ELSEVIER Chemico-Biological Interactions 100 (1996) 277-288

Cadmium uptake by Caco-2 cells. Effect of some milk components

Luis Mata, Lourdes Sanchez, Miguel Calve*

Departamento ale Tecnologia y Bioquimica a’e 10s Alimentos, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain

Received 17 November 1995; revised 4 March 1996; accepted 8 March 1996

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The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 pM of metal. Supplementation of the culture medium with lO?/ bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 100/o human milk was similar to that with serum-free medium. &Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 x lo4 Vmol. Interaction of lactofer- rin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture me- dium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing 8-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.

Keywords: Cadmium; Milk; Lactoferrin; Metallothionein; Caco-2 cells

Abbreviations: SFM, serum-free medium; FCS, medium with 1% fetal calf serum; Lf, lactoferrin; Lg. /3-lactoglobulin. l Corresponding author. Tel.: +34 76 761583; Fax: +34 76 761612; E-mail: mcalvo@mvet.unizar.es

0009-2797,96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII: SOOOS-2797(96)03706-S

278 L. Mata et al. / Chemico-Biological Interactions 100 (19%) 277-288

1. Introduction

Cadmium is a highly toxic metal which may accumulate in the body of humans and animals. Liver and kidney are the organs where it is mainly deposited in the long-term [l]. For the general population, diet is the most important source of cad- mium, although smokers also accumulate a great proportion of cadmium through the respiratory airways [2].

In humans and animals, only a small portion of dietary cadmium is absorbed by the intestinal mucosa, though there is a high variability in absorption due to physio- pathological and nutritional factors [ 1,3]. Cadmium absorption consists of two prin- cipal steps; uptake of the metal from lumen into the mucosa and transport of cadmi- um from the mucosa into blood. The intestinal mucosa plays a role in the control of cadmium absorption at the second stage, due to the existence of metallothionein, a protein which is able to bind most cadmium in the intestinal mucosa. Afterwards, the cadmium bound to metallothionein is partly eliminated by the normal renewal of mucosa [4]. A large number of studies have been carried out using jejunal segments in situ or in vitro, though the location of the intestinal section where cad- mium is absorbed has not been elucidated yet. It has been found in in vivo experi- ments, that orally administered cadmium in rats is mainly retained in the duodenum [5]. However, it has also been reported that cadmium is mostly retained in the distal small intestine [6].

The effect of metallothionein on cadmium absorption has been mainly studied using rat intestine, by stimulating the synthesis of this protein with zinc or with cad- mium [S]. Furthermore, an established cell line from human colon adenocarcinoma, Caco-2, has shown the capacity to synthesize metallothionein after zinc exposure [7]. They found that metallothionein production was greater at early stages in Caco-2 culture, before cells were completely differentiated. Likewise, Caco-2 cells have been used to study the transport of several nutrients [8,9] and also of essential metals for the organism, like zinc [lo] and iron [l 11.

On the other hand, milk and milk-based products are essential in the diet of in- fants and children. Despite the relatively low levels of cadmium in these products, they are the main source of this metal for those groups and a significant source, al- though less important, for adults [12]. Furthermore, dairy products have been shown to increase the intestinal absorption of heavy metals such as cadmium [ 131, and although the mechanism is still unknown, it has been suggested that it could be due to the association of cadmium with some milk components [14].

The aim of the present work has been to study the effect of human and bovine skim milk and some milk proteins, such as lactoferrin and &lactoglobulin, on cadmi- um uptake by Caco-2 cells. The effect of cadmium on metallothionein synthesis by this cell line has also been studied.

2. Materials and metbods

2.1. Cell culture

Caco-2 cells were kindly donated by Dr J.H. Brock (Department of Immunology,

L. Mata et al. / Chemico-Biological Interactions 100 (19%) 277-288 219

Glasgow University, UK). Cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Dorset, UK) supplemented with 10% fetal calf serum (Flow, Rickmansworth, UK), 1% nonessential amino acids (Flow, Rickmansworth, UK), 1 mgil bovine insulin (Sigma, Dorset, UK) and antibiotics, 50 000 Uil penicillin and 50 mgil streptomycin (Sigma, Dorset, UK). Cells were nor- mally grown in 25-cm2 tissue culture flasks to confluence and seeded into 24well tissue culture plates at a density of 4 x lo4 cells/cm*. Cells were maintained at 37°C in an atmosphere of 5% CO2 and 90% relative humidity. Cell viability was determined by trypan blue exclusion [15].

2.2. Preparation of cadmium-containing media

A stock solution of cadmium acetate (8.89 mM of cadmium) was sterilized through a 0.22~c(rn filter (Millipore, Molsheim, France) and kept at 4°C for use in all the assays. To obtain the desired concentration of cadmium in each experiment, the appropriate amount of the stock solution was added into the culture medium. The actual concentration of cadmium in all media was determined by atomic absorp- tion spectrometry (Perkin Elmer, model 2100, Norwalk, CT). The culture medium was checked before metal addition and no detectable amount of cadmium was found. Media containing lactoferrin with an iron-saturation of 30% (Fina Research, Seneffe, Belgium) or /3-lactoglobulin (Sigma, Dorset, UK) were prepared by adding the appropriate amount of protein from a sterile concentrated solution (125 and 555 PM, respectively) into the serum-free culture medium.

Samples of human or bovine milk were incubated with cadmium at a concentra- tion of 89 PM for 12 h at 4°C and added to the serum-free medium at 10%. Media supplemented with milk were sterilized through a 0.22~pm filter (Millipore, Molsheim, France).

2.3. Cadmium uptake by Caco-2 cells

Studies on cadmium uptake were carried out on 24well tissue culture plates with the cell monolayer slightly before confluence. Medium was removed from the wells and the cells were gently washed with serum-free medium and incubated for 1 h in fresh medium. The cells were then washed once and the samples to be assayed were added to the wells (2 ml/well) and incubated for different times.

After the incubation, the medium was removed and the monolayer was washed three times with Hanks’ balanced salt solution (HBSS). Finally, the wells were wash- ed with a solution of 1 mM EDTA in phosphate saline buffer (PBS) to remove the metal non-specifically bound to the cellular surface and to the plate walls. To deter- mine cadmium uptake, cells were removed with 0.3 M NaOH (1 ml/well). The pro- tein content of the cell suspension was determined by Bradford’s method [la] using bovine serum albumin as standard. Cadmium concentration was analyzed by atomic absorption spectrometry.

2.4. Interaction of lactoferrin or &lactoglobulin with cadmium

Lactoferrin or @-lactoglobulin were dissolved in sodium acetate 25 mM pH 6.5 at

280 L. Mata et al. / Chemico-Biological Interactions 100 (19%) 277-288

a concentration of 31 and 139 PM, respectively, and incubated with cadmium at 37°C for 1 h, at concentrations ranging from 9 to 176 PM. Then, the protein solu- tions incubated with the metal (3 ml) were added to the ultrafiltration chambers with a molecular weight cut off of 3000 (Filtron, Northborough, MA) and centrifuged at 3000 x g for 45 min. The concentration of cadmium in the filtrate and the retentate was analyzed by atomic absorption spectrometry and the concentration of protein was measured using the absorptivity at 280 nm as 1.20 and 0.96 cm2/g for lactofer- r-in and fl-lactogfobulin, respectively. The amount of cadmium bound to the protein was assumed to be that present in the retentate minus that in the filtrate. To estimate the amount of the metal non-specifically bound to the wall and the membrane of the ultrafiltration chamber, free-protein solutions added with cadmium were treated in the same conditions. The stoichiometric binding constant was obtained by a graphi- cal procedure, in which the value of cadmium bound to protein per the concentra- tion of free cadmium was extrapolated to the limiting value as free cadmium concentration approaches zero [17]. The molecuar weight was assumed to be 18 000 and 80 000 for monomeric /3-lactoglobulin and lactoferrin, respectively.

2.5. Measurement of metallothionein synthesis by Caco-2 cells

Caco-2 cells grown in 75-cm2 flasks were used slightly before confluence for the experiments for metallothionein synthesis. The culture medium was removed and the cells were washed with serum-free medium and incubated in this medium for 1 h. The cells were then washed once and media containing a range of different cadmium concentrations (0,4, 20 and 40 PM) were added to the flasks. After 24 h of incuba- tion, the medium was removed and the cells were washed three times with PBS. Finally, the monolayer was rinsed with 1 mM EDTA in PBS and the cells were detached by trypsinization (0.25% trypsin for 5 min at 37°C). To inhibit trypsin ac- tivity, medium containing 10% of fetal calf serum was added to the flasks. The cell suspension was centrifuged at 100 x g for 5 min, washed three times with PBS and sonicated for 1 min. Then, the suspension was heated at 80°C for 2 min and the cytosol was obtained by centrifugation at 90 000 x g for 1 h at 4°C.

The concentration of total protein in the cytosol was determined by Bradford’s method [16] using bovine serum albumin as standard. Metallothionein concentra- tion was determined by a cadmium-saturation method [ 181 in which the cytosols were incubated with an excess of cadmium to saturate the metal binding sites of metallothionein. The excess of cadmium was removed by addition of hemoglobin to the cytosols which was eliminate by heating and centrifugation, and this procedure was repeated several times. The cadmium concentration in the supernatant of the last centrifugation was determined by atomic absorption spectrometry. The concen- tration of metallothionein was calculated by assuming that 6 mol of cadmium bind to 1 mol of protein and with a molecular weight for metallothionein of 6050.

In addition, cytosols (1 ml) were chromatographed onto a Sephadex G-75 super- fine column (1 x 80 cm) to check whether cadmium was bound to metallothionein or to low molecular weight substances that could be present in the cytosols increas- ing the apparent concentration of metallothionein. The elution was carried out at

L. Mata et al. /Chemico-Biological Interactions 100 (19%) 277-288 281

a flow of 10 ml/h with 10 mM Tris-HCl pH 8.0 and fractions of 2 ml were collected. Cadmium concentration was analyzed by atomic absorption spectrometry. Blue dex- tran and ovine metallothionein isolated from liver according to Whanger [ 19) were chromatographed in the same conditions to estimate the void volume of the column and the elution volume for metallothionein.

3. Results

The study of cadmium uptake has been carried out using a human intestinal cell line (Caco-2). The levels of cadmium used in this work did not affect the cell viability being in all cases above 95%, except with 40 PM cadmium concentration at which the viability was 80%.

3.1. Cadmium uptake by Caco-2 cells

Cadmium uptake by the cells has been found to be lower when they were grown in medium supplemented with 1% of fetal calf serum than in serum-free medium (Fig. l), the difference being statistically significant (P < 0.05) for concentrations of cadmium in the medium > 2 pM. In both cases, cadmium uptake by Caco-2 cells increased with the concentration of cadmium added into the medium.

0 2 4 6 8 10 12 14 16

Cadmium wncenuabon (JIM)

Fig. 1. Uptake of cadmium by Caco-2 cells incubated in serum-free culture medium, as control (0) or culture medium with 1% fetal calf serum (0). Cells were exposed to cadmium in a range of concentrations from 2.2 to 16 &l for 24 h under normal culture conditions. At the end of the experiment, cells were removed and cadmium in the cells was determined by atomic absorption spectroscopy. Each point represents the mean f SD. of six replicates from two independent experiments. Significance of the data was analyxed by ANOVA using the SchetWs method, the values marked with an asterisk differ significantly from the groups incubated in serum-free medium (P < 0.05).

282 L. Mote et al. / Chemico-Biological Interactions 100 (19%) 277-288

2 6

Time (h)

24

Fig. 2. Uptake of cadmium by Caco-2 cells incubated in serum-free culture medium (solid bars), culture medium containing 1% fetal calf serum (open bars), culture medium containing 10% bovine milk (dotted bars) or culture medium containing 10% human milk (hatched bars). Cells were exposed to 8.14 * 0.72 PM of cadmium for 2,6 and 24 h under normal culture conditions. At the end of each incubation time cells were removed and intracellular cadmium was determined by atomic absorption spectroscopy. Each point represents the mean f S.D. of six replicates from two independent experiments. Significance of the data was analyzed by ANOVA using the Scheffe’s method. aSigniticantly different from the values of up take by the cells cultured in serum-free medium (P < 0.05); bSigniticantly different with respect to the cells cultured in medium containing 1% fetal calf serum (P < 0.05).

1.4 7

i

SFM (Es Lf 0.31 Lf 0.62 if 1.25 Lf 1.87 Lg 5.55

Fig. 3. Uptake of cadmium by Caco-2 cells incubated in culture medium containing 1% fetal calf serum, lactoferrin (0.31.0.62, 1.25 and 1.87 pM) or g-lactoglobulin (5.55 PM) for 24 h under normal culture con- ditions. All media contained 7.90 + 0.30 CM of cadmium. At the end of the experiment, cells were remov- ed and cadmium in the cells was determined by atomic absorption spectroscopy. Each point represents the mean f SD. of nine replicates from three independent experiments. Significance of the data was analyzed by ANOVA using the Scheffe’s method. BSignificantly different with respect to the cells cul- tured in serum-free medium (P < 0.05); bSigniticantly different with respect to the cells cultured in medi- um containing 1% fetal calf serum (P < 0.05). SFM, serum-free medium; FCS, medium with 1% fetal calf serum; Lf, lactoferrin; Lg, &lactogfobulin. The numbers on the abscissa represent the concentration bM) of lactoferrin or @-lactoglobulin in the medium.

L. Mata et al. / Chemico-Biological Interactions 100 (19%) 277-288

K’a-5x 1O’M’

0 20 40 60 80 101

Free cadmium (PM)

Fig. 4. Binding of cadmium to &lactoglobulin as assessed by equilibrium dialysis. &Lactoglobulin solu- tions of 139 CM were incubated with cadmium at a concentration between 9 and 176 rA4 and centrifuged in ultrafiltration chambers (molecular weight cut off of 3000). Concentration of free cadmium was con- sidered as that present in the compartment of the filtrate. Cadmium bound to &lactoglobulin was estimated as the total cadmium in the compartment of retentate minus free. cadmium. In the plot, bound cadmium represents moles of the bound cadmium per mol of &lactogJobulin. Each point is the mean of two determinations.

The effect of human or bovine milk on cadmium uptake by Caco-2 cells has also been studied. After 24 h of incubation, the amount of cadmium retained by the cells was significantly lower (P < 0.05) when cultured in the medium supplemented with 10% bovine milk than in serum-free medium or medium with 10% human milk. Cad- mium uptake by the cells cultured in medium supplemented with loo/o bovine milk was similar to that internalized by those grown in 1% fetal calf serum. In all cases, cadmium uptake increased in a linear rate with time (Fig. 2).

Lactoferrin and B-lactoglobulin, two milk proteins, were added to the serum-free medium at different concentrations to study their effect on cadmium uptake by Caco-2 cells (Fig. 3). Cadmium uptake was lower from the medium containing lac- toferrin than from that containing @-lactoglobulin or the protein-free medium (P < 0.05). Despite lactoferrin decreasing the uptake of cadmium by Caco-2 cells, this effect does not seem to be dose-dependent within the range of lactoferrin concentra- tions used in this work. On the other hand, the uptake of cadmium by the cells grown in the medium supplemented with /3-lactoglobulin was higher (P < 0.05) than that in the medium supplemented with 1% of fetal calf serum.

The interaction of &lactoglobulin and lactoferrin with cadmium was studied us- ing the equilibrium dialysis technique. The plot obtained for the interaction of &lactoglobulin with cadmium is shown in Fig. 4 from which the stoichiometric

284 L. Mata el al. / Chemico-Biological Interactions 100 (19%) 277-288

0.a

0 0

10 20 30 40 50 60 70 a0 90

VOLUME [ml)

Fig. 5. Fractionation of cytosols obtained from Caco-2 cells exposed to different concentrations of cadmi- um: 4 (0), 20 (A) and 40 pM (0) for 24 h. Caco-2 cells were maintained in normal culture conditions. The heat-stable fraction was obtained from the cytosols by heating at 80°C for 2 mm and centrifuging at 90 000 x g. Then, the fraction was chromatographed on a Sephadex G-75 column eluting with 10 mM Tris-HCI pH 8.0. The dotted line represents the absorbance at 254 nm of the cytosol obtained from cells exposed to 40 CM of cadmium. The concentration of cadmium in each fraction was determined by atomic absorption spectroscopy.

binding constant was calculated and found to be 5 x lo4 l/mol. The binding of cadmium to lactoferrin was negligible.

3.2. Effect of cadmium on metallothionein synhesis

Caco-2 cells were exposed to different concentrations of cadmium (0, 4, 20 and 40 PM) to induce the synthesis of metallothionein. Gel filtration chromatography of the heat-stable fraction of cytosols showed three peaks of cadmium (Fig. 5). The height of the second peak, eluting at the volume corresponding to metallothionein, increased with the concentration of cadmium added to the culture medium. Al- though the chromatographic profile showed a peak of cadmium eluting with the low molecular weight compounds, this peak contained a small amount of metal com- pared to the second peak, which contained between 70 and 75% of total cadmium. The absorbance at 254 nm, the wavelength at which metallothionein exhibits a maxi- mum of absorbance, was very low in the fraction corresponding to metallothionein, indicating that a relatively low amount of protein is able to bind a great amount of cadmium.

To quantify the amount of metallothionein contained in the cytosol, a cadmium- saturation method was used (Fig. 6). The values were corrected for the concentration of the total protein in the cytosol and they indicated that the concentration of metal-

L. Mata et al. / Chemico-Biological Interactions 100 (19%) 277-288 285

T T T

0 4 20 40

Cadmium concentration (PM)

Fig. 6. Concentration of metallothionein in the cytosols obtained from Caco-2 cells exposed to cadmium at different concentrations (0,4,20 and 40 PM) for 24 h. Caco-2 cells were maintained in normal culture conditions.The cytosols obtained in the same way as described in the legend of Fig. 5 were assayed for metallothionein using a cadmium-saturation method. The results are presented as the concentration of metallothionein per milligram of total protein in the cytosol.

lothionein in the cytosol is directly related to the amount of cadmium in the medium. However, at concentrations of cadmium in the medium above 40 PM, the levels of metallothionein were only slightly higher than those found in the cells exposed to 20 PM of cadmium.

4. Discussion

In this work, we found that cadmium uptake by Caco-2 cells increases with the concentration of the metal in the culture medium, showing a saturable behaviour. It is known that in a first step, cadmium binds to the anionic sites of the cell mem- brane through a non-specific electrostatic interaction [20]. However, the proportion of cadmium bound to the cell surface compared with that internalized becomes negligible after relatively long periods of incubation [20], as those used in our experi- ments. Furthermore, in the present work, the cadmium bound to the cell membrane was removed with EDTA to determine only the metal internalized by the cells. Therefore, the saturation behaviour observed in the uptake of cadmium by Caco-2 cells could be due to the binding of the metal to some intracellular protein rather than a saturable binding to the cell membrane.

Metallothionein is a protein rich in cysteine, inducible under certain conditions and capable of binding seven atoms of cadmium per molecule. This protein is syn- thesized in the cells of a large number of organs, mainly liver, kidney and intestine,

286 L. Mata et al. / Chemico-Biological Interacrions 100 (1996) 277-288

and it binds cadmium and other metals, thus avoiding their toxic effect [21]. Conse- quently, we investigated if Caco-2 cells synthesized metallothionein in our culture conditions. The chromatographic profile obtained when fractionating the cytosols of cells exposed to cadmium showed that there was synthesis of metallothionein, a process which increased with the concentration of cadmium in the medium. How- ever, the concentration of metallothionein did not increase further at concentrations of cadmium above 20 pM. These results indicate that cadmium induces the metallo- thionein synthesis in Caco-2 cells and agree with those found when Caco-2 cells were exposed to zinc, which also induced metallothionein synthesis [7]. The cell line used in our work shows a similar behaviour in response to cadmium as the rat intestinal mucosa [4]. It was observed that mucosal cells were able to synthesize metallothion- ein in the presence of cadmium and accumulated this metal bound to metallothion- ein. Therefore, the use of Caco-2 cells to study the influence of different nutrients on cadmium uptake seems to be appropriate.

Caco-2 cells cultured in a medium containing 40 PM of cadmium showed signs of toxicity with dead cells appearing in the medium and rounded cells starting to detach from the cell monolayer. Thereby, the limit of cadmium tolerance by Caco-2 cells seems to be in the range of 20-40 PM. As we have found in this work, the cells were not able to synthesize more metallothionein at cadmium concentrations greater than 20 PM, with a high proportion of free cadmium remaining inside the cell, which may exert a toxic effect.

Supplementation of the medium with fetal calf serum decreased the uptake of cad- mium by the cells with respect to serum-free medium, indicating that some com- ponents of fetal calf serum could hinder the internalization of cadmium. In a study using cells of renal origin it was observed that calf serum also inhibited cadmium uptake in a concentration-dependent manner [22]. This effect was also found by these authors when they supplemented the medium only with albumin.

We also studied the effect of milk secretions on cadmium uptake by Caco-2 cells and we found that when medium was supplemented with 10% bovine milk, the metal uptake diminished by 25%. Nevertheless, we did not find the same effect when the cells were incubated in medium supplemented with human milk, which showed an uptake of cadmium similar to that observed with serum-free medium. This finding may be explained by a different distribution of cadmium among the components of milk in both species. In fact, we found in a previous work that cadmium in human milk is mainly associated with substances with a molecular weight lower than 10 000, while in bovine milk most cadmium is associated with a compound of a molecular weight over 70 000 [23]. Thus, in the medium supplemented with bovine milk, cad- mium could be associated with macromolecules that can not be internalized by the cells, while in human milk, it is associated with small molecules which are easily ab- sorbed. In contrast, in experiments carried out with rats [14] it has been found that bovine milk enhances cadmium absorption. It is possible that cadmium binds to small molecules formed during digestion of milk in the intestinal tract, or migrates to other molecules present in milk, which might be more easily absorbed.

Lactoferrin is an iron-binding protein present in human and bovine milk. The ca- pacity of this protein to bind other metals [24] led us to investigate its role in the

L. Mata et al. / Chemico-Biological Interactions 100 (19%) 277-288 287

uptake of cadmium by Caco-2 cells. Our results indicate that this protein inhibits cadmium uptake when compared with a free-protein medium or a medium contain- ing &lactoglobulin, another protein present in bovine milk. It would be possible that lactoferrin could bind the cadmium present in the medium avoiding its interaction with the cell membrane. However, this mechanism does not seem probable since we have found that lactoferrin binds cadmium to a very low extent.

Another mechanism which could explain the inhibitory effect of lactoferrin on cadmium uptake, would involve the binding of lactoferrin to the cell surface through an electrostatic interaction. Lactoferrin is a basic protein and may interact with the anionic sites of the membrane in a similar way to that described for the metal cations [25]. Furthermore, lactoferrin exhibits a high capacity to interact with a large num- ber of proteins, and it has been shown to interact non-specifically with the mem- brane of Caco-2 cells [26]. Thus, lactoferrin would bind to the cell surface hampering the interaction of cadmium with the membrane and its subsequent internalization.

On the other hand, fl-lactoglobulin, an acidic protein, has not shown any effect on cadmium uptake by Caco-2 cells. This protein has a low affinity for cadmium if compared with serum albumin [27] and other low molecular weight substances pres- ent in the culture medium, such as amino acids [28]. This may explain why cadmium uptake is higher when the medium is supplemented with fl-lactoglobulin than with fetal calf serum, rich in albumin. The results shown in this work suggest that the in- teraction of some milk components with the membrane of intestinal cells may be an important factor in the absorption of cadmium. Furthermore, the inhibitory effect of lactoferrin may be due to a reduction of the cell surface charge, rather than any other mechanism.

Acknowkdgement

This investigation was supported by grant AL1 934417 from CICTY (Spain) and L.M. was beneficiary of the fellowship BCB 3391 from Diputacion General de Aragon.

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