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Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures
Neurochemistry International 62 (2013) 1012–1019
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Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures Nils Meyer a, Yvonne Koehler a,b, Ketki Tulpule a,b, Ralf Dringen a,b,⇑ a b
Centre for Biomolecular Interactions Bremen, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany Centre for Environmental Research and Sustainable Technology, Leobener Strasse, D-28359 Bremen, Germany
a r t i c l e
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Article history: Received 30 January 2013 Received in revised form 6 March 2013 Accepted 15 March 2013 Available online 29 March 2013 Keywords: Arsenic Astrocytes Export GSH Mrp1
a b s t r a c t Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2 h with arsenate in concentrations of up to 10 mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a timeand concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Arsenic is a toxic and carcinogenic metalloid that contaminates the ground water of many East Asian countries (Ahmed et al., 2006; Buschmann et al., 2007; Nickson et al., 1998; Phuong et al., 2012; Yadav et al., 2012). In fact, the drinking water of about 100 million people contains arsenic in concentrations higher than the upper limit concentration of 10 lg/L proposed by the World Health Organisation (Ahmed et al., 2006). The most abundant arsenic species in drinking water are the pentavalent inorganic arsenate and, under anaerobic conditions, the trivalent inorganic arsenite (Phuong et al., 2012; Watanabe and Hirano, in press). Chronic exposure to arsenic species in sublethal doses has been connected with black-foot disease, diabetes mellitus, some liver diseases, anemia and pigmentation changes (Vahidnia et al., 2007). In addition, Abbreviations: DMA, dimethylarsinic acid; GSH, glutathione; GSSG, glutathione disulfide; GSx, total glutathione; H33342, Hoechst 33342; IB, incubation buffer; LDH, lactate dehydrogenase; Mrp1, multidrug resistance protein 1; PBS, phosphatebuffered saline; PI, propidium iodide. ⇑ Corresponding author at: Centre for Biomolecular Interactions Bremen, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany. Tel.: +49 421 218 63230; fax: +49 421 218 63244. E-mail address: [email protected] (R. Dringen). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.03.014
peripheral neuritis and sensory neuropathy as well as impaired neuropsychological functioning and cognitive development have been associated with chronic arsenic intoxication (O’Bryant et al., 2011; Rosado et al., 2007; Vahidnia et al., 2007). After oral application of arsenate or arsenite, these inorganic species as well as dimethylarsinic acid (DMA) have been found in the brains of mice (Juarez-Reyes et al., 2009; Wang et al., 2011). As brain microvessels are completely covered by astroglial endfeet (Mathiisen et al., 2010), astrocytes are likely to be the first parenchymal cells of the brain that encounter arsenic species that have crossed the blood-brain barrier. Astrocytes have various functions in the brain (Parpura et al., 2012). Among them the protection of other brain cells against the toxic potential of oxidants, xenobiotics and metals is of special importance (Scheiber and Dringen, 2013; Schmidt and Dringen, 2012; Tiffany-Castiglioni et al., 2011). In many of these protective functions the cellular antioxidant glutathione (GSH) is involved. Within astrocytes this tripeptide delivers electrons for the reduction of peroxides and is also a substrate for the formation of conjugates with xenobiotics (Dringen et al., 2005; Schmidt and Dringen, 2012). In addition, astrocytes provide GSH precursors to neurons in a process that involves the export of GSH, mainly via multidrug resistance protein 1 (Mrp 1) (Hirrlinger et al., 2002; Minich et al., 2006), and subsequent cleavage of the
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extracellular GSH via the astrocytic c-glutamyl transpeptidase (Dringen et al., 1997; Dringen et al., 1999) and a neuronal aminopeptidase (Dringen et al., 2001). GSH is also involved in the cellular metabolism of arsenic, as the reduction potential of GSH is used by the glutathione-S-transferaseomega to perform different steps in the biotransformation of arsenic species (Chowdhury et al., 2006; Zakharyan et al., 2001; Zakharyan et al., 2005). In addition, GSH forms complexes with trivalent arsenic species, although it is still a matter of debate whether such complexes are substrates for biotransformation processes and/or whether they are used to export arsenic from the cell (Kala et al., 2000; Thomas, 2007; Watanabe and Hirano, in press). Inorganic arsenic species are known to affect astrocytic functions and have been reported to compromise the viability of cultured astrocytes and to cause DNA damage (Catanzaro et al., 2010; Jin et al., 2004; Zhao et al., 2012), to induce the synthesis of heat shock proteins (Catanzaro et al., 2010; Fauconneau et al., 2002), to inhibit glutamate metabolism (Zhao et al., 2012) and to increase the cellular GSH content (Sagara et al., 1996; Tulpule et al., 2012). In order to investigate whether arsenate is accumulated by astrocytes and whether it may affect the GSH export from astrocytes, we exposed cultured primary astrocytes to arsenate. Here we report that cultured astrocytes efficiently accumulate arsenate and that the presence of arsenate causes a time- and concentration-dependent stimulation of Mrp1-mediated GSH export from viable astrocytes. This process may contribute to the known adverse effects of arsenic compounds on brain functions.
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1 mM MgCl2, 5.4 mM KCl, 0.8 mM Na2HPO4, 145 mM NaCl, pH 7.4) and incubated at 37 °C with 200 lL incubation medium (IB supplemented with the compounds indicated in the Figures and the Table). For experiments that address the phosphate-dependence of arsenate accumulation, cells were washed and incubated with 1 mM arsenate in IB containing 0 mM or 0.8 mM phosphate. After collecting the medium at the end of the desired incubation period, the cells were washed with 1 mL ice-cold phosphate-buffered saline (PBS; 10 mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl). For quantification of cellular total glutathione contents (GSx = amount of GSH plus twice the amount of glutathione disulfide (GSSG)) or of cellular GSSG contents, the cells were lysed in 500 lL 1% (w/v) sulfosalicylic acid on ice. Media samples were diluted 1:1 with 1% sulfosalicylic acid for quantification of the extracellular GSx or GSSG contents. 2.4. Determination of cell viability, protein and glutathione contents
2. Materials and methods
Cell viability was analysed by investigating the membrane permeability of the cells. After a given incubation, the activity of the cytosolic enzyme lactate dehydrogenase (LDH) in the incubation medium and the permeation of the fluorescent dye propidium iodide (PI) into the cells were determined as previously described (Dringen et al., 1998; Scheiber et al., 2010). The protein content per well of the culture was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard protein. The contents of GSx and GSSG in media samples and cell lysates were determined by using a microtiter plate-based modification of the colorimetric Tietze method as described previously (Hirrlinger and Dringen, 2005).
2.1. Materials
2.5. Quantification of cellular arsenic content
Mono potassium arsenate and sodium arsenite were obtained from Sigma–Aldrich (Steinheim, Germany). Dimethylarsinic acid (DMA) was purchased from Roth (Karlsruhe, Germany). Dulbecco’s modified Eagle’s medium and penicillin/streptomycin solution were from Gibco/Invitrogen (Darmstadt, Germany) and fetal calf serum from Biochrom (Berlin, Germany). Bovine serum albumin, NADH and NADPH were obtained from AppliChem (Darmstadt, Germany). Glutathione reductase was from Roche Diagnostics (Mannheim, Germany) and argon was purchased from Linde (München, Germany). Other chemicals of the highest purity available were purchased from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany) or Sigma–Aldrich (Steinheim, Germany). Sterile cell culture plates and unsterile 96-well microtiter plates were obtained from Sarstedt (Nümbrecht, Germany).
The cells in wells of 24-well dishes were lysed in 200 lL 50 mM NaOH and 100 lL of this lysate was incubated with 100 lL of a 1:1 mixture of 35% H2O2 and 65% HNO3 (suprapur) at 65 °C for 60 min. Subsequently, the samples were heated at 85 °C overnight to ash the organic part of the sample. The residues of the ashing procedure were dissolved in 100 lL 1% HNO3 and further diluted with 1% HNO3 if required. The arsenic content of the solution was quantified by graphite furnace atomic absorption spectroscopy using a Varian (Darmstadt, Germany) AA-240Z spectrophotometer and a Varian GTA-120 graphite tube atomizer equipped with a Varian PSD-120 programmable sample dispenser. The Varian SpectrAA 5.01 software was employed to control the instruments and for data analysis. The optimized instrumental conditions for the quantification of arsenic were: wavelength: 193.7 nm, spectral bandwith: 0.5 nm, lamp current: 10 mA, activated Zeeman background correction, protective gas: argon, atomization temperature: 2600 °C, absorbance measurement: peak area. The total injected sample volume contained 20 lL of sample and 2 lL 2.5 mg/mL Pd(NO3)2 as modifier. The peak area of the signal obtained was proportional to the amounts of As2O3 standards in the range between 1.334 and 13.34 pmol arsenic, with correlation coefficients above 0.999.
2.2. Astrocyte cultures Astrocyte-rich primary cultures were prepared from the brains of newborn Wistar rats as previously described (Hamprecht and Löffler, 1985). The cells were suspended in culture medium (90% Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, 1 mM pyruvate, 20 units/mL penicillin G, 20 lg/mL streptomycin sulfate) and 300,000 cells were seeded in 1 mL culture medium per well of 24-well plates. The cultures were incubated in a Sanyo (Osaka, Japan) incubator at 37 °C with 10% CO2 in a humidified atmosphere. The cultures were used at an age between 14 and 28 days. The medium was renewed every 7 days and 1 day prior to the experiments. 2.3. Experimental incubation of cells Cells were washed twice with 1 mL pre-warmed (37 °C) incubation buffer (IB: 20 mM HEPES, 5 mM D-glucose, 1.8 mM CaCl2,
2.6. Presentation of data Data are presented as means ± SD of values that were obtained in three experiments performed on independently prepared astrocyte cultures. PI stainings were performed twice with identical outcomes and representative images are shown. Analysis of significance between groups of data was performed by ANOVA followed by the Bonferroni post-hoc test and the levels of significance compared to controls are indicated as ⁄p < 0.05, ⁄⁄p < 0.01 or ⁄⁄⁄ p < 0.001. Statistical comparison of two groups of data was
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performed by the paired t-test and the level of significance is indicated as #p < 0.05. p > 0.05 was considered as not significant. 3. Results 3.1. Effects of arsenate on the viability of cultured astrocytes To investigate the consequences of a treatment of astrocytes with arsenate astrocyte-rich primary cultures were incubated with arsenate in concentrations of 0.1 mM or 1 mM for up to 6 h. These conditions did not increase extracellular LDH activity (data not shown) and did not compromise membrane integrity that would lead to an increased number of PI-positive cells (Fig. 1). Even an exposure of astrocytes to 10 mM arsenate for 2 h did not result in an increased extracellular LDH activity (data not shown) or in the appearance of PI-positive cells (Fig. 1H), while most cells in cultures that had been treated for 2 or 6 h with silver nitrate as positive control were PI-positive (Fig. 1J and T). Also an incubation of astrocytes for 6 h with arsenate in concentrations higher than 1 mM did not cause an increase in the number of PI-positive cells (Fig. 1P and R). However, this treatment lowered the total number of cells that remained attached to the cell culture dish as shown by the H33342 staining of the nuclei (Fig. 1O and Q), indicating that under these conditions arsenate caused detachment of cells. 3.2. Quantification of the arsenic content of cultured astrocytes Untreated astrocytes or control cells that had been incubated without arsenate did not contain any detectable amounts of
arsenic (Fig. 2A). In contrast, the arsenic contents of cells that had been incubated for 2 h with arsenate increased almost linearly with the concentration of arsenate applied in the concentration range of up to 10 mM (Fig. 2A). On incubation of cells with 1 mM arsenate the cellular arsenic content was 7.3 ± 1.6 nmol/mg protein (Table 1) which was comparable to the arsenic content of cultured astrocytes that were treated with 1 mM arsenite. On the other hand, cultures exposed to 1 mM DMA contained only around 1 nmol arsenic/mg protein (Table 1). Phosphate transporters have been discussed to mediate cellular uptake of arsenate (Calatayud et al., 2010; Watanabe and Hirano, in press). To test whether arsenate accumulation by astrocytes is affected by the presence of phosphate, the cells were incubated for 2 h with 1 mM arsenate in the absence (0 mM) or the presence of 0.8 mM phosphate. These conditions did not affect the cell viability (data not shown). The specific arsenic content of cells that had been incubated under standard condition (0.8 mM phosphate) was 4.5 ± 0.9 nmol/mg. Compared to this value, the specific arsenic content of astrocytes that had been exposed to arsenate in the absence of phosphate was significantly (p < 0.001, n = 3, t-test) elevated to 21.8 ± 1.5 nmol/mg. 3.3. Effects of arsenate on the glutathione content of cultured astrocytes Incubation of astrocyte-rich primary cultures without arsenate resulted in a slow extracellular GSx accumulation that had a rate of around 2.3 nmol/(h mg protein). Application of arsenate in concentrations of 0.1 mM or 1 mM accelerated this time-
Fig. 1. Effect of arsenate on the membrane integrity of cultured astrocytes. The cells were incubated for 2 h (A–H) or 6 h (K–R) with arsenate in the concentrations indicated or for 2 h (I and J) or 6 h (S and T) with 100 lM AgNO3 as positive control for cell damage. While the nuclei of all cells present are identified by H33342 staining, PI staining indentifies the nuclei of cells that have permeabilised membranes. The scale bar in panel B represents 50 lm and applies to all panels.
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Fig. 2. Cellular arsenic content of cultured astrocytes after exposure to arsenate. The cells were incubated for 2 h with arsenate in the concentrations indicated (A) or with 1 mM arsenate in the absence or the presence of 50 lM MK571 (B) and cellular arsenic contents were determined. The cultures contained an initial protein content of 147 ± 6 lg protein per well. n.d not detectable.
Table 1 Effect of different arsenic species on the cell viability, the arsenic content and the glutathione content of cultured astrocytes. Compound
none Arsenate Arsenite DMA
Extracellular LDH activity (% of initial cellular LDH activity)
As content (nmol/mg)
GSx content (nmol/mg) Extracellular
Cellular
Sum
GSSG content (nmol GSx/mg) Extracellular
Cellular
Sum
2.9 ± 1.5 3.3 ± 0.7 4.5 ± 0.1 3.1 ± 0.8
n.d 7.3 ± 1.6 7.6 ± 0.8 1.1 ± 0.1
5.0 ± 1.0 25.8 ± 7.7⁄⁄⁄ 33.9 ± 9.8⁄⁄⁄ 8.3 ± 1.6
27.3 ± 7.4 14.1 ± 2.2⁄ 5.2 ± 0.5⁄⁄⁄ 26.0 ± 5.6
32.3 ± 8.3 39.5 ± 9.7 39.1 ± 10.3 34.3 ± 7.2
0.2 ± 0.1 3.4 ± 0.7⁄⁄⁄ 7.0 ± 0.7⁄⁄⁄ 0.3 ± 0.2
0.2 ± 0.2 0.2 ± 0.2 0.2 ± 0.1 0.2 ± 0.2
0.4 ± 0.3 3.6 ± 0.9⁄⁄⁄ 7.3 ± 0.8⁄⁄⁄ 0.5 ± 0.3
The cells were incubated for 2 h without (none; n = 9) or with 1 mM of arsenate, arsenite or DMA (n = 3) and the extracellular LDH activity, the specific arsenic content of the cells as well as extracellular and cellular contents of GSx and GSSG were determined. The cultures contained an initial cellular GSx content of 42 ± 10 nmol per mg protein and initial protein contents of 149 ± 24 lg (GSx quantifications) or 147 ± 6 lg (determination of arsenic contents) protein per well. Significant differences (ANOVA) compared to the control condition (none) are indicated by ⁄p < 0.05 and ⁄⁄⁄p < 0.001. n.d, not detectable.
dependent increase in the extracellular GSx content (Fig. 3A) and the corresponding decrease in the cellular GSx content (Fig. 3B), while the sum of extracellular plus cellular GSx remained almost constant (Fig. 3C). Compared to the values determined for the control condition (absence of arsenate), the extracellular GSx contents were significantly increased after 4 h and 1 h of incubation with 0.1 mM and 1 mM arsenate, respectively (Fig. 3A). After 6 h of incubation with 1 mM arsenate only 15% of the initial GSx content remained detectable within the cells (Fig. 3B). The detailed investigation of the concentration-dependent effect of arsenate on extracellular and cellular GSx contents of astrocytes after 2 h of incubation revealed that maximal extracellular accumulation and maximal cellular loss of GSx was obtained after exposure to around 3 mM arsenate (Fig. 4). Significant differences in the extracellular and cellular GSx content compared to the control condition were found at 0.3 mM or 1 mM arsenate, respectively, while the sum of extracellular plus cellular GSx content remained constant within statistical errors for all concentrations of arsenate applied (Fig. 4 triangles). Unexpectedly, the presence of arsenate in a concentration of 10 mM was less efficient to lower cellular GSx contents and to stimulate extracellular GSx accumulation than 3 mM arsenate (Fig. 4). Arsenate was applied as mono potassium salt to the cells. To exclude that the elevated potassium concentration may contribute to the observed increased accumulation of extracellular GSx in arsenate-treated astrocyte cultures, the cells were exposed for 2 h with IB supplemented with 10 mM potassium chloride or potassium
arsenate. None of these conditions affected the cell viability (data not shown). Compared to the extracellular GSx content of cultures that had been incubated under control conditions (absence of additional potassium chloride or potassium arsenate; 5.5 ± 1.3 nmol GSx/mg), the extracellular GSx content of arsenate-treated astrocytes was significantly (p < 0.001, n = 3, ANOVA) elevated to 16.0 ± 2.4 nmol/mg, while presence of additional 10 mM potassium chloride did not alter the content of extracellular GSx (5.5 ± 0.4 nmol/mg). 3.4. Effects of different arsenic species on the GSH content of astrocytes The incubation of astrocytes for 2 h with 1 mM arsenate significantly lowered the cellular GSx content and increased the extracellular GSx content five-fold, but did not compromise the cell viability as the extracellular LDH activity was not increased compared to controls (absence of arsenic compounds) (Table 1). An even stronger extracellular GSx accumulation in viable astrocyte cultures was induced by the presence of 1 mM arsenite (Table 1), while 1 mM DMA did not affect the extracellular or cellular GSx content compared with controls (absence of arsenic) (Table 1). None of the arsenic species applied significantly increased the specific cellular GSSG content. However, 13% and 22% of the extracellular GSx contents of astrocytes that had been incubated with arsenate and arsenite, respectively, represented GSSG (Table 1), reflecting most likely extracellular oxidation of some of the large amounts of GSH that had been exported under these conditions.
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Fig. 4. Concentration-dependent effects of arsenate on the GSx content of cultured astrocytes. The cells were incubated for 2 h with the indicated concentrations of arsenate and the extracellular GSx content, the cellular GSx content and the sum (R) of extracellular plus cellular GSx contents were determined. The cultures contained an initial cellular GSx content of 45 ± 8 nmol per mg protein (indicated by the dashed line) and an initial protein content of 126 ± 41 lg protein per well. Significant differences (ANOVA) compared to the control condition (absence of arsenate) are indicated.
plus cellular GSx contents was not significantly influenced by arsenate and/or MK571 (Fig. 5C). None of these conditions affected the cell viability as indicated by the absence of any significant increase in extracellular LDH activity compared to controls (Fig. 5D). Quantification of cellular arsenic contents for these conditions revealed almost identical arsenic contents of around 7 nmol/mg for astrocytes that had been exposed to 1 mM arsenate, irrespective of the absence or presence of MK571 (Fig. 2B). 4. Discussion
Fig. 3. Time-dependent effects of arsenate on the glutathione content of cultured astrocytes. Cells were incubated for up to 6 h without or with 0.1 mM or 1 mM arsenate and the extracellular GSx content (A), the cellular GSx content (B) and the sum (R) of extracellular plus cellular GSx contents (C) were determined. The cultures contained an initial cellular GSx content of 48 ± 9 nmol per mg protein (indicated in panel C by the dashed line) and an initial protein content of 121 ± 45 lg protein per well. Significant differences (ANOVA) compared to the control condition (absence of arsenate) are indicated.
3.5. Effect of Mrp1 inhibition on the arsenate-induced export of GSH Mrp1 is the transporter predominantly responsible for the basal export of GSH from astrocytes (Hirrlinger and Dringen, 2005; Hirrlinger et al., 2002; Minich et al., 2006). To investigate whether the arsenate-stimulated GSH export from cultured astrocytes is also mediated by Mrp1, astrocytes were incubated with or without 1 mM arsenate in the absence or the presence of 50 lM of the Mrp1-inhibitor MK571. The significant increase in extracellular and the significant decrease in cellular GSx contents that were observed for the presence of arsenate were completely prevented in presence of MK571 (Fig. 5A and B), while the sum of extracellular
Viable cultured astrocytes efficiently accumulate arsenate and arsenite as demonstrated by the strong increase in the specific arsenic content of the treated cells, while only small amounts of cellular arsenic were detected in astrocytes exposed to DMA. On incubation of cells with arsenate the cellular arsenic content increased proportionally to the concentration of arsenate applied, suggesting that the transporter responsible for the uptake of arsenate into astrocytes has an apparent high KM value for arsenate. Transporters that are likely to be responsible for the cellular uptake of arsenate are sodium-dependent phosphate transporters, while hexose transporters and aquaporins are considered to transport arsenite into cells (Calatayud et al., 2010; Thomas, 2007; Watanabe and Hirano, in press). The hypothesis that a phosphate transporter is involved in arsenate uptake by astrocytes is strongly supported by the fourfold increase in the specific arsenic content of astrocytes that had been exposed to arsenate in the absence of phosphate compared to the values obtained after exposure of the cells to arsenate in the presence of 0.8 mM phosphate. Despite the high specific arsenic content of cells exposed to arsenate or arsenite, cultured astrocytes appear to be rather resistant against acute toxicity of these inorganic arsenic compounds as demonstrated by the lack of increase in extracellular LDH activity and by the absence of PI-positive cells. However, arsenate in concentrations higher than 1 mM caused detachment of cells during prolonged exposure which is consistent with literature data (Jin et al., 2004; Catanzaro et al., 2010). Oxidative stress has been reported to be involved in arsenate toxicity for other cell types (Hughes, 2002; Liu et al., 2001b; Yen et al., 2011). However, the
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Fig. 5. Effect of MK571 on the arsenate-induced GSH efflux from cultured astrocytes. The cells were incubated for 2 h without or with 1 mM arsenate in the absence or the presence of 50 lM MK571 and the extracellular GSx content (A), the cellular GSx content (B), the sum (R) of extracellular plus cellular GSx contents (C) and the extracellular activity of LDH as indicator for a potential loss of cell viability (D) were determined. The cultures contained an initial cellular GSx content of 45 ± 7 nmol per mg protein (indicated by the dashed line in panel C) and an initial cellular protein content of 136 ± 35 lg protein per well. Significant differences (ANOVA) compared to the control condition (absence of arsenate) are indicated by asterisks, while the significance of differences between data obtained from cells that had been incubated with arsenate in the absence or presence of MK571 (paired t-test) is indicated by hashes.
high antioxidative potential of astrocytes (Dringen et al., 2005) is likely to prevent a potential rapid increase in the production of reactive oxygen species in arsenate-treated astrocytes, thereby contributing to the observed resistance of astrocytes against acute arsenate toxicity. This view is supported by the high ratio of GSH/ GSSG that was maintained in astrocytes despite the exposure to 1 mM arsenate or arsenite for 2 h, demonstrating that the cells did not suffer from a severe oxidative stress under those conditions. Additional processes that may help to prevent acute arsenate toxicity in astrocytes are binding of internalized arsenate to proteins (Thomas et al., 2001) and/or its transformation to methylated derivatives (Steinmaus et al., 2005, Watanabe and Hirano, in press). Cultured astrocytes accumulate only low amounts of DMA which is consistent with literature data demonstrating that DMA exposure is not toxic to astrocytes (Jin et al., 2004; Catanzaro et al., 2010). Cultured astrocytes released GSH with a basal rate of 2.3 ± 0.3 nmol/(h mg protein), which is similar to specific GSH export rates previously reported for these cells (Minich et al., 2006; Scheiber and Dringen, 2011). Application of arsenate to astrocytes caused a time- and concentration-dependent acceleration of the GSH export from viable astrocytes, which was accompanied by a matching increase in extracellular GSH levels, with a halfmaximal effect observed for around 0.3 mM arsenate. The sum of extracellular and cellular GSH was not influenced by the presence of arsenate, suggesting that the presence of arsenate selectively
modulates the distribution of GSH between the cellular and extracellular compartment and does not deplete astrocytic GSH by conjugation as reported for alkylated fumarates and halogenated acetates (Schmidt and Dringen, 2009; Schmidt and Dringen, 2010; Schmidt et al., 2011). An increase in the specific cellular GSx content that was reported for astrocytes exposed to arsenate or arsenite (Sagara et al., 1996; Tulpule et al., 2012) was not observed under the conditions used here, as the basal medium used for the incubations did not contain the amino acids required for GSH synthesis. A potential contribution of the elevated extracellular potassium content in the observed stimulated GSH export after application of potassium arsenate can be excluded, as supplementation of the incubation buffer with additional 10 mM potassium chloride did not affect GSH export, consistent with literature data reporting that even depolarization of cultured astrocytes with 50 mM potassium chloride does not affect GSH export (Hirrlinger et al., 2002). Mrp1 is the transporter responsible for the arsenate-accelerated astrocytic GSH export, as the Mrp1 inhibitor MK571 (Minich et al., 2006) completely prevented the arsenate-induced loss in cellular GSH and the accelerated accumulation of extracellular GSH. This is consistent with a recent report showing that MK571 inhibits at least in part the arsenate-induced GSH efflux from erythrocytes (Yildiz and Cakir, 2012). For cultured astrocytes, the stimulation of Mrp1-mediated GSH export has recently also been reported for antiretroviral protease inhibitors (Brandmann et al., 2012;
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Arend et al., 2013) and formaldehyde (Tulpule and Dringen, 2011). Thus, arsenate is a new member of a group of structurally very diverse molecules that stimulate Mrp1-mediated GSH export from astrocytes. Conjugation of cellular GSH with arsenic species and subsequent export of such conjugates has been discussed as a pathway that helps to eliminate arsenic species from cells (Kala et al., 2000; Thomas, 2007; Watanabe and Hirano, in press). In liver endothelial cells, the accumulation of arsenite has been reported to substantially increase in the presence of MK571 (Liu et al., 2001a), suggesting that in these cells Mrp-mediated export of arsenic-GSH complexes contributes to the elimination of arsenic compounds. However, for cultured astrocytes an export of such complexes is unlikely to contribute substantially to the observed stimulated GSH export. The arsenate-induced Mrp1-mediated increase in extracellular GSH contents amounted to around 20 nmol/mg protein. If an Mrp1-mediated export of a labile arsenic-GSH conjugate via Mrp1 would be involved in the stimulated GSH export from astrocytes, the specific cellular arsenic content of MK571-treated astrocytes should have increased strongly. However, as astrocytes that had been co-incubated with arsenate and MK571 did not differ in their specific arsenic content compared to arsenate-treated astrocytes in the absence of the inhibitor, an involvement of a potential export of a labile arsenic-GSH complex in the observed accelerated GSH export can be excluded. Thus, arsenate is likely to affect Mrp1-mediated GSH export directly either by binding to the transporter or by recruitment of Mrp1proteins from intracellular vesicles into the plasma membrane, as reported for bilirubin-treated astrocytes (Gennuso et al., 2004). Astrocytes have a key function in the antioxidative protection and in the GSH homeostasis of the brain (Hirrlinger and Dringen, 2010; Schmidt and Dringen, 2012). As the protection provided by astrocytes to neurons against hydrogen peroxide or NO is abolished in GSH-deprived astrocytes (Drukarch et al., 1997; Gegg et al., 2005), an arsenate- or arsenite-induced accelerated GSH loss from astrocytes may impair their antioxidative and protective functions. In addition, as astrocytes supply neighbouring neurons with precursors for GSH synthesis in a process that involves the export of astrocytic GSH by Mrp1 (Hirrlinger et al., 2002; Minich et al., 2006), a stimulation of GSH export from astrocytes by arsenics could also lead to an increase in the GSH content of neighboring neurons. Incorporated arsenate is primarily converted to organic derivatives in the liver (Bolt and Stewart, 2010; Juarez-Reyes et al., 2009; Wang et al., 2011) by a pathway that includes reduction to trivalent arsenic species and successive methylation reactions (Steinmaus et al., 2005; Watanabe and Hirano, in press). These reactions prevent that large amounts of inorganic arsenics will be encountered by other organs, including the brain. Indeed, the amounts of inorganic arsenics in the brains of mice after oral treatment with arsenate or arsenite are lower than those found for DMA (JuarezReyes et al., 2009; Wang et al., 2011) and DMA did not stimulate GSH export from cultured astrocytes. Whether a direct effect of arsenate on the GSH export from astrocytes may occur in brain remains to be elucidated. The levels of inorganic arsenic species reported in brain tissue of animals exposed to arsenics for a shortterm appear to be very low (Juarez-Reyes et al., 2009; Wang et al., 2011) compared to the concentrations of arsenate used in our study. However, already rather low concentrations of arsenate can strongly affect the GSH metabolism of brain cells. For example, presence of 5 lM arsenate in amino acid-containing culture medium has been reported to strongly increase within 24 h the specific cellular GSH content of cultured astrocytes (Tulpule et al., 2012). Thus, a chronic intoxication by consumption of arsenic-contaminated drinking water is likely to increase the risk that brain cells will encounter sufficient amounts of inorganic arsenic species,
which subsequently will interfere with the GSH metabolism of the brain. Such effects may contribute to the observed oxidative stress in brain (Yen et al., 2011) and to the neurological complications reported for intoxications with arsenic compounds (O’Bryant et al., 2011; Rosado et al., 2007; Vahidnia et al., 2007). References Ahmed, M.F., Ahuja, S., Alauddin, M., Hug, S.J., Lloyd, J.R., Pfaff, A., Pichler, T., Saltikov, C., Stute, M., van Geen, A., 2006. Epidemiology. Ensuring safe drinking water in Bangladesh. Science 314, 1687–1688. Arend, C., Brandmann, M., Dringen, R., 2013. The antiretroviral protease inhibitor ritonavir accelerates glutathione export from cultured primary astrocytes. Neurochem. Res. 38, 732–741. Bolt, H.M., Stewart, J.D., 2010. Arsenic: metabolism and transport mechanisms in human hepatocytes. Arch. Toxicol. 84, 1–2. Brandmann, M., Tulpule, K., Schmidt, M.M., Dringen, R., 2012. 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Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures Nils Meyer a, Yvonne Koehler a,b, Ketki Tulpule a,b, Ralf Dringen a,b,⇑ a b
Centre for Biomolecular Interactions Bremen, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany Centre for Environmental Research and Sustainable Technology, Leobener Strasse, D-28359 Bremen, Germany
a r t i c l e
i n f o
Article history: Received 30 January 2013 Received in revised form 6 March 2013 Accepted 15 March 2013 Available online 29 March 2013 Keywords: Arsenic Astrocytes Export GSH Mrp1
a b s t r a c t Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2 h with arsenate in concentrations of up to 10 mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a timeand concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Arsenic is a toxic and carcinogenic metalloid that contaminates the ground water of many East Asian countries (Ahmed et al., 2006; Buschmann et al., 2007; Nickson et al., 1998; Phuong et al., 2012; Yadav et al., 2012). In fact, the drinking water of about 100 million people contains arsenic in concentrations higher than the upper limit concentration of 10 lg/L proposed by the World Health Organisation (Ahmed et al., 2006). The most abundant arsenic species in drinking water are the pentavalent inorganic arsenate and, under anaerobic conditions, the trivalent inorganic arsenite (Phuong et al., 2012; Watanabe and Hirano, in press). Chronic exposure to arsenic species in sublethal doses has been connected with black-foot disease, diabetes mellitus, some liver diseases, anemia and pigmentation changes (Vahidnia et al., 2007). In addition, Abbreviations: DMA, dimethylarsinic acid; GSH, glutathione; GSSG, glutathione disulfide; GSx, total glutathione; H33342, Hoechst 33342; IB, incubation buffer; LDH, lactate dehydrogenase; Mrp1, multidrug resistance protein 1; PBS, phosphatebuffered saline; PI, propidium iodide. ⇑ Corresponding author at: Centre for Biomolecular Interactions Bremen, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany. Tel.: +49 421 218 63230; fax: +49 421 218 63244. E-mail address: [email protected] (R. Dringen). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.03.014
peripheral neuritis and sensory neuropathy as well as impaired neuropsychological functioning and cognitive development have been associated with chronic arsenic intoxication (O’Bryant et al., 2011; Rosado et al., 2007; Vahidnia et al., 2007). After oral application of arsenate or arsenite, these inorganic species as well as dimethylarsinic acid (DMA) have been found in the brains of mice (Juarez-Reyes et al., 2009; Wang et al., 2011). As brain microvessels are completely covered by astroglial endfeet (Mathiisen et al., 2010), astrocytes are likely to be the first parenchymal cells of the brain that encounter arsenic species that have crossed the blood-brain barrier. Astrocytes have various functions in the brain (Parpura et al., 2012). Among them the protection of other brain cells against the toxic potential of oxidants, xenobiotics and metals is of special importance (Scheiber and Dringen, 2013; Schmidt and Dringen, 2012; Tiffany-Castiglioni et al., 2011). In many of these protective functions the cellular antioxidant glutathione (GSH) is involved. Within astrocytes this tripeptide delivers electrons for the reduction of peroxides and is also a substrate for the formation of conjugates with xenobiotics (Dringen et al., 2005; Schmidt and Dringen, 2012). In addition, astrocytes provide GSH precursors to neurons in a process that involves the export of GSH, mainly via multidrug resistance protein 1 (Mrp 1) (Hirrlinger et al., 2002; Minich et al., 2006), and subsequent cleavage of the
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extracellular GSH via the astrocytic c-glutamyl transpeptidase (Dringen et al., 1997; Dringen et al., 1999) and a neuronal aminopeptidase (Dringen et al., 2001). GSH is also involved in the cellular metabolism of arsenic, as the reduction potential of GSH is used by the glutathione-S-transferaseomega to perform different steps in the biotransformation of arsenic species (Chowdhury et al., 2006; Zakharyan et al., 2001; Zakharyan et al., 2005). In addition, GSH forms complexes with trivalent arsenic species, although it is still a matter of debate whether such complexes are substrates for biotransformation processes and/or whether they are used to export arsenic from the cell (Kala et al., 2000; Thomas, 2007; Watanabe and Hirano, in press). Inorganic arsenic species are known to affect astrocytic functions and have been reported to compromise the viability of cultured astrocytes and to cause DNA damage (Catanzaro et al., 2010; Jin et al., 2004; Zhao et al., 2012), to induce the synthesis of heat shock proteins (Catanzaro et al., 2010; Fauconneau et al., 2002), to inhibit glutamate metabolism (Zhao et al., 2012) and to increase the cellular GSH content (Sagara et al., 1996; Tulpule et al., 2012). In order to investigate whether arsenate is accumulated by astrocytes and whether it may affect the GSH export from astrocytes, we exposed cultured primary astrocytes to arsenate. Here we report that cultured astrocytes efficiently accumulate arsenate and that the presence of arsenate causes a time- and concentration-dependent stimulation of Mrp1-mediated GSH export from viable astrocytes. This process may contribute to the known adverse effects of arsenic compounds on brain functions.
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1 mM MgCl2, 5.4 mM KCl, 0.8 mM Na2HPO4, 145 mM NaCl, pH 7.4) and incubated at 37 °C with 200 lL incubation medium (IB supplemented with the compounds indicated in the Figures and the Table). For experiments that address the phosphate-dependence of arsenate accumulation, cells were washed and incubated with 1 mM arsenate in IB containing 0 mM or 0.8 mM phosphate. After collecting the medium at the end of the desired incubation period, the cells were washed with 1 mL ice-cold phosphate-buffered saline (PBS; 10 mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl). For quantification of cellular total glutathione contents (GSx = amount of GSH plus twice the amount of glutathione disulfide (GSSG)) or of cellular GSSG contents, the cells were lysed in 500 lL 1% (w/v) sulfosalicylic acid on ice. Media samples were diluted 1:1 with 1% sulfosalicylic acid for quantification of the extracellular GSx or GSSG contents. 2.4. Determination of cell viability, protein and glutathione contents
2. Materials and methods
Cell viability was analysed by investigating the membrane permeability of the cells. After a given incubation, the activity of the cytosolic enzyme lactate dehydrogenase (LDH) in the incubation medium and the permeation of the fluorescent dye propidium iodide (PI) into the cells were determined as previously described (Dringen et al., 1998; Scheiber et al., 2010). The protein content per well of the culture was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard protein. The contents of GSx and GSSG in media samples and cell lysates were determined by using a microtiter plate-based modification of the colorimetric Tietze method as described previously (Hirrlinger and Dringen, 2005).
2.1. Materials
2.5. Quantification of cellular arsenic content
Mono potassium arsenate and sodium arsenite were obtained from Sigma–Aldrich (Steinheim, Germany). Dimethylarsinic acid (DMA) was purchased from Roth (Karlsruhe, Germany). Dulbecco’s modified Eagle’s medium and penicillin/streptomycin solution were from Gibco/Invitrogen (Darmstadt, Germany) and fetal calf serum from Biochrom (Berlin, Germany). Bovine serum albumin, NADH and NADPH were obtained from AppliChem (Darmstadt, Germany). Glutathione reductase was from Roche Diagnostics (Mannheim, Germany) and argon was purchased from Linde (München, Germany). Other chemicals of the highest purity available were purchased from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany) or Sigma–Aldrich (Steinheim, Germany). Sterile cell culture plates and unsterile 96-well microtiter plates were obtained from Sarstedt (Nümbrecht, Germany).
The cells in wells of 24-well dishes were lysed in 200 lL 50 mM NaOH and 100 lL of this lysate was incubated with 100 lL of a 1:1 mixture of 35% H2O2 and 65% HNO3 (suprapur) at 65 °C for 60 min. Subsequently, the samples were heated at 85 °C overnight to ash the organic part of the sample. The residues of the ashing procedure were dissolved in 100 lL 1% HNO3 and further diluted with 1% HNO3 if required. The arsenic content of the solution was quantified by graphite furnace atomic absorption spectroscopy using a Varian (Darmstadt, Germany) AA-240Z spectrophotometer and a Varian GTA-120 graphite tube atomizer equipped with a Varian PSD-120 programmable sample dispenser. The Varian SpectrAA 5.01 software was employed to control the instruments and for data analysis. The optimized instrumental conditions for the quantification of arsenic were: wavelength: 193.7 nm, spectral bandwith: 0.5 nm, lamp current: 10 mA, activated Zeeman background correction, protective gas: argon, atomization temperature: 2600 °C, absorbance measurement: peak area. The total injected sample volume contained 20 lL of sample and 2 lL 2.5 mg/mL Pd(NO3)2 as modifier. The peak area of the signal obtained was proportional to the amounts of As2O3 standards in the range between 1.334 and 13.34 pmol arsenic, with correlation coefficients above 0.999.
2.2. Astrocyte cultures Astrocyte-rich primary cultures were prepared from the brains of newborn Wistar rats as previously described (Hamprecht and Löffler, 1985). The cells were suspended in culture medium (90% Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, 1 mM pyruvate, 20 units/mL penicillin G, 20 lg/mL streptomycin sulfate) and 300,000 cells were seeded in 1 mL culture medium per well of 24-well plates. The cultures were incubated in a Sanyo (Osaka, Japan) incubator at 37 °C with 10% CO2 in a humidified atmosphere. The cultures were used at an age between 14 and 28 days. The medium was renewed every 7 days and 1 day prior to the experiments. 2.3. Experimental incubation of cells Cells were washed twice with 1 mL pre-warmed (37 °C) incubation buffer (IB: 20 mM HEPES, 5 mM D-glucose, 1.8 mM CaCl2,
2.6. Presentation of data Data are presented as means ± SD of values that were obtained in three experiments performed on independently prepared astrocyte cultures. PI stainings were performed twice with identical outcomes and representative images are shown. Analysis of significance between groups of data was performed by ANOVA followed by the Bonferroni post-hoc test and the levels of significance compared to controls are indicated as ⁄p < 0.05, ⁄⁄p < 0.01 or ⁄⁄⁄ p < 0.001. Statistical comparison of two groups of data was
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performed by the paired t-test and the level of significance is indicated as #p < 0.05. p > 0.05 was considered as not significant. 3. Results 3.1. Effects of arsenate on the viability of cultured astrocytes To investigate the consequences of a treatment of astrocytes with arsenate astrocyte-rich primary cultures were incubated with arsenate in concentrations of 0.1 mM or 1 mM for up to 6 h. These conditions did not increase extracellular LDH activity (data not shown) and did not compromise membrane integrity that would lead to an increased number of PI-positive cells (Fig. 1). Even an exposure of astrocytes to 10 mM arsenate for 2 h did not result in an increased extracellular LDH activity (data not shown) or in the appearance of PI-positive cells (Fig. 1H), while most cells in cultures that had been treated for 2 or 6 h with silver nitrate as positive control were PI-positive (Fig. 1J and T). Also an incubation of astrocytes for 6 h with arsenate in concentrations higher than 1 mM did not cause an increase in the number of PI-positive cells (Fig. 1P and R). However, this treatment lowered the total number of cells that remained attached to the cell culture dish as shown by the H33342 staining of the nuclei (Fig. 1O and Q), indicating that under these conditions arsenate caused detachment of cells. 3.2. Quantification of the arsenic content of cultured astrocytes Untreated astrocytes or control cells that had been incubated without arsenate did not contain any detectable amounts of
arsenic (Fig. 2A). In contrast, the arsenic contents of cells that had been incubated for 2 h with arsenate increased almost linearly with the concentration of arsenate applied in the concentration range of up to 10 mM (Fig. 2A). On incubation of cells with 1 mM arsenate the cellular arsenic content was 7.3 ± 1.6 nmol/mg protein (Table 1) which was comparable to the arsenic content of cultured astrocytes that were treated with 1 mM arsenite. On the other hand, cultures exposed to 1 mM DMA contained only around 1 nmol arsenic/mg protein (Table 1). Phosphate transporters have been discussed to mediate cellular uptake of arsenate (Calatayud et al., 2010; Watanabe and Hirano, in press). To test whether arsenate accumulation by astrocytes is affected by the presence of phosphate, the cells were incubated for 2 h with 1 mM arsenate in the absence (0 mM) or the presence of 0.8 mM phosphate. These conditions did not affect the cell viability (data not shown). The specific arsenic content of cells that had been incubated under standard condition (0.8 mM phosphate) was 4.5 ± 0.9 nmol/mg. Compared to this value, the specific arsenic content of astrocytes that had been exposed to arsenate in the absence of phosphate was significantly (p < 0.001, n = 3, t-test) elevated to 21.8 ± 1.5 nmol/mg. 3.3. Effects of arsenate on the glutathione content of cultured astrocytes Incubation of astrocyte-rich primary cultures without arsenate resulted in a slow extracellular GSx accumulation that had a rate of around 2.3 nmol/(h mg protein). Application of arsenate in concentrations of 0.1 mM or 1 mM accelerated this time-
Fig. 1. Effect of arsenate on the membrane integrity of cultured astrocytes. The cells were incubated for 2 h (A–H) or 6 h (K–R) with arsenate in the concentrations indicated or for 2 h (I and J) or 6 h (S and T) with 100 lM AgNO3 as positive control for cell damage. While the nuclei of all cells present are identified by H33342 staining, PI staining indentifies the nuclei of cells that have permeabilised membranes. The scale bar in panel B represents 50 lm and applies to all panels.
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Fig. 2. Cellular arsenic content of cultured astrocytes after exposure to arsenate. The cells were incubated for 2 h with arsenate in the concentrations indicated (A) or with 1 mM arsenate in the absence or the presence of 50 lM MK571 (B) and cellular arsenic contents were determined. The cultures contained an initial protein content of 147 ± 6 lg protein per well. n.d not detectable.
Table 1 Effect of different arsenic species on the cell viability, the arsenic content and the glutathione content of cultured astrocytes. Compound
none Arsenate Arsenite DMA
Extracellular LDH activity (% of initial cellular LDH activity)
As content (nmol/mg)
GSx content (nmol/mg) Extracellular
Cellular
Sum
GSSG content (nmol GSx/mg) Extracellular
Cellular
Sum
2.9 ± 1.5 3.3 ± 0.7 4.5 ± 0.1 3.1 ± 0.8
n.d 7.3 ± 1.6 7.6 ± 0.8 1.1 ± 0.1
5.0 ± 1.0 25.8 ± 7.7⁄⁄⁄ 33.9 ± 9.8⁄⁄⁄ 8.3 ± 1.6
27.3 ± 7.4 14.1 ± 2.2⁄ 5.2 ± 0.5⁄⁄⁄ 26.0 ± 5.6
32.3 ± 8.3 39.5 ± 9.7 39.1 ± 10.3 34.3 ± 7.2
0.2 ± 0.1 3.4 ± 0.7⁄⁄⁄ 7.0 ± 0.7⁄⁄⁄ 0.3 ± 0.2
0.2 ± 0.2 0.2 ± 0.2 0.2 ± 0.1 0.2 ± 0.2
0.4 ± 0.3 3.6 ± 0.9⁄⁄⁄ 7.3 ± 0.8⁄⁄⁄ 0.5 ± 0.3
The cells were incubated for 2 h without (none; n = 9) or with 1 mM of arsenate, arsenite or DMA (n = 3) and the extracellular LDH activity, the specific arsenic content of the cells as well as extracellular and cellular contents of GSx and GSSG were determined. The cultures contained an initial cellular GSx content of 42 ± 10 nmol per mg protein and initial protein contents of 149 ± 24 lg (GSx quantifications) or 147 ± 6 lg (determination of arsenic contents) protein per well. Significant differences (ANOVA) compared to the control condition (none) are indicated by ⁄p < 0.05 and ⁄⁄⁄p < 0.001. n.d, not detectable.
dependent increase in the extracellular GSx content (Fig. 3A) and the corresponding decrease in the cellular GSx content (Fig. 3B), while the sum of extracellular plus cellular GSx remained almost constant (Fig. 3C). Compared to the values determined for the control condition (absence of arsenate), the extracellular GSx contents were significantly increased after 4 h and 1 h of incubation with 0.1 mM and 1 mM arsenate, respectively (Fig. 3A). After 6 h of incubation with 1 mM arsenate only 15% of the initial GSx content remained detectable within the cells (Fig. 3B). The detailed investigation of the concentration-dependent effect of arsenate on extracellular and cellular GSx contents of astrocytes after 2 h of incubation revealed that maximal extracellular accumulation and maximal cellular loss of GSx was obtained after exposure to around 3 mM arsenate (Fig. 4). Significant differences in the extracellular and cellular GSx content compared to the control condition were found at 0.3 mM or 1 mM arsenate, respectively, while the sum of extracellular plus cellular GSx content remained constant within statistical errors for all concentrations of arsenate applied (Fig. 4 triangles). Unexpectedly, the presence of arsenate in a concentration of 10 mM was less efficient to lower cellular GSx contents and to stimulate extracellular GSx accumulation than 3 mM arsenate (Fig. 4). Arsenate was applied as mono potassium salt to the cells. To exclude that the elevated potassium concentration may contribute to the observed increased accumulation of extracellular GSx in arsenate-treated astrocyte cultures, the cells were exposed for 2 h with IB supplemented with 10 mM potassium chloride or potassium
arsenate. None of these conditions affected the cell viability (data not shown). Compared to the extracellular GSx content of cultures that had been incubated under control conditions (absence of additional potassium chloride or potassium arsenate; 5.5 ± 1.3 nmol GSx/mg), the extracellular GSx content of arsenate-treated astrocytes was significantly (p < 0.001, n = 3, ANOVA) elevated to 16.0 ± 2.4 nmol/mg, while presence of additional 10 mM potassium chloride did not alter the content of extracellular GSx (5.5 ± 0.4 nmol/mg). 3.4. Effects of different arsenic species on the GSH content of astrocytes The incubation of astrocytes for 2 h with 1 mM arsenate significantly lowered the cellular GSx content and increased the extracellular GSx content five-fold, but did not compromise the cell viability as the extracellular LDH activity was not increased compared to controls (absence of arsenic compounds) (Table 1). An even stronger extracellular GSx accumulation in viable astrocyte cultures was induced by the presence of 1 mM arsenite (Table 1), while 1 mM DMA did not affect the extracellular or cellular GSx content compared with controls (absence of arsenic) (Table 1). None of the arsenic species applied significantly increased the specific cellular GSSG content. However, 13% and 22% of the extracellular GSx contents of astrocytes that had been incubated with arsenate and arsenite, respectively, represented GSSG (Table 1), reflecting most likely extracellular oxidation of some of the large amounts of GSH that had been exported under these conditions.
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Fig. 4. Concentration-dependent effects of arsenate on the GSx content of cultured astrocytes. The cells were incubated for 2 h with the indicated concentrations of arsenate and the extracellular GSx content, the cellular GSx content and the sum (R) of extracellular plus cellular GSx contents were determined. The cultures contained an initial cellular GSx content of 45 ± 8 nmol per mg protein (indicated by the dashed line) and an initial protein content of 126 ± 41 lg protein per well. Significant differences (ANOVA) compared to the control condition (absence of arsenate) are indicated.
plus cellular GSx contents was not significantly influenced by arsenate and/or MK571 (Fig. 5C). None of these conditions affected the cell viability as indicated by the absence of any significant increase in extracellular LDH activity compared to controls (Fig. 5D). Quantification of cellular arsenic contents for these conditions revealed almost identical arsenic contents of around 7 nmol/mg for astrocytes that had been exposed to 1 mM arsenate, irrespective of the absence or presence of MK571 (Fig. 2B). 4. Discussion
Fig. 3. Time-dependent effects of arsenate on the glutathione content of cultured astrocytes. Cells were incubated for up to 6 h without or with 0.1 mM or 1 mM arsenate and the extracellular GSx content (A), the cellular GSx content (B) and the sum (R) of extracellular plus cellular GSx contents (C) were determined. The cultures contained an initial cellular GSx content of 48 ± 9 nmol per mg protein (indicated in panel C by the dashed line) and an initial protein content of 121 ± 45 lg protein per well. Significant differences (ANOVA) compared to the control condition (absence of arsenate) are indicated.
3.5. Effect of Mrp1 inhibition on the arsenate-induced export of GSH Mrp1 is the transporter predominantly responsible for the basal export of GSH from astrocytes (Hirrlinger and Dringen, 2005; Hirrlinger et al., 2002; Minich et al., 2006). To investigate whether the arsenate-stimulated GSH export from cultured astrocytes is also mediated by Mrp1, astrocytes were incubated with or without 1 mM arsenate in the absence or the presence of 50 lM of the Mrp1-inhibitor MK571. The significant increase in extracellular and the significant decrease in cellular GSx contents that were observed for the presence of arsenate were completely prevented in presence of MK571 (Fig. 5A and B), while the sum of extracellular
Viable cultured astrocytes efficiently accumulate arsenate and arsenite as demonstrated by the strong increase in the specific arsenic content of the treated cells, while only small amounts of cellular arsenic were detected in astrocytes exposed to DMA. On incubation of cells with arsenate the cellular arsenic content increased proportionally to the concentration of arsenate applied, suggesting that the transporter responsible for the uptake of arsenate into astrocytes has an apparent high KM value for arsenate. Transporters that are likely to be responsible for the cellular uptake of arsenate are sodium-dependent phosphate transporters, while hexose transporters and aquaporins are considered to transport arsenite into cells (Calatayud et al., 2010; Thomas, 2007; Watanabe and Hirano, in press). The hypothesis that a phosphate transporter is involved in arsenate uptake by astrocytes is strongly supported by the fourfold increase in the specific arsenic content of astrocytes that had been exposed to arsenate in the absence of phosphate compared to the values obtained after exposure of the cells to arsenate in the presence of 0.8 mM phosphate. Despite the high specific arsenic content of cells exposed to arsenate or arsenite, cultured astrocytes appear to be rather resistant against acute toxicity of these inorganic arsenic compounds as demonstrated by the lack of increase in extracellular LDH activity and by the absence of PI-positive cells. However, arsenate in concentrations higher than 1 mM caused detachment of cells during prolonged exposure which is consistent with literature data (Jin et al., 2004; Catanzaro et al., 2010). Oxidative stress has been reported to be involved in arsenate toxicity for other cell types (Hughes, 2002; Liu et al., 2001b; Yen et al., 2011). However, the
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Fig. 5. Effect of MK571 on the arsenate-induced GSH efflux from cultured astrocytes. The cells were incubated for 2 h without or with 1 mM arsenate in the absence or the presence of 50 lM MK571 and the extracellular GSx content (A), the cellular GSx content (B), the sum (R) of extracellular plus cellular GSx contents (C) and the extracellular activity of LDH as indicator for a potential loss of cell viability (D) were determined. The cultures contained an initial cellular GSx content of 45 ± 7 nmol per mg protein (indicated by the dashed line in panel C) and an initial cellular protein content of 136 ± 35 lg protein per well. Significant differences (ANOVA) compared to the control condition (absence of arsenate) are indicated by asterisks, while the significance of differences between data obtained from cells that had been incubated with arsenate in the absence or presence of MK571 (paired t-test) is indicated by hashes.
high antioxidative potential of astrocytes (Dringen et al., 2005) is likely to prevent a potential rapid increase in the production of reactive oxygen species in arsenate-treated astrocytes, thereby contributing to the observed resistance of astrocytes against acute arsenate toxicity. This view is supported by the high ratio of GSH/ GSSG that was maintained in astrocytes despite the exposure to 1 mM arsenate or arsenite for 2 h, demonstrating that the cells did not suffer from a severe oxidative stress under those conditions. Additional processes that may help to prevent acute arsenate toxicity in astrocytes are binding of internalized arsenate to proteins (Thomas et al., 2001) and/or its transformation to methylated derivatives (Steinmaus et al., 2005, Watanabe and Hirano, in press). Cultured astrocytes accumulate only low amounts of DMA which is consistent with literature data demonstrating that DMA exposure is not toxic to astrocytes (Jin et al., 2004; Catanzaro et al., 2010). Cultured astrocytes released GSH with a basal rate of 2.3 ± 0.3 nmol/(h mg protein), which is similar to specific GSH export rates previously reported for these cells (Minich et al., 2006; Scheiber and Dringen, 2011). Application of arsenate to astrocytes caused a time- and concentration-dependent acceleration of the GSH export from viable astrocytes, which was accompanied by a matching increase in extracellular GSH levels, with a halfmaximal effect observed for around 0.3 mM arsenate. The sum of extracellular and cellular GSH was not influenced by the presence of arsenate, suggesting that the presence of arsenate selectively
modulates the distribution of GSH between the cellular and extracellular compartment and does not deplete astrocytic GSH by conjugation as reported for alkylated fumarates and halogenated acetates (Schmidt and Dringen, 2009; Schmidt and Dringen, 2010; Schmidt et al., 2011). An increase in the specific cellular GSx content that was reported for astrocytes exposed to arsenate or arsenite (Sagara et al., 1996; Tulpule et al., 2012) was not observed under the conditions used here, as the basal medium used for the incubations did not contain the amino acids required for GSH synthesis. A potential contribution of the elevated extracellular potassium content in the observed stimulated GSH export after application of potassium arsenate can be excluded, as supplementation of the incubation buffer with additional 10 mM potassium chloride did not affect GSH export, consistent with literature data reporting that even depolarization of cultured astrocytes with 50 mM potassium chloride does not affect GSH export (Hirrlinger et al., 2002). Mrp1 is the transporter responsible for the arsenate-accelerated astrocytic GSH export, as the Mrp1 inhibitor MK571 (Minich et al., 2006) completely prevented the arsenate-induced loss in cellular GSH and the accelerated accumulation of extracellular GSH. This is consistent with a recent report showing that MK571 inhibits at least in part the arsenate-induced GSH efflux from erythrocytes (Yildiz and Cakir, 2012). For cultured astrocytes, the stimulation of Mrp1-mediated GSH export has recently also been reported for antiretroviral protease inhibitors (Brandmann et al., 2012;
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Arend et al., 2013) and formaldehyde (Tulpule and Dringen, 2011). Thus, arsenate is a new member of a group of structurally very diverse molecules that stimulate Mrp1-mediated GSH export from astrocytes. Conjugation of cellular GSH with arsenic species and subsequent export of such conjugates has been discussed as a pathway that helps to eliminate arsenic species from cells (Kala et al., 2000; Thomas, 2007; Watanabe and Hirano, in press). In liver endothelial cells, the accumulation of arsenite has been reported to substantially increase in the presence of MK571 (Liu et al., 2001a), suggesting that in these cells Mrp-mediated export of arsenic-GSH complexes contributes to the elimination of arsenic compounds. However, for cultured astrocytes an export of such complexes is unlikely to contribute substantially to the observed stimulated GSH export. The arsenate-induced Mrp1-mediated increase in extracellular GSH contents amounted to around 20 nmol/mg protein. If an Mrp1-mediated export of a labile arsenic-GSH conjugate via Mrp1 would be involved in the stimulated GSH export from astrocytes, the specific cellular arsenic content of MK571-treated astrocytes should have increased strongly. However, as astrocytes that had been co-incubated with arsenate and MK571 did not differ in their specific arsenic content compared to arsenate-treated astrocytes in the absence of the inhibitor, an involvement of a potential export of a labile arsenic-GSH complex in the observed accelerated GSH export can be excluded. Thus, arsenate is likely to affect Mrp1-mediated GSH export directly either by binding to the transporter or by recruitment of Mrp1proteins from intracellular vesicles into the plasma membrane, as reported for bilirubin-treated astrocytes (Gennuso et al., 2004). Astrocytes have a key function in the antioxidative protection and in the GSH homeostasis of the brain (Hirrlinger and Dringen, 2010; Schmidt and Dringen, 2012). As the protection provided by astrocytes to neurons against hydrogen peroxide or NO is abolished in GSH-deprived astrocytes (Drukarch et al., 1997; Gegg et al., 2005), an arsenate- or arsenite-induced accelerated GSH loss from astrocytes may impair their antioxidative and protective functions. In addition, as astrocytes supply neighbouring neurons with precursors for GSH synthesis in a process that involves the export of astrocytic GSH by Mrp1 (Hirrlinger et al., 2002; Minich et al., 2006), a stimulation of GSH export from astrocytes by arsenics could also lead to an increase in the GSH content of neighboring neurons. Incorporated arsenate is primarily converted to organic derivatives in the liver (Bolt and Stewart, 2010; Juarez-Reyes et al., 2009; Wang et al., 2011) by a pathway that includes reduction to trivalent arsenic species and successive methylation reactions (Steinmaus et al., 2005; Watanabe and Hirano, in press). These reactions prevent that large amounts of inorganic arsenics will be encountered by other organs, including the brain. Indeed, the amounts of inorganic arsenics in the brains of mice after oral treatment with arsenate or arsenite are lower than those found for DMA (JuarezReyes et al., 2009; Wang et al., 2011) and DMA did not stimulate GSH export from cultured astrocytes. Whether a direct effect of arsenate on the GSH export from astrocytes may occur in brain remains to be elucidated. The levels of inorganic arsenic species reported in brain tissue of animals exposed to arsenics for a shortterm appear to be very low (Juarez-Reyes et al., 2009; Wang et al., 2011) compared to the concentrations of arsenate used in our study. However, already rather low concentrations of arsenate can strongly affect the GSH metabolism of brain cells. For example, presence of 5 lM arsenate in amino acid-containing culture medium has been reported to strongly increase within 24 h the specific cellular GSH content of cultured astrocytes (Tulpule et al., 2012). Thus, a chronic intoxication by consumption of arsenic-contaminated drinking water is likely to increase the risk that brain cells will encounter sufficient amounts of inorganic arsenic species,
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