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Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions† Martine Cuillel,abc Mireille Chevallet,abc Peggy Charbonnier,abc Caroline Fauquant,abc Isabelle Pignot-Paintrand,de Josiane Arnaud,fg Doris Cassio,hi Isabelle MichaudSoret*abc and Elisabeth Mintz*abc Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated the early responses to sub-toxic doses of CuO-NP and compared them to equivalent doses of Cu added as salt to see if there is a specific nano-effect related to Cu homeostasis in hepatocytes. The expression of the genes encoding the Cu-ATPase ATP7B, metallothionein 1X, heme oxygenase 1, heat shock protein 70, superoxide dismutase 1, glutamate cysteine ligase modifier subunit, metal responsive element-binding transcription factor 1 and zinc transporter 1 was analyzed by qRT-PCR. These genes are known to be involved in response to Cu, Zn and/or oxidative stresses. Except for MTF1, ATP7B and SOD1, we clearly observed an up regulation of these genes expression in CuO-NP treated cells, as compared to CuCl2. In addition, ATP7B trafficking from the Golgi network to the bile canaliculus membrane was observed in WIF-B9 cells, showing a need for Cu detoxification. This shows an increase in the intracellular Cu concentration, probably due to Cu

Received 20th September 2013 Accepted 20th November 2013

release from endosomal CuO-NP solubilisation. Our data show that CuO-NP enter hepatic cells, most probably by endocytosis, bypassing the cellular defence mechanism against Cu, thus acting as a Trojan

DOI: 10.1039/c3nr05041f

horse. Altogether, this study suggests that sub-toxic CuO-NP treatments induce successively a Cu

overload, a Cu–Zn exchange on metallothioneins and MTF1 regulation on both Cu and Zn homeostasis.

1. Introduction Copper is an essential element in all living cells as a cofactor of many enzymatic reactions; to full this function at catalytic sites, copper must be ionic and cycle between Cu(I) and Cu(II), the two redox states favoured by the intracellular environment. However, these free metal ions can be the source of radical oxygen species;1 therefore, copper homeostasis is tightly regulated by a series of proteins that exchange the ion to bring it safely to its nal place, i.e. catalytic sites of various

cuproenzymes (Scheme 1), avoiding accumulation of intracellular free ionic copper and toxicity.2,3 Since the 1990s, the rapid expansion of nanotechnology has led to massive production of engineered nanoparticles (NP), as


CEA, iRTSV, LCBM, 38054, Grenoble, France. E-mail: [email protected]; elisabeth. [email protected]

b c

CNRS, UMR 5249, 38000, Grenoble, France

Univ. Grenoble Alpes, UMR 5249, 38000, Grenoble, France


INPGrenoble, IMBM, LMGP, 38016, Grenoble, France


CNRS, UMR 5628, 38016, Grenoble, France


CHU de Grenoble, IBP, UBNH, 38000, Grenoble, France


INSERM, U1055, 38000, Grenoble, France


INSERM, UMR S757, 91405, Orsay, France


Univ. Paris 11, UMR S757, 91405, Orsay, France

† Electronic supplementary 10.1039/c3nr05041f



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Scheme 1 Copper homeostasis in hepatocytes. Cu(I) enters through hCTR1 (copper transporter 1), binds to GSH (glutathione) and/or to MET1 (metallothionein 1), and is transferred to 3 cuproenzymes: CCO (cytochrome c oxidase), SOD1 (superoxide dismutase 1) and CP (ceruloplasmin) that is secreted into the blood circulation.

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opposed to naturally occurring ultrane particles, both denominations referring to particles with at least one dimension smaller than 100 nm.4 Such a small size promotes reactions with cells with a potential toxicity that depends on their chemical composition and this has prompted the new nanotoxicology eld.5,6 Of major concern for the society is the potential toxicity of widespread inorganic NP.7 The question of an interference of copper NP with metal homeostasis is tackled in the present work. Indeed, copper is used worldwide for various industrial purposes such as electrical wires, electronics, construction, transportation8 and recently, its antiseptic properties have been the matter of new uses in hospitals.9 Copper nanoparticles have industrial applications in gas sensing,10 high-performance batteries,11 water cleanup12 and also as bactericides.13 Tons of Cu-NP, including their oxidation products Cu2O-NP and CuO-NP,14 are produced that will eventually be released into the environment, perturbing the soil and aquatic organisms15–19 and possibly inducing long-term health problems in the human population.20,21 In vivo studies report inammation of organs, including stomach, lung, brain, kidney, spleen and liver.22–28 Interestingly, in rodents, the worst effects were found at the liver, whether the Cu-NP were nasally instilled or eaten.27,28 Cu- and CuO-NP induced toxicity has also been studied in various cell lines29–40 where oxidative stress,33 inammation41 or genotoxicity42 was elicited, at least in epithelial cells. CuO-NP are susceptible to dissolution and a few studies on rodents compare the effects of NP to those of copper salt.22,27 The toxicity of CuO-NP has also been compared in vitro to that of copper ions that appear from their dissolution in the culture medium or to that of added copper salt.34,43 These studies show that Cu- and CuO-NP are more toxic than the dissolved metal ions, suggesting that the nano-structure plays a role on its own. The size, the shape and the dispersion of the NP in biological media are also important built-in features determining their toxicity.36–39,43–45 Based on these data and bearing in mind that the effects of copper ions have been observed independently on various cells and tissues,46–49 we decided to study the effects of CuO-NP on metal homeostasis in hepatocytes. To this end, we focused on the early responses of cells to sub-toxic doses of CuO-NP (i.e. conditions that do not induce cell mortality), using two different lines derived from hepatocytes, HepG2 cells for their common use in in vitro toxicology and WIF-B9 cells for their remarkable ability to adopt the complex polarity of hepatocytes in vivo.50 Altogether our data bring the rst demonstration that metal oxide nanoparticles such as CuO-NP interfere not only with copper homeostasis, as revealed by the need for detoxication by ATP7B, but also with zinc homeostasis, as revealed by up regulation of ZnT1. This work supplements the recent work on HepG2 cells that reports early oxidative stress and pro-inammatory responses at sub-toxic doses, i.e. 10 mg mL 1 for 6 h,39 and reinforces the idea that the nanoeffect is here due to a Trojan horse mechanism, as proposed very recently.51

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2. 2.1

Experimental CuO-NP preparation

For industrial purposes, a lot of efforts have been put on Cu-NP preparation from water dissolved CuSO4 and interestingly enough, polyvinylpyrrolidone (PVP), a non-ionic surfactant that binds Cu2+, was found useful to control the size and the dispersion of the Cu-NP population52,53 and to protect it against oxidation.54 In addition, PVP is widely used as a pharmaceutical excipient55 and is considered as a non-toxic polymer.56 PVP could therefore be used as a surfactant to prevent agglomeration of the CuO-NP in the culture medium. The CuO-NP used in this work ( 7). * p < 0.05, ** p < 0.005 versus control, ANOVA, Tukey's test.

Scheme 2

CuO-NP disturb Cu and Zn homeostasis.

salt,47 for 4 or 8 h incubations of HepG2 cells (Fig. S3†). Namely, MET1X, HMOX1, and HSPA6 were up regulated (2- to 30-fold), whereas GCLM and ZnT1 mRNA increase was hardly signicant (less than 2-fold) and no change was observed for MTF1, SOD1 or ATP7B mRNA, even at 250 mM, at variance with previous data on zebrash hepatocytes.29 A specic production of metallothionein has been observed in HepG2 cells in response to a 48 h incubation with 300 mg mL 1 (or 470 mM) copper.49 The increase in MET1X mRNA aer 6 h observed here conrms that the phenomenon starts early,46 even at 60 mM (3.8 mg mL 1). As regards the effects of CuO-NP, there was at 250 mM a clearcut up regulation of the transcription of MET1X, HMOX1, HSPA6, GCLM and ZnT1, to 38-, 18-, 15-, 3.5- and 2.8-fold, respectively. However, CuO-NP had no effect on MTF1, SOD1 or ATP7B, neither at 60 mM, nor at 250 mM, at variance with previous data on zebrash hepatocytes.29 MET1X, HMOX1 and HSPA6 were also found to respond to 60 mM CuO-NP. The up regulation of HMOX1 expression under these conditions conrms the recent observation that the amount of the heme oxygenase 1 protein has increased aer 6 h in contact with 20 mg mL 1 CuO-NP (or 315 mM equivalent copper).39 HSPA6 up regulation was further checked by proteomic analysis of HepG2

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cells aer 24 h in 250 mM CuO-NP which revealed a spot corresponding to the HSP70 protein (data not shown). Interestingly, the increase in both MET1X and ZnT1 mRNA shows that copper interferes with zinc homeostasis, especially as it is brought by CuO-NP. Interestingly, there was no increase in the expression of ATP7B aer 6 h incubation. This is in agreement with previous data showing that copper induced trafficking of ATP7B does not require de novo synthesis.66 Combination of our data and those of the literature on HepG2 cells46,47 shows that the effect of CuO-NP is systematically higher than that of the copper salt at equivalent copper concentration, whether the responses are measured at 4–6 h or 24 h (Fig. S3†). This is consistent with the idea that copper is being delivered inside the cells by a Trojan horse mechanism which bypasses the cell defence.

4. Conclusions When copper increases in hepatocytes, two phenomena occur rapidly (Scheme 1). One is at the plasma membrane, where vesicles are formed that internalize hCtr1, thereby stopping copper entry;71 the other one is at the trans-Golgi network, where vesicles are formed that move towards the canalicular membrane. Thanks to the Cu-ATPase ATP7B, these vesicles are lled with copper that will eventually be excreted into the bile (Fig. 4).72 Among the genes chosen to follow the responses of HepG2 cells, ve were up regulated, especially as copper was in the CuO-NP state. The following events could occur that would tentatively explain the results, as shown in Scheme 2: (1) endocytosis delivers CuO-NP to endo/lysosomes where dissolution occurs; (2) disruption of lysosomes induces copper ion overload in the cell; (3) Cu(II) is reduced by GSH, which is oxidised into GSSG;

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therefore more GSH is needed and more glutamate cysteine ligase (GCLM);73 (4) Cu(I) is stabilized by forming Cu2GS5 complexes74 and displaces Zn(II) from constitutive Zn-bound metallothioneins;75 (5) dissociated Zn(II) ions bind MTF1;76 and (6) MTF1 migrates to the nucleus, binds to the metal responsive elements of MET1X and ZnT1 promoters, up regulating their expression among other MRE-regulated genes.77 Reinforcing this description are the following points, mainly concerning Zn homeostasis and the homeostatic role of metallothioneins. MTF1 is constitutively active,78 it is a Zn-nger protein that binds MRE,76 and there is no need for its own up regulation under the mild conditions studied here. However, Zn was found to be the only metal ion promoting the binding of MTF1 to MRE in vitro,79 so that in our experiments, copper cannot be responsible for MET1X and ZnT1 up regulation by itself, although this was previously proposed in other cells.78 The contradiction was only apparent and has been explained later by a role for metallothioneins between copper and MTF1.75 Indeed, Cu(I) has a higher affinity for thiols than Zn(II), so that when it becomes available in the cell, the homeostatic Zn(II)bound metallothioneins release Zn and Zn activates MTF1 which in turn up regulates MET1X and ZnT1. In addition, HMOX1 is known to be a highly inducible gene that responds to many stresses, among them is an imbalance in redox homeostasis80 which could come from the imbalance of the apothionein–metallothionein equilibrium.81 As regards oxidative stress, there was no up regulation of SOD1, the enzyme involved in detoxication of reactive oxygen species, showing that these sub-toxic conditions are mild oxidative stress conditions. Finally, although very little is known about what induces HSPA6 expression, it is known to respond to intracellular Zn increase.82 Hepatocytes react to sub-toxic amounts of CuO-NP by detecting intracellular copper increase. Genes involved in copper and zinc homeostasis and cell stress are up regulated in response to CuO-NP. Interestingly, their up regulation can be tentatively explained by an increase in intracellular zinc that could be displaced from Zn(II)-bound metallothioneins by Cu(I), the reduced form of copper ions that are released from CuO-NP by dissolution. Altogether our data demonstrate for the rst time the interference of CuO-NP with metal homeostasis under sub-toxic conditions. Because they enter the cells by endocytosis, a Trojan-horse like mechanism bypassing the cell defences, CuONP can be foreseen as a copper pool that could induce long-term toxicity despite the adaptation of the cells described here. Interestingly, the lowest concentration used here, 60 mM or 3.8 mg mL 1, is only about 4-fold the physiological range for copper in human serum, 11–22 mM or 0.7–1.4 mg mL 1. Since the level of MET1X mRNA is very sensitive to CuO-NP, we could try to use it as a biomarker to determine the lowest concentration of NP that would affect it for short times and also for longer times to mimic chronic exposures. Taking a 2-fold increase in Met1X mRNA as a criterion for non-negligible biological effects, we could try to measure in the future a threshold for exposition to these NP in terms of concentration and time duration. The follow-up of CuO-NP in hepatocytes submitted to low-dose chronic exposure should also be performed to measure the

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intracellular CuO-NP lifetime and to see if hepatocytes can recover a basal copper status.

Acknowledgements We thank Cl´ ement Levin and Wael Traboulsi who both participated as undergraduate students at the beginning of the programme and Am´ elie Harel for efficient technical assistance for quantitative RT-PCR. We acknowledge the CEA Toxicology Program for the stimulating environment provided and for the nancial support (NanoBioMet and NanoStress project grants); the other members of these projects are thanked for their fruitful discussions. We also acknowledge the SERENADE LabEx and the ARCANE LabEx to whom we are associated. We are grateful to Ignacio Sandoval (CIBEREHD, Madrid, Spain) for his generous gi of an anti-ATP7B polyclonal antibody and to Caroline Desvergnes and the LABM at CEA-Grenoble for some ICP-MS analyses.

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Nanoscale, 2014, 6, 1707–1715 | 1715

Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions.

Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disr...
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