ToxSci Advance Access originally published online on January 25, 2006
Toxicological Sciences 2006 91(1):70-81; doi:10.1093/toxsci/kfj117
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Chronic Exposure to Methylated Arsenicals Stimulates Arsenic Excretion Pathways and Induces Arsenic Tolerance in Rat Liver Cells
* Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan; and Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA
1 To whom correspondence should be addressed at Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan. Fax: x81-88–655–3051. E-mail: teruaki{at}ph.bunri-u.ac.jp.
Received December 14, 2005; accepted January 19, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although inorganic arsenicals are toxic and carcinogenic in humans, inorganic arsenite has recently emerged as a highly effective chemotherapeutic agent for acute promyelocytic leukemia (APL). Inorganic arsenicals are enzymatically methylated to monomethylarsonic acid (MMAsV), dimethylarsinic acid (DMAsV), and trimethylarsine oxide (TMAsVO) in mammals. We examined the effects of chronic exposure to methylated arsenicals on arsenic tolerance by using rat normal liver TRL 1215 cells. TRL 1215 cells were exposed for 20 weeks to MMAsV, DMAsV, or TMAsVO at levels that produced submicromolar cellular concentrations of arsenic. On chronic exposure to these methylated arsenicals, the cells acquired tolerance to acute arsenic cytolethality. Cellular arsenic uptake was reduced in these cells compared to passage-matched control cells. The long-term arsenic exposure increased glutathione S-transferase (GST) activity and cellular glutathione (GSH) levels. Glutathione S-transferase, multidrug resistance-associated proteins (Mrps; efflux transporters encoded by Mrp genes), and P-glycoprotein [P-gp; efflux transporter encoded by multidrug resistance gene (MDR)] had also increased in these cells at the transcript and protein levels. The depletion of cellular GSH and the inhibition of Mrps and P-gp functions increased cellular arsenic uptake and reduced arsenic tolerance in these cells. These results indicate that chronic exposure to methylated arsenicals induces a generalized arsenic tolerance that is caused by increased arsenic excretion. Because accumulation of methylated arsenicals may occur in patients with chronic arsenic poisoning and arsenic-treated APL patients, this study may provide important information regarding chronic arsenic poisoning and the latent risk of developing multidrug resistance in APL therapy using inorganic arsenite.
Key Words: arsenic; monomethylarsonic acid; dimethylarsinic acid; glutathione; multidrug resistance–associated proteins; tolerance.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inorganic arsenicals are metalloids that are widely distributed in soil and water, and they are known human carcinogens (NRC, 1999
). Humans encounter inorganic arsenicals in drinking water from wells drilled into arsenic-rich strata or in burning of coal containing arsenicals, and chronic arsenic poisoning has occurred in many countries in the world (NRC, 1999). In humans and some experimental animals, inorganic arsenicals are rapidly absorbed from the gastrointestinal tract and enzymatically methylated in the liver or other organs into organic arsenicals, such as monomethylarsonic acid (MMAsV) and dimethylarsinic acid (DMAsV) (Hindmarsh and McCurdy, 1986; Yamauchi and Yamamura, 1979). MMAsV and DMAsV are the major organic pentavalent arsenic metabolites in human urine after the exposure to inorganic arsenical (Yamauchi and Yamamura, 1979). DMAsV is the ultimate metabolite in humans, while DMAsV is further methylated to trimethylarsine oxide (TMAsVO) in some rodents (Fig. 1) (Yamauchi and Yamamura, 1984). It is believed that methylation of inorganic arsenicals results in a reduction in general toxicity, as indicated by their increased in vivo lethal dose in 50% of a population (LD50) and in vitro cytolethal concentration in 50% of a population (LC50) (Sakurai et al., 1998, 2002a, 2002b). However, recent studies have increasingly suggested that the methylation is not a universal detoxification mechanism. For instance, the exposure to DMAsV induces cellular DNA damage and promotes or causes tumors (Sordo et al., 2001; Wei et al., 1999, 2002). It has also been reported that highly toxic trivalent methylated arsenicals may be produced in the mammalian body by reducing pentavalent methylated arsenicals (Kala et al., 2000; Mandal et al., 2001; Mass et al., 2001). The details on the effects of chronic exposure to DMAsV have not yet been clarified. Furthermore, much less is known concerning the in vitro toxic potential or mechanisms of the other methylated arsenic metabolites, such as MMAsV and TMAsVO. We recently reported that MMAsV was not highly toxic in mammalian cells, but it induced cell death when cellular reduced glutathione (GSH) was depleted (Sakurai et al., 2004, 2005). It has been reported that inorganic arsenicals are largely and rapidly methylated in mammals (Hindmarsh and McCurdy, 1986), and methylated arsenicals are accumulated during chronic arsenic poisoning in the human body (Yamauchi, 2000). A trivalent inorganic arsenic compound, arsenic trioxide, has recently been used with remarkable success in treatment of acute promyelocytic leukemia (APL) (Chen et al., 1997; Shen et al., 1997). Clinically, multiple high doses of inorganic arsenite by intravenous injection (10 mg/day) for at least 28 consecutive days are needed to induce remission in APL patients (Shen et al., 1997), and the accumulation of the methylated forms of arsenicals occurs in these patients (Fukai et al., 2003). Therefore, defining the mechanisms of chronic arsenic poisoning and the toxic side effects of arsenical use in APL therapy requires further studies on the effects of the chronic exposure to methylated arsenicals.
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In this study, using rat liver cells, we demonstrated that chronic exposure to MMAsV, DMAsV, and TMAsVO induced tolerance to the acute cytolethality of inorganic and organic arsenicals in vitro. This study may not only aid in understanding chronic arsenic poisoning but could also have implications for arsenical use in APL therapy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Sodium arsenite, sodium arsenate, and dimethylarsinic acid (DMAsV) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Monomethylarsonic acid (MMAsV) was obtained from Tri Chemical Laboratory Inc. (Yamanashi, Japan). Trimethylarsine oxide (TMAsVO) was synthesized from trimethylarsine using hydrogen peroxide as described elsewhere (Sakurai et al., 1998
). These arsenicals were recrystallized twice and their purities were >99.9% as determined by gas chromatography–mass spectrometry (Sakurai et al., 2002b). Endotoxin contamination of these arsenicals was <0.0000003% (wt/wt) as determined by the endotoxin-specific limulus test (Seikagaku Co., Tokyo, Japan). L-Buthionine-(S,R)-sulfoximine (BSO; an inhibitor of 
–glutamylcysteine synthetase, which decreases cellular GSH levels), verapamil [an inhibitor of multidrug resistance–associated proteins (Mrps) and multidrug resistance gene (MDR)-encoded P-glycoprotein (P-gp)], probenecid (an inhibitor of Mrps), sodium taurocholate (an inhibitor of Mrp3 and P-gp) and sodium glycocholate hydrate (an inhibitor of Mrp3) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Cell culture conditions.
TRL 1215 cells are nontumorigenic adhesive rat epithelial liver cells originally derived from the liver of 10-day-old Fisher F344 rats (Zhao et al., 1997) and were cultured in William's medium E (Sigma) supplemented with 10% fetal bovine serum, 2 mM glutamine and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) under a humidified atmosphere of 5% CO2/95% air at 37°C.
Arsenic analysis.
Cellular arsenic contents were analyzed by hydride generation coupled with atomic absorption spectrometry (AAS) using SpestraAA-220 (Varian Australia Pty Ltd., Mulgrave, Victoria, Australia) (Kojima et al., 2004; Ohta et al., 2004). The results are expressed as nanograms of the cellular arsenic contents per milligram of cellular protein determined by BCA protein assay (Pierce Co., Rockford, IL, USA) with bovine serum albumin as a standard. Cellular arsenic concentrations were calculated using total cell numbers and estimated cell density (2.4 x 10–9 cm3/cell) as measured using a hemacytometer counting chamber (Mizoguchi and Hara, 1996).
Chronic arsenic exposure.
TRL 1215 cells were chronically exposed to MMAsV (1.3 mM), DMAsV (0.7 mM) or TMAsVO (10 mM) for 20 weeks in the 25 cm2 tissue culture flasks. These concentrations of MMAsV and DMAsV were one-tenth of acute cytotoxic concentrations (LC50 values for 48 h in vitro incubation) of these arsenicals in TRL 1215 cells (Kojima et al., 2004; Sakurai, 2002, 2003; Sakurai et al., 2002b, 2004). TMAsVO was not cytotoxic even at a concentration greater than 10 mM (Kojima et al., 2004; Sakurai et al., 1998). Control cells were incubated with medium alone for 20 weeks.
Assay for acute arsenic cytolethality.
Control cells and chronic methylated arsenical-exposed cells were isolated by trypsinization, washed twice, and resuspended in fresh medium. Cells (1 x 104/100 µl/well) were plated in flat-bottomed 96-well tissue culture plates and allowed to adhere to the plate for 24 h, at which time the medium was removed and replaced with fresh medium containing arsenite, arsenate, MMAsV, DMAsV, or TMAsVO. Cells were then incubated with these arsenicals for an additional 48 h. After incubation, cells were washed twice with warmed phosphate-buffered saline (PBS; pH = 7.4) to remove non-adherent dead cells, and cellular viability was determined by AlamarBlue assay, which is similar to MTT assay and measures metabolic integrity (Sakurai et al., 2002b). Briefly, after incubation with arsenicals and replacement with 100 µl fresh medium, 10 µl/well AlamarBlue solution (Iwaki Grass Co., Chiba, Japan) was added directly to the wells, incubated at 37°C for 4 h, and the absorbance at 570 nm (reference as 600 nm) was measured by a microplate reader model 550 (Bio-Rad Laboratories, Hercules, CA, USA). Data are expressed as relative metabolic integrity using the values from untreated cells as 100%.
Stability of arsenic tolerance.
To define the stability of acquired arsenic tolerance in cells chronically exposed to methylated arsenicals, the cells were passed in an arsenic-free medium for an additional 8 weeks, and then acute cytolethality of arsenicals was assessed. This arsenic-free incubation returned the cellular arsenic contents in chronic methylated arsenical-exposed cells to the control level (<0.2 ng/mg cellular protein; n = 3) determined by hydrogen generation coupled with AAS.
Assay for glutathione S-transferase (GST) activity.
Cellular GST activity was measured by the method of Lee et al. (1989) using 1-chloro-2,4-dinitrobenzene and GSH as substrates. Cells were isolated by trypsinization, rinsed twice with PBS (pH = 7.4), resuspended in 400 µl of 100 mM potassium phosphate buffer (pH = 6.8), and sonicated for 10 s on ice. Cellular debris was removed by centrifugation, and 50 µl of the supernatant of each cell was then mixed with 850 µl of 100 mM sodium phosphate buffer including 1 mM ethylenediamine tetraacetate (EDTA, pH = 6.5), 50 µl of 20 mM GSH, and 50 µl of 20 mM 1-chloro-2,4-dinitrobenzene at room temperature, and the absorbance at 340 nm was continuously measured for 2 min. Data are expressed as specific GST activity (nmol/min) per mg of cellular protein determined by BCA protein assay.
Assay for cellular reduced glutathione (GSH) levels.
Cellular GSH levels were measured by the method of Hissin and Hilf (1976) using o-phthaldialdehyde as a substrate. Cells were isolated by trypsinization, rinsed twice with PBS (pH = 7.4), and digested in 150 µl of ice-cold 800 mM perchloric acid including 8 mM EDTA. Cellular debris was removed by centrifugation, and 100 µl of the supernatant of each cell was then added into 2 ml of 100 mM sodium phosphate buffer (pH = 8.0), including 5 mM EDTA and 1 mg/ml o-phthaldialdehyde, and was kept for 15 min at room temperature in the dark. The fluorescence intensity of the sample solutions was measured at excitation and emission wavelengths of 350 nm and 425 nm, respectively. Aliquots of GSH were used to construct a standard curve, and the results are expressed as GSH nmol per mg of cellular protein determined by BCA protein assay.
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis.
Control cells and chronic methylated arsenical-exposed cells were isolated by trypsinization and rinsed twice with PBS (pH = 7.4). Total RNA in control cells and chronic methylated arsenical-exposed cells was isolated using ISOGEN (Nippon Gene Co., Tokyo, Japan), according to the manufacturer's instructions. The template cDNA for PCR was synthesized using reverse transcriptase M-MLV (Wako). Polymerase chain reaction was performed using TaKaRa PCR Thermal Cycler model TP 2000 (TaKaRa Bio, Shiga, Japan). The primer sequences for the GST-
gene (GST-) (5'-ATGAGAAGTTTATACAAAGTCC-3'; 5'-GATCTAAAATGCCTTCGGTG-3'), GST-µ gene (GST-µ) (5'-ATGCCTATGATACTGGGATACTGG-3'; 5'-AGGTCTTGTGAGGAAGCGGCTG-3'), GST-
gene (GST-) (5'-GATGGGGTGGAGGACCTTCGATGC-3'; 5'-CTGAGGCGAGCCACATAGGCAGAG-3'), multidrug resistance gene (MDR1) (5'-CTCACCAAGCGACTCCGATACATG-3'; 5'-GATAATTCCTGTGCCAAGGTTTGCTAC-3'), multidrug resistance associated protein 1 gene (MRP1) (5'-GGAAGACAAAGATTCT-AGTGTTGGACG-3'; 5'-AGATATGCCAGAGATCAGTTC-3'), MRP2 (5'-TGCAGCCTCCATAACCATGAG-3'; 5'-GATGCCTGCCATTGGACCTA-3'), and MRP3 (5'-CCAACCCATGAACCCCAA-3'; 5'-GCACATCTAGGTCAGCTAGCAGG-3') were obtained from Nippon EGT Co. (Toyama, Japan). The primer for ß-actin gene (ß-actin) was purchased from R&D Systems (Minneapolis, MN, USA). Polymerase chain reaction products (25 cycles for GST-, GST-µ, and GST-; 30 cycles for ß-actin; 40 cycles for MDR1, MRP1, MRP2, and MRP3) were visualized by ultraviolet illumination after electrophoresis through 2% agarose gel, with 0.5 µg/ml ethidium bromide at 50 V at 1 h, and scanned using Printgraph AE-6911CX (Atto, Tokyo, Japan).
Western-blot analysis.
Control cells and chronic methylated arsenical-exposed cells were isolated by trypsinization, rinsed twice with PBS (pH = 7.4), and pelleted in lysis buffer (62.5 mM Tris-HCl, pH 6.8, 10% grycerol, 4% sodium dodecyl sulfate, 100 mM dithiothreitol, and 0.005% bromophenol blue). Total protein (20 µg for GST-, GST-µ, and GST-; 40 µg for Mrp1, Mrp2, Mrp3, and P-gp) was subjected to electrophoresis on Tris-glycine polyacrylamide gels (4–20%), followed by electrophoretic transfer to polyvinylidene difluoride membrane for 1 h. The membrane was blocked in 0.5% skim milk in TPBS (81 mM sodium dihydrogenphosphate, 19 mM disodium hydrogenphosphate dihydrate, 100 mM sodium chloride, and 0.1% Tween 20) for over night at 4°C, followed by incubation with the appropriate primary antibody (1:1000), the rabbit polyclonal antibody against GST- (Oxford Biomedical Research, Oxford, MI, USA), against GST-µ (Oxford Biomedical Research), and against GST- (Oxford Biomedical Research), or the goat polyclonal antibody against Mrp1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), against Mrp2 (Santa Cruz), against Mrp3 (Santa Cruz), and against P-gp (Santa Cruz), in TPBS including 1 mg/ml bovine serum albumin for 1 h at room temperature. After four-times wash with TPBS, the membranes were incubated in secondary antibody (1:5000), the peroxidase-conjugated goat polyclonal antibody against rabbit immunoglobulin G-Fc or the peroxidase-conjugated rabbit polyclonal antibody against goat immunoglobulin G-Fc (Chemicon International Inc., Temecula, CA, USA), for 1 h at room temperature, followed by another four-times wash with TPBS. Immunoreactive proteins were detected by chemiluminescence using ECL Western Blotting Detection Reagents (Amersham Biosciences Co., Piscataway, NJ, USA).
Assay for cellular arsenic uptake in chronic methylated arsenical-exposed cells.
To deplete the cellular arsenicals accumulated by chronic methylated arsenical exposure, chronic methylated arsenical-exposed cells were washed and preincubated in an arsenic-free medium for 1 week. This arsenic-free incubation returned the cellular arsenic contents in chronic methylated arsenical-exposed cells to the control level (<0.2 ng/mg cellular protein; n = 3) determined by hydrogen generation coupled with AAS. Control cells and chronic methylated arsenical-exposed cells that were grown in flat-bottomed 75 cm2 tissue culture flask were exposed to 5 µM arsenite, 50 µM arsenate, or 1 mM DMAsV for 48 h. After exposure, the amounts of cellular arsenicals in these cells were analyzed by hydride generation coupled with AAS. The results are expressed as nanograms of the cellular arsenic contents per milligram of cellular protein determined by BCA protein assay, and cellular arsenic concentrations were calculated using total cell numbers and estimated cell density (2.4 x 10–9 cm3/cell) as measured using a hemacytometer counting chamber (Mizoguchi and Hara, 1996).
The chemical species of cellular arsenicals in the cells exposed to 10 µM arsenite for 48 h was determined by high performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS). Cells were lysed with 0.5 ml of 2 M sodium hydroxide and were transferred into polypropylene tube. Cell lysates in the tubes were heated at 80°C for 2 h in a water bath, cooled, and neutralized with hydrochloric acid. The aqueous solution was made a volume of 10 ml with distilled water and was filtered through a 0.20-µm filter. For HPLC-ICP MS analysis (Shraim et al., 2001), 10 µl of a sample solution was applied to a reversed-phase C18 column (Cadenza CD-C18; Imtakt Co., Kyoto, Japan) connected to an Liquid Chromatograph pump (Waters Co., Milford, MA, USA) with a mobile phase of 5 mM tetrabutylammonium hydroxide, 3 mM malonic acid, and 5% methanol at flow rate of 0.6 ml/min. The outlet of the HPLC system was coupled directly to the inlet of the ICP MS (HP 4500, Hewlett-Packard Company, Palo Alto, CA, USA), and signals at m/z 75 and 77 (corresponding to arsenicals and ArCl, respectively) were monitored.
Statistics.
Statistical evaluations in experiments were expressed as the arithmetic mean ± SEM and performed by analysis of variance (ANOVA) followed by Dunnett's multiple comparison test or the Student's t-test as appropriate (Zar, 1999). A value of p < 0.05 was considered significant in all cases.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chronic Methylated Arsenical Exposure Induced Tolerance to Acute Cytolethality of Arsenite, Arsenate, and DMAsV in Rat Liver TRL 1215 Cells
In this study, TRL 1215 cells were incubated with MMAsV (1.3 mM), DMAsV (0.7 mM), TMAsVO (10 mM), or the medium alone (control) for 20 weeks. Arsenic contents in the cells exposed to MMAsV, DMAsV, or TMAsVO for 20 weeks were 135.4 ± 12.0, 41.8 ± 2.5, or 543.8 ± 12.2 ng/mg cellular protein (121.8 ± 10.8, 37.6 ± 2.3, or 489.4 ± 11.0 nM; n = 3), respectively, determined by AAS. The concentrations of methylated arsenicals had no effect on the viability of TRL 1215 cells during the 20 weeks of incubation. Thus, the experimental conditions produced submicromolar levels of cellular arsenicals that were not cytolethal. Arsenicals were not methylated or demethylated in TRL 1215 cells (Sakurai et al., 2005
; present study). Chronic exposure to MMAsV, DMAsV, or TMAsVO did not induce malignant transformation in TRL 1215 cells. These chronic methylated arsenical-exposed cells were further exposed to various concentrations of arsenicals for an additional 48 h, and cell viability was then assessed by AlmarBlue assay. As the results, arsenite, arsenate, and DMAsV showed significant cytolethality in both control cells and chronic methylated arsenical-exposed cells (Tables 1, 2, and 3), although MMAsV and TMAsVO were not cytotoxic even at over 10 mM (data not shown).
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As shown in Table 1, chronic MMAsV- or TMAsVO-exposed cells clearly acquired tolerance to the acute cytolethality of arsenite. The LC50 values for arsenite in chronic MMAsV- or TMAsVO-exposed cells was 4.5-fold or 3.4-fold higher than the LC50 values in control cells, respectively. Cells chronically exposed to MMAsV or TMAsVO also became tolerant to the acute cytolethality of arsenate and DMAsV (Tables 2 and 3). Chronic MMAsV- or TMAsVO-exposed cells had a LC50 for arsenate 17.5-fold or 4.4-fold higher, and had a LC50 for DMAsV 3.3-fold or 2.2-fold higher than control cells, respectively.
On the other hand, there was no difference in the LC50 values for arsenite and arsenate between chronic DMAsV-exposed cells and control cells (Tables 1 and 2). However, chronic DMAsV-exposed cells were tolerant to DMAsV compared to control cells, as the LC50 value was 2.3-fold higher (Table 3).
Effects of Arsenic Withdrawal from Cells Chronically Exposed to Methylated Arsenicals on Acute Cytolethality of Arsenite, Arsenate, and DMAsV
To define the stability of acquired tolerance in chronic methylated arsenical-exposed cells, these cells were passed in an arsenic-free medium for an additional 8 weeks, and the acute cytolethality of arsenite, arsenate, or DMAsV in these cells was then measured. This arsenic-free incubation returned the cellular arsenic contents in chronic methylated arsenical-exposed cells to the control level (<0.2 ng/mg cellular protein). As shown in Tables 1, 2, and 3, the additional 8 weeks of arsenic-free incubation decreased tolerance to the acute cytolethality of arsenite, arsenate, and DMAsV in chronic MMAsV- or TMAsVO-exposed cells. However, chronic DMAsV-exposed cells still showed significant tolerance to arsenate, and they maintained tolerance to the acute cytolethality of DMAsV after incubation in an arsenic-free medium for 8 weeks.
Effects of Cellular GSH Depletion and Inhibition of Mrps and P-gp on Arsenic Tolerance Induced by Chronic Methylated Arsenical Exposure
We observed the effects of cellular GSH depletion on the arsenic tolerance induced by chronic exposure to methylated arsenicals. BSO (25 µM; an inhibitor of cellular GSH synthesis) was not cytotoxic either to control cells or to chronic methylated arsenical-exposed cells. However, it significantly increased the acute cytolethality of arsenite and arsenate (Tables 1, 2, 4, and 5) and reversely decreased the acute cytolethality of DMAsV in both control cells and chronic methylated arsenical-exposed cells (Tables 3 and 6).
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It is known that Mrps and P-gp are xenobiotic export transporters. We also examined the effect of verapamil (50 µM; an inhibitor of Mrps and P-gp), probenecid (500 µM; an inhibitor of Mrps), taurocholate (500 µM; an inhibitor of Mrp3 and P-gp), and glycocholate (500 µM; an inhibitor of Mrp3), on the tolerance induced by chronic exposure to methylated arsenicals. These inhibitors at these concentrations did not influence the cell viability. As a result, they significantly decreased tolerance to the acute cytolethality of arsenite, arsenate, and DMAsV in both control cells and chronic methylated arsenical-exposed cells (Tables 4, 5, and 6).
Changes in Cellular GST Activity and Cellular GSH Levels by Chronic Methylated Arsenical Exposure
Cellular GST activity and GSH levels in chronic methylated arsenical-exposed cells were assessed. As shown in Table 7, chronic exposure to MMAsV, DMAsV or TMAsVO significantly increased cellular GST activity in TRL 1215 cells. Chronic exposure to MMAsV and TMAsVO also markedly increased cellular GSH levels in TRL 1215 cells, although only chronic DMAsV exposure decreased them (Table 7).
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Chronic Methylated Arsenical Exposure Increased the Expression of GST-
, MRPs, and P-gpAs shown in Figure 2, RT-PCR analysis confirmed increased expression of the genes encoding GST-µ, GST-
, MRP1, MRP2, MRP3, and MDR1, and decreased expression of the gene encoding GST- in the cells that have undergone chronic exposure to MMAsV, DMAsV, or TMAsVO. The expression of ß-actin, which was used to standardize load, was similar in control cells and chronic methylated arsenical-exposed cells.
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To determine whether chronic exposure to methylated arsenicals also increased the above gene products at the protein level, the extracts of chronic MMAsV-, DMAsV-, or TMAsVO-exposed cells were analyzed for the expression of GST-
, GST-µ, GST-, Mrp1, Mrp2, Mrp3, and P-gp (MDR gene product) by Western-blot analysis. Figure 3 shows that the chronic exposure to MMAsV, DMAsV, or TMAsVO increased the expression of GST-µ, GST-, Mrp1, Mrp2, Mrp3, and P-gp, although it decreased the expression of GST-.
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Cellular Arsenic Uptake in Chronic Methylated Arsenical-Exposed Cells
Additional studies measured the arsenic contents that accumulated in chronic methylated arsenical-exposed cells versus control cells over a 48-h exposure period. To excrete the cellular arsenicals accumulated by chronic methylated arsenical exposure, chronic methylated arsenical-exposed cells were washed and preincubated in an arsenic-free medium for 1 week. This arsenic-free incubation returned the cellular arsenic contents in chronic methylated arsenical-exposed cells to the control level (<0.2 ng/mg cellular protein). Control cells and chronic methylated arsenical-exposed cells were then exposed to 5 µM arsenite, 50 µM arsenate, or 1 mM DMAsV for 48 h, and the amounts of cellular arsenicals were measured. As a result, cellular arsenic content in chronic methylated arsenical-exposed cells that acquired arsenic tolerance was significantly decreased when they were acutely exposed to arsenite, arsenate, or DMAsV (Tables 8, 9, and 10).
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To assess whether inhibition of export transporters P-gp and Mrps affects arsenic uptake, we measured cellular arsenic contents of the cells acutely exposed to arsenite, arsenate, or DMAsV in the presence or absence of BSO, verapamil, probenecid, taurocholate, or glycocholate. Treatment with BSO and probenecid notably increased the cellular uptake of arsenite in both control cells and all of chronic methylated arsenical-exposed cells, and treatment with verapamil increased it in only chronic DMAsV-exposed cells (Table 8).
BSO, probenecid, and verapamil significantly increased the cellular uptake of arsenate in both control cells and all of chronic methylated arsenical-exposed cells (Table 9).
When cells were exposed to DMAsV with BSO, cellular arsenic contents in both control cells and chronic methylated arsenical-exposed cells did not change (Table 10). Verapamil and taurocholate increased cellular uptake of DMAsV in both the control cells and all of the chronic methylated arsenical-exposed cells. Probenecid increased cellular uptake of DMAsV in control cells, chronic MMAsV-exposed cells, and chronic DMAsV-exposed cells (Table 10). Glycocholate also significantly increased them in chronic MMAsV- or DMAsV-exposed cells (Table 10).
We also observed the chemical species of cellular arsenicals in these cells acutely exposed to inorganic arsenite. Control cells and chronic methylated arsenical-exposed cells were incubated with 10 µM arsenite for 48 h, and cellular arsenic species were determined by HPLC-ICP MS. As shown in Figure 4, cellular arsenic contents in chronic methylated arsenical-exposed cells was significantly decreased comparing with that in control cells, and these cells did not metabolize arsenite within 48 h exposure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study demonstrates that tolerance to the acute cytolethality of arsenite, arsenate, and DMAsV is induced in mammalian cells by chronic exposure to nontoxic levels of MMAsV, DMAsV, and TMAsVO. Some researchers have reported that arsenic tolerance can be induced in mammalian cells by acute exposure to cytotoxic levels of arsenicals or by exposure to progressively increasing levels of arsenicals (Lee and Ho, 1994
; Ovelgönne et al., 1995). Thus, it is likely that arsenic exposure selects for arsenic-tolerant cells by eliminating sensitive subpopulations from the total cell pool. However, a different process may be occurring in humans exposed to arsenicals. Humans are chronically exposed to low levels of inorganic arsenicals in the environment (NRC, 1999). Methylated arsenicals are formed from inorganic arsenicals by enzymatic methylation (Hindmarsh and McCurdy, 1986; Yamauchi and Yamamura, 1979) and accumulate during chronic arsenic poisoning in the human body (Yamauchi, 2000). Urinary concentrations of methylated arsenicals in patients with chronic arsenic poisoning in Inner Mongolia, China, were in the micromolar range (Yamauchi, 2000). The cytolethality of arsenicals may depend on their cellular uptake. In this study, we demonstrated that the cellular arsenic contents of the cells that were exposed to micromolar to millimolar levels of methylated arsenicals for 20 weeks were in the submicromolar range. Thus, we studied the effects of chronic exposure to submicromolar cellular levels of methylated arsenicals on arsenic sensitivity in rat liver cells under our experimental conditions. The cellular concentration of methylated arsenicals that are formed from inorganic arsenicals by enzymatic methylation in the liver cells may be in the submicromolar range (Sakurai et al., 2005; Yamauchi, 2000). Therefore, the use of submicromolar cellular arsenic concentrations for in vitro experiments may at least partially reflect the in vivo conditions and prove helpful in defining mechanisms involved in chronic arsenic toxicity.
Chronic exposure to MMAsV or TMAsVO induced significant tolerance to the acute cytolethality of inorganic arsenite and arsenate and enhanced the ability to excrete cellular arsenicals; however, it did not affect the ability to metabolize them. Thus, this arsenic tolerance appears to be based on increased arsenic efflux. Chronic methylated arsenical-exposed cells that acquired tolerance to inorganic arsenicals had high levels of intracellular GSH, and cellular GSH depletion by BSO increased cellular uptake and acute cytolethality of inorganic arsenicals in these cells. Thus, cellular GSH may play an important role in tolerance to inorganic arsenicals. It has been reported that GSH may decrease the cytolethality of arsenicals through some processes, possibly through its role as an antioxidant, by directly binding arsenicals and thereby reducing their toxic potential or through enhanced efflux of arsenical–GSH conjugates (Romach et al., 2000). It has also been suggested that GST catalyzes the formation of arsenical-GSH conjugates (Dey et al., 1996; Kala et al., 2000; Liu et al., 2001; Sakurai et al., 2002b), and these conjugates are pumped out from the cells by ATP-dependent cell membrane export transporters such as Mrp1 and Mrp2 (Kala et al., 2000, 2004).
In the present study, we showed that chronic exposure to submicromolar methylated arsenicals increased GST activity in TRL 1215 cells. The upregulation of GST and Mrps in chronic methylated arsenical-exposed cells was confirmed at both transcript and protein levels. Additionally, the inhibition of Mrps functions by verapamil and probenecid significantly decreased tolerance to inorganic arsenicals in chronic methylated arsenical-exposed cells and increased their cellular uptake. These results strongly suggest that GST and Mrps induced by chronic methylated arsenical exposure play a role in the reduction of cellular arsenite and arsenate by increasing the efflux of arsenical–GSH conjugates. On the other hand, chronic DMAsV exposure did not induce significant tolerance to the acute cytolethality of inorganic arsenite and arsenate. Chronic DMAsV exposure decreased cellular GSH levels, although it increased the expression levels of GST and Mrps. It is suggested that the depletion of cellular GSH by chronic DMAsV exposure might counteract a possible manifestation of tolerance to inorganic arsenite and arsenate, because GSH depletion sensitizes the cells to the lethal effects of inorganic arsenicals.
Chronic methylated arsenical exposure also induced significant tolerance to the acute cytolethality of a toxic organic arsenic metabolite, DMAsV. Chronic methylated arsenical exposure significantly decreased the cellular uptake of DMAsV; thus, the tolerance to DMAsV in chronic methylated arsenical-exposed cells also seems to result in a large part from increased arsenic efflux, leading to reduced cellular arsenic burden. We previously demonstrated that DMAsV required cellular GSH to induce cytolethality (Sakurai, 2002, 2003; Sakurai et al., 1998, 2002b, 2004, 2005). In the present study, cellular GSH depletion by BSO decreased the acute cytolethality of DMAsV in both control cells and chronic methylated arsenical-exposed cells, and BSO did not affect the uptake of DMAsV in these cells. Chronic DMAsV-exposed cells showed tolerance to DMAsV, although their intracellular GSH level was lower than that of control cells. These findings indicate that cellular GSH may not be related to DMAsV efflux. On the other hand, verapamil (an inhibitor of Mrps and P-gp), probenecid (an inhibitor of Mrps), taurocholate (an inhibitor of Mrp3 and P-gp), or glycocholate (an inhibitor of Mrp3) reduced the ability of control cells and chronic methylated arsenical-exposed cells to survive DMAsV exposure and the increased cellular content of DMAsV. Chronic methylated arsenical exposure increased the expression of the genes encoding MRP1, MRP2, MRP3, and MDR. It has been reported that Mrp1 and Mrp2 cleared GSH conjugates of many compounds from the cells (Kala et al., 2000, 2004; Liu et al., 2001); however, Mrp3 did not efficiently transport these GSH conjugates (Hirohashi et al., 2000; Kool et al., 1999). MDR-encoded P-gp has also been shown to participate in the GSH-independent excretion of various xenobiotics (Kala et al., 2000, 2004; Liu et al., 2001). Additionally, mdr1a/1b double knockout mice, which lack expression of P-gp, showed increased sensitivity to acute arsenic toxicity with increased arsenic uptake in tissues (Liu et al., 2002). Therefore, the tolerance to the acute cytolethality of DMAsV induced by chronic methylated arsenical exposure appears to be based on the induction of GSH-independent arsenic excretion pathways such as Mrp3 and P-gp. Further experiments will be needed to clarify the relative contributions of some kinds of cell membrane transporters to the efflux of various arsenic compounds including arsenical–GSH conjugates from mammalian cells.
The stability of arsenic tolerance induced by chronic methylated arsenical exposure depended on the type of arsenicals. Acquired tolerance in chronic MMAsV- or TMAsVO-exposed cells was reversed by incubating the cells in an arsenic-free medium for a protracted period. This suggests that chronic exposure to MMAsV or TMAsVO induced temporary changes and that the effect could be reversed by removing the arsenicals. However, chronic DMAsV-exposed cells were tolerant to arsenate and retained the tolerance to DMAsV even after incubation in an arsenic-free medium. Thus, arsenic tolerance induced by chronic DMAsV exposure might be due to a more stable change in the cells. It has been frequently reported that the genotoxicity of DMAsV is much higher than those of other methylated arsenicals (Sordo et al., 2001). Further research is required to clarify the effects of chronic methylated arsenical exposure and acquired arsenic tolerance.
In summary, chronic exposure to submicromolar cellular levels of MMAsV, DMAsV, and TMAsVO activated the GST, MRP, and MDR genes, enhanced arsenic efflux, and resulted in tolerance to acute arsenic cytolethality in rat liver cells. It had been reported that inorganic arsenite could also induce arsenic tolerance (Ishikawa et al., 1996; Kimura et al., 2005; Liu et al., 2001). These findings may prove helpful in defining the mechanisms involved in chronic arsenic poisoning and in the long-term chemotherapeutic use of inorganic arsenite in APL patients. In particular, it is indicated that not only the therapeutically given inorganic arsenite, but also its methylated metabolites have the potency to induce arsenic tolerance. Furthermore, this chronic exposure may render tumor cells resistant to various chemotherapeutic agents by enhancing efflux, as there may be a potential for development of multidrug resistance in APL therapy using inorganic arsenite.
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ACKNOWLEDGMENTS |
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We express our thanks to Yasuhiro Shinkai and Prof. Yoshito Kumagai (University of Tsukuba, Ibaraki, Japan) for their technical advice for Western-blot analysis, to Dr. Masumi H. Sakurai (Azabu University, Kanagawa, Japan) for her technical advice for HPLC-ICP MS, and to Chihiro Kawata, Kouichirou Matsuda, Tomoe Sakoda, and Hiroki Soejima (Tokushima Bunri University, Tokushima, Japan) for their excellent technical assistance. This work was supported in part by the Intramural Research Program of the National Institute of Health, National Cancer Institute, Center for Cancer Research.
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