Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

Toxicology and Applied Pharmacology 231 (2008) 308–317

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells Martine Lévesque a,1, Corine Martineau a,1, Catherine Jumarie b, Robert Moreau a,⁎ a b

Laboratoire du Métabolisme Osseux, BioMed, Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Quebec, Canada H3C 3P8 Laboratoire de Toxicologie Cellulaire des Métaux, TOXEN, Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Quebec, Canada H3C 3P8

a r t i c l e

i n f o

Article history: Received 17 December 2007 Revised 15 April 2008 Accepted 21 April 2008 Available online 29 April 2008 Keywords: Osteoblasts Cadmium Cytotoxicity Calcium Magnesium Voltage-dependent calcium channel

a b s t r a c t Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of 109Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd2+ species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels. © 2008 Elsevier Inc. All rights reserved.

Introduction Bone is a dynamic tissue, continuously remodeled at coordinated rates. Under normal conditions, cells called osteoclasts are responsible for old bone degradation (designated as the resorption process), while other cells, the osteoblasts, proceed with new tissue deposition (termed bone formation). Osteoblastic cells ensure bone formation and mineralization through the secretion of bone matrix components (type I collagen and non-collagenous proteins) and also provide essential factors for osteoclastic differentiation, such as macrophage-

⁎ Corresponding author. Département des Sciences Biologiques, Université du Québec à Montréal, CP 8888, succ Centre-Ville, Montreal, Quebec, Canada H3C 3P8. Fax: +1 514 987 4647. E-mail address: [email protected] (R. Moreau). 1 Both authors have contributed equally to this work. 0041-008X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2008.04.016

colony stimulating factor (M-CSF) and the receptor activator of NF-kappaB ligand (RANKL). Therefore, osteoblasts not only play a central role in bone formation but also regulate bone resorption (Mackie, 2003). In this respect, adequate osteoblastic proliferation, differentiation, secretory functions and rate of apoptosis are crucial for both the formation and resorption processes, thereby maintaining normal bone remodeling. Disruption of this equilibrium may lead to loss of bone mass and to the development of osteoporosis with the associated higher risks of fractures. Exposure to cadmium (Cd) was first associated with bone disease in the 1940s; Japan witnessed an outbreak of itai–itai disease, manifested by severe renal dysfunction with parallel osteomalacic and osteoporotic lesions (Pier, 1975). Although numerous studies have since suggested that exposure to Cd may increase the risk of osteoporosis in humans (Staessen et al., 1999; Alfven et al., 2000; Jarup and Alfven, 2004; Jin et al., 2004) and experimental animals (Katsuta et al.,

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1994; Regunathan et al., 2003; Brzoska and Moniuszko-Jakoniuk, 2004, 2005), the link between Cd and osteoporosis remains poorly understood. Two hypotheses are generally proposed for the bone toxicity of Cd: indirect and direct mechanisms. The indirect mechanisms of toxicity involve Cd-induced damage to the liver, kidney and intestine with the resulting impacts on bone (Nogawa et al., 1987; Kjellstrom, 1992). However, Cd has been shown to accumulate in bone (Ogoshi et al., 1992; Hunder et al., 2001) and studies have demonstrated severe adverse bone effects in some populations environmentally exposed to Cd without evidence of lesion to the kidney (Wang et al., 2003). Also, epidemiological studies have shown that this heavy metal may promote skeletal alterations at significantly lower levels than previously anticipated (Staessen et al., 1999; Alfven et al., 2000; Jarup and Alfven, 2004). Therefore, how Cd may directly affect bone cell functions becomes a critical question that clearly deserves further investigation. In accordance with the idea that Cd may impair bone remodeling equilibrium, Cd has been shown in vitro to alter the functions and viability of osteoblasts (Angle et al., 1993; Iwami and Moriyama, 1993; Long, 1997b; Kaneki et al., 2000; Coonse et al., 2007) as well as osteoclasts (Iwami and Moriyama, 1993; Wilson et al., 1996). However, the cellular mechanisms responsible for Cd-induced bone alterations are still not understood. It should be noted that cell sensitivity to Cd's deleterious effects is first related to the uptake capacity and related levels of cellular accumulation. Since Cd is a non-essential metal, the existence of membrane transport systems specifically devoted to the cellular uptake of Cd is unlikely and it is generally believed that Cd may use transport mechanisms for essential metals. Voltage-dependent Ca channels (VDCCs) have been shown to be responsible for Cd uptake in renal, pituitary and hepatic cells (Hinkle et al., 1987, 1992; Long, 1997b; Endo et al., 2002). The iron transporter NRAMP2/DMT-1 has been found to participate in Cd uptake by intestinal cells (Elisma and Jumarie, 2001; Park et al., 2002; Bannon et al., 2003), and “TRP-melastatin-related” (TRPM) channels 6 and 7 to transport Cd (Monteilh-Zoller et al., 2003; Li et al., 2006). However, at the present time, no data are available about which uptake mechanisms are involved in Cd accumulation in osteoblastic cells. The aim of the present study was to characterize the cellular uptake and cytotoxicity of Cd in human osteoblast-like MG-63 cells, and to obtain insights into the transport mechanisms responsible for cellular accumulation. Given that calcium (Ca) and magnesium (Mg) are important ions for bone metabolism, the respective impacts of these metals on the observed parameters were also investigated. Materials and methods Cell culture. Human osteoblast-like MG-63 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in a 1:1 mixture of phenol-free DMEM/Ham's F12 medium (DMEM/F12; Sigma, Oakville, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS; Cansera, Etobicoke, Ontario, Canada), 2 mM L-glutamine (Invitrogen, Burlington, Ontario, Canada), 100 U/mL penicillin and 100 µg/mL streptomycin (both from Invitrogen). Cells were maintained in a 5% CO2 atmosphere at 37 °C and were harvested weekly with 0.05% Trypsin–0.02% EDTA solution (Invitrogen). Cytotoxicity assays. Cells were seeded in 96-well plates (Sarstedt, Montréal, Québec, Canada) at a 3000 cells/cm2 density. After 6 days of culture, the cells were serum-starved for 24 h and then incubated with increasing concentrations of Cd or Mn in the serumfree culture medium in the absence or presence of 50 ng/mL of platelet-derived growth factor BB (PDGF-BB; Sigma), or of various concentrations of bovine serum albumin (BSA) or ionic channel modulators (Sigma). In order to investigate the effects of calcium (Ca) and magnesium (Mg) on Cd-induced cytotoxicity, experiments were performed using Ca- and Mg-free DME/F12 supplemented with well-controlled levels of added CaCl2 or MgCl2. To study the possible involvement of VDCCs in Cd uptake and the resulting Cdinduced cytotoxicity, membrane depolarization was induced by increasing the potassium concentration from 5 mM to 50 mM in serum-free DMEM/F12 medium. Some viability measurements were performed in the presence of channel modulators with appropriate controls using the vehicle alone. Cytotoxicity was determined by microtiter tetrazolium (MTT) assays. Briefly 4 h before the end of cell treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide (MTT; final concentra-

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tion of 0.5 mg/mL) (Sigma, Oakville, Ontario, Canada) was added to the incubation medium. At the end of the incubation period, the medium was aspired and the formazan crystals were dissolved in DMSO. Absorbance was measured at 575 nm using a Tecan SpectraFluor Plus spectrophotometer (Esbe Scientific Industries Inc., Canada). Data are expressed as the ratio of absorbance of treated cells relative to untreated control cells. Measurement of Cd and Ca uptake. Cells were seeded in 35-mm Petri dishes (Sarstedt) at a cell density of 3000 cells/cm2. Once cell confluency was reached, the cells were serum-starved 24 h prior to the uptake experiments. Briefly, the dishes were rinsed 4 times with 2 mL of chloride buffer at room temperature before being exposed to 0.5 µM 109Cd (0.15 µCi/mL, specific activity ranging from 3 to 4 µCi/µg) (Eckert & Ziegler, Berlin, Germany) or 0.5 mM 45Ca (2.5 µCi/mL, specific activity ranging from 7.8 to 12.6 µCi/µg) (GE Healthcare, Mississauga, Ontario, Canada) in the defined chloride or nitrate transport media containing (in mM): 10 Hepes, 4 D-glucose, 137 NaCl/NaNO3, 5.9 KCl/KNO3 with indicated concentrations of CaCl2/Ca(NO3)2 and/or MgSO4 adjusted to pH 7.4 with NaOH. Some uptake measurements were performed in the presence of channel modulators. Long-term accumulation experiments were conducted in DMEM/ F-12 medium with similar exposure conditions as for the cytotoxicity measurements. To determine the involvement of VDCCs in Cd-induced cytotoxicity, membrane depolarization was promoted by an equimolar substitution of NaCl/NaNO3 for KCl/KNO3 in the transport media. Under all conditions, the uptake process was stopped with 4 cell rinses with 2 mL of ice-cold radioisotope-free chloride buffer containing 2 mM EDTA to reduce metal adsorption at the external surface of the cell membrane. The cells were then lysed in 500 µL of NaOH 1 N, and 300-µL aliquots were used for radioactivity determination using a gamma counter for 109Cd samples (Cobra II, Canberra Packard Canada) or a beta counter (Wallac 1409 DSA, Wallac Oy, Turku, Finland) for 45Ca samples. Results are expressed as pmol of 109Cd or nmol of 45Ca per mg protein. Protein quantification. Protein contents were quantified according to Bradford (1976) with BioRad reagent (Mississauga, Ontario, Canada) using BSA (0–100 µg) as the calibration curve. Briefly, 50-µL aliquots of diluted samples were sampled in 96-well microplates, and the absorbance was read at 595 nm using a Tecan SpectraFluor Plus spectrophotometer. Statistical analysis. All experiments were performed on at least three independent cell cultures, each time in duplicate. Cell viability as a function of increasing concentration of Cd was analyzed according to the dose-response Eq. (1) y = ymin +

ymax −ymin 1 + 10ðlog

LC50−XÞTHillslope

ð1Þ

where Ymax and Ymin are the maximal and minimal ratios of cell viability, respectively, and LC50 is the concentration of Cd for which a cell viability ratio of 0.5 is observed. The kinetic parameters of 109Cd uptake were determined by analyses of the onetime point measurements at 3 min (v3) according to the modified Michaelis–Menten Eq. (2)

v3 =

h i h i Vmax 109 Cd h i + kD 109 Cd Km + 109 Cd + ½Cd

ð2Þ

where Vmax and Km have their usual meaning, [109Cd] was set at 0.5 µM, while [Cd] increased from 0 to 100 µM, and kD represents all the non-specific contributions to the 3-min uptake data. The apparent constant of inhibition (Ki) of 109Cd uptake by Mg was determined by non-linear regression analyses of the one-time point measurements at 3 min (v3) according to the following non-competitive inhibition Eq. (3) v3 =

h i Vmax + KD 109 Cd 1 + ð½I=Ki Þ

ð3Þ

where [I] is the concentration of Mg in the exposure medium, ranging from 0 to 20 mM, and KD[109Cd] represents the non-inhibitable component of 109Cd uptake. Because the uptake data analyzed according to Eq. (3) were corrected for the non-specific contribution (kD[109Cd] in Eq. (2)), the residual uptake (KD[109Cd]) that still takes place in the presence of Mg then represents the specific but Mg-insensitive component of 109Cd uptake (then the different kD and KD in Eqs. (2) and (3)). Non-linear regression analyses were performed using Prism 4 software (GraphPad Software, San Diego, California, USA). The errors associated with the parameter values given in the text represent the standard error of regression (SER). Statistical analyses were performed with the unpaired student's Student's t-test or ANOVA with Dunnett's or Bonferoni's post-test.

Results Cadmium-induced loss in cell viability and related cellular accumulation To first establish the optimal experimental conditions for characterizing the effects of Cd on osteoblasts, MG-63 cell viability was

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measured using the MTT assay as a function of time and increasing concentrations of Cd in the serum-free culture medium. As shown in Fig. 1a, a significant (P b 0.05) reduction in cell viability was observed as early as 3 h with exposure to 100 µM Cd. Longer exposure times resulted in much lower levels of cell viability. A typical sigmoid doseresponse curve was obtained following a 24-h exposure; data analyses according to Eq. (1) revealed an LC50 value (Cd concentration for which cell viability is half the control value) of 18 ± 1 µM. The observed cytotoxic effect was specific to Cd since the addition of up to 200 µM MnCl2 to the culture medium had no significant effect on MG-63 cell viability, whereas small reductions (18–24%) were observed with 200 to 250 µM (P b 0.001) (Fig. 1b). A 24-h exposure to 20 µM Cd resulted in marked changes in MG-63 cell morphology and a loss of adherence typical of cell death (Fig. 1c). Furthermore, the presence of albumin (BSA) in the incubation medium, which likely binds Cd and thus lowers metal availability for uptake, alleviated in a concentration-dependent manner Cd-induced loss in cell viability with significant effects observed at 1 mg/mL or higher BSA levels (P b 0.001) (Fig. 1d). Because Cd cytotoxicity is expected to be related to the levels of cellular accumulation, 109Cd uptake in MG-63 cells was characterized. According to the time-dependent properties of the dose-response curves observed in Fig. 1a, the cellular accumulation of 0.5 µM 109Cd markedly increased with the time of exposure (Fig. 2a).

Characterization of the cellular uptake of cadmium Because Cd readily accumulated in the osteoblasts, we further investigated the kinetic parameters of the initial uptake of Cd. As shown in Fig. 2b, the 3-min cellular accumulation of 0.5 µM 109Cd decreased as a function of increasing levels of unlabeled Cd used as a competitive inhibitor of tracer uptake. Data analyses according to the modified Michaelis–Menten Eq. (2) gave the following parameter values: Km = 13.5 ± 2.7 µM; Vmax = 262 ± 59 pmol 109Cd/3 min/mg protein, and kD = 8 ± 1 pmol 109Cd/3 min/µM/mg protein. Note that kD represents all non-specific contributions to total uptake levels (possibly including binding). The impact of inorganic metal speciation on 109Cd uptake has been studied using a nitrate medium optimizing [Cd2+] over chlorocomplex formation. Indeed, contrary to Cl−, NO−3 does not bind as much Cd; changing Cl− for NO−3 increases to 80% the relative level of dissolved Cd present as Cd2+ compared to 14% in the chloride medium (Elisma and Jumarie, 2001). Fig. 2c shows that the total uptake of 109Cd was significantly higher under nitrate exposure conditions, whereas the non-specific uptake (measured in the presence of 100 µM unlabeled Cd) remained unmodified. Following correction for the nonspecific contribution to the total uptake data, a 1.8-fold increase in specific uptake was estimated, whereas [Cd2+] increased 5.7-fold in the nitrate medium. These results are consistent with the existence of a preferential specific uptake mechanism for Cd2+ species, although

Fig. 1. Cadmium-induced cytotoxicity in MG-63 cells. a) Cell viability dose-response curve as a function of increasing Cd concentrations in the culture medium following a 3- (○), 6- (Δ), 9- (□) or 24-h (●) exposure. b) Dose-response of cell viability as a function of increasing Mn concentration in the culture medium following a 24-h exposure. c) Phase-contrast photograph (100× magnification) of cells incubated without or with 20 µM Cd for 24 h. d) Cell viability following a 24-h exposure to 30 µM Cd and increasing concentrations of BSA in the medium. Data are expressed as mean ± SEM of viability ratio relative to the control evaluated on 3 to 5 independent cell cultures.

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concentration in the culture medium from 1 (normal concentration in the culture medium) to 0.1 mM shifted the dose-response curve of cell viability to the left, demonstrating an increased cellular sensitivity (two-way ANOVA, P b 0.02) to the cytotoxic effect of Cd with a reduction in the LC50 value from 22 ± 2 to 15 ± 2 µM (Fig. 3b). However, variations in Ca concentrations in the culture medium did not modify the long-term accumulation levels of 109Cd (Fig. 3c). Effects of voltage-dependent calcium channel modulators on the accumulation of cadmium Previous studies have shown the involvement of VDCCs in Cd uptake in kidney cells (Flanagan and Friedman, 1991), hepatocytes (Blazka and Shaikh, 1991) and pituitary cells (Hinkle et al., 1987). Since

Fig. 2. Accumulation of cadmium in MG-63 cells. a) Time-course of cellular accumulation of 0.5 µM 109Cd in the culture medium. b) 3-min uptake of 0.5 µM 109 Cd as a function of increasing concentrations of unlabeled Cd in the chloride medium. c) 3-min uptake of 0.5 µM 109Cd in the chloride or nitrate media in the absence (Total) or the presence (NS) of 100 µM unlabeled Cd. Data are mean ± SEM evaluated on 2 to 5 independent experiments. ⁎⁎Significant difference (P b 0.01) compared to the corresponding chloride condition.

accumulation levels and [Cd2+] were not directly correlated. Since we observed that Cd uptake in MG-63 cells occurs via specific uptake mechanisms and may lead to cell death, we further investigated the possible involvement of relevant ionic channels in Cd uptake in the osteoblast cells. Effects of calcium on the cellular accumulation and cytotoxicity of cadmium Cadmium has an ionic radius similar to that of Ca, leading to the concept of ionic mimicry; numerous studies have suggested that Cd may enter the cell via Ca transport systems (Hinkle et al., 1987; Blazka and Shaikh, 1991; Flanagan and Friedman, 1991). In order to investigate whether Cd/Ca interaction may occur for uptake in MG-63 cells, 109 Cd uptake measurements were conducted in the presence or absence of Ca in the incubation medium. As shown in Fig. 3a, the specific 3-min uptake of 109Cd (corrected for non-specific accumulation) was reduced in the presence of 2.5 mM Ca both in the chloride and nitrate media, suggesting that the cellular uptake of Cd partially involved Ca transport mechanisms. Consequently, a reduction in Ca

Fig. 3. Effect of calcium on the accumulation and cytotoxicity of cadmium. a) 3-min uptake of 0.5 µM 109Cd in the chloride or nitrate media containing 1.2 mM Mg without or with 2.5 mM Ca. Specific cellular accumulation of 109Cd was determined as described in Materials and methods and results were corrected for the non-specific uptake (kD × [109Cd]). ⁎⁎Significant difference (P b 0.01) compared to the corresponding control condition without Ca. b) Dose-response of cell viability as a function of increasing Cd concentrations in the culture medium containing 0.8 mM Mg with normal (1 mM) (●) or low (0.1 mM) (○) Ca levels. Data are mean ± SEM relative to control cell viability measured in the absence of Cd of at least 3 independent experiments. c) 24-h cellular accumulation of 0.5 µM 109Cd in serum-free DME/F12 culture medium containing standard (1 mM) or excess (5 mM) Ca levels with normal (0.8 mM) Mg levels. Data are mean ± SEM evaluated on 3 to 6 independent experiments.

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BayK8644, a VDCC agonist (Fig. 5b), did not reveal any differences in the sensitivity of the MG-63 cells to Cd. Therefore, our results indicate that VDCCs do not contribute significantly to the accumulation of Cd in MG-63 cells. Effects of canonical transient receptor potential (TRPC) channel modulators on cadmium accumulation To further characterize the cellular uptake of Cd in the MG-63 cells, we tested the possible involvement of non-selective Ca channels such as TRPCs. We have previously shown that MG-63 cells express numerous TRPCs (Labelle et al., 2007). Incubation of the MG-63 cells with thapsigargin and PDGF led to significant capacitative Ca entry (CCE) related to the activation of TRPC channels. We also observed that SKF96365 and 2-APB, two well-known inhibitors of TRPC channels, prevented the stimulation of CCE. As shown in Fig. 6a, the initial accumulation of 109Cd was not affected by either thapsigargin or SKF96365, added alone or in combination to the uptake medium. Moreover, SKF96365 did not modify cell sensitivity to Cd cytotoxicity (Fig. 6b). Similarly, the presence of 50 ng/mL PDGF-BB in the culture medium, which stimulates MG-63 cell proliferation and activates TRPC channels (Labelle et al., 2007), did not modify cell response to Cd (LC50 = 15.8 ± 3.8 vs 15.9 ± 5.9 µM without and with PDGF, respectively) (Fig. 6c). However, 2-APB lowered cell sensitivity to Cd (LC50 of 22.8 ± 3.2 vs 51.0 ± 11.0 µM in the absence or the presence of 100 µM 2-APB, respectively, two-way ANOVA P b 0.0001) (Fig. 7a). Consequently, 2APB decreased the 24-h cellular accumulation of 109Cd in a dosedependent manner with a significant effect observed for 50 µM and higher concentrations (Fig. 7b). Effect of magnesium on the accumulation of cadmium As 2-APB has been shown to also inhibit the magnesium (Mg) conductance of TRPM7 channels (Jiang et al., 2003; Li et al., 2006), we also investigated the effect of Mg on Cd uptake. As shown in Fig. 8a, a

Fig. 4. Involvement of VDCC in cellular accumulation of 109Cd and cadmium-induced cytotoxicity. a) 3-min uptake of 0.5 mM 45Ca in standard nitrate medium (Na) or in the presence of 100 mM potassium in the absence (K) or in the presence of 100 µM nifedipine (K + Nifedipine). Cellular accumulation data were corrected for the nonspecific uptake. ⁎Significant difference (P b 0.05) compared to the corresponding Na condition. b) 3-min uptake of 0.5 µM 109Cd in the Ca-free chloride or nitrate standard media (Na) containing 1.2 mM Mg or in the presence of 100 mM (K) potassium. Cellular accumulation data were corrected for the non-specific uptake. ⁎⁎Significant difference (P b 0.0005) compared to the corresponding chloride condition. c) Dose-response of cell viability determined by the MTT assay as a function of increasing Cd concentrations in normal (5 mM) (●) or high (50 mM) (○) potassium media. Data are expressed as mean ± SEM evaluated on 2 to 5 independent experiments.

Ca lowered the specific 3-min uptake of 109Cd in MG-63 cells (Fig. 3a), we investigated the possible implication of VDCCs in the accumulation of Cd in the MG-63 cells. We have previously reported that MG-63 cells express functional L-type VDCCs (Labelle et al., 2007). We now show that high potassium-induced membrane depolarization, which promotes activation of VDCC, led to a 2.2-fold increase in 45Ca uptake in these cells (Fig. 4a). This stimulation was prevented in the presence of 100 µM nifedipine, a VDCC blocker. However, depolarizing conditions did not significantly modify the initial 3-min uptake of 109 Cd in the Ca-free chloride or nitrate media (Fig. 4b). Furthermore, the presence of either 100 µM verapamil or 100 µM nifedipine did not modify 109Cd accumulation (data not shown). Consequently, MTT assays performed in the presence of high potassium concentrations (50 mM) in the culture medium (Fig. 4c), or verapamil (Fig. 5a) or

Fig. 5. Involvement of VDCC in cadmium-induced cytotoxicity. Dose-response of cell viability determined by the MTT assay as a function of increasing Cd concentrations in the culture medium in the absence or presence of different concentrations of Verapamil (a) or Bay K8644 (b). Data are expressed as mean ± SEM evaluated on 5 to 7 independent experiments.

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according to Eq. (3) gave an apparent Ki value of 0.7 ± 0.4 mM and a KD value of 35.7 ± 1.5 pmol 109Cd/3 min/µM/mg protein (Fig. 9a). Note that testing the effect of Mg on 109Cd uptake using only one tracer concentration (0.5 µM) does not allow competitive and non-competitive interactions to be discriminated. The latter was assumed simply to reduce the number of parameters in curve fitting and to allow a better precision in the apparent Ki (I50) estimation. Both Mg and Ca only partially inhibited the specific uptake of Cd without evidence for additive effect (Fig. 9b). Maximum inhibition (37%) was observed with 5 mM Mg and 5 mM Ca, but did not show consistent additivity compared to inhibition by Mg (about 23%) and Ca (32%) separately. Discussion The cellular mechanisms responsible for Cd-induced bone alterations are not yet completely understood, although a growing body of evidence indicates that Cd bone toxicity occurs by direct mechanisms on bone cells. Since cell susceptibility to the deleterious effects of Cd on osteoblast functions is first related to uptake capacity and cellular accumulation, we undertook the characterization of Cd uptake by osteoblastic cells. Since Cd is a non-essential metal, the existence of membrane systems specifically devoted to the cellular uptake of Cd is unlikely and it is generally believed that Cd may use the transport mechanisms for essential metals. The cellular accumulation and toxicity of Cd in osteoblast-like human cell line MG-63 have been studied under various conditions, namely standard culture conditions and high-potassium-induced depolarizing conditions, in addition to several channel modulators. Neither depolarization nor VDCC and TRPC modulators had any significant effect, but low levels of Ca and Mg, as well as 2-APB, which may inhibit TRPM7 channels, were found to exacerbate and minimize Cd uptake and cytotoxicity, respectively.

Fig. 6. Involvement of TRPC channels in the cellular accumulation of 109Cd and in cadmium-induced cytotoxicity. a) 3-min uptake of 0.5 µM 109Cd in the Ca-free chloride or nitrate media containing 1.2 mM Mg in the absence or in the presence of 0.1 µM thapsigargin (Tg) and 5 µM SKF96365 (SKF) added alone or in various combinations. Accumulation data are corrected for the non-specific uptake. b) Dose-response of cell viability determined by the MTT assay as a function of increasing Cd concentrations in the culture medium in the presence of various concentrations of SKF96365 or vehicle alone. c) Dose-response of cell viability determined by the MTT assay as a function of increasing Cd concentrations in the culture medium in the absence (Control) or the presence of 50 ng/mL PDGF-BB. Inset: stimulation of cell proliferation by 50 ng/mL PDGF-BB determined by the MTT assay. ⁎Significant difference (P b 0.05) compared to the condition without PDGF. Data are expressed as mean ± SEM evaluated on 3 to 4 independent experiments.

30% decrease was observed in the initial 3-min specific uptake of 0.5 µM 109Cd in the presence of 2.5 mM Mg in the nitrate medium, suggesting Cd/Mg interactions for uptake processes. As a result, a significant increase in cellular sensitivity to the cytotoxic effect of Cd was observed in the absence of Mg (Fig. 8b) with a reduction in the LC50 value from 21.4 ± 2.4 to 6.3 ± 0.8 µM (two-way ANOVA P b 0.0001). Contrary to what was observed with Ca, the presence of 2.5 mM Mg in the culture medium reduced the cellular accumulation of 109Cd over a 24-h exposure (Fig. 8c). Note that 2-APB did not modify 109Cd uptake in the presence of Mg, but significantly reduced uptake levels in the absence of Mg (Fig. 8d), clearly showing that 2-APB inhibits the Mgsensitive component of Cd uptake. In order to further characterize the Cd/Mg interaction, the 3-min uptake of 0.5 µM 109Cd was measured in the presence of increasing concentrations of Mg (Fig. 9a). Data analyses

Fig. 7. Effect of the 2-APB as an inhibitor of both TRPC and TRPM7 on the accumulation and cytotoxicity of cadmium. a) Dose-response of cell viability determined by the MTT assay as a function of increasing Cd concentrations in the culture medium in the presence of various concentrations of 2-APB or vehicle alone. Data are expressed as mean ± SEM evaluated on 3 to 5 independent experiments. Significant difference (twoway ANOVA, P b 0.0001) between conditions without and with 100 µM 2-APB. b) 24-h cellular accumulation of 0.5 µM 109Cd in serum-free DMEM/F12 culture medium in the presence of various concentrations of 2-APB. ⁎⁎⁎Significant difference (Bonferoni, P b 0.001) compared to the corresponding condition without 2-APB.

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Fig. 8. Effect of magnesium and of 2-APB as an inhibitor of TRPM7 on the accumulation and cytotoxicity of cadmium. a) 3-min uptake of 0.5 µM 109Cd in the nitrate medium containing 2.5 mM Ca in the absence or the presence of 2.5 mM Mg. Data are corrected for the non-specific contribution. ⁎Significant difference (P b 0.05) compared to condition without Mg. b) Doseresponse of cell viability determined by the MTT assay as a function of increasing Cd concentration in serum-free DME/F12 culture medium containing 1 mM Ca in the absence (○) or in the presence of 0.8 mM Mg (●). c) 24-h cellular accumulation of 0.5 µM 109Cd in serum-free DME/F12 culture medium containing 1 mM Ca in the absence or the presence of 2.5 mM Mg. ⁎Significant difference (P b 0.05) compared to condition without Mg. d) 3-min uptake of 0.5 µM 109Cd in the nitrate medium containing 2.5 mM Ca in the absence or presence of 2.5 mM Mg and various concentrations of 2-APB or vehicle alone. Data are corrected for the non-specific contribution to total uptake and are expressed as mean± SEM evaluated on 3 independent experiments. δSignificant difference (P b 0.05) compared to the respective control condition without 2-APB; ⁎Significant difference (P b 0.05) compared to condition without Mg.

Time- and dose-dependent cytotoxicity effect of Cd on MG-63 cells It has long been thought that the impact of Cd on bone was related to the disruption of vitamin D metabolism (Jarup et al., 1998). Only recently, it has been established that this metal exerts deleterious direct effects on bone cells (Dohi et al., 1993; Wilson et al., 1996; Long, 1997a,b). Here we show that Cd may affect the viability of the human osteoblast-like MG-63 cells in a time- and dose-dependent manner with a typical sigmoid doseresponse curve following a 24-h exposure. Under these in vitro conditions, an LC50 value of 18 µM has been estimated (Fig. 1a), and cell swelling and detachment were observed (Fig.1c). This value is comparable to what has been reported in pituitary GH4C1 (Hinkle et al., 1987), enterocytic-like Caco-2 (Huynh-Delerme et al., 2005) and hepatoma-derived HepG2 (Dehn et al., 2004) cells. Moreover, the addition of BSA to the culture medium significantly reduced Cd toxicity (Fig. 1d). BSA binds Cd (DelRaso et al., 2003; Pham et al., 2004), thus reducing its availability for uptake and consequently the levels of cellular accumulation. Involvement of specific mechanisms of high affinity in Cd uptake in MG-63 cells

Fig. 9. Characterization of Mg-sensitive component of Cd uptake. a) 3-min uptake of 0.5 µM 109 Cd as a function of increasing concentrations of Mg (0 to 20 mM) in the Ca-free nitrate medium. b) 3-min uptake of 109Cd in the presence or absence of 5 mM Mg, 5 mM Ca or both in the nitrate medium. Significant difference (P b 0.001) versus control without Ca and Mg. Data are expressed as mean± SEM evaluated on at least 3 independent experiments.

Mechanisms of Cd uptake have been studied in many cell types (Hinkle et al., 1987; Jumarie et al., 1997; Pham et al., 2004; Bergeron and Jumarie, 2006). Fig. 2a shows that MG-63 cells greatly accumulate Cd (up to 500 pmol/mg protein following a 24-h exposure in the culture medium), clearly establishing that toxicity is related to cellular accumulation. Furthermore, a specific system of high affinity (Km about 13 µM) has been characterized (Fig. 2b). Similar Km values ranging from 3.5 to 19.3 µM have been reported in human intestinal crypt cells HIEC (Bergeron and Jumarie, 2006), primary cultures of rat hepatocytes (Pham et al., 2004), human enterocytic-like Caco-2 cells (Jumarie et al., 1997), rat alveolar type II cells, and human lung carcinoma A549 cells

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(Jumarie, 2002). To get insights into metal species preferentially taken up, chloride was changed for nitrate, which does not bind as much Cd to increase the level of total dissolved metal recovered as aquo ion Cd2+ (Jumarie et al., 2001). As expected, the non-specific uptake remained similar regardless of the medium used, whereas the specific uptake (i.e., total corrected for the non-specific) was greatly enhanced under nitrate exposure conditions, indicating a more efficient uptake of the Cd2+ species. This preferential uptake of Cd2+ has been reported in enterocytic-like Caco-2 cells (Elisma and Jumarie, 2001). Therefore, the transport mechanisms for Cd probably involve cationic channels, at least in part. Note that although our kinetic data failed to reveal any heterogeneity, more than one inorganic Cd species may be taken up. This is further supported by the absence of direct correlation between the increase in 109Cd uptake in the nitrate medium and the increase in [Cd2+]. Evidence of Ca/Cd interactions for voltage-independent uptake mechanisms Although Cd permeability through Ca channels remains unquantified, a number of studies have suggested Cd/Ca interactions for uptake mechanisms in various cell types (Przelecka and Mrozinska, 2002; Raynal et al., 2005; Gagnon et al., 2007). We found the specific uptake of 0.5 µM Cd to be partially inhibited in the presence of 2.5 mM Ca (Fig. 3a). Contrary to what may be expected, a similar level of inhibition was obtained in the chloride and nitrate media (note that nitrate conditions dramatically increase [Cd2+] but do not significantly change Ca speciation, which remains mainly as Ca2+ over 80% of the total Ca). These results are consistent with the lack of direct correlation between [Cd2+] and the uptake levels previously observed (Fig. 2c) but may also suggest a much higher affinity for the Cd2+ species, whose concentrations would far exceed the Km value (in the range of nM), thus leading to saturation conditions. Although Cd uptake was only partially inhibited by Ca, an increase in Cd cytotoxicity was clearly observed when the Ca level in the incubation medium was lowered to 0.1 mM (Fig. 3b). Our results clearly show the presence of a Cd/Ca interaction for uptake processes in MG-63 cells. Various Ca channels have been shown to be expressed in osteoblastic cells and they could be responsible for the uptake of Cd in MG-63 cells. A widely expressed class of Ca channels is the voltagedependent channel family or VDCCs. These channels are sensitive to membrane depolarization, selective to Ca2+ (Catterall, 2000) and are expressed in osteoblastic cells (Barry et al., 1995; Barry, 2000). Furthermore, their involvement in Cd uptake has been demonstrated in other cell types (Hinkle et al.,1987,1992; Endo et al., 2002). However, our results failed to reveal any effect of membrane depolarization on Cd uptake (Fig. 4b) or toxicity (Fig. 4c) in the MG-63 cells, although Ca uptake was clearly stimulated under membrane depolarization conditions (Fig. 4a). The lack of VDCC involvement in Cd uptake was further supported by the lack of effect of either verapamil (a VDCC inhibitor) or Bay K8644 (a VDCC activator) in MG-63 cell sensitivity to Cd (Fig. 5). These results argue against the involvement of VDCCs in Cd uptake in osteoblastic cells. Further studies are needed to identify the transport mechanism that may be subjected to Cd/Ca interactions. TRPC channels are not likely to be involved in the uptake of Cd in MG-63 cells

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of sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA) pumps, leading to the release of Ca and the subsequent activation of CCE in the MG-63 cells (Labelle et al., 2007). The lack of a thapsigargin effect on Cd uptake levels suggests that TRPCs are not mainly involved in Cd uptake processes in the MG-63 cells. Furthermore, SKF96365, a TRPC inhibitor, remained without effect regardless of the medium used, and did not modify Cd cytotoxicity (Figs. 6a,b). Also, PDGF, a well-known TRPC activator (Labelle et al., 2007) did not affect Cd cytotoxicity (Fig. 6c). Possible involvement of TRPM channels in the Mg-sensitive uptake component of Cd Magnesium deficiency is not uncommon in the general population: its intake has decreased in the past years especially in the Western world. There is increasing consideration about the physiological importance of Mg2+ ions in clinical medicine, nutrition and physiology. A negative correlation has been shown between Mg status and illnesses such as alcoholism, eclampsia, hypertension, atherosclerosis, cardiac diseases, diabetes, and asthma (Laires et al., 2004). Magnesium accounts for up to 0.5–1% of bone ash and is therefore not a trace element in the skeleton. Interestingly, Mg has also been reported to be a competitive inhibitor of Cd uptake (Guiet-Bara et al., 1990). Although numerous TRP channels mainly transport the Ca2+ cation, it has been demonstrated that some TRPs, including TRPM6 and TRPM7 (Schlingmann et al., 2007), have a high affinity for the Mg2+ ion. To verify whether TRPMs are involved in Cd accumulation (and then cytotoxicity) in osteoblast-like cells, uptake experiments were performed with 2-APB, a TRPC and TRPM inhibitor. Both the initial 3-min uptake in the Mg-free nitrate medium and the 24-h accumulation levels in the culture medium (Figs. 8d and 7b) were significantly inhibited. In accordance, the treatments of cells with 2-APB resulted in an increase of LC50 values (Fig. 7a). Since TRPCs were found not to be involved, these results suggest that Cd may be transported by TRPM channels. This is further supported by the observation that both the uptake and cytotoxicity of Cd are highly sensitive to the presence of Mg (Fig. 8). Note that Mg depletion increased the uptake of Cd in renal epithelial MDCK cells, suggesting a protective role of Mg against Cd toxicity (Quamme, 1992). Also, Mg supplementation has been shown to reduce tissue accumulation of Cd (Boujelben et al., 2006; Djukic-Cosic et al., 2006). Mg deprivation enhances and Mg supplementation diminishes the embryotoxic and teratogenic effects of Cd, and competition between Cd and Mg for a carrier mechanism has been proposed (Luo et al., 1993). The apparent Ki of around 0.7 mM (Fig. 9a) is similar to the value reported for TRPM7 affinity for Mg (Harteneck, 2005; Li et al., 2006). The high KD value obtained clearly shows the existence of Mginsensitive uptake of Cd. This Mg-insensitive component of Cd uptake may be inhibitable by other cations. However, the absence of additive effects between Ca and Mg on Cd uptake suggests that the Mgsensitive and Ca-sensitive components of Cd uptake are related to the same transport mechanism. Indeed, contrary to other Mg-specific transporters such as the newly identified MagT channels (Goytain and Quamme, 2005), TRPM7 channels are known to be permeable to both ions (Harteneck, 2005; Li et al., 2006). Cadmium exposure and bone metabolism

Other channels known to transport Ca are the “transient receptor potential” or TRP channels. First identified in Drosophila melanogaster, their mammalian equivalents have been identified and are now intensively studied (Pedersen et al., 2005). The TRP channel family includes 3 main classes: canonical (TRPC), melastatin-related (TRPM) and vanilloids (TRPV), with the best characterized being the TRPCs. Numerous TRPCs are associated with capacitative Ca entry (CCE) (Pedersen et al., 2005), which is induced by the release of Ca from intracellular stores, mainly from the endoplasmic reticulum (ER). To determine whether Cd can enter the cells through TRPCs, transport measurements were done in the presence of thapsigargin, an inhibitor

Exposure to Cd has been associated with disruption of the equilibrium between bone resorption and formation, and with the subsequent loss in bone mass as well as the development of osteoporosis. This could be the result of an imbalance between the apoptotic and proliferative processes, which determines the size of osteoclast or osteoblast populations dedicated to bone resorption or formation (Xing and Boyce, 2005). Our results show that exposure to Cd reduces osteoblast viability, which may lead to abnormal remodeling patterns with a deficit in bone formation, resulting in net bone loss as observed in osteoporosis. Moreover, we observed that

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Cd uptake and cytotoxicity are enhanced in osteoblastic cells under low Ca and Mg exposure conditions in vitro. It is known that low levels of Ca intake are positively correlated with low bone density, and that high levels of Ca intake are beneficial for preventing excessive bone loss (Cumming, 1990). Also, epidemiological studies provide links between insufficient dietary Mg intake in humans and low bone mass and osteoporosis (for a review, Rude and Gruber, 2004). Therefore, in addition to their direct involvement in bone metabolism, Ca and Mg deficiencies may also contribute to an increase in the deleterious effects of Cd on these cells. Some studies have demonstrated severe adverse effects of Cd on bone tissue in environmentally exposed populations, with skeletal alterations at significantly lower levels than previously anticipated (Staessen et al., 1999; Alfven et al., 2000; Jarup and Alfven, 2004). Also, in vitro exposure levels required to exert biochemical effects are most often lower that the LC50 values estimated for cell viability. In accordance, exposures to non-cytotoxic concentrations of Cd have been shown to decrease collagen synthesis and to alter intracellular Ca2+ levels in the rat osteoblast-like ROS 17/2.8 cells (Long, 1997a,b), also to reduce alkaline phosphatase activity in the mouse osteoblastlike MC3T3-E1 cells (Iwami and Moriyama, 1993) as well as bone nodulation in primary rat calvarial osteoblast cultures (Kaneki et al., 2000). Here we show for the first time significant levels of Cd accumulation in osteoblastic cells exposed to the non-cytotoxic concentration of 0.5 µM Cd (56 µg/L) used for 109Cd uptake experiments, which nearly compares with Cd blood levels (1.5 µg/L) measured in smokers (Jarup et al., 1998). At this concentration, we have characterized the kinetic properties of Cd uptake and revealed the inhibitory effects of Ca and Mg. These data give insights into the effects of Cd on osteoblast functions. In conclusion, our results show that even though a Ca carrier is involved in Cd uptake in osteoblasts, VDCC and TRPC channels are unlikely candidates since specific antagonists and/or activators failed to modify Cd uptake. However, the inhibitory effect of Mg or 2-APB suggests that some TRPM channels might be of importance. A thorough investigation is needed to determine exactly which TRPM channels are responsible for the cellular uptake of Cd in osteoblastic cells. Acknowledgments This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds Québécois de la Recherche sur la Nature et les Technologies. References Alfven, T., Elinder, C.G., Carlsson, M.D., Grubb, A., Hellstrom, L., Persson, B., Pettersson, C., Spang, G., Schutz, A., Jarup, L., 2000. Low-level cadmium exposure and osteoporosis. J. Bone Miner. Res. 15, 1579–1586. Angle, C.R., Thomas, D.J., Swanson, S.A., 1993. Osteotoxicity of cadmium and lead in HOS TE 85 and ROS 17/2.8 cells: relation to metallothionein induction and mitochondrial binding. Biometals 6, 179–184. Bannon, D.I., Abounader, R., Lees, P.S., Bressler, J.P., 2003. Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells. Am. J. Physiol., Cell Physiol. 284, C44–C50. Barry, E.L., 2000. Expression of mRNAs for the alpha 1 subunit of voltage-gated calcium channels in human osteoblast-like cell lines and in normal human osteoblasts. Calcif. Tissue Int. 66, 145–150. Barry, E.L., Gesek, F.A., Froehner, S.C., Friedman, P.A., 1995. Multiple calcium channel transcripts in rat osteosarcoma cells: selective activation of alpha 1D isoform by parathyroid hormone. Proc. Natl. Acad. Sci. U. S. A. 92, 10914–10918. Bergeron, P.M., Jumarie, C., 2006. Characterization of cadmium uptake in human intestinal crypt cells HIEC in relation to inorganic metal speciation. Toxicology 219, 156–166. Blazka, M.E., Shaikh, Z.A., 1991. Differences in cadmium and mercury uptakes by hepatocytes: role of calcium channels. Toxicol. Appl. Pharmacol. 110, 355–363. Boujelben, M., Ghorbel, F., Vincent, C., Makni-Ayadi, F., Guermazi, F., Croute, F., El Feki, A., 2006. Lipid peroxidation and HSP72/73 expression in rat following cadmium chloride administration: interactions of magnesium supplementation. Exp. Toxicol. Pathol. 57, 437–443. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

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