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Cellular Uptake of Trivalent Arsenite and Pentavalent Arsenate in KB Cells Cultured in Phosphate-Free Medium
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
136, 243–249 (1996)
0031
Cellular Uptake of Trivalent Arsenite and Pentavalent Arsenate in KB Cells Cultured in Phosphate-Free Medium RONG-NAN HUANG AND TE-CHANG LEE Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, Republic of China Received April 24, 1995; accepted September 26, 1995
Cellular Uptake of Trivalent Arsenite and Pentavalent Arsenate in KB Cells Cultured in Phosphate-Free Medium. HUANG, R. N., AND LEE, T. C. (1996). Toxicol. Appl. Pharmacol. 136, 243–249. Trivalent arsenite (As(III)) and pentavalent arsenate (As(V)) have been shown to have differential uptake mechanisms. In regular RPMI 1640 medium, As(III) was about 40-fold more toxic to KB oral epidermoid carcinoma cells. However, the cytotoxicity and intracellular accumulation of As(V) were dramatically enhanced, equalling those of As(III) when cells were grown in phosphatefree RPMI medium. As(V) uptake was dose-dependently inhibited by phosphate, mersalyl acid (a membrane sulfhydryl agent), and energy poisons, such as sodium azide and potassium cyanide. These results suggest that As(V) and phosphate share a common transport system. In contrast, As(III) uptake was not affected by the above agents. However, the initial uptake rates of As(III) were linearly correlated with its extracellular concentrations, suggesting that As(III) uptake is probably accomplished through simple diffusion. Our results also show that As(III) and As(V) are excreted from KB cells at a comparable rate, and at least half of As(V) is reduced to the more toxic As(III) prior to excretion into the medium. Therefore, the toxicity of As(V) may in part result from its reduction to As(III). q 1996 Academic Press, Inc.
Arsenic is a ubiquitous chemical widely distributed in food, water, air, and soil (Knowles and Benson, 1983; NAS, 1977). Arsenic exposure, either due to geochemical enrichment or industrial processes, is accompanied by many severe toxicological and pathological problems, such as increased prevalence of hypertension (Chen et al., 1995) and increased risks for lung, nonmelanotic skin, and liver cancers (Chen et al., 1985; IARC, 1980; Leonard and Lauwerys, 1980; Mabuchi et al., 1979; Pinto et al., 1977; Pinto and Nelson, 1976). Arsenic exists as many chemical forms. Inorganic trivalent arsenite (As(III)) and pentavalent arsenate (As(V)) are of most importance in causing toxicological problems. Since As(III) is much more toxic than As(V) (Bertolero et al., 1987; Fischer et al., 1985; Lee et al., 1985), most toxicological studies have focused on As(III). However, As(V) is the predominant form in nature (Del Razo et al., 1990), but its
toxicity has not been well elucidated. Understanding the differential toxic mechanisms of As(III) and As(V) is critically important in assessing the risk from arsenic exposure. Recently, arsenic transport has been extensively studied in bacterial systems (Carlin et al., 1995; Dey and Rosen, 1995; Gladysheva et al., 1994; Ji et al., 1994; Ji and Silver, 1992a,b; Oden et al., 1994; Silver and Ji, 1994). Basically, intracellular As(III) was extruded by an ATPase-dependent pump and As(V) was reduced to As(III) by an arsenate reductase prior to being released from the cells. Most of these studies were conducted to understand the cellular mechanism of arsenic excretion. However, the differential toxic effects of As(III) and As(V) on mammalian cells have usually been attributed to differences in cellular uptake of the two forms of arsenic (Bertolero et al., 1987; Fischer et al., 1985; Lerman et al., 1983; Vahter and Marafante, 1983). As(V), structurally similar to inorganic phosphate, has been shown to compete with phosphate in a number of metabolic reactions (Kamiya et al., 1983; Kenney and Kaplan, 1988; Thiel, 1988; Aposhian, 1989). Although As(V) uptake has been shown to be mediated through phosphate channels in some organisms (Chen et al., 1990; Kenney and Kaplan, 1988; Rosenberg et al., 1977; Aposhian, 1989; Willsky and Malamy, 1980), the results of several reports are controversial (Budd and Craig, 1981; Fullmer and Wasserman, 1985; Grillo and Gibson, 1979; Thiel, 1988). Unfortunately, no data are available on the uptake of As(III). Phosphate is a major inorganic salt in all cell culture media and may be a limiting factor in determining arsenic uptake in regular medium. To better understand the mechanism of arsenic uptake, we therefore studied the cellular uptake of As(III) and As(V) using phosphate-free medium. Since arsenic is a unique human carcinogen, a human oral epidermal carcinoma KB cell line was adopted in these experiments.
MATERIALS AND METHODS Chemicals and cell cultures. Trivalent sodium arsenite (NaAsO2 , As(III)), pentavalent sodium arsenate (Na3AsO4r7H2O, As(V)), and dimeth-
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0041-008X/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ylarsinic acid (DMA)1 were commercial products of Merck (E. Merck, AG). Monosodium acid methane arsonate (MMA) was purchased from Chem Service (Chem Service, Wester Chester, PA), and ethylenediaminetetraacetic acid (EDTA) and mersalyl acid from Sigma (Sigma, St. Louis, MO). The reagents used for cell culture, including fetal bovine serum (FBS), RPMI 1640 regular and phosphate-free media, glutamine, and antibiotics, were obtained from Gibco (Life Technologies, Grand Island, NY). KB cells, obtained from American Type Culture Collection (Rockville, MD), were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. The cultures were maintained at 377C in a humidified incubator with 5% carbon dioxide in air. Cytotoxicity assay. The cytotoxicity of As(III) and As(V) to KB cells was determined by a colony formation assay as described previously (Lee et al., 1989a). In brief, 5 1 105 cells were plated in a 60-mm petri dish. After a 24-hr incubation, the cultures were treated with drugs for 2 hr in freshly prepared regular or phosphate-free RPMI medium. The cells were trypsinized, replated in regular RPMI medium at a cell density of 400–500 cells/60-mm dish in triplicate, and then incubated for 10 days without medium change. The colonies were then fixed, stained, and counted as described (Lee et al., 1989a). The relative survival was calculated by dividing the number of colonies in arsenic-treated cultures by that in untreated cultures. The plating efficiency of untreated KB cells was 57.6 { 3.6%. Effect of phosphate ion on arsenic uptake. Arsenic uptake was assayed as described previously (Wang and Lee, 1993). In brief, KB cells were plated at a density of 3–5 1 105 cells/60-mm dish and incubated at 377C for 36–48 hr. The cells were treated with As(III) or As(V) for the time indicated, in medium containing various concentrations of phosphate. At the end of treatment, cells were washed five times with phosphate-buffered saline containing 1 mM EDTA and harvested by trypsinization. An aliquot of cells was subjected to arsenic content determination with an atomic absorption spectrophotometer (AAS, Hitachi Z-8000, Tokyo, Japan) equipped with a hydride formation system (Hitachi HSF-2, Tokyo, Japan) as described previously (Wang and Lee, 1993). To investigate the effect of phosphate on As(V) uptake, KB cells were incubated with 200 mM As(V) in RPMI 1640 medium containing 0–800 mg/liter (5.63 mM) sodium phosphate for 30 min. As(V) accumulation in KB cells was then determined as described (Wang and Lee, 1993). Effects of sulfhydryl modifier and energy poisons on the uptake of arsenic in KB cells. A sulfhydryl modifier (mersalyl acid) and energy poisons such as sodium azide (NaN3) and potassium cyanide (KCN) were used to characterize the uptake mechanism of arsenic. KB cells were pretreated with mersalyl acid (0–320 mM) for 10 min, or NaN3 (0–40 mM) or KCN (0–40 mM) for 60 min in phosphate-free medium, and subsequently coincubated with arsenic for 30 min. The arsenic uptake of KB cells was then determined as described above. Kinetic analysis of As(III) uptake in KB cells. Uptake kinetics of As(III) were analyzed by incubating KB cells for various times and As(III) concentrations in regular RPMI medium. The cellular uptake of As(III) was then determined as described above. The initial velocity of As(III) uptake at different concentrations was obtained by fitting the data with a polynomial model and calculating the uptake rate at time zero. Arsenic excretion. To determine arsenic excretion, KB cells were first treated with arsenic (200 mM) for 30 min in phosphate-free medium, rinsed once with medium, and then incubated in drug-free medium for the time indicated. Arsenic remaining in cells was then determined as described.
1
Abbreviations used: DMA, dimethylarsinic acid; MMA, monosodium acid methane arsonate; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; AAS, atomic absorption spectrophotometer; HPLC, highpressure liquid chromatography.
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FIG. 1. Cytotoxicity of As(III) and As(V) to KB cells in regular and phosphate-free medium. KB cells were treated with various concentrations of As(III) (s, l) or As(V) (L, l) either in regular (solid symbols) or in phosphate-free (open symbols) RPMI medium for 2 hr and cell survival was determined as described under Materials and Methods. Bar, SD of at least three independent experiments.
Analysis of arsenic derivatives. Logarithmically growing KB cells were exposed to 1 mM As(III) or As(V) in phosphate-free medium for 30 min, washed once with drug-free medium, and then incubated in complete RPMI medium for 2 hr. At the end of treatment, arsenic derivatives in culture medium were separated by a nucleosil 10B HPLC column (Phenomenex, Torrance, CA) using phosphate buffer (50 mM, pH 6.75) at a flow rate of 0.5 ml/min. Arsenic derivatives were detected by an online connected AAS as described (Wang and Lee, 1993). Standard arsenic derivatives were prepared in complete RPMI medium.
RESULTS
Cytotoxicity of As(III) and As(V) Mammalian cells grown in regular culture medium were generally more sensitive to As(III) than to As(V). For a 2hr treatment, the concentrations for As(III) and As(V) required for 50% inhibition of colony-forming ability of KB cells were 100 and 4000 mM, respectively (Fig. 1). Regular RPMI 1640 medium contains 800 mg/liter Na2HPO4 (5.63 mM). When the experiments were performed using phosphate-free RPMI medium, the toxicity of As(III) did not change (Fig. 1). According to doses to kill 50% of cells, the cytotoxic effect of As(V), however, was enhanced 40-fold, to reach the same cytotoxicity as As(III) (Fig. 1). Effect of Phosphate on the Uptake of Arsenic in KB Cells Since phosphate may inhibit As(V) uptake, we examined the cellular uptake of As(III) and As(V) in regular and phosphate-free medium. When KB cells were treated with As(III), the uptake of arsenic was independent of the pres-
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and 3C revealed that As(V) uptake was dose-dependently inhibited by both NaN3 and KCN pretreatment (p £ 0.05, according to regression analysis in both cases). Pretreatment of KB cells with NaN3 or KCN showed only slight to no effect on As(III) uptake (p £ 0.123 and 0.162, respectively). These results suggest that biological energy is required for As(V) uptake, whereas As(III) uptake is energy independent. Kinetic Analysis of As(III) Uptake in KB Cells
FIG. 2. Effects of phosphate ion on uptake of arsenic in KB cells. (A) KB cells were treated with As(III) or As(V) as described in the legend to Fig. 1 and cellular content of arsenic was determined as described under Materials and Methods. Symbols are the same as Fig. 1. (B) KB cells were exposed to As(V) in RPMI medium containing various concentrations of phosphate ion for 30 min. The cellular uptake of As(V) was then determined as described under Materials and Methods. Bar, SD of at least three independent experiments.
A kinetic study was performed to further clarify the uptake mechanism of As(III). As shown in Fig. 4, As(III) uptake rapidly and time-dependently increased during the first 20 min and reached a plateau within 30 min. By fitting the data with a polynomial model (R 2 ú 0.997 for all concentrations), the initial velocity (v0) of As(III) uptake could be obtained. The initial velocity of As(III) uptake was linearly correlated to its concentration (Fig. 4, inset). Thus, As(III) uptake obviously follows first-order reaction kinetics up to 1000 mM and probably occurs through simple diffusion.
ence of phosphate in the medium (Fig. 2A). However, in regular RPMI medium, no arsenic was detected in KB cells exposed to As(V) at doses lower than 500 mM (Fig. 2A). In contrast, KB cells could take up As(V) as efficiently as As(III) when they were incubated in phosphate-free RPMI medium (Fig. 2A). To confirm the inhibitory effect of phosphate on As(V) uptake, the As(V) accumulation in KB cells was determined by incubating the cells with medium containing various concentrations of sodium phosphate. As shown in Fig. 2B, As(V) uptake in KB cells was dosedependently inhibited by sodium phosphate. Sodium phosphate at a dose of 10 mg/liter (70 mM) resulted in 50% inhibition of As(V) uptake. Effects of Sulfhydryl Modifier and Energy Poisons on the Uptake of Arsenic in KB Cells Mersalyl acid, a sulfhydryl modifier which cannot permeate the plasma membrane (Tsuchiya and Ochi, 1994), was used to elucidate the role of sulfhydryl groups in As(III) and As(V) uptake. As shown in Fig. 3A, the uptake of As(V), but not of As(III), was dose-dependently inhibited by mersalyl acid pretreatment (p £ 0.05, according to regression analysis). This result shows that the requirement for sulfhydryl groups in the plasma membrane for As(V) uptake is similar to that of phosphate uptake (Suzuki et al., 1990; Yamaguchi and Kimoto, 1992). Since carrier-mediated transport of a drug is usually coupled to biological energy, energy poisons such as NaN3 and KCN were used to determine whether energy is necessary for the uptake of As(III) and As(V). The results of Figs. 3B
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FIG. 3. Effects on As(III) and As(V) uptake by mersalyl acid, NaN3 , and KCN in KB cells. Results are expressed as percentage of control. Bar, SD of at least three independent experiments. Open column, As(III); shaded column, As(V).
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FIG. 4. Uptake kinetics of As(III) in KB cells. KB cells were treated with As(III) and cellular arsenic content was determined with AAS as described under Materials and Methods. Bar, SD of at least three independent experiments. Inset, the initial uptake rate (v0) was plotted against As(III) concentration.
Arsenic Excretion As(V) was found to be almost as toxic as As(III) to KB cells in phosphate-free medium. Therefore, we determined whether As(III) and As(V) have similar retention times in KB cells. We fed the cells with As(III) or As(V) in phosphate-free medium for 30 min. Afterward, the arsenic excretion was performed in regular medium. The results in Fig. 5 show that excretion of both As(III) and As(V) from KB cells occurred in a time-dependent manner. Approximately 80% of arsenic taken up was excreted into regular medium within 2 hr. There was no significant difference between excretion rates of As(III) and As(V) in KB cells. Since arsenic excretion was performed in regular RPMI medium, the presence of phosphate did not interfere with the release of arsenic from the cells.
FIG. 5. Time course of arsenic excretion in KB cells. After feeding KB cells with 200 mM As(III) (l) or As(V) (l) in phosphate-free medium for 30 min, the cells were incubated in regular medium for various times. The arsenic remaining in cells was determined as described under Materials and Methods. Bar, SD of at least three independent experiments.
line b), whereas more than half the amount of excreted arsenic was reduced to As(III) in medium from As(V)-treated cells (Fig. 6, line c). These results confirmed that As(V) can be rapidly reduced to As(III) in KB cells. DISCUSSION
As(V) has been shown to be at least 10-fold less toxic than As(III) in terms of cell survival or genotoxicity (Bertolero et
Reduction of As(V) to As(III) in KB Cells Since As(V) is suspected to exert its toxicity following reduction to As(III) (Jonnalagadda and Rao, 1993; Tamaki and Frankenberger, 1992), we analyzed which arsenic species were released into the medium by using an HPLC– AAS system. Four major arsenic metabolites (standards) could be well separated by a nucleosil 10B HPLC column (Fig. 6, line a). Within a 2-hr incubation, no methylated arsenic derivatives were detectable in medium from KB cells treated with As(III) or As(V). Only an As(III) peak was observed in medium from As(III)-treated KB cells (Fig. 6,
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FIG. 6. HPLC chromatogram for arsenic derivatives excreted in culture medium from KB cells. The culture medium from As(III)- or As(V)-treated KB cells was collected and analyzed by an HPLC–AAS technique as described under Materials and Methods. (a) Standard chromatogram of As(III), MMA, DMA, and As(V) (26.7 mM arsenic of each derivative). (b) Excreted arsenic profile of culture medium of KB cells treated with As(III). (c) Excreted arsenic profile of culture medium of KB cells treated with As(V). The ordinate scale is arbitrary units (AU) of atomic absorbance at 193.7 nm.
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al., 1987; Fischer et al., 1985; Lee et al., 1985). The discrepancy has usually been explained by less effective absorption of As(V) than As(III) (Bertolero et al., 1987; Fischer et al., 1985; Lerman et al., 1983; Vahter and Marafante, 1983). However, by using phosphate-free medium, our data demonstrate that As(V) can be as toxic as As(III) (Fig. 1) and can be taken up by cells as effectively as As(III) (Fig. 2A). It is likely that the normally lower cytotoxic activity of the As(V) is due to interference with its uptake by phosphate ion. Indeed, our results also demonstrate that the transport of As(V) is very similar to that of phosphate, i.e., both are energydependent and require sulfhydryl groups (Fig. 3) (Chen et al., 1990; Suzuki et al., 1990; Yamaguchi and Kimoto, 1992), and that it is highly sensitive to inhibition by phosphate (Fig. 2A). Therefore, As(V) shares with phosphate a common transport system in KB cells, as suggested in some prokaryotic cells (Rosenberg et al., 1977; Willsky and Malamy, 1980) and eukaryotic systems (Kenney and Kaplan, 1988). Drugs can be transported into cells through simple passive diffusion or carrier-mediated uptake systems (Tunnicliff, 1994). The present results show that the initial rates of As(III) uptake were linearly correlated with As(III) concentrations (Fig. 4, inset), indicating that As(III) transport does not involve a readily saturated carrier system. Moreover, As(III) uptake was not inhibited by a membrane sulfhydryl modifier or energy poisons (Fig. 3). These results support the view that As(III) can be freely taken by cells through simple diffusion. As(III), a well-recognized sulfhydryl agent, exerts its toxic effects by reacting with thiols in the cells (Knowles and Benson, 1983; Sunderman, 1979), while As(V), structurally similar to phosphate, can substitute for phosphate in a number of biochemical reactions (Kamiya et al., 1983; Kenney and Kaplan, 1988; Thiel, 1988; Aposhian, 1989; Willsky and Malamy, 1980). Since both As(III) and As(V) could be detected in medium from As(V)-treated cultures (Fig. 6), at least some of the toxic effects of As(V) might result from its reduction to As(III). The reduction of As(V) to As(III) did not spontaneously occur in the medium, since no As(III) was detected after incubation of As(V) in cell-free medium for 2 hr (data not shown). These results indicate that KB cells can effectively reduce As(V) to As(III) and efficiently excrete both into the medium. Arsenate reductase encoded by plasmids has been well characterized and can confer arsenate resistance to Escherichia coli and Staphylococcus aureus (Gladysheva et al., 1994; Ji et al., 1994). In bacterial systems, excretion of arsenic is governed by an active, plasmid-encoded ATP-driven oxyanion extrusion pump (Carlin et al., 1995; Dey and Rosen, 1995; Ji and Silver, 1992a,b; Oden et al., 1994; Silver and Ji, 1994). Whether a similar pumping system exists in eukaryotic sys-
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tems remains to be investigated. Recent studies have shown that arsenic resistance in Leishmania is associated with overexpression of a ltpgpA-encoded P-glycoprotein A (Detke et al., 1989; Katakura and Chang, 1989; Papadopoulou et al., 1994). This P-glycoprotein is a membrane pump removing a variety of compounds from cells (Chin et al., 1993; Dano, 1973; Dey et al., 1994; Gottesman and Pastan, 1993). An arsenic-resistant Chinese hamster ovary cell line (SA7), established in this laboratory (Lee et al., 1989b), was also demonstrated to have a higher ability to excrete As(III) (Wang and Lee, 1993). However, its molecular pumping mechanism remains to be elucidated. Metabolic methylation of arsenic compounds in vivo has generally been considered to be a major detoxification mechanism of arsenic (Bertolero et al., 1981; Marafante et al., 1985). However, no methylated arsenic was detected in medium from KB cells exposed to either As(III) or As(V). In the literature, some studies report that treatment of HeLa S3 and Hep G2 cells with As(V) for 24 hr resulted in methylated arsenic derivatives in cells as well as in cultured medium (Peel et al., 1991), but this was not found in BALB/3T3 cells treated with either As(III) or As(V), even for 72 hr (Bertolero et al., 1987). The explanations for these controversial results are not clear at the present time. Since exposure of As(III) to air can result in its oxidation to As(V) (Bushee et al., 1984), As(V) would be the dominant and stable species in well water (Del Razo et al., 1990) drunk by residents in endemic areas of black foot disease. Recent reports have shown that As(V), but not As(III), suppresses the expression of involucrin (Kachinskas et al., 1993, 1994). The expression of involucrin is a characteristic feature of terminal differentiated keratinocytes. Thus, As(V), in addition to reduction to As(III), may also have a unique pathological effect on cells through other mechanisms (Aposhian, 1989; Kachinskas et al., 1993, 1994; Westheimer, 1987). Our present findings show that As(V) can be reduced to As(III) and is as toxic as As(III) in phosphate-free medium (Figs. 1 and 2A). Thus, further investigation of arsenate reductase in human tissues may help us to understand the toxic and carcinogenic effects of arsenic. ACKNOWLEDGMENTS The authors thank Drs. K. Y. Jan, Sho Tone Lee, and Cathy Fletcher for carefully reading the manuscript. This work was supported by Academia Sinica and a grant (NSC 83-0203-B-001-102) from the National Science Council, Republic of China.
REFERENCES Aposhian, H. V. (1989). Biochemical toxicology of arsenic. Rev. Biochem. Toxicol. 10, 265–299. Bertolero, F., Marafante, E., Rade, J. E., Pietra, R., and Sabbioni, E. (1981). Biotransformation and intracellular binding of arsenic in tissues of rabbits
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after intraperitoneal administration of 74As labelled arsenite. Toxicology 20, 35–44. Bertolero, F., Pozzi, G., Sabbioni, E., and Saffiotti, U. (1987). Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation. Carcinogenesis 8, 803–808. Budd, K., and Craig, S. R. (1981). Resistance to arsenate toxicity in the blue-green alga Synechococcus leopoliensis. Can. J. Bot. 59, 1518–1521. Bushee, D. S., Krull, I. S., Demko, P. R., and Smith, S. B. J. (1984). Trace analysis and speciation for arsenic anions by HPLC-hydride generationinductively coupled plasma emission spectroscopy. J. Liq. Chromatogr. 7, 861–876. Carlin, A., Shi, W., Dey, S., and Rosen, B. P. (1995). The ars operon of Escherichia coli confers arsenical and antimonial resistance. J. Bacteriol. 177, 981–986. Chen, C. J., Chuang, Y. C., Lin, T. M., and Wu, H. Y. (1985). Malignant neoplasms among residents of a blackfoot disease-endemic area in Taiwan: High-arsenic artesian well water and cancers. Cancer Res. 45, 5895– 5899. Chen, C. J., Hsueh, Y. M., Lai, M. S., Shyu, M. P., Chen, S. Y., Wu, M. M., Kuo, T. L., and Tai, T. Y. (1995). Increased prevalence of hypertension and long-term arsenic exposure. Hypertension 25, 53–60. Chen, M. L., King, R. S., and Armbrecht, H. J. (1990). Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats. J. Cell. Physiol. 143, 488–493. Chin, K. V., Pastan, I., and Gottesman, M. M. (1993). Function and regulation of the human multidrug resistance gene. Adv. Cancer Res. 60, 157– 180. Dano, K. (1973). Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochim. Biophys. Acta 323, 466–483. Del Razo, L. M., Arellano, M. A., and Cebrian, M. E. (1990). The oxidation states of arsenic in well-water from a chronic arsenicism area of northern Mexico. Environ. Pollut. 64, 143–153. Detke, S., Katakura, K., and Chang, K. P. (1989). DNA amplification in arsenite-resistant Leishmania. Exp. Cell Res. 180, 161–170. Dey, S., Papadopoulou, B., Haimeur, A., Roy, G., Grondin, K., Dou, D., Rosen, B. P., and Ouellette, M. (1994). High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system. Mol. Biochem. Parasitol. 67, 49–57. Dey, S., and Rosen, B. P. (1995). Dual mode of energy coupling by the oxyanion-translocating ArsB protein. J. Bacteriol. 177, 385–389. Fischer, A. B., Buchet, J. P., and Lauwerys, R. R. (1985). Arsenic uptake, cytotoxicity and detoxification studied in mammalian cells in culture. Arch. Toxicol. 57, 168–172. Fullmer, C. S., and Wasserman, R. H. (1985). Intestinal absorption of arsenate in the chick. Environ. Res. 36, 206–217. Gladysheva, T. B., Oden, K. L., and Rosen, B. P. (1994). Properties of the arsenate reductase of plasmid R773. Biochemistry 33, 7288–7293. Gottesman, M. M., and Pastan, I. (1993). Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62, 385–427. Grillo, J. F., and Gibson, J. (1979). Regulation of phosphate accumulation in the unicellular cyanobacterium Synechococcus. J. Bacteriol. 140, 508– 517. IARC (1980). Carcinogenesis of arsenic compounds. IARC Monograph on Evaluation of Carcinogenic Risks, Vol. 23, pp. 37–141. IARC, Lyon. Ji, G., and Silver, S. (1992a). Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258. Proc. Natl. Acad. Sci. USA 89, 9474–9478.
m4715$7704
01-05-96 13:00:55
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Ji, G., and Silver, S. (1992b). Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258. J. Bacteriol. 174, 3684–3694. Ji, G., Garber, E. A., Armes, L. G., Chen, C. M., Fuchs, J. A., and Silver, S. (1994). Arsenate reductase of Staphylococcus aureus plasmid pI258. Biochemistry 33, 7294–7299. Jonnalagadda, S. B., and Rao, P. V. (1993). Toxicity, bioavailability and metal speciation. Comp. Biochem. Physiol. C 106, 585–595. Kachinskas, D. J., Philips, M. A., Qin, Q., and Rice, R. H. (1993). Arsenate suppression of involucrin expression in malignant human keratinocytes. FASEB J. 7, A1230. Kachinskas, D. J., Phillips, M. A., Qin, Q., Stokes, J. D., and Rice, R. H. (1994). Arsenate perturbation of human keratinocyte differentiation. Cell Growth Differ. 5, 1235–1241. Kamiya, K., Cruse, W. B., and Kennard, O. (1983). The arsonomethyl group as an analogue of phosphate. An X-ray investigation. Biochem. J. 213, 217–223. Katakura, K., and Chang, K. P. (1989). H DNA amplification in Leishmania resistant to both arsenite and methotrexate. Mol. Biochem. Parasitol. 34, 189–191. Kenney, L. J., and Kaplan, J. H. (1988). Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger. J. Biol. Chem. 263, 7954–7960. Knowles, F. C., and Benson, A. A. (1983). The biochemistry of arsenic. Trends Biochem. Sci. 8, 178–180. Lee, T. C., Oshimura, M., and Barrett, J. C. (1985). Comparison of arsenicinduced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6, 1421– 1426. Lee, T. C., Ko, J. L., and Jan, K. Y. (1989a). Differential cytotoxicity of sodium arsenite in human fibroblasts and Chinese hamster ovary cells. Toxicology 56, 289–299. Lee, T. C., Wei, M. L., Chang, W. J., Ho, I. C., Lo, J. F., Jan, K. Y., and Huang, H. (1989b). Elevation of glutathione levels and glutathione Stransferase activity in arsenic-resistant Chinese hamster ovary cells. In Vitro Cell. Dev. Biol. 25, 442–448. Leonard, A., and Lauwerys, R. R. (1980). Carcinogenicity, teratogenicity and mutagenicity of arsenic. Mutat. Res. 75, 49–62. Lerman, S. A., Clarkson, T. W., and Gerson, R. J. (1983). Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state. Chem.-Biol. Interact. 45, 401–406. Mabuchi, K., Lilienfeld, A. M., and Snell, L. M. (1979). Lung cancer among pesticide workers exposed to inorganic arsenicals. Arch. Environ. Health 34, 312–320. Marafante, E., Vahter, M., and Envall, J. (1985). The role of the methylation in the detoxication of arsenate in the rabbit. Chem.-Biol. Interact. 56, 225–238. NAS (1977). Medical and biologic effects of environmental pollutants: Arsenic. Natl. Acad. Sci., Washington, DC. Oden, K. L., Gladysheva, T. B., and Rosen, B. P. (1994). Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione. Mol. Microbiol. 12, 301–306. Papadopoulou, B., Roy, G., Dey, S., Rosen, B. P., and Ouellette, M. (1994). Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance. J. Biol. Chem. 269, 11980–11986. Peel, A. E., Marzin, B. D., and Erb, F. (1991). Cellular uptake and biotransformation of arsenic V in transformed human cell lines HeLa S3 and Hep G2 . Toxicol. in Vitro 5, 165–168.
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Tsuchiya, N., and Ochi, T. (1994). Mechanism for the decrease in the accumulation of cadmium (Cd) in Cd-resistant Chinese hamster V79 cells. Arch. Toxicol. 68, 325–331. Tunnicliff, G. (1994). Amino acid transport by human erythrocyte membranes. Comp. Biochem. Physiol. Comp. Physiol. 108, 471–478. Vahter, M., and Marafante, E. (1983). Intracellular interaction and metabolic fate of arsenite and arsenate in mice and rabbits. Chem.-Biol. Interact. 47, 29–44. Wang, H. F., and Lee, T. C. (1993). Glutathione S-transferase pi facilitates the excretion of arsenic from arsenic-resistant Chinese hamster ovary cells. Biochem. Biophys. Res. Commun. 192, 1093–1099. Westheimer, F. H. (1987). Why nature chose phosphates. Science 235, 1173–1178. Willsky, G. R., and Malamy, M. H. (1980). Effect of arsenate on inorganic phosphate transport in Escherichia coli. J. Bacteriol. 144, 366–374. Yamaguchi, T., and Kimoto, E. (1992). Inhibition of phosphate transport across the human erythrocyte membrane by chemical modification of sulfhydryl groups. Biochemistry 31, 1968–1973.
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Cellular Uptake of Trivalent Arsenite and Pentavalent Arsenate in KB Cells Cultured in Phosphate-Free Medium RONG-NAN HUANG AND TE-CHANG LEE Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, Republic of China Received April 24, 1995; accepted September 26, 1995
Cellular Uptake of Trivalent Arsenite and Pentavalent Arsenate in KB Cells Cultured in Phosphate-Free Medium. HUANG, R. N., AND LEE, T. C. (1996). Toxicol. Appl. Pharmacol. 136, 243–249. Trivalent arsenite (As(III)) and pentavalent arsenate (As(V)) have been shown to have differential uptake mechanisms. In regular RPMI 1640 medium, As(III) was about 40-fold more toxic to KB oral epidermoid carcinoma cells. However, the cytotoxicity and intracellular accumulation of As(V) were dramatically enhanced, equalling those of As(III) when cells were grown in phosphatefree RPMI medium. As(V) uptake was dose-dependently inhibited by phosphate, mersalyl acid (a membrane sulfhydryl agent), and energy poisons, such as sodium azide and potassium cyanide. These results suggest that As(V) and phosphate share a common transport system. In contrast, As(III) uptake was not affected by the above agents. However, the initial uptake rates of As(III) were linearly correlated with its extracellular concentrations, suggesting that As(III) uptake is probably accomplished through simple diffusion. Our results also show that As(III) and As(V) are excreted from KB cells at a comparable rate, and at least half of As(V) is reduced to the more toxic As(III) prior to excretion into the medium. Therefore, the toxicity of As(V) may in part result from its reduction to As(III). q 1996 Academic Press, Inc.
Arsenic is a ubiquitous chemical widely distributed in food, water, air, and soil (Knowles and Benson, 1983; NAS, 1977). Arsenic exposure, either due to geochemical enrichment or industrial processes, is accompanied by many severe toxicological and pathological problems, such as increased prevalence of hypertension (Chen et al., 1995) and increased risks for lung, nonmelanotic skin, and liver cancers (Chen et al., 1985; IARC, 1980; Leonard and Lauwerys, 1980; Mabuchi et al., 1979; Pinto et al., 1977; Pinto and Nelson, 1976). Arsenic exists as many chemical forms. Inorganic trivalent arsenite (As(III)) and pentavalent arsenate (As(V)) are of most importance in causing toxicological problems. Since As(III) is much more toxic than As(V) (Bertolero et al., 1987; Fischer et al., 1985; Lee et al., 1985), most toxicological studies have focused on As(III). However, As(V) is the predominant form in nature (Del Razo et al., 1990), but its
toxicity has not been well elucidated. Understanding the differential toxic mechanisms of As(III) and As(V) is critically important in assessing the risk from arsenic exposure. Recently, arsenic transport has been extensively studied in bacterial systems (Carlin et al., 1995; Dey and Rosen, 1995; Gladysheva et al., 1994; Ji et al., 1994; Ji and Silver, 1992a,b; Oden et al., 1994; Silver and Ji, 1994). Basically, intracellular As(III) was extruded by an ATPase-dependent pump and As(V) was reduced to As(III) by an arsenate reductase prior to being released from the cells. Most of these studies were conducted to understand the cellular mechanism of arsenic excretion. However, the differential toxic effects of As(III) and As(V) on mammalian cells have usually been attributed to differences in cellular uptake of the two forms of arsenic (Bertolero et al., 1987; Fischer et al., 1985; Lerman et al., 1983; Vahter and Marafante, 1983). As(V), structurally similar to inorganic phosphate, has been shown to compete with phosphate in a number of metabolic reactions (Kamiya et al., 1983; Kenney and Kaplan, 1988; Thiel, 1988; Aposhian, 1989). Although As(V) uptake has been shown to be mediated through phosphate channels in some organisms (Chen et al., 1990; Kenney and Kaplan, 1988; Rosenberg et al., 1977; Aposhian, 1989; Willsky and Malamy, 1980), the results of several reports are controversial (Budd and Craig, 1981; Fullmer and Wasserman, 1985; Grillo and Gibson, 1979; Thiel, 1988). Unfortunately, no data are available on the uptake of As(III). Phosphate is a major inorganic salt in all cell culture media and may be a limiting factor in determining arsenic uptake in regular medium. To better understand the mechanism of arsenic uptake, we therefore studied the cellular uptake of As(III) and As(V) using phosphate-free medium. Since arsenic is a unique human carcinogen, a human oral epidermal carcinoma KB cell line was adopted in these experiments.
MATERIALS AND METHODS Chemicals and cell cultures. Trivalent sodium arsenite (NaAsO2 , As(III)), pentavalent sodium arsenate (Na3AsO4r7H2O, As(V)), and dimeth-
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ylarsinic acid (DMA)1 were commercial products of Merck (E. Merck, AG). Monosodium acid methane arsonate (MMA) was purchased from Chem Service (Chem Service, Wester Chester, PA), and ethylenediaminetetraacetic acid (EDTA) and mersalyl acid from Sigma (Sigma, St. Louis, MO). The reagents used for cell culture, including fetal bovine serum (FBS), RPMI 1640 regular and phosphate-free media, glutamine, and antibiotics, were obtained from Gibco (Life Technologies, Grand Island, NY). KB cells, obtained from American Type Culture Collection (Rockville, MD), were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. The cultures were maintained at 377C in a humidified incubator with 5% carbon dioxide in air. Cytotoxicity assay. The cytotoxicity of As(III) and As(V) to KB cells was determined by a colony formation assay as described previously (Lee et al., 1989a). In brief, 5 1 105 cells were plated in a 60-mm petri dish. After a 24-hr incubation, the cultures were treated with drugs for 2 hr in freshly prepared regular or phosphate-free RPMI medium. The cells were trypsinized, replated in regular RPMI medium at a cell density of 400–500 cells/60-mm dish in triplicate, and then incubated for 10 days without medium change. The colonies were then fixed, stained, and counted as described (Lee et al., 1989a). The relative survival was calculated by dividing the number of colonies in arsenic-treated cultures by that in untreated cultures. The plating efficiency of untreated KB cells was 57.6 { 3.6%. Effect of phosphate ion on arsenic uptake. Arsenic uptake was assayed as described previously (Wang and Lee, 1993). In brief, KB cells were plated at a density of 3–5 1 105 cells/60-mm dish and incubated at 377C for 36–48 hr. The cells were treated with As(III) or As(V) for the time indicated, in medium containing various concentrations of phosphate. At the end of treatment, cells were washed five times with phosphate-buffered saline containing 1 mM EDTA and harvested by trypsinization. An aliquot of cells was subjected to arsenic content determination with an atomic absorption spectrophotometer (AAS, Hitachi Z-8000, Tokyo, Japan) equipped with a hydride formation system (Hitachi HSF-2, Tokyo, Japan) as described previously (Wang and Lee, 1993). To investigate the effect of phosphate on As(V) uptake, KB cells were incubated with 200 mM As(V) in RPMI 1640 medium containing 0–800 mg/liter (5.63 mM) sodium phosphate for 30 min. As(V) accumulation in KB cells was then determined as described (Wang and Lee, 1993). Effects of sulfhydryl modifier and energy poisons on the uptake of arsenic in KB cells. A sulfhydryl modifier (mersalyl acid) and energy poisons such as sodium azide (NaN3) and potassium cyanide (KCN) were used to characterize the uptake mechanism of arsenic. KB cells were pretreated with mersalyl acid (0–320 mM) for 10 min, or NaN3 (0–40 mM) or KCN (0–40 mM) for 60 min in phosphate-free medium, and subsequently coincubated with arsenic for 30 min. The arsenic uptake of KB cells was then determined as described above. Kinetic analysis of As(III) uptake in KB cells. Uptake kinetics of As(III) were analyzed by incubating KB cells for various times and As(III) concentrations in regular RPMI medium. The cellular uptake of As(III) was then determined as described above. The initial velocity of As(III) uptake at different concentrations was obtained by fitting the data with a polynomial model and calculating the uptake rate at time zero. Arsenic excretion. To determine arsenic excretion, KB cells were first treated with arsenic (200 mM) for 30 min in phosphate-free medium, rinsed once with medium, and then incubated in drug-free medium for the time indicated. Arsenic remaining in cells was then determined as described.
1
Abbreviations used: DMA, dimethylarsinic acid; MMA, monosodium acid methane arsonate; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; AAS, atomic absorption spectrophotometer; HPLC, highpressure liquid chromatography.
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FIG. 1. Cytotoxicity of As(III) and As(V) to KB cells in regular and phosphate-free medium. KB cells were treated with various concentrations of As(III) (s, l) or As(V) (L, l) either in regular (solid symbols) or in phosphate-free (open symbols) RPMI medium for 2 hr and cell survival was determined as described under Materials and Methods. Bar, SD of at least three independent experiments.
Analysis of arsenic derivatives. Logarithmically growing KB cells were exposed to 1 mM As(III) or As(V) in phosphate-free medium for 30 min, washed once with drug-free medium, and then incubated in complete RPMI medium for 2 hr. At the end of treatment, arsenic derivatives in culture medium were separated by a nucleosil 10B HPLC column (Phenomenex, Torrance, CA) using phosphate buffer (50 mM, pH 6.75) at a flow rate of 0.5 ml/min. Arsenic derivatives were detected by an online connected AAS as described (Wang and Lee, 1993). Standard arsenic derivatives were prepared in complete RPMI medium.
RESULTS
Cytotoxicity of As(III) and As(V) Mammalian cells grown in regular culture medium were generally more sensitive to As(III) than to As(V). For a 2hr treatment, the concentrations for As(III) and As(V) required for 50% inhibition of colony-forming ability of KB cells were 100 and 4000 mM, respectively (Fig. 1). Regular RPMI 1640 medium contains 800 mg/liter Na2HPO4 (5.63 mM). When the experiments were performed using phosphate-free RPMI medium, the toxicity of As(III) did not change (Fig. 1). According to doses to kill 50% of cells, the cytotoxic effect of As(V), however, was enhanced 40-fold, to reach the same cytotoxicity as As(III) (Fig. 1). Effect of Phosphate on the Uptake of Arsenic in KB Cells Since phosphate may inhibit As(V) uptake, we examined the cellular uptake of As(III) and As(V) in regular and phosphate-free medium. When KB cells were treated with As(III), the uptake of arsenic was independent of the pres-
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and 3C revealed that As(V) uptake was dose-dependently inhibited by both NaN3 and KCN pretreatment (p £ 0.05, according to regression analysis in both cases). Pretreatment of KB cells with NaN3 or KCN showed only slight to no effect on As(III) uptake (p £ 0.123 and 0.162, respectively). These results suggest that biological energy is required for As(V) uptake, whereas As(III) uptake is energy independent. Kinetic Analysis of As(III) Uptake in KB Cells
FIG. 2. Effects of phosphate ion on uptake of arsenic in KB cells. (A) KB cells were treated with As(III) or As(V) as described in the legend to Fig. 1 and cellular content of arsenic was determined as described under Materials and Methods. Symbols are the same as Fig. 1. (B) KB cells were exposed to As(V) in RPMI medium containing various concentrations of phosphate ion for 30 min. The cellular uptake of As(V) was then determined as described under Materials and Methods. Bar, SD of at least three independent experiments.
A kinetic study was performed to further clarify the uptake mechanism of As(III). As shown in Fig. 4, As(III) uptake rapidly and time-dependently increased during the first 20 min and reached a plateau within 30 min. By fitting the data with a polynomial model (R 2 ú 0.997 for all concentrations), the initial velocity (v0) of As(III) uptake could be obtained. The initial velocity of As(III) uptake was linearly correlated to its concentration (Fig. 4, inset). Thus, As(III) uptake obviously follows first-order reaction kinetics up to 1000 mM and probably occurs through simple diffusion.
ence of phosphate in the medium (Fig. 2A). However, in regular RPMI medium, no arsenic was detected in KB cells exposed to As(V) at doses lower than 500 mM (Fig. 2A). In contrast, KB cells could take up As(V) as efficiently as As(III) when they were incubated in phosphate-free RPMI medium (Fig. 2A). To confirm the inhibitory effect of phosphate on As(V) uptake, the As(V) accumulation in KB cells was determined by incubating the cells with medium containing various concentrations of sodium phosphate. As shown in Fig. 2B, As(V) uptake in KB cells was dosedependently inhibited by sodium phosphate. Sodium phosphate at a dose of 10 mg/liter (70 mM) resulted in 50% inhibition of As(V) uptake. Effects of Sulfhydryl Modifier and Energy Poisons on the Uptake of Arsenic in KB Cells Mersalyl acid, a sulfhydryl modifier which cannot permeate the plasma membrane (Tsuchiya and Ochi, 1994), was used to elucidate the role of sulfhydryl groups in As(III) and As(V) uptake. As shown in Fig. 3A, the uptake of As(V), but not of As(III), was dose-dependently inhibited by mersalyl acid pretreatment (p £ 0.05, according to regression analysis). This result shows that the requirement for sulfhydryl groups in the plasma membrane for As(V) uptake is similar to that of phosphate uptake (Suzuki et al., 1990; Yamaguchi and Kimoto, 1992). Since carrier-mediated transport of a drug is usually coupled to biological energy, energy poisons such as NaN3 and KCN were used to determine whether energy is necessary for the uptake of As(III) and As(V). The results of Figs. 3B
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FIG. 3. Effects on As(III) and As(V) uptake by mersalyl acid, NaN3 , and KCN in KB cells. Results are expressed as percentage of control. Bar, SD of at least three independent experiments. Open column, As(III); shaded column, As(V).
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FIG. 4. Uptake kinetics of As(III) in KB cells. KB cells were treated with As(III) and cellular arsenic content was determined with AAS as described under Materials and Methods. Bar, SD of at least three independent experiments. Inset, the initial uptake rate (v0) was plotted against As(III) concentration.
Arsenic Excretion As(V) was found to be almost as toxic as As(III) to KB cells in phosphate-free medium. Therefore, we determined whether As(III) and As(V) have similar retention times in KB cells. We fed the cells with As(III) or As(V) in phosphate-free medium for 30 min. Afterward, the arsenic excretion was performed in regular medium. The results in Fig. 5 show that excretion of both As(III) and As(V) from KB cells occurred in a time-dependent manner. Approximately 80% of arsenic taken up was excreted into regular medium within 2 hr. There was no significant difference between excretion rates of As(III) and As(V) in KB cells. Since arsenic excretion was performed in regular RPMI medium, the presence of phosphate did not interfere with the release of arsenic from the cells.
FIG. 5. Time course of arsenic excretion in KB cells. After feeding KB cells with 200 mM As(III) (l) or As(V) (l) in phosphate-free medium for 30 min, the cells were incubated in regular medium for various times. The arsenic remaining in cells was determined as described under Materials and Methods. Bar, SD of at least three independent experiments.
line b), whereas more than half the amount of excreted arsenic was reduced to As(III) in medium from As(V)-treated cells (Fig. 6, line c). These results confirmed that As(V) can be rapidly reduced to As(III) in KB cells. DISCUSSION
As(V) has been shown to be at least 10-fold less toxic than As(III) in terms of cell survival or genotoxicity (Bertolero et
Reduction of As(V) to As(III) in KB Cells Since As(V) is suspected to exert its toxicity following reduction to As(III) (Jonnalagadda and Rao, 1993; Tamaki and Frankenberger, 1992), we analyzed which arsenic species were released into the medium by using an HPLC– AAS system. Four major arsenic metabolites (standards) could be well separated by a nucleosil 10B HPLC column (Fig. 6, line a). Within a 2-hr incubation, no methylated arsenic derivatives were detectable in medium from KB cells treated with As(III) or As(V). Only an As(III) peak was observed in medium from As(III)-treated KB cells (Fig. 6,
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FIG. 6. HPLC chromatogram for arsenic derivatives excreted in culture medium from KB cells. The culture medium from As(III)- or As(V)-treated KB cells was collected and analyzed by an HPLC–AAS technique as described under Materials and Methods. (a) Standard chromatogram of As(III), MMA, DMA, and As(V) (26.7 mM arsenic of each derivative). (b) Excreted arsenic profile of culture medium of KB cells treated with As(III). (c) Excreted arsenic profile of culture medium of KB cells treated with As(V). The ordinate scale is arbitrary units (AU) of atomic absorbance at 193.7 nm.
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al., 1987; Fischer et al., 1985; Lee et al., 1985). The discrepancy has usually been explained by less effective absorption of As(V) than As(III) (Bertolero et al., 1987; Fischer et al., 1985; Lerman et al., 1983; Vahter and Marafante, 1983). However, by using phosphate-free medium, our data demonstrate that As(V) can be as toxic as As(III) (Fig. 1) and can be taken up by cells as effectively as As(III) (Fig. 2A). It is likely that the normally lower cytotoxic activity of the As(V) is due to interference with its uptake by phosphate ion. Indeed, our results also demonstrate that the transport of As(V) is very similar to that of phosphate, i.e., both are energydependent and require sulfhydryl groups (Fig. 3) (Chen et al., 1990; Suzuki et al., 1990; Yamaguchi and Kimoto, 1992), and that it is highly sensitive to inhibition by phosphate (Fig. 2A). Therefore, As(V) shares with phosphate a common transport system in KB cells, as suggested in some prokaryotic cells (Rosenberg et al., 1977; Willsky and Malamy, 1980) and eukaryotic systems (Kenney and Kaplan, 1988). Drugs can be transported into cells through simple passive diffusion or carrier-mediated uptake systems (Tunnicliff, 1994). The present results show that the initial rates of As(III) uptake were linearly correlated with As(III) concentrations (Fig. 4, inset), indicating that As(III) transport does not involve a readily saturated carrier system. Moreover, As(III) uptake was not inhibited by a membrane sulfhydryl modifier or energy poisons (Fig. 3). These results support the view that As(III) can be freely taken by cells through simple diffusion. As(III), a well-recognized sulfhydryl agent, exerts its toxic effects by reacting with thiols in the cells (Knowles and Benson, 1983; Sunderman, 1979), while As(V), structurally similar to phosphate, can substitute for phosphate in a number of biochemical reactions (Kamiya et al., 1983; Kenney and Kaplan, 1988; Thiel, 1988; Aposhian, 1989; Willsky and Malamy, 1980). Since both As(III) and As(V) could be detected in medium from As(V)-treated cultures (Fig. 6), at least some of the toxic effects of As(V) might result from its reduction to As(III). The reduction of As(V) to As(III) did not spontaneously occur in the medium, since no As(III) was detected after incubation of As(V) in cell-free medium for 2 hr (data not shown). These results indicate that KB cells can effectively reduce As(V) to As(III) and efficiently excrete both into the medium. Arsenate reductase encoded by plasmids has been well characterized and can confer arsenate resistance to Escherichia coli and Staphylococcus aureus (Gladysheva et al., 1994; Ji et al., 1994). In bacterial systems, excretion of arsenic is governed by an active, plasmid-encoded ATP-driven oxyanion extrusion pump (Carlin et al., 1995; Dey and Rosen, 1995; Ji and Silver, 1992a,b; Oden et al., 1994; Silver and Ji, 1994). Whether a similar pumping system exists in eukaryotic sys-
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tems remains to be investigated. Recent studies have shown that arsenic resistance in Leishmania is associated with overexpression of a ltpgpA-encoded P-glycoprotein A (Detke et al., 1989; Katakura and Chang, 1989; Papadopoulou et al., 1994). This P-glycoprotein is a membrane pump removing a variety of compounds from cells (Chin et al., 1993; Dano, 1973; Dey et al., 1994; Gottesman and Pastan, 1993). An arsenic-resistant Chinese hamster ovary cell line (SA7), established in this laboratory (Lee et al., 1989b), was also demonstrated to have a higher ability to excrete As(III) (Wang and Lee, 1993). However, its molecular pumping mechanism remains to be elucidated. Metabolic methylation of arsenic compounds in vivo has generally been considered to be a major detoxification mechanism of arsenic (Bertolero et al., 1981; Marafante et al., 1985). However, no methylated arsenic was detected in medium from KB cells exposed to either As(III) or As(V). In the literature, some studies report that treatment of HeLa S3 and Hep G2 cells with As(V) for 24 hr resulted in methylated arsenic derivatives in cells as well as in cultured medium (Peel et al., 1991), but this was not found in BALB/3T3 cells treated with either As(III) or As(V), even for 72 hr (Bertolero et al., 1987). The explanations for these controversial results are not clear at the present time. Since exposure of As(III) to air can result in its oxidation to As(V) (Bushee et al., 1984), As(V) would be the dominant and stable species in well water (Del Razo et al., 1990) drunk by residents in endemic areas of black foot disease. Recent reports have shown that As(V), but not As(III), suppresses the expression of involucrin (Kachinskas et al., 1993, 1994). The expression of involucrin is a characteristic feature of terminal differentiated keratinocytes. Thus, As(V), in addition to reduction to As(III), may also have a unique pathological effect on cells through other mechanisms (Aposhian, 1989; Kachinskas et al., 1993, 1994; Westheimer, 1987). Our present findings show that As(V) can be reduced to As(III) and is as toxic as As(III) in phosphate-free medium (Figs. 1 and 2A). Thus, further investigation of arsenate reductase in human tissues may help us to understand the toxic and carcinogenic effects of arsenic. ACKNOWLEDGMENTS The authors thank Drs. K. Y. Jan, Sho Tone Lee, and Cathy Fletcher for carefully reading the manuscript. This work was supported by Academia Sinica and a grant (NSC 83-0203-B-001-102) from the National Science Council, Republic of China.
REFERENCES Aposhian, H. V. (1989). Biochemical toxicology of arsenic. Rev. Biochem. Toxicol. 10, 265–299. Bertolero, F., Marafante, E., Rade, J. E., Pietra, R., and Sabbioni, E. (1981). Biotransformation and intracellular binding of arsenic in tissues of rabbits
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after intraperitoneal administration of 74As labelled arsenite. Toxicology 20, 35–44. Bertolero, F., Pozzi, G., Sabbioni, E., and Saffiotti, U. (1987). Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation. Carcinogenesis 8, 803–808. Budd, K., and Craig, S. R. (1981). Resistance to arsenate toxicity in the blue-green alga Synechococcus leopoliensis. Can. J. Bot. 59, 1518–1521. Bushee, D. S., Krull, I. S., Demko, P. R., and Smith, S. B. J. (1984). Trace analysis and speciation for arsenic anions by HPLC-hydride generationinductively coupled plasma emission spectroscopy. J. Liq. Chromatogr. 7, 861–876. Carlin, A., Shi, W., Dey, S., and Rosen, B. P. (1995). The ars operon of Escherichia coli confers arsenical and antimonial resistance. J. Bacteriol. 177, 981–986. Chen, C. J., Chuang, Y. C., Lin, T. M., and Wu, H. Y. (1985). Malignant neoplasms among residents of a blackfoot disease-endemic area in Taiwan: High-arsenic artesian well water and cancers. Cancer Res. 45, 5895– 5899. Chen, C. J., Hsueh, Y. M., Lai, M. S., Shyu, M. P., Chen, S. Y., Wu, M. M., Kuo, T. L., and Tai, T. Y. (1995). Increased prevalence of hypertension and long-term arsenic exposure. Hypertension 25, 53–60. Chen, M. L., King, R. S., and Armbrecht, H. J. (1990). Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats. J. Cell. Physiol. 143, 488–493. Chin, K. V., Pastan, I., and Gottesman, M. M. (1993). Function and regulation of the human multidrug resistance gene. Adv. Cancer Res. 60, 157– 180. Dano, K. (1973). Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochim. Biophys. Acta 323, 466–483. Del Razo, L. M., Arellano, M. A., and Cebrian, M. E. (1990). The oxidation states of arsenic in well-water from a chronic arsenicism area of northern Mexico. Environ. Pollut. 64, 143–153. Detke, S., Katakura, K., and Chang, K. P. (1989). DNA amplification in arsenite-resistant Leishmania. Exp. Cell Res. 180, 161–170. Dey, S., Papadopoulou, B., Haimeur, A., Roy, G., Grondin, K., Dou, D., Rosen, B. P., and Ouellette, M. (1994). High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system. Mol. Biochem. Parasitol. 67, 49–57. Dey, S., and Rosen, B. P. (1995). Dual mode of energy coupling by the oxyanion-translocating ArsB protein. J. Bacteriol. 177, 385–389. Fischer, A. B., Buchet, J. P., and Lauwerys, R. R. (1985). Arsenic uptake, cytotoxicity and detoxification studied in mammalian cells in culture. Arch. Toxicol. 57, 168–172. Fullmer, C. S., and Wasserman, R. H. (1985). Intestinal absorption of arsenate in the chick. Environ. Res. 36, 206–217. Gladysheva, T. B., Oden, K. L., and Rosen, B. P. (1994). Properties of the arsenate reductase of plasmid R773. Biochemistry 33, 7288–7293. Gottesman, M. M., and Pastan, I. (1993). Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62, 385–427. Grillo, J. F., and Gibson, J. (1979). Regulation of phosphate accumulation in the unicellular cyanobacterium Synechococcus. J. Bacteriol. 140, 508– 517. IARC (1980). Carcinogenesis of arsenic compounds. IARC Monograph on Evaluation of Carcinogenic Risks, Vol. 23, pp. 37–141. IARC, Lyon. Ji, G., and Silver, S. (1992a). Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258. Proc. Natl. Acad. Sci. USA 89, 9474–9478.
m4715$7704
01-05-96 13:00:55
toxas
Ji, G., and Silver, S. (1992b). Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258. J. Bacteriol. 174, 3684–3694. Ji, G., Garber, E. A., Armes, L. G., Chen, C. M., Fuchs, J. A., and Silver, S. (1994). Arsenate reductase of Staphylococcus aureus plasmid pI258. Biochemistry 33, 7294–7299. Jonnalagadda, S. B., and Rao, P. V. (1993). Toxicity, bioavailability and metal speciation. Comp. Biochem. Physiol. C 106, 585–595. Kachinskas, D. J., Philips, M. A., Qin, Q., and Rice, R. H. (1993). Arsenate suppression of involucrin expression in malignant human keratinocytes. FASEB J. 7, A1230. Kachinskas, D. J., Phillips, M. A., Qin, Q., Stokes, J. D., and Rice, R. H. (1994). Arsenate perturbation of human keratinocyte differentiation. Cell Growth Differ. 5, 1235–1241. Kamiya, K., Cruse, W. B., and Kennard, O. (1983). The arsonomethyl group as an analogue of phosphate. An X-ray investigation. Biochem. J. 213, 217–223. Katakura, K., and Chang, K. P. (1989). H DNA amplification in Leishmania resistant to both arsenite and methotrexate. Mol. Biochem. Parasitol. 34, 189–191. Kenney, L. J., and Kaplan, J. H. (1988). Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger. J. Biol. Chem. 263, 7954–7960. Knowles, F. C., and Benson, A. A. (1983). The biochemistry of arsenic. Trends Biochem. Sci. 8, 178–180. Lee, T. C., Oshimura, M., and Barrett, J. C. (1985). Comparison of arsenicinduced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6, 1421– 1426. Lee, T. C., Ko, J. L., and Jan, K. Y. (1989a). Differential cytotoxicity of sodium arsenite in human fibroblasts and Chinese hamster ovary cells. Toxicology 56, 289–299. Lee, T. C., Wei, M. L., Chang, W. J., Ho, I. C., Lo, J. F., Jan, K. Y., and Huang, H. (1989b). Elevation of glutathione levels and glutathione Stransferase activity in arsenic-resistant Chinese hamster ovary cells. In Vitro Cell. Dev. Biol. 25, 442–448. Leonard, A., and Lauwerys, R. R. (1980). Carcinogenicity, teratogenicity and mutagenicity of arsenic. Mutat. Res. 75, 49–62. Lerman, S. A., Clarkson, T. W., and Gerson, R. J. (1983). Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state. Chem.-Biol. Interact. 45, 401–406. Mabuchi, K., Lilienfeld, A. M., and Snell, L. M. (1979). Lung cancer among pesticide workers exposed to inorganic arsenicals. Arch. Environ. Health 34, 312–320. Marafante, E., Vahter, M., and Envall, J. (1985). The role of the methylation in the detoxication of arsenate in the rabbit. Chem.-Biol. Interact. 56, 225–238. NAS (1977). Medical and biologic effects of environmental pollutants: Arsenic. Natl. Acad. Sci., Washington, DC. Oden, K. L., Gladysheva, T. B., and Rosen, B. P. (1994). Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione. Mol. Microbiol. 12, 301–306. Papadopoulou, B., Roy, G., Dey, S., Rosen, B. P., and Ouellette, M. (1994). Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance. J. Biol. Chem. 269, 11980–11986. Peel, A. E., Marzin, B. D., and Erb, F. (1991). Cellular uptake and biotransformation of arsenic V in transformed human cell lines HeLa S3 and Hep G2 . Toxicol. in Vitro 5, 165–168.
AP: Tox
DIFFERENTIAL UPTAKE MECHANISM OF ARSENIC Pinto, S. S., and Nelson, K. W. (1976). Arsenic toxicology and industrial exposure. Annu. Rev. Pharmacol. Toxicol. 16, 95–100. Pinto, S. S., Enterline, P. E., Henderson, V., and Varner, M. O. (1977). Mortality experience in relation to a measured arsenic trioxide exposure. Environ. Health Perspect. 19, 127–130. Rosenberg, H., Gerdes, R. G., and Chegwidden, K. (1977). Two systems for the uptake of phosphate in Escherichia coli. J. Bacteriol. 131, 505– 511. Silver, S., and Ji, G. (1994). Newer systems for bacterial resistances to toxic heavy metals. Environ. Health Perspect. 3, 107–113. Sunderman, F. W. (1979). Mechanism of metal carcinogens. Biol. Trace Elem. Res. 1, 53–58. Suzuki, M., Capparelli, A. W., Jo, O. D., and Yanagawa, N. (1990). Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamide. Biochim. Biophys. Acta 1021, 85–90. Tamaki, S., and Frankenberger, W. J. (1992). Environmental biochemistry of arsenic. Rev. Environ. Contam. Toxicol. 124, 79–110. Thiel, T. (1988). Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis. J. Bacteriol. 170, 1143–1147.
m4715$7704
01-05-96 13:00:55
toxas
249
Tsuchiya, N., and Ochi, T. (1994). Mechanism for the decrease in the accumulation of cadmium (Cd) in Cd-resistant Chinese hamster V79 cells. Arch. Toxicol. 68, 325–331. Tunnicliff, G. (1994). Amino acid transport by human erythrocyte membranes. Comp. Biochem. Physiol. Comp. Physiol. 108, 471–478. Vahter, M., and Marafante, E. (1983). Intracellular interaction and metabolic fate of arsenite and arsenate in mice and rabbits. Chem.-Biol. Interact. 47, 29–44. Wang, H. F., and Lee, T. C. (1993). Glutathione S-transferase pi facilitates the excretion of arsenic from arsenic-resistant Chinese hamster ovary cells. Biochem. Biophys. Res. Commun. 192, 1093–1099. Westheimer, F. H. (1987). Why nature chose phosphates. Science 235, 1173–1178. Willsky, G. R., and Malamy, M. H. (1980). Effect of arsenate on inorganic phosphate transport in Escherichia coli. J. Bacteriol. 144, 366–374. Yamaguchi, T., and Kimoto, E. (1992). Inhibition of phosphate transport across the human erythrocyte membrane by chemical modification of sulfhydryl groups. Biochemistry 31, 1968–1973.
AP: Tox