Copper overload affects copper and iron metabolism in Hep-G2 cells

M. Arredondo,1 V. Cambiazo,2 L. Tapia,2 M. González-Agüero,2 M. T. Núñez,3 R. Uauy,1 and M. González2

1Microminerals Laboratory and 2Bioinformatic and Gene Expression Laboratory, Institute of Nutrition and Food Technology and 3Faculty of Science, Department of Biology, University of Chile, Casillo 138-11 Santiago, Chile

Submitted 16 July 2003 ; accepted in final form 20 February 2004


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Divalent metal transporter #1 (DMT1) is responsible for intestinal nonheme Fe apical uptake. However, DMT1 appears to have an additional function in Cu transport in intestinal cells. Because the liver has an essential role in body Cu homeostasis, we examined the potential involvement of Cu in the regulation of DMT1 expression and activity in Hep-G2 cells. Cells exposed to 10 µM Cu exhibited a 22-fold increase in Cu content and a twofold decrease in Fe content compared with cells maintained in 0.4 µM Cu. 64Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in Km compared with cells grown in 10 µM Cu. The decreased Km may represent an adaptive response to Cu deficiency. Cells treated with >50 µM Cu, showed an eightfold increase in cytosolic metallothionein. DMT1 protein decreased (35%), suggesting that intracellular Cu caused a reduction of DMT1 protein levels. Our data indicate that, as a result of Cu overload, Hep-G2 cells reduced their Fe content and their DMT1 protein levels. These findings strongly suggest a relationship between Cu and Fe homeostasis in Hep-G2 cells in which Cu accumulation downregulates DMT1 activity.

uptake; iron; copper; divalent metal transporter #1; metallothionein


THE HEPATOCYTE PLAYS a central role in copper (Cu) excretion and in the control of systemic Cu homeostasis. To maintain a physiological level of plasmatic Cu (10–20 µM; see Refs. 33 and 58), hepatic tissue removes Cu from the circulation by rapidly trapping the metal in chelating Cu proteins, whereas Cu excess is excreted in bile (12, 22, 45). At the cellular level, Cu uptake mechanism involves the high-affinity Cu transporters 1 and 2 (CTR1/2; see Ref. 61). In the cytoplasm, free Cu rarely exists, because it can readily generate free radicals or oxidize cellular components through its redox activity (4, 17). Therefore, Cu is immediately transferred to glutathione (GSH) and metallothionein (MT); the latter is a cytosolic protein that binds up to 12 atoms of Cu and increase its cellular level in response to Cu exposure (5, 26, 38, 41, 44). GSH and MT, along with Cu chaperones, provide efficient and specific mechanisms for safe intracellular storage and transport of this metal (25, 35). During these processes, it is critical to evaluate the effect of extracellular Cu fluctuations on the rate of Cu transport and on the level of cellular accumulation of Cu and other trace metals.

The metabolic links between Cu, Zn, and Fe have been described previously (for a review, see Refs. 15 and 23). Examples of their interplay are found in processes such as Fe uptake (11), ceruloplasmin ferroxidase activity (23), MT induction (6, 16, 44, 48), and Cu/Zn-superoxide dismutase activity (24, 46). Recently, a new link between Cu and Fe metabolism was established by the finding that divalent metal transporter #1 (DMT1), the principal, if not the only, transporter of ferrous Fe from the intestinal lumen in the enterocyte (32), is also a Cu1+ transporter in intestinal cells (2). Because different tissues express DMT1 (20, 27), it was of interest to know whether the interrelation between Cu and DMT1 holds for other tissues as well. In this work, we tested the hypothesis that DMT1 participates actively in Cu metabolism in hepatic cells. We used the human hepatoblastoma Hep-G2 cell line to evaluate cellular adaptation to sub-, iso-, and supraphysiological Cu exposure. We observed that extracellular Cu fluctuations affected the cellular levels of Cu and MT. Cu loading decreased Fe and Zn cell levels and diminished the expression of the protein DMT1. The findings reported here shed new light on the Cu-Fe relationship and help to understand the basic mechanisms of hepatic Cu absorption.


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Reagents.

All buffer solutions were filtered through Chelex 100 (Sigma, St. Louis, MO) to minimize the levels of contaminating heavy metals. 64CuSO4 was prepared by Comisión Chilena de Energía Nuclear (Santiago, Chile) and was used at the setting point (1–2 h after preparation). Monoclonal antibody to MT I/II (E9) was obtained from DAKO (Carpinteria, CA). Anti-mouse or anti-rabbit IgG conjugated to fluorescein was purchased from Sigma. Peroxidase-conjugated antibodies were obtained from Amersham Life Science (Buckinhamshire, UK).

Cell culture and Cu treatments.

Hep-G2 cells, a model of hepatic cells (9, 51, 52), were cultured as described previously (19). Briefly, cells were incubated at 37°C in a 5% CO2 atmosphere and grown in plastic cell culture flasks (Nalge Nunc) containing DMEM and 10% (vol/vol) FBS. The medium was changed every 3 days. Concentrations of elements in the standard culture medium were 0.44 Cu, 2.69 Fe, and 3.80 µM Zn (19). Cu was considered to be at a subphysiological concentration. For Cu treatments, Cu was supplemented to the culture medium, as Cu-His complex (1:10 ratio; see Ref. 19). Cu supplements of 10 or >50 µM were used to reach iso- or supraphysiological Cu concentrations, respectively. For 64Cu uptake, cells were plated in sixwell plates at 0.6 x 106 cells/well. After Cu treatments, relative survival of cells was evaluated by using trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays (10). The cells were plated in 24-well plates at 0.1 x 106 cells/well and treated 24 h after plating. Hep-G2 cells showed >90% viability after Cu treatment. For all of the experiments, variables were tested in triplicate samples, and the analyses of each sample were repeated at least two times.

Uptake studies.

Uptake experiments were carried out as reported (3, 56, 57), except that Hep-G2 cells were preexposed for 48 h to 10 or 100 µM Cu as a Cu-His complex or maintained without supplement of the metal. Briefly, the cells were washed two times with PBS, pH 7.0, and 50 µM CaCl2 (buffer A) and then incubated for different periods of time (0–60 min) with buffer A containing different 64Cu concentrations. After incubation, uptake was stopped by washing the cells with 4 x 2 ml PBS and 1 mM EDTA at 4°C. 64Cu uptake was linear during the first 60 min of incubation, and the apparent kinetic parameters were calculated from uptake values obtained at 30 min.

Quantification of cellular Cu, Fe, and Zn.

Cells were processed as described (3, 10, 19). Briefly, for cell-associated metal content determination, 1–2 x 106 Hep-G2 cells were washed two times with PBS and harvested by tripsinization. Cells were sedimented by centrifugation at 3,500 rpm in a Beckman GS-15R centrifuge for 5 min, resuspended in 1 ml PBS, and repelleted. The pellet was disrupted by incubation with 0.2 ml buffer A containing 0.1 N NaOH and 0.1% Triton X-100 to yield a homogenate (H1) fraction. Aliquots of H1 were kept for protein determination by Bradford assay, and the remaining fraction was mixed (1:1, vol/vol) with nitric acid (Merck Suprapure) and digested at room temperature for 48 h. Total-reflection X-ray fluorescence spectrometry (TXRF) analysis was performed by using a Seifert EXTRA-II spectrometer (Rich. Seifert, Ahrensburg, Germany). For each case, one-fifth of the digested sample volumes was standardized with 1 µg/ml of Se (ICP selenium standard solution; Merck, Darmstadt, Germany; see Ref. 19). The concentration was obtained using the following equation:

where C is the concentration of a given element, A is the integrated peak area in the spectrum, f is the relative sensitivity, the index x denotes each element analyzed, and Se is the element chosen as internal standard. The TXRF permits measurement of the simultaneous cellular concentration of different elements using a minimal amount of sample (19, 41).

Preparation of MT-enriched fraction and Western blot analysis.

Cell extract for MT analysis was prepared as previously described (50). Briefly, pellets of Hep-G2 cells (5 x 106) were homogenized by mechanical breakage at 4°C in 200 µl extraction buffer (150 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, and 10 mM Tris·HCl, pH 7.5). The homogenate was centrifuged at 12,000 g for 15 min, and the supernatant was boiled (5 min) and centrifuged again. The supernatant was lyophilized and resuspended in deionized distilled water, and the proteins were carboxymethylated by adding DTT and fresh-made 1 M iodoacetamid. SDS-PAGE was performed according to Laemmli (30) with the modifications described (37). Western blot assays were performed as described (7) using the monoclonal antibody anti-MT I/II (1:100; see Ref. 56). Densitometric analysis was performed using the Quantity One software (Bio-Rad) and was expressed as arbitrary units.

Immunofluorescence analysis.

Treated and control cells grown on coverslips were fixed in methanol at –20°C for 10 min and processed as described (18). Briefly, cells were incubated for 1 h in blocking solution (1% BSA in PBS) and 2 h with a monoclonal antibody against MT I/II (1:200). Fluorescein-conjugated rabbit IgG against mouse IgG previously centrifuged at 126,000 g in a Beckman Airfuge was used as secondary antibodies. Cells were rinsed in PBS, mounted in DABCO/Mowiol, and examined with a confocal microscope (x63 objectives; Carl Zeiss Germany).

DMT1 immunodetections.

The polyclonal monospecific antibody anti-iron-responsive element (IRE)-containing isoform of DMT1 (DMT1-IRE) was raised in rabbits against the peptide SKGLLTEEATRGYVK, which corresponds to the COOH-terminal segment of the IRE form of human DMT1 (1). DMT1 was detected by Western blot analysis in a Hep-G2 cellular extract, using purified anti-DMT1 antibody diluted to 0.5 µg/ml and a peroxidase-based secondary antibody (Pierce, Rockford, IL). The intensity of the individual bands was determined using Quantity One software (Bio-Rad). The antibody recognized a protein band of ~66-kDa apparent mass that was not evident when either the primary or the secondary antibody was omitted from the procedure. As a gel loading control, the membranes were reprobed with a monoclonal anti-actin antibody (Sigma).

Statistical analysis.

Variables were tested in triplicates, and the experiments were repeated at least two times. Variability among experiments was <20%. One-way ANOVA was used to test differences in means, and the Bonferroni post hoc t-test was used for comparisons (GraphPad InStat software). Differences were considered significant at P < 0.05.


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Cu concentration in cell media.

Cu concentration in the plasma is 10–20 µM, but only 10–30% of this amount, which is bound to amino acids and albumin, is actually available to the cells (37, 47). In our standard culture medium, the amount of Cu (0.4 µM) was considered a subphysiological concentration of the metal with respect to the plasma. To mimic iso- and supraphysiological extracellular Cu concentrations, the medium was supplemented with 10 and >50 µM Cu-His, respectively.

Cellular metal content.

When Hep-G2 cells were treated with a cultured media supplemented with 10 µM Cu-His for 48 h, the cellular Cu content rose 22-fold from 1.30 ± 0.5 to 28.8 ± 4.1 nmol Cu/mg protein (P < 0.01). Cellular exposure to 100 µM Cu-His produced only a minor further increment (1.3-fold) relative to that measured at 10 µM Cu-His. Thus, when extracellular Cu increased from 0.4 to 100 µM, the cellular Cu content increased 28-fold (P < 0.001; Fig. 1). The viability of the cells was unaffected by this Cu treatment, as determined by trypan blue and MTT assay (data not shown). These results indicate that, when Cu exposure is >10 µM, uptake/efflux adaptive processes and not storage protects the cells from the cellular Cu excess (25, 42). We further investigated whether increments in the cellular Cu affect the content of other trace metals. Cellular Fe and Zn decreased from 4.9 ± 0.8 to 2.3 ± 0.2 nmol Fe/mg protein (2.1-fold; P < 0.01) and 14.9 ± 0.1 to 9.2 ± 0.2 nmol Zn/mg protein (1.6-fold; P < 0.01) when the extracellular Cu increased from 0.4 to 100, respectively (Fig. 1, inset). Thus Cu exposure affected primarily the cellular Cu content, but, to a lesser degree, it also affected the Fe and Zn content, providing further evidence of their metabolic interaction.



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Fig. 1. Total content of Cu, Zn, and Fe in Hep-G2 cells exposed to different extracellular Cu concentrations. Cu ({blacksquare}), Fe, and Zn contents were determined by total-reflection X-ray fluorescence spectrometry. Values correspond to the averages of 3 different samples. Inset shows cellular levels of Fe and Zn.

 
Intracellular MT content.

Intracellular MT, a protein that stores and sequesters excess Cu, is part of a mechanism that controls the intracellular Cu content (41, 51). Here, we show that MT is localized both in cytoplasm and nucleus, as it has been described by others (8). MT exhibited a notorious change in its relative abundance and subcellular distribution after 48 h of exposure to 100 µM Cu. This effect was not observed when cells were exposed to physiological Cu concentration, up to 10 µM (data no shown). A representative view of the immunostaining pattern of untreated cells showed the presence of MT in both nucleus and cytoplasm of Hep-G2 cells (Fig. 2A1). Interestingly, Hep-G2 cells exposed to an excess of Cu displayed an enhanced emission of fluorescence in the cytoplasm (Fig. 2A2). In addition, a densitometric analysis of the MT protein abundance showed an 8.2-fold increase in these cells compared with untreated cells (Fig. 2B, lanes 1 and 2, respectively). These results suggest that the increment of immunoreactive protein signal reflects a change in the relative abundance of MT in cells exposed to Cu excess. When cells were exposed to 10 µM Cu, no detectable changes on MT distribution or content were observed (data not shown).



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Fig. 2. Metallothionein distributions in Hep-G2 cells exposed to Cu. A: cells were stained with anti-MT I/II antibody (dilution 1:200). Cells were seeded over coverslips and maintained without Cu supplement (1) or with 100 µM Cu-His (2) for 48 h. Representative images are shown. B: pellets of untreated cells (lane 1) and cells treated with Cu-His (lane 2) were analyzed by Western blot with an anti-MT I/II antibody (dilution 1:100).

 
64Cu uptake studies.

The control of metal uptake constitutes the first step in the regulation of intracellular Cu. We determined the kinetic parameters of 64Cu uptake by Hep-G2 cells after 48 h of exposure to 0.4, 10, or 100 µM Cu (Fig. 3). In agreement with previous reports (3, 57), our results indicate that the 64Cu uptake rate of Hep-G2 cells is a saturable process, and the level of uptake is within previously reported ranges for liver cells (49, 52). A double-reciprocal analysis of uptake data (Fig. 3, inset) showed that cells grown in the absence of Cu-His supplement had a apparent Km value (0.70 ± 0.20 µM) considerably lower than cells pretreated with 10 or 100 µM Cu-His (1.48 ± 0.2 and 1.33 ± 0.23 µM, respectively) for 48 h (P < 0.02). The Vmax values were 6.85 ± 0.57, 7.11 ± 0.30, and 5.33 ± 0.29 pmol 64Cu·min–1·mg protein–1 for cells exposed to sub-, iso-, and supraphysiological Cu concentrations, respectively. These results suggest that Cu exposure affects its uptake kinetics. The changes in the cellular Cu content observed at different Cu exposure conditions appear to affect the uptake process, whereas the velocity of the event remains unchanged.



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Fig. 3. 64Cu uptake experiments in Hep-G2 cells exposed to Cu. Cells preexposed for 48 h to 0.4 ({blacksquare}), 10 ({bullet}), or 100 ({blacktriangledown}) µM Cu were incubated for 30 min with different concentrations of 64Cu-His. Mean value of 64Cu uptake (n = 3) from a representative experiment is shown. Inset shows the kinetic parameters obtained in cells grown at 0.4, 10, and 100 µM.

 
DMT1 protein content in Hep-G2 cells.

The results presented above on 64Cu uptake suggest a decrease in the number of Cu transporters along with a more efficient incorporation of the metal. Therefore, we assessed whether the differences in Cu transport observed in Hep-G2 cells exposed to Cu (0.4–50 µM) were a consequence of changes in the content of DMT1. The relative abundance of DMT1 was determined by Western blot analysis, using a polyclonal monospecific antibody raised against a carboxyl-terminal peptide of the protein. The results indicated that the abundance of DMT1 was higher at a low level of extracellular Cu concentration and decreased when extracellular Cu was augmented (Fig. 4A). Densitometric analysis showed that DMT1 decreased by 35% between cells incubated with 0.4 and 50 µM Cu (P < 0.01) for 48 h (Fig. 4B). This result suggests that during the adaptation process to Cu exposure, Hep-G2 cells modify the relative contribution of Cu transporters involved in Cu uptake.



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Fig. 4. DMT1 protein level in Hep-G2 cells exposed to Cu. A: Western blots of DMT1. Hep-G2 cells were grown in 24-mm plates for 4 days and then incubated with Cu-His (range 0.4–50 µM) for 2 days. Cellular extracts (30 µg protein) were subjected to Western immunodetection of DMT1 using a rabbit anti-DMT1, IRE isoform. As a gel loading control, the membranes were reprobed with anti-actin antibody. B: results of densitometric analysis of 3 different DMT1 immunodetections.

 

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In this report, we have used Hep-G2 cells to examine the process of uptake and storage of Cu during cellular adaptation (3, 34) to sub-, iso-, and supraphysiological concentrations of extracellular Cu. Our results indicate that increases in the cellular Cu concentration are not directly proportional to extracellular Cu-His exposition. Although the addition of 10 µmol/l Cu-His resulted in an apparently significant increase in cellular Cu, further increases in Cu-His in the media induced MT expression, as expected (26, 39, 50), but did not result in additional increases in Cu content. Considering that in our experimental condition cells reached a plateau at 5–10 µM both in 64Cu uptake and Cu content, we propose that the capacity of Hep-G2 to accumulate Cu reaches a plateau near the physiological concentration of the metal. At higher Cu concentration, the hepatocytes contain unique Cu-binding proteins (i.e., MT) that are more effective at binding and accumulate Cu. The actions of these proteins along with the process of Cu efflux protect cells from intracellular Cu excess (34, 38, 43).

At supraphysiological Cu exposure, total Fe content decreased twofold with respect to the control cells, indicating that this metal was affected in the opposite direction (Fig. 1, inset). Because DMT1 is able to transport both metals (2), the decreasing Fe content may be the consequence of a competition with Cu for DMT1-dependent Fe uptake. This possibility is consistent with previous observations that DMT1 is involved in Fe and Cu1+ transporter (2, 20) and that Fe content decreases in cells exposed to Cu excess (21, 54, 55). Likewise, we found that Cu uptake was inversely related to cellular Fe content, in agreement with recent reports describing increased Cu uptake in desferrioxamine-treated cells (2, 60). Interestingly, several reports indicated that DMT1 could be upregulated by Fe deficiency (20, 53, 59), through an IRE-dependent mechanism (14). Thus an increment of Cu content in Caco-2 cells was associated with lower cellular Fe levels and with upregulation of DMT1 expression (2, 21); both responses are involved in the homeostatic regulation that increases Fe uptake. Therefore, an intimate relationship exists between intestinal Cu and Fe homeostasis. Although the molecular mechanisms that connect Cu and Fe uptake are unknown at present, our data are in line with the idea that DMT1 is a common link. In fact, the 55Fe uptake/transport uptake increased after 8 days of Cu exposure in Caco-2 cells (21). Here, we show that, as a consequence of Cu exposure, Hep-G2 cells undergo an increase in Cu but a decreasing in Fe content. This observation is consistent with a competition between Fe and Cu uptake, as observed in intestinal cells (2, 28). Because the mRNA levels of DMT1 are regulated by cellular Fe levels (1, 17), the increase in DMT1 mRNA expression observed in Caco-2 cells exposed to high Cu concentrations most likely is a reflection of the diminished Fe content. However, the mass of DMT1 transporter in Hep-G2 cells decreased under Cu overload, despite the decrease in the content of Fe. This change in the level of DMT1 protein is conflicting with the effect observed in Caco-2 cells deprived of Fe. We do not have an explanation of the mechanisms that are operating to reduce DMT1 levels. It may be that Cu decreases the translation of DMT1 mRNA or accelerates the degradation of the DMT1 protein. Cu could also regulate DMT1 activity as a feedback mechanism to reduce cellular Cu accumulation.

Consistent with a change in the level of DMT1 transporter, we observed a change in 64Cu uptake when the cells were overloaded with Cu. An increase in the Km value was observed in cells incubated with high extracellular Cu concentrations. This modification might be mediated by changes in the relative contribution of DMT1 and human CTR1 to Cu uptake. Alternatively, the changes in DMT1 might not affect Cu uptake in Hep-G2, and the proposed changes in transport could all be the result of CTR1-mediated processes. The plasma membrane Cu transporter human CTR1, with a Km of 1–5 µM (13, 29, 31), has been proposed as responsible for high-affinity Cu uptake in human cells (20). Our data suggest that the decreasing number of DMT1 transporters along with a more efficient 64Cu uptake might be explained as a relative rise in the contribution of CTR1 to Cu uptake in Hep-G2 cells exposed to Cu. Regarding CTR1 expression, studies (29) have shown that its abundance remains unchanged, under Cu treatments, in different cell lines. These results vary from those reported previously (40), in which internalization and degradation of human CTR1 in transfected human cell lines was observed. Whether CTR1 and DMT1 are coregulated during cellular adaptation to Cu exposure needs to be elucidated.


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This work was supported by Fondo Nacional de Desarrollo Cientifico j Technológico grants 1030618 and 1000852 (to M. González), 1010693 (to V. Cambiazo), and 1010657 (to M. T. Nuñez), by a Conisión Técnica Asesora–Centro De Investigación Minera y Metalúrgica Grant (to M. González and M. Arredondo), and by Grant P99–031F (to the Millennium Institute for Advanced Studies in Cell Biology and Biotechnology). M. González-Agüero is a recipient of Steckel fellowship.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. González, INTA, Universidad de Chile. Casilla 138–11, Santiago, Chile (E-mail: mgonzale{at}inta.cl).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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