Is There a Trojan-Horse Effect during Magnetic Nanoparticles and

Aug 24, 2012 - This biological effect also known as the Trojan- horse effect has been mentioned in the literature but remains poorly investigated, exc...
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Is There a Trojan-Horse Effect during Magnetic Nanoparticles and Metalloid Cocontamination of Human Dermal Fibroblasts? Melanie Auffan,†,‡,* Jerome Rose,†,‡ Olivier Proux,‡,§ Armand Masion,†,‡ Wei Liu,†,‡ Laila Benameur,‡, ∥ Fabio Ziarelli,⊥ Alain Botta, ∥ Corinne Chaneac,‡,# and Jean-Yves Bottero†,‡ †

Aix-Marseille Université, CNRS, IRD, CEREGE UM34, UMR 7330, 13545 Aix en Provence, France. GDRi iCEINT, international Consortium for the Environmental Implication of Nanotechnology, France § OSU de Grenoble, UMS CNRS, 38400, St Martin d’Hères, France ∥ IMBE, UMR 7263, CNRS Aix-Marseille université, Facultés de Médecine et de Pharmacie, 13385 Marseille, France ⊥ Fédération Sciences Chimiques FR-CNRS 1739, Aix-Marseille Univ, 13013 Marseille, France # LCMC, Collège de France, université Pierre-et-Marie-Curie, 75231 Paris, France ‡

S Supporting Information *

ABSTRACT: This study investigates the issue of nanoparticles/pollutants cocontamination. By combining viability assays, physicochemical and structural analysis (to probe the As speciation and valence), we assessed how γFe2O3 nanoparticles can affect the cytotoxicity, the intra- and extracellular speciation of As(III). Human dermal fibroblasts were contaminated with γFe2O3 nanoparticles and As(III) considering two scenarios: (i) a simultaneous coinjection of the nanoparticles and As, and (ii) an injection of the nanoparticles after 24 h of As adsorption in water. In both scenarios, we did not notice significant changes on the nanoparticles surface charge (zeta potential ∼ −10 mV) nor hydrodynamic diameters (∼950 nm) after 24 h. We demonstrated that the coinjection of γFe2O3 nanoparticles and As in the cellular media strongly affects the complexation of the intracellular As with thiol groups. This significantly increases at low doses the cytotoxicity of the As nonadsorbed at the surface of the nanoparticles. However, once As is adsorbed at the surface the desorption is very weak in the culture medium. This fraction of As strongly adsorbed at the surface is significantly less cytotoxic than As itself. On the basis of our data and the thermodynamics, we demonstrated that any disturbance of the biotransformation mechanisms by the nanoparticles (i.e., surface complexation of thiol groups with the iron atoms) is likely to be responsible for the increase of the As adverse effects at low doses.

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toward both algae and crustaceous.4 Limbach et al. also observed that NPs could enter the cells by a Trojan-horse type mechanism and induced an oxidative stress.5 Iron oxide NPs have a strong affinity for metalloids and metals in water and soils. Their adsorption efficiency and the adsorption mechanisms depend on their size, crystal structure, and surface energy.6−11 For instance, the number of arsenic and cobalt atoms adsorbed per unit of surface strongly increases for 6−8 nm maghemite (γFe2O3) NPs (As(III/V)/nm2, 22−34 Co(II)/nm2)10−13 compared to larger particles (20 and 300 nm). This strong affinity for pollutants is not only related to the increase in specific surface area (SSA) as size decreases but also to a significant increase in surface reactivity for NPs smaller than 20 nm.6 These reactivity and SSA effects both imply that manufactured iron oxide NPs can modify the bioavailability and

INTRODUCTION To date, several questions are raised about the remarkable properties of nanoparticles (NPs) in terms of (photo)catalytic, optical, magnetic, electronic, physicochemical properties, and surface reactivity. Regarding the enhancement of the surface properties, the large proportion of surface atoms, surface curvature, surface defects, and the excess of surface energy are usually involved.1 Whatever the origin of the enhanced surface reactivity is, once NPs are released in natural systems they may react with pollutants, cross-biological barriers and modify the bioavailability/bioaccessibility of the associated pollutants. Then the issue of NPs as contaminant carriers needs to be investigated. This biological effect also known as the Trojanhorse effect has been mentioned in the literature but remains poorly investigated, except for intentional effects as drug delivery.2 Zhang et al. (2007) found that studied the amount of Cd2+ accumulated in the viscera and gills of fish increase from 9 to 22 μg/g presence of 10 μg/L of TiO2 NPs.3 Others found that the presence of nC 60 decreases the toxicity of pentachlorophenol and increases the toxicity phenanthrene © 2012 American Chemical Society

Received: Revised: Accepted: Published: 10789

June 20, 2012 August 23, 2012 August 24, 2012 August 24, 2012 dx.doi.org/10.1021/es302493s | Environ. Sci. Technol. 2012, 46, 10789−10796

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Two cocontamination scenarios of cells with Nm and As(III) were assessed. In all of these treatments, the pH of the stock solutions and suspensions were adjusted to 7.3−7.4 to avoid any change in the pH of the fibroblast culture media. First, Nm (50 mg/L) and As (25, 50, 100, and 300 μM) were separately but simultaneously injected in the fibroblasts culture medium. Samples regarding this coinjection experiment are called [As]+[Nm]. For the second scenario, As(III) was put in contact with Nm in water at pH 7 for 24 h (following the protocol detailed in ref 6) and then injected in the fibroblasts culture media. The final concentrations in the fibroblast culture media are 50 mg/L of Nm, and 25, 50, 100, and 300 μM of total As. Samples regarding this preadsorption/injection experiment are called [AsNm]. For the preadsorption in water, 500 mg/L of Nm were incubated with 0.25 mM, 0.5 mM, 1 mM, and 3 mM of As(III). On the basis of the adsorption isotherm in water published by Auffan et al. (2008), the Nm adsorption capacity reach a plateau for 13.4 ± 0.1 μmol of As/m2.6 Physicochemical Characterization. A thorough physicochemical characterization of the Nm has already been published 6,15,16 and is summarized in the Supporting Information. In the current study, we assessed the colloidal stability of Nm in the abiotic DMEMc after treatment with As (following both the [As]+[Nm] and [AsNm] scenarios, 24 h, 50 mg/L of Nm) by measuring the hydrodynamic diameters and the zeta potentials using a Zetasizer NanoZ and the Mastersizer 3000 (Malvern, UK). Adsorption and desorption experiments were performed in abiotic DMEMc to quantify the amounts of As retained at the surface of Nm in conditions close to the two cocontamination scenarios. First, an adsorption isotherm of As onto Nm was performed in the abiotic DMEMc. This isotherm corresponds to the coinjection scenario ([As]+[Nm]). Nm (50 mg/L) were incubated with 25, 50, 100, and 300 μM of As. After 24 h, the suspensions were ultracentrifuged (550 000 g during 2 h) and the supernatants were analyzed by ICP-MS (Thermo X series II model, equipped with a collision cell). A second adsorption/desorption experiment was performed to match the preadsorption/injection scenario ([AsNm]). First, 500 mg/L of Nm were incubated with 0.25, 0.5, 1, and 3 mM of As. After 24 h of adsorption, each batch was diluted 10 times in abiotic DMEMc. The final concentrations of this 24 h desorption experiment were 50 mg/L of Nm, and 25−300 μM of As. The suspensions were then ultracentrifuged (550 000 g during 2 h) and the supernatants analyzed by ICP-MS. Cytotoxicity Assay. The metabolic activity of cells was evaluated using the WST-1 assay.17 It is based on cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells to form a yellow formazan dye. A lower absorbance (at 450 nm) than the negative control is therefore interpreted as a decrease in cell viability. After the treatments, the cells were rinsed 3 times using a phosphate buffer and treated for 30 min with WST-1. The absorbance was measured using a microtiter plate reader (Multiskan RC, Labsystems). Four replicates were used for each condition, and the statistical significance was determined using the Student’s t-test. Assessment of the As Speciation. Arsenic K-edge X-ray Absorption Spectroscopy (XAS) experiments were performed at the European Synchrotron Radiation Facility (ESRF, France) on the FAME beamline (BM30b) equipped with a Si(220) monochromator.18,19 Samples were pressed into thin pellets and cooled down to ca. 10 K with liquid Helium to improve the

toxic effects of pollutants during NPs/pollutants cocontamination. To assess the effects of such cocontaminations, we used the maghemite NPs/arsenite pair. Arsenic is a trace element, whose fate and bioavailability for plants, organisms, and humans strongly depend on its speciation. As(III) can impair protein functions or interact with DNA resulting in single-strand breaks.12 Once assimilated, most mammals methylate inorganic arsenic via alternating reduction of As(V) to As(III) and addition of a methyl group. This biomethylation has long been considered as one major detoxification process even if it might generate toxic transient species. The role of thiolated compounds such as glutathione (GSH) is of high importance in the different biocellular transformation. For instance, depletion of GSH increases the clastogenic effects of arsenite13 and has a decisive effect on As2O3-induced apoptosis.14 In the present study, we will discuss (i) how the cocontamination of cells by manufactured iron oxide NPs and As disturbs the intraand extracellular speciation of As and (ii) how it modifies the arsenic cytotoxicity toward human dermal fibroblasts. Two cocontamination scenarios were considered to emphasize different mechanisms of interactions between As and the NPs (e.g., surface adsorption, complexation, competition between NPs and As for molecules of the cellular media). The first scenario is a simultaneous coinjection of As and NPs in the cellular media, whereas the second scenario is an injection of NPs after 24 h of As adsorption in water. By combining viability assays, physicochemical, and structural analysis (to probe the As speciation and valence), we demonstrated that the presence of γFe2O3 nanoparticles in the cellular media contaminated with As strongly affects the complexation of the intracellular As with thiol groups. This increases the cytotoxicity of the As nonadsorbed at the surface of the NPs. However, once As is strongly adsorbed and weakly desorbed from the surface of the NPs its cytotoxicity significantly decreases.

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EXPERIMENTAL SECTION Normal Human Fibroblast Culture. Normal human fibroblasts were isolated by the outgrowth method using infant foreskin obtained after circumcisions. Fibroblasts were incubated in Dulbecco’s Modified Eagle’s Medium (DMEMc) complemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 μg/mL gentamicin (Eurobio, France) at 37 °C in a 5% CO2 humidified atmosphere for 24 h before incubation with the following treatments. Using 13C NMR, we estimated the concentrations of the individual components of the DMEMc (Supporting Information). It is mainly composed of NaCl, NaHCO3, glucose, and ∼2 mM of thiolated compounds. Maghemite Nanoparticles and Arsenite Treatments. Fibroblasts were first incubated with 25, 50, 100, and 300 μmol/L of arsenite. As(III) is introduced in the cellular media via a stock solution of 4 mM of NaHAsO3. Samples regarding this arsenite injection experiment are called [As]. Fibroblasts were also incubated with 25, 50, and 75 mg/L of maghemite nanoparticles (Nm) from a 2.4 g/L of Nm stock suspension. These particles are synthesized via aqueous coprecipitation of Fe2+ and Fe3+ to form magnetite Fe3O4, followed by an oxidation to maghemite γFe2O3 under acidic conditions. Nm are well crystallized, roughly spherical with a mean diameter of 6 ± 1 nm (Supporting Information) and a specific surface area of 172 m2/g (N2 adsorption and BET analysis). 10790

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spectrum quality by decreasing thermal motions of atoms, and minimizing radiation damages.20 XAS spectra were scanned above the edge (11.87 keV) in transmission mode for the reference compounds and in fluorescence mode for the biological samples. Three to five scans were collected for each sample. Standard procedures for pre-edge subtraction, normalization, polynomial removal, and wave vector conversion using the IFEFFIT software package21 were done to obtain the XANES (X-ray absorption near edge structure) and EXAFS (extended X-ray absorption fine structure) spectra. For each atomic shell, the interatomic distance (R), the coordination number (CN), and mean squared displacement (ss2) were adjusted. The amplitude reduction factor and the threshold energy position were fit to the data from reference compounds (As2O3, As2S3, and FeAsO4) and fixed for all subsequent analyses.

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RESULTS Physicochemical Behavior of Nm and As Adsorption within Abiotic Conditions. First, we assessed how the colloidal stability of the Nm was affected in the DMEMc by the presence of As in both exposure scenarios. After 24 h in the DMEMc, Nm aggregates with mean hydrodynamic diameters of 0.9 ± 0.2 μm are formed. The zeta potential of Nm in the DMEMc is about −17 ± 0.9 mV. After 24 h of contact with arsenic in the DMEMc following the two exposure scenarios, the size of the aggregates are quasi similar with mean hydrodynamic diameters of 0.4 ± 0.1 μm and 0.9 ± 0.2 μm respectively for the [AsNm] and [As]+[Nm]. The zeta potential are also similar at the end of the [AsNm] scenario (−11 ± 0.15 mV), and the [As]+[Nm] scenario (−10 ± 0.4 mV). Consequently, an aggregation of the Nm occurs in the DMEMc and this colloidal destabilization (surface charge or hydrodynamic diameter) is not affected by the As adsorption onto Nm. Despite this colloidal destabilization, we do not notice any change in the Nm crystal structure after 24 h in the abiotic DMEMc using XANES data at the Fe K-edge and X-ray diffraction (Supporting Information). Adsorption and desorption experiments were performed in abiotic DMEMc to estimate the As retention efficiency of Nm in the two exposures scenarios. It is noteworthy that these adsorption isotherms are performed in abiotic conditions and do not take into account the cells activity that can modify the Nm adsorption efficiency. The adsorption isotherm of As onto Nm in the abiotic DMEMc (coinjection scenario [As]+[Nm]) is presented in Figure 1. After 24 h, 2.1, 4.0, 7.8, and 8.3 μmol/m2 of As are retained at the surface of Nm (initial concentration: 50 mg/L of Nm and 25, 50, 100, and 300 μM of As). This matches well the adsorption isotherm performed in water (pH 7 ± 0.2, 0.01 M NaCl, 22−25 °C, 24 h) published previously.6 On the basis of the surface saturation obtained in ref 6 the Nm surface coverage by As in DMEMc is estimated at 16 ± 3%, 30 ± 6%, 59 ± 10%, 63 ± 12%, respectively for 25, 50, 100, and 300 μM of As initial concentrations. The adsorption/desorption experiment performed to reproduce in abiotic conditions the preadsorption/injection scenario ([AsNm]) is shown in Figure 1. The amounts of As retained at the surface of Nm after this two stages-experiment is not significantly lower than the ones obtained after a simple adsorption in DMEMc. We estimated that 1.1, 2.0, 2.5, 5.4 μmol/m2 of As remain at the surface, which corresponds to 8 ± 2%, 15 ± 3%, 19 ± 4%, and 41 ± 8% of surface coverage to be compared with the initial state. This highlights the strong

Figure 1. Adsorption of As onto Nm in abiotic DMEMc during 24 h of incubation (◊). Adsorption of As onto Nm (500 mg/L) in water at pH 7 ± 0.2 for 24 h (■) followed by a desorption in abiotic DMEMc (□) for 24 h (dilution factor for the desorption experiment: 10). The logarithmic fit corresponds to the adsorption isotherm of As onto Nm in water at pH 7 ± 0.2 (0.01 M NaCl, 30 mg/L of Nm, 22−25 °C, 24 h) previously published.

energy of the As adsorption onto Nm and that ligands competing for Fe sites (e.g., 1.1 mM of phosphate, or 39 mM of carbonate) do not significantly decrease the adsorption capacity. To study the adsorption mechanisms of As onto Nm in the abiotic DMEMc, the Nm surface was saturated with As. After 24 h, the liquid and solid phases were separated by ultracentrifugation and the Nm were analyzed by XANES at the As K-edge. A shift in the white line position is related to the nature of the first atomic shell (e.g., O for As2O3 and S for As2S3) but also to the oxidation state of As (e.g., As(III) for As2O3 and As(V) for As2O5) (Figure 2). Using linear combination of the XANES spectra of reference compounds (As2O3, As2O5, As2S3), we estimated the percentage of As(III) and As(V) at the Nm surface. In DMEMc 83% of As adsorbed at the surface of Nm remained As(III) while 13% was oxidized into As(V) (part A of Figure 2). Modeling of the EXAFS oscillations enable to further examining the adsorption mechanisms. EXAFS oscillations relate to the atomic environment of As viz. the number, the nature, and the distance of atoms surrounding As up to 4−5 Å. In the abiotic DMEMc, the first coordination sphere of As adsorbed onto Nm is attributed to 3.0 ± 20% oxygen atoms at 1.74 ± 0.02 Å (Figure 2, Table 1). These bond length values can be directly compared with the ones obtained for the As(V) and As(III) references. For As(V) mineral As(V)-O distance is in a 1.67−1.69 Å (1.67 Å for CaZnAsO4OH, 1.69 Å for FeAsO4, 2H2O, 1.69 Å for (Fe,Zn)(AsO4)2(OH, H2O)2) and for organometallic forms the As(V)-O is 1.61 Å for cacodylic acid, 1.69 Å for O-arsanilic acid, 1.68 Å for 4-hydroxy-3nitrobenzenearsenic acid. Regarding the As(III) mineral the As(III)-O distance is in a 1.77−180 Å range: 1.77 Å for the As2O3, 1.80 Å for NaAsO2, 1.79 for PbFe4As10O22, 1.77 Å for CaFeSbAs2O7, 1.79 Å for (Fe2, Zn)12(OH)6(AsO3)6(AsO3, HOSiO3)2).22,23 The 1.74 ± 0.02 Å corroborates the slight oxidation of As(III) onto As(V) at the Nm surface observed by 10791

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XANES. The second coordination sphere corresponds to 2.0 ± 20% iron atoms at 3.31 ± 0.02 Å (Figure 2, Table 1). This As− Fe distance is close to the average As(III)−Fe(III) interatomic distance of 3.33 ± 0.02 Å attributed to As(III)−O-Fe linkages through double-corner-sharing previously observed for As(III) adsorbed onto Nm in water.6 Cytotoxicity over the Different Cocontamination Scenarios. The effects of Nm and As(III) on the fibroblasts mitochondrial activity were first assessed separately (part A of Figure 3). Between 25 to 75 mg/L of Nm, no significant changes of the cell survival was observed. Consequently, if a decrease in cell viability occurs during cocontamination with Nm (50 mg/L) and As (both [As]+[Nm] or [AsNm] scenarios), the cytotoxicity will not be generated by the Nm themselves. On the opposite, significant dose−responses effects were observed following As(III) exposure ([As]). The lowest concentration tested (25 μM) does not decrease the cell viability, whereas for 300 μM of As(III), almost 80% are not viable (part A of Figure 3). This is consistent with Burnichon et al. (2003), who did not observe significant cell death up to 25 μM of As(III) under similar culture conditions.24 Regarding the preadsorption/injection exposure scenario ([AsNm]), after 24 h the cytotoxic effects are less important than As(III) alone (part A of Figure 3). No change of the cell viability was observed up to 100 μM, whereas a decrease by 55% of the survival appeared for a total As concentration of 300 μM. Once expressed as a function of the arsenic equilibrium concentrations, the percentages of viable cells in the [As] and [AsNm] scenarios match (part B of Figure 3). These equilibrium concentrations obtained in abiotic conditions (Figure 1) inform about the distribution of As in the [AsNm] scenario at the time of the injection in the cellular media. Different trends are observed with the coinjection exposure scenario ([As]+[Nm]) since cytotoxic effects are more important at low concentrations than for [AsNm] or [As]. For doses below 100 μM, the cell viability is decreased by 40− 45% compared to the 65−75% for As alone or the 80% for the [AsNm] scenario (part A of Figure 3). In contrast with the [AsNm] scenario, once the viability data are expressed as a function of the As equilibrium concentration the cytotoxicity of [As]+[Nm] do not match the [As] scenario (part B of Figure 3). However, at high doses (300 μM), these differences in viability disappear and all scenarios induce similar effects (parts A and B of Figure 3). Speciation of As(III) in the Intra- and Extracellular Medium in Absence of Nm. As K-edge XANES spectra of the intra- and extracellular compartments of fibroblasts incubated with 20 μM of As(III) during 24 h are compared

Figure 2. (A) Arsenic K-edge XANES data of As adsorbed at the surface of Nm after 24 h in the abiotic DMEMc and in water (from ref 6). The experimental data are compared to XANES spectra of reference compounds (As2O3, As2O5, and As2S3). (B) EXAFS spectra at the As K-edge, (C) their corresponding Fourier transform. The structural parameters deduced from the fits are given in Table 1.

Table 1. Structural Parameters Deduced from the EXAFS Analysis at the As K-Edge. The Parameters Correspond to the Fits Presented in Figures 2 and 4

standard reference compounds abiotics conditions

As2S3 As2O3 As in DMEM As+Nm in DMEM As+Nm in water

biotics conditions

intracellular [As] extracellular [As]

atomic shells

CN

R

ss2

χ2

− − − − − − − − −

3.0 3.0 3.0 3.0 2.0 3.2 1.7 2.5 3.0

2.29 1.81 1.79 1.74 3.31 1.76 3.33 2.27 1.80

0.003 0.002 0.003 0.008 0.023 ref 6

123 3872 471 21

0.003 0.004

202 1076

As As As As As As As As As

10792

S1 O1 O1 O1 Fe O1 Fe S1 O1

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Figure 3. (A) Fibroblasts viability over a 24 h period determined by the WST-1 assay expressed as a function of the total arsenic (full line) and maghemite (dotted line) concentrations. (B) Viability expressed as a function of the As equilibrium concentration measured by ICP-MS in abiotic DMEMc. Statistical significance (t-test) of the assay: * P < 0.05; ** P < 0.01; *** P < 0.001. The viability data at As concentration higher than 300 μM of As comes from ref 24.

Figure 4. (A) Arsenic K-edge XANES spectra of As in the extra- and intracellular media of human dermal fibroblasts following 24 h exposure to 20 μM of As(III). The experimental data are compared to XANES spectra of reference compounds (As2O3, As2O5, and As2S3). (B) EXAFS spectra at the As K-edge, and (C) their corresponding Fourier transforms of the intra- and extracellular media of fibroblasts exposed to 20 μM of As(III). The structural parameters deduced from the fits are given in Table 1. (D) XANES spectra of As localized in the extra- and intracellular media of fibroblasts following 24 h exposure to 500 μM As(III) and 50 mg/L of Nm coinjected simultaneously ([As]+[Nm]).

in part A of Figure 4. A 1.4 eV shift in the edge is observed between both compartments. In the intracellular medium, the white line is at same position than the As2S3 reference compounds, whereas in the extracellular medium it is at the same position as As2O3. This first observation indicates a strong interaction between As(III) and thiolated molecules within fibroblasts.

From part B of Figure 4, it is noteworthy that the EXAFS oscillation at 5.5−6 Å−1 is negative for As in the extracellular media and the As2O3 reference compound, whereas it is positive for As in the intracellular media and the As2S3 reference. The first peak of the RDF of As in the intracellular compartment corresponds to As atoms surrounded by 2.5 ± 20% sulfur atoms at 2.27 ± 0.02 Å (part C of Figure 4). This is 10793

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in agreement with the first peak of As2S3 RDF for which As is surrounded by 3.0 ± 20% sulfur atoms at 2.29 ± 0.02 Å. For As in the extracellular compartment, the first peak of the RDF corresponds to As atoms surrounded by 3.0 ± 20% oxygen atoms at 1.80 ± 0.02 Å. All our attempts to improve the fit residue by adding As−O−C contributions corresponding to As methylated species failed. Both EXAFS and XANES data highlight that As initially introduced in the extracellular media in the As(III) form is entirely complexed by thiolated molecules inside the cells. Speciation of As(III) Coinjected with Nm in the Fibroblast Medium. To study the effects of Nm on the As biotransformation, As K-edge XANES experiments were performed in the intra- and extracellular compartments of fibroblasts cocontaminated with 500 μM of As and 50 mg/L of Nm ([As]+[Nm]) during 24 h. Using such a high As concentration was the only way to detect and assess the speciation of As in the intracellular compartment using XAS. A 0.3 eV shift on the XANES of As in the extra- and intracellular media is observed. Within the fibroblasts, the white line is centered on the As2O5 reference compounds with a shoulder at the position of the As2O3 edge. In the extracellular medium, an opposite trend appears (part D of Figure 4). Using linear combination of XANES spectra of reference compounds (As2O3, As2O5, As2S3), the As(III)/As(V) ratio were estimated at 90/10 and 50/50 for the extra- and intracellular media, respectively. The addition of As2S3 reference compounds in the linear combination fit does not improve the fit residue. These results highlight that in presence of Nm (i) an oxidation of As(III) into As(V) occurs inside the fibroblasts, and (ii) the As was not complexed by thiolated molecules as observed in absence of Nm.

accounts for more than 90% of the nonprotein thiols31 and is usually present in millimolar concentrations (∼1−10 mM) inside cells.32 Thus, the As-(SR)3 complex observed within cells by XAS is likely to be As-(GS)3. This thiolation of arsenite has long been considered as a detoxification mechanism. It was established that arsenic-resistant cell lines manifest higher levels of GSH and glutathione S-transferase (GST).27 However, thiol groups of proteins can also interact with arsenite. It was previously demonstrated that galectin and thioredoxin peroxidase of the CHO (Chinese Hamster Ovary) fibroblasts33 or human metallothionein34 act as As-binding proteins. Disturbance of the As Biotransformation Processes by Nm. Nm act as efficient As carriers in cell culture medium (Figure 1). Whereas the presence of ligands competing for Fe sites (as PO4 or thiolated compounds) slightly desorbs the As from the surface, between 3.2 to 5 As atoms/nm2 remained attached at the surface of the Nm in our experimental conditions. XAS data show that at high surface coverage, the As−O3 complexes adsorbed at the surface of Nm in DMEMc are surrounded by 2.0 ± 20% Fe atoms at 3.31 ± 0.02 Å (Figure 2). This is in agreement with previous works showing that As(III) is adsorbed onto Nm via reactive tridentate hexanuclear corner-sharing surface complexes at low surface coverage and monodentate trinulear complexes at high surface coverage.6,8 We assessed whether this strong affinity between the Nm surface and As modifies the aggregation states of the Nm in the [AsNm], and the [As]+[Nm] scenarios. Different aggregation states could have affected the dose delivered to the cells, the uptake, and consequently the cytotoxicity profiles. In both scenarios, we did not notice significant difference on the Nm surface charge (zeta potential ∼ −10 mV) nor hydrodynamic diameters (∼950 nm) after 24 h in the abiotic culture medium. Whereas the aggregation state of Nm is not affected by As, the strong surface reactivity of Nm for As in biological media has implications in terms of cytotoxicity. First, our results highlight that the adsorption of arsenic at the surface of Nm is a physicochemical mechanism reducing its cytotoxicity. Indeed, cells exposed to As in the preadsorption/injection [AsNm] scenario are more viable that cells exposed only to As (part A of Figure 3). Moreover, once expressed as function of the As equilibrium concentration, the toxicity obtained in the two scenarios match (part B of Figure 3). These results are corroborated by the weak desorption of As in the abiotic DMEM solution (Figure 1). Then, we demonstrated that the coinjection of Nm and low amounts of As disturbs the As intracellular speciation (Figure 4) and increases the As cytotoxic effects (Figure 3). 100% of As is complexed with SH groups inside cells in absence of Nm, whereas 100% of the As (III and V) is surrounded by O in the first coordination sphere in presence of Nm (Figure 4). It is noteworthy that, to ensure analysis of intracellular As, the As concentrations used in part D of Figure 4 ([As]+[Nm] coinjection experiment) corresponds to a lethal concentration (500 μM). It can be pointed out that the absence of As−SHx in presence of Nm might be due to the strong disturbance of the biological activity for high As doses. However, several studies observed that, even at lethal As concentrations, As binds to GSH. For instance, Munro et al. (2008) showed the predominance of As tris-sulfur species within HepG2 cells treated with 1 mM of As(III) or 20 mM of As(V).29 Delnomdedieu et al. (1994) also observed the binding of

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DISCUSSION Biotransformation of Arsenite in Absence of Nm. All organisms, from bacteria to mammals, exhibit mechanisms to transport and detoxify As(III). A common feature is that As(OH)3 is taken into cells by passive diffusion through aquaporine and then methylated.25 This uptake is cell typedependent and fibroblasts were found efficient compared to other cells.26 From our XAS results, it clearly appears that As introduced initially under As(III) form has been taken up and transformed inside the cells. The presence of 2.5 ± 20% sulfur atoms at 2.25 ± 0.02 Å from As indicated that 80−100% of As is complexed by 3 thiol groups in the intracellular compartment. However, these As-(SR)3 complexes were not observed in the extracellular media. The 3.0 ± 20% oxygen atoms at 1.80 ± 0.02 Å from As indicated that 100% of As remained in a As− O3 reduced state outside the cells with oxygen in the first coordination sphere. The effects of the incubation time and As concentration on its speciation in the extracellular media were assessed by XAS in abiotic DMEMc. Whatever the duration (15 min, 24 h) and/or the initial concentrations (20 μmol/L, 4 mmol/L), the As speciation in DMEMc did not change and matches the As speciation in the extracellular compartment. Given the detection limit of XAS for identifying minor Ascontaining species,27 we estimate that less than 5% of extracellular As can exist under the As-(SR)x form. The observed intracellular As-(SR)3 complexes observed are consistent with previously reported XAS results obtained for As analyzed in earthworms28 or human hepatoma cells.29 This thiolation is part of the arsenic methylation process in which glutathione (GSH) has an absolute requirement.30 GSH 10794

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We are grateful to the machine and beamline groups whose outstanding efforts have made these experiments possible.

As(III) by intracellular GSH for As doses between 0.1 and 0.4 mM.35 The different intracellular speciation of As in presence or not of Nm can be due to competitive effects between As and thiolated compounds (as GSH or proteins) at the Nm surface. Abiotic experiments (Figure 1) show that, for low As concentrations (25 and 50 μM), 16 ± 1% and 30 ± 2% of the Nm surface is covered by As. This leaves 70 to 85% of the Nm surface available to interact with SH groups. However, for 100 and 300 μM of As, only 37 to 41% of the surface is noncovered by As. Consequently, competitive effects between −SH and As are expected to be more intense for low As doses. Moreover, the kinetic of competition of −SH and As for the surface of Nm seems to play a great role when As and Nm are simultaneously added to the cell culture. Such an affinity between SH groups and Nm has been demonstrated for cysteine or dimercaptosuccinic acid. Fe−S bounds (distances 2.21 ± 0.02 Å) are formed at the surface and are never or weakly displaced inside human dermal fibroblasts.16 Using XAS at the Fe K-edge (data not shown), we were not able to detect Fe−S bounds at the surface of the Nm. This is related to the low percentage of Fe atoms localized at the surface of the Nm (20−25%) and the steric effects of the GSH or proteins in solution. Thermodynamics also corroborate the As and Nm competitive effects toward SH groups since the log K for Fe− GSH (log K = 5.1) or the Fe−cysteine (log K = 6.2) complexes36 are much lower than the As(III)−cysteine or As(III)−GSH complexes (log K[As(H−1Cys)3] = 29.8, log K[As(H−2Cys(OH)2)]− = 12.0, log K[As(H−2Gs)3]3− = 32.0, log K[As(H−3GS)|(OH)2]2− = 10.0) 37 or the Fe−O−As surface species (log K[FeH2AsO3] = 38.4 and log K[FeHAsO3−] = 33.0).38 Consequently, even if the magnetic nanoparticles act as efficient contaminant carriers, a Trojan-horse effect has not be observed in our experimental conditions because, once As is adsorbed at the surface, its cytotoxicity is lower than As alone. However, we highlighted that any disturbances of the intracellular biotranformation mechanisms of As by the nanoparticles via adsorption or complexation are likely to be responsible for the increase of the As adverse effects observed at low doses.

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ASSOCIATED CONTENT

S Supporting Information *

(i) TEM of Nm, (ii) XANES and X-ray diffraction at the Fe Kedge of Nm before and after incubation within abiotic DMEMc, and (iii) estimated composition of the DMEMc. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: auff[email protected], phone: +33 442 971 543, fax: +33 442 971 559. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the CNRS and CEA for funding the GDRi iCEINT, and the financial support from the French National Agency. The authors also thank Florence Chaspoul for her help with the ICP-MS analysis. The XAS work was performed on BM30b CRG beamline at the European Synchrotron Radiation Facilities, Grenoble, France. 10795

dx.doi.org/10.1021/es302493s | Environ. Sci. Technol. 2012, 46, 10789−10796

Environmental Science & Technology

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dx.doi.org/10.1021/es302493s | Environ. Sci. Technol. 2012, 46, 10789−10796