Effect of Lead Exposure and Accumulation on Copper Homeostasis in Cultured C6 Rat Glioma Cells

Toxicology and Applied Pharmacology 158, 41– 49 (1999) Article ID taap.1999.8657, available online at http://www.idealibrary.com on

Effect of Lead Exposure and Accumulation on Copper Homeostasis in Cultured C6 Rat Glioma Cells Yongchang Qian,* Ginger Mikeska,† Edward D. Harris,* Gerald R. Bratton,† and Evelyn Tiffany-Castiglioni† ,1 *Department of Biochemistry and Biophysics, †Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843 Received November 3, 1998; accepted February 22, 1999

biochemical processes may result from intracellular Pb accumulation, as demonstrated in astroglia by atomic absorption spectroscopy (Rowles et al., 1989; Tiffany-Castiglioni et al., 1986, 1987) and X-ray diffraction electron microscopy (Holtzman et al., 1987), as well in other neural cells by flow cytometry or nuclear magnetic resonance with Ca-sensitive probes (Shao and Suszkiw, 1991; Tomsig and Suszkiw, 1991). Few studies have examined the mechanisms of intracellular Pb deposition in neural cells, even though potentially critical intracellular Pb targets, such as protein kinase C and calmodulin, are affected in vitro (Cox and Harrison, 1983; Habermann et al., 1983; Markovac and Goldstein, 1988; Vig et al., 1989). In culture, astroglia are comparatively more resistant to Pb cytotoxicity than are neurons and oligodendroglia (Holtzman et al., 1987; Rowles et al., 1989; Tiffany-Castiglioni et al., 1986, 1987), and they accrue Pb at an intracellular concentration that is estimated to exceed the extracellular level by up to four orders of magnitude (Tiffany-Castiglioni, 1993). Pb accumulation by astroglia is not accompanied by overt cytotoxic effects. This observation is consistent with the hypothesis that astroglial cells may function as Pb sinks to protect neurons in the central nervous system (Holtzman et al., 1984). Possible mechanisms by which astroglia tolerate the presence of intracellular Pb include elevated glutathione content of the cytoplasm (Legare et al., 1993) and the induction of specific proteins in Pb-exposed astroglial cells (Opanashuk and Finkelstein, 1995a). However, neither Pb– glutathione complexes nor Pb-binding proteins have as yet been isolated from Pb-adapted astroglial cells. One specific effect of Pb accumulation on glial cells is the intracellular accumulation of copper (Cu), which may therefore be a mechanism of neurotoxicity in Pb exposure. We have shown that in cultured astroglia exposed to a high Pb dose (100 mM), intracellular Cu content increases initially but then returns to normal levels after Pb exposure is discontinued, even though intracellular Pb remains elevated (Rowles et al., 1989). However, the effects of repeated low-level Pb exposure on Cu content in glia have not been measured. Furthermore, we have demonstrated that significant Cu accumulation occurs in the cerebellum and forebrain of guinea pigs exposed to low levels of Pb in utero (Sierra et al., 1989) or during early postnatal

Effect of Lead Exposure and Accumulation on Copper Homeostasis in Cultured C6 Rat Glioma Cells. Qian, Y., Mikeska, G., Harris, E. D., Bratton, G. R., and Tiffany-Castiglioni, E. (1999). Toxicol. Appl. Pharmacol. 158, 41– 49. C6 rat glioma cells resemble rat astroglia in culture in that both cell types accumulate lead (Pb) intracellularly from the medium. As such, C6 cells are a model for Pb accumulation by the brain. In this study, an increase in intracellular Pb accumulation induced by p-chloromercuribenzoate (PCMB) after exposure to 10 mM Pb acetate suggests a role for sulfhydryl groups in Pb retention. Stimulation of Pb accumulation by nifedipine suggests the entry of Pb into these cells by a novel path. Most of the intracellular Pb from exposure for 7 days to 1 mM Pb was associated with highmolecular weight components in cytosol. Pb exposure increased the abundance of three proteins with the following characteristics on two-dimensional gels: 81 kDa with pI of 5.6, 81 kDa with pI of 4.9, and 71 kDa with pI of 5.6. The levels of five other proteins, ranging in size from 37-41 kDa with pIs of 6.0-6.8 declined. Exposed C6 cells accumulated copper (Cu) intracellularly, and Cu accumulation after Pb exposure was shown by kinetic analysis with 67Cu to result from an increased uptake and a decreased efflux for Cu. Pb-exposed cells also showed increased Cu binding to membranes, which is consistent with the increase of Cu uptake. These data indicate that intracellular Pb interacts with high molecular weight proteins in C6 cells, and exposure also alters membrane transport properties for copper. © 1999 Academic Press Key Words: lead accumulation; sulfhydryl groups; nifedipine; copper transport; protein induction; glioma cells.

Exposure of rat astroglial cells to lead (Pb) in culture results in numerous biochemical alterations, including reduced basal respiratory rate (Holtzman et al., 1984, 1987), elevated cytosolic glutathione levels (Legare et al., 1993), reduced glutamine synthetase activity (Engle and Volpe, 1990; Ro¨nnba¨ck and Hansson, 1992; Sierra and Tiffany-Castiglioni, 1991), transient copper accumulation (Rowles et al., 1989; TiffanyCastiglioni et al., 1987), and novel protein induction (Opanashuk and Finkelstein, 1995a). The effect of Pb on these 1 To whom correspondence should be addressed at Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, TX 77843-4458.

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0041-008X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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development (Rowles et al., unpublished results). These data taken together implicate Cu accumulation by glia as a possible factor in the mechanisms by which Pb injures developing brain cells. The C6 rat glioma cell line, which expresses both properties of oligodendroglia (de Vellis and Brooker, 1972) and astroglia (Bissell et al., 1985; Pishak and Phillips, 1980), has been used in Pb and Cu metabolism and toxicity studies (de Vellis et al., 1987; Qian et al., 1995, 1996; Tiffany-Castiglioni et al., 1988). Like astroglia, C6 cells accumulate Pb intracellularly (TiffanyCastiglioni et al., 1988). Unlike astroglia, they can easily be cultured in quantities sufficient for biochemical analysis. Furthermore, short-term Pb exposure (10 mM Pb for 1–2 h) stimulates Cu accumulation in C6 cells without altering the kinetics of copper uptake (Qian et al., 1995). The effects of longer term exposure would therefore be of interest. Thus, C6 cells are a desirable model for astroglia, with which to study Pb-induced neurotoxicity. In the present study, we characterized short-term (up to 4 h) Pb accumulation and long-term (1 week) Pb localization in C6 cells. We also compared cytosolic protein patterns from control and Pb-exposed C6 cells by two-dimensional acrylamide gel electrophoresis in order to identify proteins potentially induced in glial cells by Pb exposure. In addition, we assessed the effects of long-term (1 week), low-level Pb exposure on copper uptake and efflux. METHODS Cell culture. C6 rat glioma cells were routinely cultured in 75-cm 2 flasks (Corning) at 37°C under 5% CO 2 with a mixture (1:1, v/v) of Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12, Sigma) supplemented with 10% fetal bovine serum (FBS, GIBCO/BRL), as previously described (Qian et al., 1995). The medium was changed every 2 or 3 days. Cells were subcultured into new flasks or dishes after detachment by incubation with Puck’s saline solution containing 1 mM EDTA (Tiffany-Castiglioni et al., 1988). For each of the following assays, cells were seeded at high density in 35-mm tissue culture dishes (Corning) and grown for 2–3 days to a final density of about 3 3 10 6 cells/dish. Pb accumulation. C6 cells cultured in 35-mm dishes were rinsed in situ with 2 ml of Dulbecco’s phosphate-buffered Saline (DPBS, Irvine Scientific) at room temperature for 30 s after removal of the old medium. Cultures were preincubated in serum-free medium for 30 min at 37°C in the presence of 50 mM nifedipine or 200 mM p-chloromercuribenzoate (PCMB) before Pb treatment began. Controls received serum-free medium during the preincubation period. The nifedipine and PCMB were washed out with DPBS, and the cultures were then exposed to 2 ml of serum-free medium containing 10 mM Pb acetate. The Pb-containing medium was removed after 10, 20, 50, 120, and 240 min at 37°C, and the cells were rinsed with 2 ml of 150 mM NaCl (pH was adjusted to 4.0 with 0.1N HCl) at room temperature for 30 s. The cells were harvested with a cell scraper (Falcon) after adding 1 ml of 30 mM Tris z HCl (pH 7.3) containing 0.25 M sucrose, transferred to a 1.5-ml Eppendorf tube, and centrifuged at 800g for 5 min (CR412, Jouan). After the buffer was removed, the pellet was dried in a fume hood by surface air flow and stored for Pb determinations. Average protein amount was determined by the bicinchoninic acid assay (BCA, Pierce) according to Pierce’s assay protocol, for five randomly selected dishes for each analysis. Pb content was expressed as mg Pb/mg protein. Pb analysis by atomic absorbance spectroscopy (AAS). Two hundred microliters of concentrated nitric acid (Ultrex, J. T. Baker, Phillipsburg, NJ)

was added to each tube containing a dried pellet of C6 cells, and the suspension was vortexed for 20 s and then allowed to sit overnight at room temperature. The digested cells were diluted with 1.8 ml of matrix modifier. The matrix modifier contained 0.5% nitric acid and 1% ammonium phosphate in a 2:1 ratio (v/v). The diluted cell solution was vortexed for 20 s and centrifuged for 5– 6 min at 2000 rpm (800g). Total lead was measured by atomic absorption spectroscopy with a Thermo Jarrell Ash Smith-Heiftje 12 spectrometer with furnace atomizer (model 188). Determination was by injection of 10 –20 ml of digestion solution with drying, ashing, and atomization in accordance with optimum parameters for Pb suggested by the Methods Manual for Furnace Operation, Vol. II (Thermo Jarrel Ash Corp., 1993). All materials that came in contact with the samples, such as flasks and tubes, were Pb-free, disposable materials. Cell fractionation of Pb-exposed cultures. In some experiments, C6 cells grown in 75-cm 2 flask were exposed to Pb acetate (0 or 1 mM) for 1 week. Cells were passaged at a subconfluent concentration, allowed to attach to the flask for 1 day, and then fed with Pb-containing medium (DMEM/F-12 with 10% FBS). The medium was changed every 2 or 3 days for 1 week, at which time the cultures were confluent. Confluent C6 cells were washed with 150 mM NaCl (pH 4.0) and harvested into 30 mM Tris z HCl (pH 7.3), 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma) as previously described (Qian et al., 1995). The cells were transferred to 15-ml polypropylene centrifuge tubes (Corning). All procedures were carried out at 4°C unless otherwise noted. The cells were sonicated on ice for 15 s at 50 W with a Sonifier Cell Disruptor (model W185, Heat System-Ultrasonics, Inc.). The cell homogenate was centrifuged at 800g on a desktop centrifuge for 15 min to produce a supernatant fraction S1 and a pellet of unbroken cells. Membranes from the 800g supernatant (S1) were collected by ultracentrifugation (30,000g; Ti70 rotor, L8-M Ultracentrifuge, Beckman) for 30 min to obtain a second supernatant (S2) and a pellet. The membrane-containing pellet (designated total membranes) was resuspended in buffer (30 mM Tris z HCl, pH 7.3, 0.25 M sucrose, 0.5 mM PMSF). The supernatant (S2) was dialyzed against 10 mM Tris z HCl, pH 7.3, with a dialysis tubing of 12,000 –14,000 Da retention overnight at 4°C to remove unbound Pb and small molecular weight fractions. The dialyzed supernatant was designated as S3. The resuspended membrane pellet was applied to a discontinuous sucrose gradient (17%, 40%, and 80%) and separated by ultracentrifugation (40 min, 150,000g; SW40 rotor, L8-M Ultracentrifuge, Beckman), according to a modification of the procedure of Johnson and Bourne (1977). Bands appearing at interfaces of 0/17%, 17/40%, and 40/80% were siphoned with a 5-ml syringe and diluted with five volumes of buffer (0.25 M sucrose, 10 mM Tris z HCl, pH 7.3). The membranes were pelleted by ultracentrifugation (40 min, 150,000g; Ti70, L8-M Ultracentrifuge, Beckman) and resuspended in buffer (0.25 M sucrose, 10 mM Tris z HCl, pH 7.3, 0.5 mM PMSF) for subsequent analysis of membrane-bound Pb content, expressed as nanograms Pb per milligrams protein. Sephadex G-100 chromatography. The S3 supernatant (about 10 ml) was concentrated to 1 ml by dialysis (membrane retention 12,000 –14,000 Da) against polyethylene glycol (PEG, mol wt 15,000 –20,000, Sigma). The concentrated supernatant was applied to a Sephadex G-100 column (0.65 3 28 cm) equilibrated with 10 mM Tris z HCl, pH 7.3. The column was eluted with 10 mM Tris z HCl, pH 7.3, and 0.3 ml fractions were collected. Protein content in each fraction was determined by the BCA method (Pierce). Four continuous fractions were combined for Pb assay by AAS because of the small fraction volume and low amount of Pb per fraction. Two-dimensional gel analysis of proteins. Two-dimensional patterns of total cell proteins were analyzed by the procedure of O’Farrell (O’Farrell, 1975) with minor modifications. Proteins in S1 supernatant were precipitated with four volumes of chilled acetone (220°C) for 30 min at 220°C and pelleted by centrifugation (12,000g, 10 min). The proteins were vacuum-dried and dissolved in isoelectrofocusing sample buffer consisting of 9 M urea, 4% (v/v) NP-40, 2% (v/v) 2-mercaptoethanol, and 2% ampholytes (0.4% pH 3.5–10 and 1.6% pH 5–7). Protein samples (200 mg) were loaded at the tops of the focusing gels and overlaid with 10 ml 5 M urea. The upper (cathode) buffer was 40 mM NaOH, and the lower (anode) buffer was 20 mM H 3PO 4.

LEAD AND COPPER INTERACTIONS IN C6 CELLS The isoelectrofocusing gels were run in a cold room (5–10°C) for 15 h at 400 V plus 1 h at 800 V. Following focusing, the average pH gradient in the gels was determined by cutting five gels into 0.5-cm pieces according to the distance from the top of the gel, pooling comparable pieces, soaking them in double-distilled water, and measuring the pH of the water. For two-dimensional SDS electrophoresis, the gels were equilibrated for 1 h at room temperature with buffer consisting of 62.5 mM Tris z HCl, pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 10% (w/v) glycerol. The equilibrated gels were applied to SDS-PAGE gels. SDS gels consisted of a separating gel of 10% acrylamide overlaid with a stacking gel of 3% acrylamide. The SDS gels were run at 60 V for the stacking gel until the proteins penetrated the separating gel and then were changed to 120 V for the separating gel. Two-dimensional gels were stained with 0.125% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid for 4 – 8 h at room temperature. They were destained with 50% methanol and 10% acetic acid for 1 h and with 5% methanol and 7% acetic acid until the bands showed clearly. 67

Cu uptake. As previously described (Qian et al., 1995), C6 cells were cultured in 35-mm dishes and exposed for 1 week to Pb acetate (0 or 1 mM) in medium. The cultures were rinsed in situ with DPBS at room temperature for 30 s and 2 ml of fresh serum-free DMEM/F-12 medium containing 10, 20, 50, 100, and 200 nM 67CuCl 2 (carrier-free, 256 Ci/mmol, Brookhaven National Laboratory, Upton, NY) was added. After 10 min incubation at 37°C, the radioactive medium was removed. The cultures were rinsed with 2 ml of 150 mM NaCl (pH was adjusted to 4.0 with 0.1N HCl) at room temperature for 30 s and harvested with a cell scraper (Falcon) after adding 1 ml of 0.5N NaOH to each dish. The cells from each dish were transferred to a 4-ml vial for counting. The cell-retained 67Cu was determined with a gamma counter (Gamma 5500, Beckman). The absolute molar quantity of 67Cu was deduced from a standard curve of CPM vs standard 67Cu (1, 2, 5, 10, and 20 pmol), which was run alongside the unknown. Proteins were determined with the BCA method (Pierce). The uptake of 67Cu was expressed as picomoles Cu/per milligram protein. The velocity of 67Cu uptake was expressed as picomoles Cu/per milligram protein/per minute. Values for K m and V max were determined from a double reciprocal plot. 67 Cu efflux. C6 cells exposed to Pb for 1 week as described in the previous section were loaded with 10, 20, 50, 100, or 200 nM 67CuCl 2 in serum-free DMEM/F-12 medium at 37°C for 50 min and were measured for 67Cu retention after various times of efflux, as previously described (Qian et al., 1995). The quantity of 67Cu retained at zero and 5 min was used to estimate the efflux rate. As discussed elsewhere (Qian et al., 1995), measurements of K m and V max primarily reflect 67Cu release from the exchangeable pool. The 67Cu concentration at the efflux site at time zero was estimated from the concentration of 67 Cu in the medium at the time of cell loading as these concentrations are assumed to be the same (Darwish et al., 1984). This value was nearly constant during loading, because cells took up only 1–2% of the Cu initially present in the medium. Efflux velocity was expressed as picomoles Cu/per milligrams protein/per minute.

RESULTS

Pb Accumulation Because C6 glioma cells have many astroglial-like properties, including the propensity to accumulate Pb from the culture medium (Tiffany-Castiglioni et al., 1988), we investigated mechanisms of Pb accumulation. A relatively high Pb acetate concentration (10 mM) and the absence of serum in the medium were used to enhance Pb uptake by the cells in order to achieve levels detectable by AAS at early time points (Legare et al., 1998). Within 4 h of incubation, Pb accumulation by the cells reached a plateau. At 2 h, the accumulation was maximal, 4.24 6 0.56 mg Pb/mg protein (Fig. 1) and we estimated that

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FIG. 1. Pb accumulation in C6 rat glioma cells as determined by atomic absorption spectroscopy. Cells were pretreated with 50 mM nifedipine (squares) or 200 mM PCMB (triangles) in serum-free DMEM/F-12 medium at 37°C for 30 min, after which excess reagent was washed out. Cells were then exposed to 10 mM Pb acetate for the time periods indicated. Data show mean 6 SD of three samples. Means were compared by Student’s t test. **Differs significantly from control (P , 0.01); *differs significantly from control (P , 0.05).

51% of the total Pb in the medium had been absorbed by the cells. C6 cells pretreated for 30 min with 200 mM PCMB, a sulfhydryl reagent, accumulated more Pb from the medium than cells not given PCMB, suggesting that sulfhydryl groups are involved in cellular Pb accumulation. This phenomenon also showed a plateau with time. Maximum accumulation in the presence of PCMB was 5.38 6 0.28 mg Pb/mg protein, or 65% of total Pb added to the medium. In addition, cells treated for 30 min with 50 mM nifedipine, a blocker of L-type calcium channels, prior to the addition of Pb retained Pb to the same extent as cells treated with PCMB (Fig. 1). Pb Distribution We investigated the subcellular distribution of Pb in the cell fractions by atomic absorption spectroscopy. After 1 week in 1 mM Pb acetate most of the Pb (94%) taken in was found in a supernatant fraction (30,000g S2 supernatant). When the S2 supernatant fraction was dialyzed to retain particles above 12–14 kDa, nearly all of the Pb (91% of total Pb) was retained (Table 1). A preliminary characterization of the Pb-binding proteins was undertaken to identify the Pb-binding components. The S3 retentate of the S2 fraction was separated on a Sephadex G-100 column. The results (Fig. 2) show that the greatest amount of Pb recovered overlapped protein peaks with molecular weights of 66 kDa. Within the membrane compartment (Table 1), most of the Pb was located in the heavy membrane fraction, perhaps corresponding to mitochondria. Alterations in 2D Protein Patterns Induced by Pb Exposure We compared total protein patterns of Pb-treated cells with control cells by two-dimensional acrylamide gel electrophoresis. As shown in Fig. 3, the protein patterns on two-dimen-

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TABLE 1 Distribution of Pb in C6 Rat Glioma Cells Exposed to 1 mM Pb Acetate for 1 Week Cell fractions Cell S1 S2 S3 Light membranes (0/17%) Middle membranes (17/40%) Heavy membranes (40/80%)

Pb (ng)

% of Total Pb

Pb/protein (ng/mg)

480 474 453 437 18.6

100 99 94 91 4

6.25 6.46 8.22 14.2 7.10

20.7

4

11.0

43.5

9

29.2

Note. Cells were cultured in DMEM/F-12 medium supplemented with 10% FBS and containing 1 mM Pb acetate. The medium was changed every 2 or 3 days with fresh Pb-containing medium. Fraction S1 is the supernatant of a 800g centrifugation step, S2 is the supernatant of a 30,000g centrifugation step, and S3 is the retentate of the dialysis with a 12- to 14-kD retention size. The data show the results of one of two experiments that produced similar findings.

sional gels revealed several differences. Three acidic proteins, corresponding to molecular masses of 71 kDa (pI of 5.6), 81 kDa (pI of 4.9), and 81 kDa (pI of 5.6), were strongly enhanced in cells exposed to 1 mM Pb acetate for 1 week (Fig. 3B, box 1). Enhancement was greatest for the 71 kDa protein. In addition, five major protein spots showed decreased intensities in Pb-treated cells; their molecular mass and pI values ranged from 37 to 41 kDa and from 6.0 to 6.8, respectively (Fig. 3B, box 2).

control cells. Pb exposure not only increased Cu content of the soluble fraction, but also the membrane fractions, particularly the heavy fraction. In contrast, PCMB-treated cells retained over twice as much copper as controls and nearly twice as much as Pb-treated cells, but this increase occurred in the soluble fraction. The amount of Cu bound to membrane fractions in the PCMB-treated group was the same as or lower than control values. DISCUSSION

The present study provides evidence that intracellular retention and accumulation of Pb by C6 rat glioma cells can occur through two distinct mechanisms: blockage of ion channels and complexation of internal sulfhydryl groups. Both nifedipine, a blocker of L-type calcium channels, and PCMB, a sulfhydrylbinding reagent, increased Pb accumulation. The finding that 50 mM nifedipine stimulated Pb accumulation suggests a novel type of Pb transport. To date three mechanisms have been postulated for Pb entry across plasma membranes of cells: uptake into bovine chromaffin cells via L-type Ca 21 channels, a process that is inhibited by nifedipine (Simons and Pocock, 1987; Tomsig and Suszkiw, 1991); uptake into erythrocyte ghosts via an anion exchanger (Simons, 1993); and activation of a cation channel in C6 and other cell types by depletion of intracellular Ca 21 stores (Kerper and Hinkle, 1997). Whereas nifedipine inhibits the first mechanism and has no known effect

Effect of Pb Exposure on Copper Transport The kinetics of copper transport in C6 cells exposed to Pb acetate (1 mM) for 1 week were also determined. As calculated from Fig. 4A, the double-reciprocal plot gave a K m value for copper uptake of 0.20 6 0.09 mM in Pb-exposed cells (n 5 6) compared to 0.6 mM for control C6 cells. This K m value was significantly less than the values previously reported (Qian et al., 1995) for control C6 cells (0.63 mM) and cells exposed to Pb (10 mM) for 1–2 h (0.57 mM). The kinetics of copper efflux are shown in Fig. 4B: the K m for copper efflux was 0.40 6 0.06 mM in Pb-exposed cultures (n 5 6). This value is significantly larger than the K m value (0.15 mM) previously reported for control cells (Qian et al., 1995). Effect of Pb Exposure on Membrane-Bound Cu Table 2 shows the effect of Pb acetate exposure (1 mM for 1 week) on Cu-binding to membranes. Cell homogenates were separated into a soluble fraction and a membrane fraction by ultracentrifugation. The membrane fraction was further separated by discontinuous sucrose density ultracentrifugation. Pbexposed cells accumulated more 67Cu intracellularly than did

FIG. 2. Correlation of Pb content (closed circles) with protein fraction (opened circles) in cytosolic fractions from C6 glioma cells. Cells were exposed in culture to 1 mM Pb acetate in DMEM/F-12 medium with 10% FBS for 1 week. Cytosolic protein fractions were separated by Sephadex G-100 (0.65 3 28 cm) chromatography. The cytosol was dialyzed against 10 mM Tris z HCl, pH 7.3, overnight at 4°C in a membrane with a retention of 12,000 –14,000 kDa before being applied to the column equilibrated with 10 mM Tris z HCl, pH 7.3. The column was eluted with 10 mM Tris z HCl, pH 7.3, at 4°C and 0.3 ml eluant was collected in each fraction. Protein was determined by BCA in each fraction. Continuous fractions were pooled because of their low Pb content (4 fractions per sample) and analyzed for Pb content by AAS. The highest levels of Pb were found in fractions containing protein with mol wt of 66 kDa. The void volume (V 0) was 3 ml. Protein size standards were 66 kDa bovine serum albumin (BSA, Sigma) and 14 kDa chicken egg white lysozyme (Sigma).

LEAD AND COPPER INTERACTIONS IN C6 CELLS

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FIG. 3. Two-dimensional gel electrophoresis of cytosolic proteins from control and Pb-exposed C6 cells. C6 cells were exposed to 0 (A) or 1 mM Pb acetate (B) in complete medium for 1 week. Proteins from the cytosolic fraction (fraction S1 in Table 1) were applied to 4% acrylamide isoelectric focusing (IEF) gels (first dimension); 200 mg protein was loaded to each gel. Following focusing, the IEF gels were applied to SDS-PAGE gels (second dimension) consisting of a separation gel of 10% acrylamide overlaid with a stacking gel of 3% acrylamide. The horizontal dimension indicates IEF, and the vertical dimension shows SDS-PAGE. Two horizontal arrows and two vertical upward arrows indicate three major protein spots (81 kDa with pI of 5.6, 81 kDa with pI of 4.9, and 71 kDa with pI of 5.6) that were detected by Coomassie Brilliant Blue R-250 staining in Pb-exposed cells but not in controls. Molecular weight markers are indicated on the left side of the gels. Vertical downward arrows indicate the pH of the gel at four positions along the gradient.

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FIG. 4. Double reciprocal plots of initial rates of copper uptake and efflux in Pb-exposed C6 cells. The cells were cultured in DMEM/F-12 medium containing 1 mM Pb acetate for 1 week. (A) Uptake: Cells were incubated in DMEM/F-12 medium containing 10, 20, 50, 100, or 200 nM 67CuCl 2 at 37°C for 10 min. The values of kinetic parameters (K m and V max) for 67Cu uptake were calculated from the plot. Data show means 6 SD from six determinations. (B) Efflux: Cells were preloaded with 10, 20, 50, 100, and 200 nM 67 CuCl 2 in DMEM/F-12 medium at 37°C for 50 min. The velocity of 67Cu efflux was determined by subtracting 67Cu retained at zero from that retained at 5 min. Data show means 6 SD from six determinations. Therefore, K m values calculated from these plots can be compared with published control K m values. The data presented here were carried out at the same time with previously published control (Qian et al., 1995). Therefore, K m values calculated from these plots can be compared with published control K m values.

cells differentiated with dibutyryl cyclic AMP is the alteration of 5-hydroxytryptamine-stimulated [Ca 21] i oscillations, as shown by Cholewinski and Leslie (1996). These authors found that oscillations continued in the presence of nifedipine and concluded that nifedipine was without effect. However, examination of their published report indicates that nifedipine decreases the amplitude of the oscillations and alters the shapes of the individual [Ca 21] i peaks, which suggests interference with Ca 21-buffering mechanisms that contribute to the oscillations. Analysis of the quantitative effect of nifedipine on these oscillations of [Ca 21] i, including the respective contributions of extracellular Ca 21 and inositol 1,4,5-trisphosphate (InsP 3) or ryanodine-releasable pools, would be of interest in resolving the role of nifedipine in stimulating Pb accumulation. Enhancement of Pb accumulation by PCMB suggests another mechanism by which Pb content in glia may increase. Although our data do not address the biochemical mechanism by which sulfhydryl groups are involved in Pb transport, previous work in which we examined the role of sulfhydryl groups in Cu transport in C6 cells suggests that there are internal thiol and external disulfide moieties in C6 rat glioma cells (Qian et al., 1996). PCMB membrane permeability has been reported in erythrocytes with 203Hg-PCMB, and its binding to interior proteins has also been established (Knauf and Rothstein, 1971; Shapiro et al., 1970). By analogy, the internal thiols may be involved in the extrusion of accumulated Pb, and their blockage by PCMB may increase Pb accumulation. This interpretation is consistent with the view that metal accumulation in glia reflects a failure of these cells to pump out metals, rather than increased active uptake by the cell (Tiffany-Castiglioni, 1998). This view will be discussed in more detail below in the context of Cu accumulation, which was stimulated by both Pb and PCMB, indicating that Cu accumulation resulted from the

TABLE 2 Effects of Pb and PCMB on 67Cu Binding to C6 Cell Fractions Copper/protein (pmol Cu/mg protein) Membranes

on the other two, a fourth previously undescribed mechanism may be involved. Nifedipine is primarily an inhibitor of L-type Ca 21 channels, although it and other L-type Ca 21 channel blockers may have inhibitory effects on receptor-operated Ca 21 channels in rat hepatocytes (Hughes et al., 1986; Striggow and Bohnensack, 1993). C6 cells lack voltage-regulated Ca 21 channels (Imoto et al., 1996) and are thus insensitive to the doses (1-10 mM) of nifedipine typically employed to open such channels and stimulate a transient rise in intracellular Ca 21 levels ([Ca 21] i). However, C6 cells possess receptor-operated Ca 21 channels that contribute to the elevation of [Ca 21] i (Finkbeiner, 1993). One apparent action of 50 mM nifedipine on C6

Cells

Cell

Soluble fraction

Light (0/17%)

Middle (17/40%)

Heavy (40/80%)

Control Pb-treated PCMB-treated

1.92 2.41 4.65

2.06 2.59 4.87

1.45 1.84 1.45

1.98 2.23 1.46

2.69 3.44 1.76

Note. Cells were treated with or without Pb acetate (1 mM) for 1 week in DMEM/F-12 medium with 10% FBS. At the end of 1 week, some of the cultures unexposed to Pb were treated with 200 mM PCMB in serum-free DMEM/F-12 medium at 37°C for 30 min, and excess PCMB was washed out with DPBS. All cultures were then incubated in situ with 37.5 nM 67CuCl 2 in serum-free DMEM/F-12 medium at 37°C for 50 min. After the incubation, cultures were washed and harvested, and membranes were isolated. The data show the results of one of two experiments that produced similar findings.

LEAD AND COPPER INTERACTIONS IN C6 CELLS

intracellular blockage of the thiol-rich P-type ATPase that controls copper efflux (Qian et al., 1995). The greater part of Pb taken in by C6 cells was associated with soluble components of high molecular weight (. 12–14 kDa). This finding is contrary to earlier speculations that Pb is associated with either metallothionein or glutathione, which are postulated Pb detoxifying components or temporary storage sites (Tiffany-Castiglioni et al., 1996). Metallothionein has been localized to astroglial cells in brain (Young et al., 1991), and cytosolic glutathione content is elevated by Pb exposure (Legare et al., 1993). The additional observation in the present study that Pb exposure resulted in the increased abundance of two proteins of 81 kDa and one of 71 kDa, coupled with colocalization of most of the Pb with high molecular weight cytosolic components, suggests the possibility that these inducible proteins are involved in Pb detoxification or storage in C6 cells. Novel protein synthesis, including a 70-kDa protein that does not cross-react with antibodies to hsp70, has also been reported in cultured rat astroglial cells exposed to Pb (Opanashuk and Finkelstein, 1995a,b). Sequence data are needed to establish if this 70-kDa protein and the 71-kDa protein we report for C6 cells are one and the same. Protein induction may constitute a process of tolerance. Tolerance to the presence of Pb in intracellular sites appears to be a signal feature of mature astroglia, as indicated by their ability to accumulate Pb without loss of viability. The level of tolerance is imperfect to the degree that some functions of astroglia are compromised, and thus neuronal activities that are astroglial-dependent may be impaired (Tiffany-Castiglioni, 1993). The induction of specific proteins in glia by Pb, both in the present study and those of Opanashuk and Finkelstein (1995a,b), may be a key to understanding the mechanism of tolerance. Such proteins may inactivate Pb effects by sequestering it in nontoxic sites, offset other biochemical impairments induced in the cell by Pb, or stimulate Pb removal from the cell. These possibilities are supported by our recent finding that astroglia exposed to Pb acetate at the same level as C6 cells in the present study (1 mM for 7 days) develop a partial capacity for buffering total intracellular Pb and Ca levels, both of which rapidly increased within 1 day of Pb exposure but thereafter leveled off or decreased even in the face of continued Pb exposure. An adaptive response in terms of Ca 21 and Pb 21 buffering is therefore suggested (Legare et al., 1998). In the present study, the effect of Pb on Cu transport was further characterized. We have previously shown that C6 cells exposed to 10 mM Pb acetate for 1–2 h accumulated excess Cu, but the kinetics of copper uptake did not change. In contrast, the K m value for Cu uptake in these cells exposed to 1 mM Pb acetate for 1 week was about one third that of the values previously reported (Qian et al., 1995) for control C6 cells (0.63 mM) and cells exposed to 1 mM Pb for 1–2 h (0.57 mM), suggesting that long-term Pb exposure and short-term Pb exposure have different effects on copper uptake in C6 cells. Furthermore, the K m for Cu efflux after long-term Pb exposure

47

was nearly three-fold that of the K m value (0.15 mM) previously reported for control cells (Qian et al., 1995). The K m for Cu efflux under short-term Pb treatment was not previously determined. Although Pb and PCMB both decreased Cu efflux, these treatments had distinct effects on membrane-bound Cu. Pbexposed cells showed an increased amount of Cu bound to membranes, which is consistent with a decrease of K m for Cu uptake. PCMB-treated cells retained twofold as much copper as control cultures but less of the Cu was membrane-bound than in either control or Pb-treated cells. Thus, long-term Pb treatment and short-term PCMB produced opposite effects on Cu binding to membranes. On the other hand, Cu binding to membranes in short-term Pb exposure more closely resembled the PCMB effect (Qian et al., 1995). We postulate that Pb accumulation by cells in long-term exposure triggered cellular responses that altered the membrane environment with respect to Cu. Cells tend to take up Cu 1, rather than Cu 21 (Bingham et al., 1996). Therefore, the membrane environment may be more capable of metal ion reduction in Pb-treated C6 cells than in control cells in order for Cu 21 to be reduced to Cu 1. The elevation of the cytosolic glutathione content by Pb exposure is therefore of potential interest as a mechanism for metal ion reduction on the membrane (Legare et al., 1993). The finding that Pb and PCMB produced a functionally similar decrease in Cu efflux is of interest because of the presence of a known Cu-ATPase in C6 cells (Qian et al., 1997, 1998). A good candidate protein for Cu efflux is the Menkes gene product, which has been shown to be a membrane-bound Cu-ATPase (Vulpe et al., 1993). This protein is one of a family of P-type ATPases functioning as metal transporting enzymes that also includes Na 1, K 1, -ATPase and Ca 21-ATPase, the latter two of which are inhibited by Pb (Fox et al., 1991; Sandhir and Gill, 1994). Indirect evidence also exists for the inhibition of the Cu-ATPase in C6 cells: Cu efflux is inhibited by Pb, as well as by inhibitors that specifically target sulfhydryl groups and block the ATPase function (Qian et al., 1995). Pb 21 ions are attracted to proteins rich in sulphur clusters. The Menkes protein contains a sulfhydryl-rich heavy metal binding domain that is a potential target for Pb interference with metal ion homeostasis. In addition to the impairment of Cu efflux by Pb in C6 cells described, the accumulation of Cu by Pbexposed brain cells or tissues (Rowles et al., 1989; Sierra et al., 1989) implicates Cu transport as a potential mechanism of neurotoxicity in Pb exposure. When astroglia from the macular mouse model for Menkes disease are cultured in medium with normal Cu levels, the cells show a fourfold elevation of Cu levels (Kodama et al., 1991), which is identical to the elevation seen in rat astroglia exposed to Pb in culture (Rowles et al., 1989). The latter finding suggests a parallel mechanism by which abnormal Cu metabolism in astroglia may be a mechanism for neurological damage in both Menkes disease and Pb exposure.

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QIAN ET AL.

ACKNOWLEDGMENTS

S., Nishimura, M. (1991). Genetic expression of Menkes disease in cultured astrocytes of the macular mouse. J. Inherit. Metab. Dis. 14, 896 –901.

This study was supported in part by National Institutes of Health Grants RO1 ES-05871, RO1 HD-29952, and P30 ES-09106; a CVM Enhancement Grant; and a TAMU Interdisciplinary Research Grant.

Legare, M. E., Barhoumi, R., Burghardt, R. C., and Tiffany-Castiglioni, E. (1993). Low-level lead exposure in cultured astroglia: Identification of cellular targets with vital fluorescent probes. Neurotoxicology 14, 267– 272.

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