 µM and incubated at 37°C and 5% CO2 for 2 hr in a 5-ml plastic tube. These concentrations were chosen because our previous studies have shown that these concentrations caused DNA damage without being excessively toxic [Mass et al., 2001; Tennant and Kligerman, 2011]. MMS was used as a positive control for nonoxidative stress-induced DNA damage at a concentration of 50 µM. Cells treated with MMS were also incubated at 37°C and 5% CO2 for 2 hr in a 5-ml plastic tube. After treatment, the cells were transferred to a 1.5-ml microcentrifuge tube, centrifuged at 500g for 5 min, washed in 1 ml PBS, centrifuged at 500g for 5 min, and resuspended in 200 µl PBS.

Hepatocytes

Hepatocytes were exposed by adding 100 µl of each of the dilutions of stock H2O2 at a final concentration of 20 and 40 µM to individual 900-µl cell suspensions (5.0 × 105 cells per milliliter) and incubating them on ice for 15 min in a 5-ml plastic tube. Other hepatocytes were treated with 5, 10, and 15 µM MMAIII and incubated at 37°C and 5% CO2 for 1 hr in a 25-cm2 cell culture flask. MMS was used as a positive control for nonoxidative stress-induced DNA damage at a concentration of 50 µM. After treatment, the cells were transferred to a 1.5-ml microcentrifuge tube, centrifuged at 100g for 30 sec, washed in 1 ml WMI, centrifuged at 100g for 30 sec, and resuspended in 100 µl WMI.

Alkaline Single-Cell Gel Electrophoresis (Comet) Assay

The single-cell gel electrophoresis (SCGE) assay was performed as previously described [Singh et al., 1991; Tennant and Kligerman, 2011], with some modifications. Briefly, three to four replicate experiments were performed examining 100 cells per concentration. All samples had viabilities of more than 85% for lymphocytes or more than 60% for hepatocytes before exposures. Multiple samples were placed on each slide using eight-chamber Coverwell™ perfusion chambers (Grace Bio-Labs, Bend, OR). Ten microliters of splenic lymphocytes (3.75 × 104 cells per milliliter) or hepatocytes (5 × 104 cells per milliliter) were mixed with 190 µl of 0.53% low-melting-point agarose that was kept molten at 37°C. Thirty-five microliters of this mixture was pipetted into corresponding chambers on each of two slides in randomized positions. The slides were placed on ice for 5–10 min to allow the agarose to solidify. The perfusion chamber was removed, and then the slides were incubated at 4°C in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 10.0) overnight. After incubation in lysis buffer, the slides were rinsed with dH2O and immersed in denaturing electrophoresis buffer (300 mM NaOH, 1 mM EDTA) at 4°C for 20 min. Electrophoresis was performed in cold buffer (pH ≥ 13) at 25 V (1.33 V cm−1) for 30 min. Slides were placed in neutralizing buffer (0.4 M Tris-HCl, pH 7.5) for 15 min at 4°C, dehydrated in 100% ethanol for 5 min, and allowed to air dry. Slides were immediately scored after drying or stored at room temperature in the dark until scoring.

Analysis with Komet®

The SCGE samples were analyzed by staining with 5× Sybr Gold® and viewed using fluorescence microscopy as previously described [Tennant and Kligerman, 2011]. Briefly, images were collected using the 25 × objective on a Nikon Microphot FXA, a Nikon B-2H filter, and a Hammamatsu ORCA CCD (Hammamatsu City, Japan) camera connected to a personal computer. The images were analyzed using Komet® version 5.5 (Andor Technology, Morrisville, NC). Fifty images per chamber and two chambers per concentration were analyzed in each experiment. Slides were coded before analysis so that the scorer was blind to the treatment when obtaining the SCGE data. Data from each H2O2, MMAIII, and MMS experiment are a representation from three or four independent experiments. The experimental replicate was the unit of experimentation for statistical analyses.

CAT Enzymatic Activity

Cell Lysate

Cell lysates were prepared from splenic lymphocytes and hepatocytes by sonication in 1 ml CellLytic MT™ (Sigma-Aldrich) supplemented with 0.2% protease inhibitor cocktail (Sigma-Aldrich) at 4°C. The supernatant was collected and assayed for protein concentration using the Bradford Reagent according to the manufacturer's directions (Sigma-Aldrich).

Enzymatic Assay

The CAT enzymatic activity assay was performed following previously described methods (Worthington Biochemical) [Beers and Sizer, 1952], with some modifications. The assay measures the decomposition of 0.059 M H2O2 into H2O and O2 using a U800 UV spectrophotometer (Beckman Coulter, Brea, CA) interfaced with a software program developed specifically for this instrument. The spectrophotometer readings were performed at 240 nm for 90 sec at 25°C. The enzymatic activity of CAT was determined using the following equation: units/mg−1 = (ΔA240/min × 1,000)/(43.6 × mg enzyme/ml mixture), where 43.6 is the molar extinction coefficient (M−1 cm−1) of H2O2 at 240 nm.

Statistics

Statistical analyses were performed using Statgraphics Centurion® XVI software (Statpoint Technologies, Warrentown, VA) on a personal computer. A comparison of regression lines was performed to determine if (a) there was a significant positive linear increase in DNA damage with increasing arsenical concentration and (b) if the effect of Cat genotype influenced the susceptibility of the cells to H2O2 and MMAIII. The model assumes a common intercept at the dH2O control (by subtracting the controls from each data set) and then tests the null hypothesis that the slopes are equal in magnitude. The alpha level was set at 0.05 for the F-test with two degrees of freedom. A one-tailed Student's t-test was performed on the data comparing the MMS exposures with the controls and on the data comparing the levels of CAT enzymatic activity of the Cat+/+ lymphocyte samples with the Cat+/+ hepatocyte samples. A one-way ANOVA test followed by Tukey's multiple comparison test was performed on the data comparing the levels of CAT enzymatic activity and the levels of MMS-induced DNA damage of the Cat+/+, Cat+/−, and Cat−/− hepatocyte samples. To compare DNA damage at each concentration to concurrent controls, a one-tailed Dunnett's test was used [Dunnett, 1955]. For all these tests the alpha level was set at 0.05.

Electron Spin Resonance Measurements

Electron spin resonance was performed as previously described [Nesnow et al., 2002], with some modifications. Samples (500 μl) were mixed in a 1.5-ml microcentrifuge tube (concentrations as described in the figure) and transferred to a quartz glass flat cell, and then the electron spin resonance spectrum was recorded immediately. The samples were mixed with 100 mM potassium phosphate buffer, pH 7.4, treated with Chelex-100 resin to chelate metallic impurities. The spectra were recorded at room temperature with a Bruker EMX spectrometer (Billerica, MA) equipped with an ER 4122 SHQ cavity operating at 9.73 GHz (X-band) and recorded via a personal computer interfaced to the spectrometer. We used 20 mW microwave power, 1 × 104 receiver gain, a modulation frequency of 100 kHz, and modulation amplitude of 1.0 G. The time constant was 655.36 ms, and conversion time was 327.68 ms. The field scan width was 100 G with a resolution of 1,024 points and a center of 3,490. Hyperfine coupling constants were determined with the Winsim spectral simulation program [Duling, 1994].

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

Alkaline SCGE Assay: Splenic Lymphocytes

As a proof-of-concept-experiment, Cat+/+ and Cat−/− primary splenic lymphocytes were exposed to various concentrations (0, 10, 20, 30, and 40 µM) of H2O2 for 15 min on ice in a 5-ml plastic tube. The genotoxicity of H2O2 was measured via the Comet assay. Our results showed that as the concentration of H2O2 increased, the % tail DNA of Cat+/+ splenic lymphocytes increased proportionally (Fig. 2A). In addition, a similar trend was observed in Cat−/− lymphocytes. The % tail DNA of Cat−/− lymphocytes was significantly higher than in the Cat+/+ lymphocytes exposed to 40 µM H2O2 (Fig. 2A). We compared the genotoxic potential of MMAIII in Cat+/+ and Cat−/− splenic lymphocytes by exposing the lymphocytes to 0, 10, and 20 µM MMAIII for 1 hr at 37°C and 5% CO2 and measuring the % tail DNA of the exposed cells. Our results showed that as the concentration of MMAIII increased, there was a proportional increase in % tail DNA as measured by the slope of the regression lines in both cell types. In addition, at the 20 µM concentration, there was a significantly higher amount of DNA damage in the Cat−/− lymphocytes when compared with the Cat+/+ lymphocytes (P < 0.05; Fig. 2B).

image

Figure 2. Comet assay. A: Comparison of linear regression lines of percent tail DNA of Cat+/+ and Cat−/− mice primary splenic lymphocytes exposed to H2O2 (n = 3; Cat+/+ slope = 0.96, Cat−/− slope = 1.1; r2 = 0.96, P < 0.05). B: Comparison of linear regression lines of percent tail DNA of Cat+/+ and Cat−/− mice splenic lymphocytes exposed to MMAIII (n = 3; Cat+/+ slope = 0.3, Cat−/− slope = 0.4; r2 = 0.92; P < 0.05). In each case, only the highest concentration of either H2O2 or MMAIII caused a statistically significant greater response in Cat−/− lymphocytes when compared with Cat+/+ lymphocytes (*). C: Comparison of percent tail DNA of Cat+/+ and Cat−/− mice splenic lymphocytes exposed to 50 µM MMS (n = 3, P > 0.5, Student's t-test; bars represent mean ± SEM).

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The alkylating agent MMS was used as a positive control in the Comet assay for nonoxidative stress-induced DNA damage. There was no statistically significant difference between the % tail DNA of Cat+/+ and Cat−/− splenic lymphocytes after exposure to MMS (P > 0.05; Fig. 2C).

Catalase Activity

A comparison was performed on Cat+/+ splenic lymphocytes and primary hepatocytes to determine their CAT activity. Our results showed that Cat+/+ hepatocytes harbored ∼10-fold higher levels of CAT activity than did Cat+/+ splenic lymphocytes (Fig. 3), as previously reported [Schriner et al., 2000]. The results also demonstrated that Cat+/+ hepatocytes displayed higher levels of CAT enzymatic activity than both the Cat+/− (approximately twofold) and Cat−/− (∼20-fold) hepatocytes (Fig. 4) and that the Cat+/− hepatocytes displayed higher levels of CAT than Cat−/− hepatocytes (∼10-fold; P < 0.001; the differences between all the means were statistically significant; Fig. 4).

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Figure 3. Comparison of CAT enzymatic activity (U/mg) between lymphocytes and hepatocytes determined by spectrophotometrically measuring the decomposition of H2O2 (n = 4, P < 0.001, Student's t-test; top of the box represents to the 75th percentile, bottom of the box represents the 25th percentile, the line in the middle of the box represents the mean, and the error bars are SD from the mean).

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Figure 4. Comparison of CAT enzymatic activity (U/mg) determined by spectrophotometrically measuring the decomposition of H2O2 between Cat+/+, Cat+/−, and Cat−/− primary mice hepatocytes (n = 4, P < 0.001 one-way ANOVA followed by Tukey's multiple comparison test; top of the box represents to the 75th percentile, bottom of the box represents the 25th percentile, the line in the middle of the box represents the mean, and the error bars are SD from the mean).

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Alkaline SCGE Assay: Hepatocytes

As observed with the splenic lymphocytes, Cat−/− hepatocytes were more susceptible to DNA damage than both the Cat+/+ (∼3.5-fold) and Cat+/− (∼3.0-fold) hepatocytes when exposed to various concentrations of H2O2 for 15 min on ice (Fig. 5A). We also observed a much larger difference in % tail DNA between the Cat−/− hepatocytes and the Cat+/+ hepatocytes exposed to MMAIII than what we had observed with the splenic lymphocytes of comparable genotypes (Fig. 5B). There was no statistical difference in MMS-induced DNA damage among the Cat+/+, Cat+/−, and Cat−/− hepatocytes (Fig. 5C).

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Figure 5. Comet assay. A: Comparison of linear regression lines of percent tail DNA of Cat+/+, Cat+/−, and Cat−/− primary mice hepatocytes exposed to H2O2 (n = 3; Cat+/+ slope = 0, Cat+/− slope = 0.17, Cat−/− slope = 0.7; r2 = 0.78, P < 0.0001). The Cat−/− hepatocytes were significantly more sensitive to the DNA-damaging effects of 40 µM H2O2 than the hepatocytes from mice of the other two genotypes. B: Comparison of linear regression lines of percent tail DNA of Cat+/+, Cat+/−, and Cat−/− primary mice hepatocytes exposed to (A) MMAIII (n = 4; Cat+/+ slope = 0.2, Cat+/− slope = 0.66, Cat−/− slope = 1.9; r2 = 0.48, P < 0.05). At 15 µM MMAIII, Cat−/− hepatocytes were significantly more sensitive to the DNA-damaging effects of MMAIII than were hepatocytes from Cat+/+ mice. For the Cat+/− hepatocytes, only the response at the highest concentration was significantly different from the control, whereas for the Cat−/− hepatocytes, both 10 and 15 µM MMAIII concentrations were statistically different from the concurrent controls. C: Comparison of percent tail DNA of Cat+/+, Cat+/−, and Cat−/− mice hepatocytes exposed to 50 µM MMS (n = 3, P > 0.05, one-way ANOVA followed by Tukey's multiple comparison test; bars represent mean ± SEM).

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Electron Spin Resonance Measurements

A significant four-line signal was detected once H2O2 and MMAIII were mixed in 100 mM DMPO (Fig. 6A); however, neither 250 µM H2O2 (Fig. 6B) nor 1 mM MMAIII (Fig. 6C) produced a signal when incubated individually in 100 mM DMPO when compared with that of DMPO only (Fig. 6G). By spectral simulation, the hyperfine coupling constants of (A) were determined to be aN = 14.99 G and aH = 14.74 G, typical for DMPO/·OH (aN = aH = 14.9 G) [Kuppusamy and Zweier, 1989]. In the presence of 10% DMSO (Fig. 6D), the DMPO/·OH signal was decisively decreased; the spectra also contained a new species (32% relative intensity), which was identified as DMPO/·CH3 (aN = 16.16 G and aH = 23.10 G; aN = 16.4 G and aH = 23.0 G) [Kuppusamy and Zweier 1989]. This is evidence for the production of ·OH [Yue Qian et al., 2005] from the coincubation of 1 mM MMAIII and 250 µM H2O2. In the presence of 500 U ml−1 SOD, the signal was dramatically decreased (Fig. 6E), suggesting that O2· was produced, which is required for ·OH formation according to Haber-Weiss chemistry. The addition of 1,000 U ml−1 CAT (Fig. 6F) also strongly decreased the signal, which is in accordance with the cell experiments, confirming that H2O2 plays a key role in MMAIII-dependent radical formation.

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Figure 6. Electron spin resonance was used to detect the potential production of the ·OH radical by MMAIII. Electron spin resonance spectra of (A) 100 mM DMPO, 1 mM MMAIII, and 250 µM H2O2, (Asim) spectral simulation of (A), (B) as (A), without MMAIII, (C) as (A), without H2O2 (D) as (A) plus 10% (v/v) DMSO, (Dsim) spectral simulation of (D), (E) as (A) plus 500 U/ml SOD (F) as (A) plus 1,000 U/ml CAT, and (G) DMPO only.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

As we briefly summarized in the Introduction section, there is no consensus on how arsenic induces cancer or genetic damage. Our laboratory has concentrated on investigating if ROS play a major role in the genotoxicity and possible carcinogenicity of arsenic. Previous work in our laboratory demonstrated that SOD (Sod1) homozygous knockout (Sod1tm1Leb/Sod1tm1Leb) lymphocytes were more susceptible to MMAIII than wild-type (Sod1+/+) and heterozygous (Sod1+/Sod1tm1Leb) cells [Tennant and Kligerman, 2011]. These results suggested that O2· plays a role in the oxidative stress MOA of MMAIII. This led us to investigate whether ·OH, known to be produced in rats exposed to arsenite [García-Chávez et al., 2003], could be involved in the toxic MOA of MMAIII. In the experiments reported here, we verified that splenic lymphocytes that were Cat−/− and, thus, unable to produce CAT were more sensitive to the induction of DNA damage by both H2O2 and MMAIII than wild-type splenic lymphocytes. However, contrary to our expectations, the differences in sensitivity, although statistically significant, were quite small. This led us to investigate hepatocytes from wild-type mice. These were found to have a much higher level of CAT than splenic lymphocytes from the same mice. As expected, Cat−/− mice essentially had no CAT activity. Results with the hepatocytes verified the qualitative findings from the splenic lymphocytes; however, the differences in sensitivity among the genotypes to both H2O2 and MMAIII were much greater in magnitude. Thus, CAT does offer some protection from the DNA-damaging potential from both MMAIII and H2O2.

These findings along with other work previously reported in the literature [Hei et al., 1998; Liu et al., 2001; Nesnow et al., 2002] support the hypothesis that the oxidative damage pathway is an important factor in arsenic's ability to induce genetic damage. Evidence supports the involvement of H2O2 being a focal point in this process. Arsenite given to rats through intraperitoneal injection showed significantly elevated concentrations of H2O2 in their bile [Kobayashi and Hirano, 2008]. This could lead to the formation of ·OH through the Fenton reaction. In addition, the data presented here complement previous work produced by the Gandolfi group, who have shown in several studies that MMAIII transforms UROtsa cells [Bredfeldt et al., 2006] potentially through the formation of ROS-induced DNA damage [Eblin et al., 2006] as evidenced when cells were incubated with CAT or SOD after allowing the cells to recover from MMAIII exposure [Wnek et al., 2009]. The transformed cells showed tumorigenic properties for anchorage-independent growth in culture and in severe compromised immunodeficiency (SCID) mice after transplantation [Bredfeldt et al., 2006; Wnek et al., 2010]. Arsenite exposure in cell cultures induces the production of ·OH as shown by electron spin resonance spectroscopy with and without the addition of CAT [Liu et al., 2001]. In addition, the generation of ·OH radicals induced solely by dimethylarsinous acid (DMAIII) has also been demonstrated [Nesnow et al., 2002]. We hypothesized that a similar process can occur with MMAIII which might also spontaneously form ·OH presumably through a Fenton-type reaction. However, as determined with the spin-trapping experiments in this study, MMAIII alone does not produce detectable levels of ·OH, but if MMAIII and H2O2 are coincubated, a significant DMPO/·OH signal was observed, which was decreased in the presence of CAT. This effect is in accordance with our cell experiments. In the presence of 10% DMSO, the signal intensity was decreased, and the occurrence of a new species, DMPO/·CH3, was observed. This suggests that ·OH reacts with DMSO to produce ·CH3, which is then trapped by DMPO [Kuppusamy and Zweier, 1989]. We believe that the levels of H2O2 produced by MMAIII are too small to be detected using this methodology; however, when H2O2 is present, the arsenical can induce the production of ·OH in Haber-Weiss/Fenton-type chemistry. The ·OH signal also decreased when SOD was added, which suggests that the DMPO/·OH signal could originate from the production of O2·.

One of the most prevalent hypotheses to explain the induction of bladder cancer by arsenic is cytotoxicity followed by regenerative proliferation leading to hyperplasia [Cohen et al., 2002, 2007; Yokohira et al., 2010]. Although there is good support for some aspects of this hypothesis, this view fails to take into account the need for mutation in the induction of cancer [Negrini et al., 2010] and, at the same time, ignores the fact that the trivalent methylated forms of arsenic are potent clastogens [Kligerman et al., 2003; Dopp et al., 2004] and aneugens [Kligerman et al., 2005]. Chromosome breakage and changes in chromosome numbers are potent driving forces in the production of mutant cells and their progression to tumors; thus, the genotoxicity of these compounds has to be considered when developing a hypothesis on how arsenic induces cancer.

In the experiments reported here using CAT knockout mice, Cat−/− cells were more susceptible to DNA damage from both H2O2 and MMAIII than the Cat+/+ cells due to the fact that they do not harbor any significant CAT activity. In cells or animals exposed to H2O2 either directly or through metabolism, the absence of CAT activity could lead to an increase in the levels of ·OH if H2O2 reacts with free iron through the Fenton or Haber-Weiss reaction [Miller et al., 1990]. In experiments performed using CAT-overexpressing rat WEHI7.2 cells exposed to arsenic trioxide (As2O3), results showed that they were more resistant to cytotoxicity from the arsenical than wild-type or H2O2-resistant cells [Sertel et al., 2012]. The results from that work suggest that the H2O2 present in these cells could react with As2O3 to produce cytotoxic levels of ROS [Sertel et al., 2012], supporting our hypothesis that H2O2 concentrations in Cat−/− cells could react with MMAIII to produce ROS, including ·OH, which leads to genotoxicity. The ·OH radicals could then directly interact with DNA and induce strand breakage leading to clastogenicity [Kligerman and Tennant, 2007]. The results presented here could also suggest that the genotoxicity of arsenic might be due to the generation of H2O2 that reacts with Fe2+ ions producing ·OH.

Studies have shown that mutations in genes involved in the metabolism of arsenic or enzymes involved in the DNA repair pathway might render individuals more susceptible to the toxic effects of arsenicals [Hsu et al., 2011; Huang et al., 2011]. Moreover, arsenic and its metabolites have also been suggested to cause DNA damage repair inhibition [Li and Rossman, 1989; Yager and Wiencke, 1997; Hartwig and Schwerdtle, 2002]. DNA damage induction coupled with repair inhibition could amplify the genotoxic and carcinogenic properties of arsenicals. These and other studies suggest that individuals harboring a mutation that codes for a nonfunctional form of CAT and who are chronically exposed to arsenic might be more susceptible to the toxic effects of the metalloid than individuals who produce a wild-type or a functional form of the enzyme. Individuals homozygous for a single-nucleotide polymorphism (SNP) in the promoter region of the CAT gene (C-262T) have been shown to have increased transcription of the CAT mRNA [Forsberg et al., 2001]; however, the levels of enzymatic activity of individuals with the TT genotype are lower than heterozygous (CT) or CC homozygous individuals [Nadif et al., 2005]. Individuals harboring the TT genotype might be susceptible to the genotoxic effects of arsenicals if exposed. This has been demonstrated in studies performed on individuals exposed to arsenic in their drinking water in Bangladesh who harbor the TT SNP [Ahsan et al., 2003]. These individuals showed a fourfold increased risk of developing hyperkeratosis from arsenic exposure [Ahsan et al., 2003]. Furthermore, it could be inferred based on our studies that the levels of CAT activity in cells, tissues, or individuals may be used as a biomarker of possible susceptibility to arsenic exposure, as previously postulated [Banerjee et al., 2010]. Additionally, individuals with such mutations might be susceptible to other environmental agents that act through the production of H2O2.

Even though volumes of information exist on the toxicity of arsenic and its derivatives, there is no consensus on its MOA. Several hypotheses have been proposed that have attempted to clarify the MOA of this metalloid. The focus of our studies has been on whether or not oxidative stress is responsible for the genetic damage induced by arsenicals. In this study, we assessed the induction of DNA damage using the Comet assay and found that the Cat/ lymphocytes and hepatocytes exhibit higher levels of DNA strand breakage than the Cat+/+ cells exposed to MMAIII. In spin-trapping experiments, we detected the generation of ·OH via MMAIII when H2O2 was present; radical formation was diminished in the presence of CAT and DMSO; the sensitivity to SOD suggests that O2· could be the primary radical species in the spin-trapping experiment. Taken together, we propose that CAT is involved in protection against the genotoxic effects of the ·OH generated by MMAIII.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

The authors are grateful to Dr. Ye-Shih Ho (Wayne State University, Detroit, MI) for sending the original stock of catalase knockout mice to start the colony. The authors also thank the Animal Care Staff for their technical help in maintaining the catalase knockout colony. The authors thank Alan Tennant and James Campbell for sharing their technical expertise. The authors also thank John Pope and Kim Shepard at Integrated Laboratory Systems (ILS) (Research Triangle Park, NC) and Dr. Daneida Lizarraga at Maastricht University (Maastricht, The Netherlands) for consultation on mouse liver perfusion. The authors thank Ms. Mary J. Mason for her help with the manuscript. The authors also thank Drs. David M. DeMarini, David Thomas, and Eric Tokar for reviewing the manuscript before submission.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES

J.G.M.O. and A.D.K. designed the experiments, analyzed the data, and drafted the manuscript. J.G.M.O. performed all of the experiments and collected the data, except the electron spin resonance experiments, which were performed by F.L. who also helped to draft the manuscript. M.K. and R.P.M. helped with the electron spin resonance analysis and drafting of the manuscript. K.A.W. helped with the liver perfusions on mice.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. AUTHOR CONTRIBUTIONS
  9. REFERENCES
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