Metal homeostasis disruption and mitochondrial ...

Nanoscale

Metal homeostasis disruption and mitochondrial dysfunction in hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles

Journal: Manuscript ID Article Type: Date Submitted by the Author: Complete List of Authors:

Nanoscale NR-ART-07-2016-005306 Paper 04-Jul-2016 Chevallet, Mireille; CEA, BIG/LCBM; CNRS, UMR5249 Gallet, Benoit; CEA, IBS Fuchs, Alexandra; CEA, DRF/BIG Jouneau, Pierre-Henri; CEA Grenoble, INAC /SP2M / LEMMA UM, Khemary; CEA, BIG/LCBM Mintz, Elisabeth; CEA, BIG/LCBM Michaud-Soret, isabelle; Laboratoire de Chimie et Biologie des Metaux,

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Nanoscale’s latest Impact Factor is 7.394 We aspire to even higher values in future years Nanoscale Associate Editors stress very high standards for acceptance in the journal. Articles must report extremely novel, very high quality, reproducible new work of broad general interest. As a referee, our Associate Editors strongly encourage you to recommend only the best work for publication in Nanoscale. Since launch in late 2009, Nanoscale has quickly become a leading journal. We aspire for the journal to publish truly world-class research. Routine, limited novelty or incremental work – even if competently researched and reported - should not be recommended for publication. Nanoscale demands high novelty and high impact. We strongly discourage fragmentation of work into several short publications. Unnecessary fragmentation is a valid reason for rejection. Thank you very much for your assistance in evaluating this manuscript, which is greatly appreciated. With our best wishes, Chunli Bai (Editor-in-Chief) Xiaodong Chen, Serena Corr, Yves Dufrêne, Andrea Ferrari, Dirk Guldi, Xingyu Jiang, RongChao Jin, Yamuna Krishnan, Jie Liu, Xiaogang Liu, Wei Lu, Francesco Stellacci, Shouheng Sun, Jianfang Wang, Hongxing Xu, Xiao Cheng Zeng (Associate Editors) General Guidance (For further details, see the Royal Society of Chemistry’s Refereeing Procedure and Policy) Referees have the responsibility to treat the manuscript as confidential. Please be aware of our Ethical Guidelines which contain full information on the responsibilities of referees and authors. When preparing your report, please: • Comment on the originality, importance, impact and scientific reliability of the work; • State clearly whether you would like to see the paper accepted or rejected and give detailed comments (with references) that will both help the Editor to make a decision on the paper and the authors to improve it; Please inform the Editor if: • There is a conflict of interest; • There is a significant part of the work which you are not able to referee with confidence; • If the work, or a significant part of the work, has previously been published, including online publication, or if the work represents part of an unduly fragmented investigation. When submitting your report, please: • Provide your report rapidly and within the specified deadline, or inform the Editor immediately if you cannot do so. We welcome suggestions of alternative referees. If you have any questions about reviewing this manuscript, please contact the Editorial Office at [email protected]

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To Professor Yves Dufrêne, Associate Editor July 04th, 2016

Dear Sir,

Please find attached the electronic copy of a manuscript that we would like to submit for publication in Nanoscale: Metal homeostasis disruption and mitochondrial dysfunction in hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles M. Chevallet, B. Gallet, A. Fuchs, P.H. Jouneau, K.Um, E. Mintz, I. Michaud-Soret All the authors have seen and approved the submission of the manuscript, which is original and exclusively submitted to Nanoscale. Our group has expertise in metal ion homeostasis and is interested in understanding the effects of inorganic nanoparticles on metal homeostasis in human cells. We have recently published in Nanoscale an article describing the early effects of copper oxide nanoparticles on hepatocytes. This work was continued by the study of effects of zinc oxide nanoparticles on the same cell line. Interestingly we found that it involved a very different toxicity mechanism. As molecular pathways involved in ZnO-NP toxicity are still under debate, we believe this work will attract interest from Nanoscale readers. We chose to work at sub-toxic doses and to focus on early effects on metal homeostasis and redox balance. At these sub-toxic doses, massive dissolution of ZnO-NPs occurred together with accumulation of zinc ions inside the cell as shown by ICP-AES measurements and high resolution TEM coupled with EDX. Gene expression analysis highlighted zinc homeostasis disruption with only a minor implication of ROS generation. Thorough TEM analyses coupled with more classical approaches showed that mitochondria and autophagy are involved in cell survival to ZnO-NPs. We are convinced that our novel findings obtained in sub-toxic conditions are of general interest for the broad readership of Nanoscale. We hope you will share our view and consider publication of our manuscript. Please do not hesitate to contact the corresponding authors at any time. Sincerely yours,

Mireille Chevallet LCBM, CEA-Grenoble. 17, rue des Martyrs, 38054 Grenoble, France. E-mail: [email protected] Phone: +33 4 38 78 29 64 Fax: +33 4 38 78 54 87

Isabelle Michaud-Soret LCBM, CEA-Grenoble. 17, rue des Martyrs, 38054 Grenoble, France. E-mail: [email protected] Phone: +33 4 38 78 99 40 Fax: +33 4 38 78 54

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Journal Name ARTICLE Metal homeostasis disruption and mitochondrial dysfunction in hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles Received 00th January 20xx, Accepted 00th January 20xx

M. Chevalleta, B. Galletb, A. Fuchsc, P.H. Jouneaud, K.Uma, E. Mintza, I. Michaud-Soreta

DOI: 10.1039/x0xx00000x www.rsc.org/

Increased production and use of zinc oxide nanoparticles (ZnO-NPs) in consumer products has prompted the scientific community to investigate their potential toxicity, and understand their impact on the environment and organisms. Molecular mechanisms involved in ZnO-NP toxicity are still under debate and focus essentially on high dose expositions. In our study, we chose to evaluate the effect of sub-toxic doses of ZnO-NPs on human hepatocytes (HepG2) with a focus on metal homeostasis and redox balance disruptions. We showed massive dissolution of ZnO-NPs outside the cell, transport and accumulation of zinc ions inside the cell but no evidence of nanoparticle entry, even when analysed by high resolution TEM microscopy coupled with EDX. Gene expression analysis highlighted zinc homeostasis disruptions as shown by metallothionein 1X and zinc transporter 1 and 2 (ZnT1, ZnT2) over-expression. Major oxidative stress response genes, such as superoxide dismutase 1, 2 and catalase were not induced. Phase 2 enzymes in term of antioxidant response, such as heme oxygenase 1 (HMOX1) and the regulating subunit of the glutamate-cysteine ligase (GCLM) were slightly upregulated, but these observations may be linked solely to metal homeostasis disruptions, as these actors are involved in both metal and ROS responses. Finally, we observed abnormal mitochondria morphologies and autophagy vesicles in response to ZnO-NPs, indicating a potential role of mitochondria in storing and protecting cells from zinc excess but ultimately causing cell death at higher doses.

1 Introduction Zinc is an essential trace element. Not only does it act as a cellular ionic signal, but in thousands of proteins, zinc participates in enzymatic catalysis, structural organization, and/or regulation of function. Zn2+ is a non-redox metal ion but it is involved in the redox equilibrium of the cell by binding to thiolate containing sites with a very high affinity.1 In both its ionic and protein-bound form zinc is at the crossroads of all crucial decisions in the life of mammalian cells: cell growth and proliferation, differentiation or programmed cell death.2 Maintenance of zinc homeostasis is essential and deficiency as well as excess zinc is toxic for organisms and cells. For example, deregulation of free zinc has been implicated in the formation of -amyloid plaques associated with Alzheimer’s disease.3-4

a. (1)

CNRS, Laboratoire de Chimie et Biologie des Métaux (LCBM), UMR 5249, Grenoble, France. Email: [email protected] (2) CEA, BIG, LCBM, Grenoble, France. Email: [email protected] (3) Université Grenoble Alpes, LCBM, Grenoble, France. b. (1) Université Grenoble Alpes, IBS, Grenoble, France. (2) CNRS, IBS, Grenoble, France. (3) CEA, IBS, Grenoble, France. c. (1) CEA, BIG, DIR, Grenoble, France. d. (1) CEA, INAC, Minatec campus, Grenoble, France. (2) Université Grenoble Alpes, INAC-MEM-LEMMA, Grenoble, France. Electronic Supplementary Information (ESI) available: See DOI: 10.1039/x0xx00000x

To complicate things further, zinc at physiological concentrations has also been shown to be cytoprotective as a pro-antioxidant.4 The large number of proteins that are potentially dedicated to Zn2+ transport (ZnT and ZIP families) and buffering (metallothioneins) reflect the complexity and importance of zinc homeostasis.5-6 The ZnT transporters are dedicated to Zn 2+ efflux from the cytoplasm to the extracellular medium or to organelles. Zip transporters control Zn2+ import from the extracellular medium or organelle lumen into the cytoplasm. 7 Although the up-regulation of Zn2+ chelation and transport machineries appears to be directly activated by Zn2+ binding to the transcription factor MTF-1,8 there is still much to decipher in the mechanisms governing zinc homeostasis. Due to their specific physical properties linked to surface and quantum size effects,9 nanoparticles (NPs) are finding their way into a wide range of consumer products, including food packaging. This increased exposure of all living beings and their environment to NPs is driving concerns about their potential adverse impact on the environment and our well-being. Over the last decade, considerable scientific effort has been invested in research to better understand the risks of exposure to NPs.1011

ZnO nanoparticles (ZnO-NPs) are among the top5 nanoparticles produced in high tonnage (estimated global annual production as of 2010 is >30,000 tons)12 and are used in commercial

This journal is © The Royal Society of Chemistry 20xx

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applications such as antibacterial coatings or as UV absorbers in sunscreens and textiles.13-14 The literature on mammalian toxicity of ZnO-NPs published between 2009 and 2011 was recently summarized in a review. 15 In vivo studies explored the main exposure means and showed a potential risk for mammals. Through airway exposure in rats, ZnO-NPs elicited an increased Zn content in multiple organs and in particular in the liver where it was sufficient to cause damage as shown by histopathological examination.16 In skin applications, while several reviews have concluded that metal oxide NPs do not penetrate the stratum corneum,17 Adamcakova-Dodd et al.18 demonstrated that small amounts of Zn from ZnO-NPs in sunscreens can pass through the skin’s protective layers and be detected in blood and urine. Oral administration of ZnO-NPs also resulted in zinc accumulation in the liver and other organs.19 In a majority of published studies, incomplete information (or characterization) of the NPs themselves and in some cases the lack of information or studies on ion release and NP uptake by cells make it difficult to compare results. In vitro studies on different cell lines showed consensual results: ZnO-NPs are in all cases cytotoxic via the release of Zn2+ ions, reactive oxygen species (ROS) production, and mitochondria dysfunction. Some studies also revealed a genotoxic effect of ZnO-NPs.20 Others have demonstrated that autophagy is activated21-22 as well as inflammation via IL8 production23. In hepatocytes, an alteration of albumin production was highlighted23 and an important disruption in energy metabolism with an increase in both gluconeogenesis and glycogenolysis was described.24 Moreover, most studies focused solely on NP toxicity at high and toxic doses using classical tests to measure cytotoxicity, ROS production, and DNA damages without addressing the specific intracellular mechanisms and pathways related to metal homeostasis. This fact prompted us to investigate the effects of ZnO-NP exposure on metal homeostasis regulation, taking into account its particulate or ionic nature in situ in human liver-derived cells (HepG2), as the liver is the primary target for concentrating and metabolizing toxic agents. To take into account toxic effects that could be linked to the surface charge - anionic or cationic - of the nanoparticles, we chose to work with 2 types of ZnO nanoparticles, namely foetal bovine serum (FBS) -coated ZnO-NPs with a negative surface charge and silane-coated ZnO-NP with a positive surface charge. We compared the effects of these two types of characterized ZnONPs with the effects of the soluble form of zinc, using zinc acetate, and focused on the early response of metal homeostatic control and oxidative stress genes to sub-toxic doses of zinc (i.e. conditions that do not induce cell mortality). Our results clearly demonstrate no specific nano effect of the ZnO-NPs in contrast to what we previously observed with CuONPs.25 We demonstrate however that sub-toxic doses of both ionic and nanoparticulate forms of zinc induce a metal stress response with zinc homeostasis disruption and mitochondria alterations, even after short exposure times.

2 Experimental 2.1 Nanoparticles ZnO-NPs with a primary particle size of less than 100 nm were purchased from Sigma-Aldrich: an uncoated ZnO-NP powder (ref number 721077) and an aminopropyl-silane-coated ZnO-NP dispersion (ref number 544906). The uncoated ZnO-NPs were dispersed at 50 mg/mL in 50% FBS by sonication for 30 min by “1s on-1s off” pulses at 60% amplitude (750 W) in a cup-horn instrument (Vibra cell, BioBlock Scientific, France) under water cooling thermostated at 4°C to get stable and reproducible dispersion of NPs and to avoid rapid aggregation (denoted ZnONP-FBS). The ZnO-NP-silane were diluted directly in water at 50 mg/mL. The actual size of the particles was determined after dilution in water or in complete culture medium by dynamic light scattering, using a Wyatt Dynapro Nanostar instrument. A Malvern instrument Zetasizer plus was used to determine the zeta potential. The size and shape of NPs were verified by STEM on a Hitachi S5500. 2.2 Cell culture HepG2 cells were grown in modified Eagle’s medium (MEM) supplemented with 10% v/v FBS, 20 mM L-glutamine, 10 mM sodium pyruvate, 100 µg/mL streptomycin and 100 U/mL penicillin. Cells were cultured at 37°C in a humidified atmosphere with 5% CO2. 2.3 Cell viability Cells were seeded at 300 000 cells/mL in 12-well plates. They were treated with zinc acetate or ZnO-NPs on the following day and harvested after a further 24 h. Cell viability was evaluated by counting Trypan Blue stained cells in a TC20 Automated Cell Counter (BioRad). 2.4 ICP-AES Analysis of zinc was performed by inductively coupled plasma atomic emission spectroscopy (ICP-AES: ICPE-9000 from Shimadzu Scientific Instruments). HepG2 cells were treated with Zn acetate or ZnO-NPs for 6h, 24 h and 48 h or kept in complete culture medium as control. Briefly, after removing the medium and washing with PBS buffer, cells were collected using trypsin, centrifuged at 1,200 rpm for 5 min and suspended in PBS buffer. Cells were then counted using a TC20 Automated Cell Counter (BioRad) and centrifuged again. The pellets were suspended with 100 µL of pure nitric acid and mineralized overnight at 95° C in a DigiPrep (SCP Science). The internal standard Ytterbium was added and the samples were diluted in pure water qs 6.5 mL prior to analysis. A standard curve was performed using an atomic absorption standard solution of zinc (Sigma-Aldrich). To follow zinc dissolution, 90 and 900 µM zinc acetate and ZnONPs were incubated in water or in complete culture medium in cell culture plates. The plates were kept for 24 h in a cell culture incubator at 37°C and 5 % CO2. The media were retrieved at different times, filtered at 0.1 µm (PVDF, Merck Millipore LTD) and centrifuged at 150,000 g for

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45 min to sediment the ZnO-NPs. The concentration of zinc ions in the supernatant was then measured by ICP-AES. 2.5 Real-time PCR assays & measurements HepG2 cells were harvested and RNA was isolated using Absolutely RNA miniprep kit (Agilent # 400800). RNA concentration was determined using a NanoDrop spectro photometer (ND-1000). Reverse transcription was performed with the Affinity script qPCR cDNA synthesis kit (Agilent # 600559), according to the manufacturer’s instructions. Gene specific primers for MET1X were taken from.26 The other human primers were designed using DNASTAR or Primer-Blast. The designed primers are given in Table S1. Quantitative PCR was performed with Brilliant II SYBR green qPCR master mix1 (Agilent # 600828) using the primers at 200 nM. PCR conditions (primer concentrations, cDNA quantity) were optimized and PCR efficiency was determined for each target gene. PCR reaction mixtures (10 µL) were placed in the Cfx96 instrument (Bio-Rad) where they underwent the following cycling program, optimized for a 96-well block: 95°C for 15 min, immediately followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. At the end, PCR products were dissociated by incubating for 1 min at 95°C and then 30 s at 55°C, followed by a ramp up to 95°C. PCR quality and specificity were verified by analysing the dissociation curve. For each set of primers, a notemplate control (NTC) and a no-reverse-amplification control (NAC) were included. qRT-PCR reactions were run in triplicate, and quantification was performed using comparative regression (Cq determination mode). Quantitative PCR data were comparatively analysed using the Cfx software (Bio-Rad Cfx manager) with 36B4 and hHPRT amplification signals as internal “normalizers” (to correct for total RNA content). Results are expressed as the relative change in expression compared to the control. Results were the average (+/- SEM) of at least 3 independents experiments. 2.6 Electron microscopy Monolayers of HepG2 cells were fixed overnight at room temperature in a 1:1 ratio mixture of 4% paraformaldehyde, 0.4% glutaraldehyde in 0.2 M PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2) pH 7.2 and culture medium, washed in 0.1 M PHEM pH 7.2, and fixed for 30 minutes in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1M PHEM pH 7.2, washed in 0.1 M PHEM pH 7.2 and post-fixed in 1% OsO4, 1.5% potassium ferrocyanide in 0.1 M PHEM buffer for 1 h at room temperature. After 3 washes in water, post-staining was done using 0.5% uranyl acetate in 30% ethanol for 30 min at room temperature in the dark. Cells were then dehydrated in graded ethanol series, and flat-embedded using the Epoxy Embedding Medium kit (Sigma-Aldrich). Ultrathin sections (80 nm) were cut on a Leica UC7 ultra-microtome using a DiATOME 35° diamond knife and collected on formvar carbon coated 100-mesh copper grids. Sections were stained in 5% uranyl acetate in water for 10 min and in 2% lead citrate for 5 min. Images were taken on a Tecnai G2 Spirit BioTwin (FEI) at 120 kV using an ORIUS SC1000 CCD camera (Gatan).

3 Results and discussion 3.1 Nanoparticle characterization Dynamic light scattering was used to characterize the nanoparticle populations, by their hydrodynamic diameter distribution (as % of intensity). ZnO-NP-FBS had a diameter of 237 nm (Fig. S1) and a polydispersity index of 20%, ZnO-NPsilane presented a diameter of 79 nm and a polydispersity index of 19%. The zeta potentials were measured in 1mM KCl 27-28 and were of -25 mV for the ZnO-NP-FBS and 19.3 mV for the ZnONP-silane. The albumin coating accounts for the negative surface charge at physiological pH and the silane cationic coating accounts for the positive surface charge of these NPs. STEM images of coated NPs diluted in water confirmed the size of the particles (Fig. 1), the ZnO-NP-FBS were rod-shaped while the ZnO-NP-silane were more spherical. 3.2 HepG2 viability studies ZnO-NP toxicity was first studied by measuring the viability of HepG2 cells after 24 h incubation with either zinc acetate or ZnO-NP-FBS or ZnO-NP-silane in a range of concentrations from 0 to 300 µM equivalent zinc (Fig. 2). The toxicity appeared to be similar among the different forms of zinc, with a slightly higher toxicity of ZnO-NP-silane. The shape of the viability curves showed that HepG2 cells could tolerate 150 µM of zinc for 24 h without loss of viability but that beyond this dose, viability decreased drastically. These dose tolerances are in agreement with the literature for HepG2 cells29 and other cell types such as macrophages.30 In order to decipher the response of the cells, the expression of genes involved in metal homeostasis and oxidative stress was followed. 3.3 Early responses of metal homeostatic control and oxidative stress genes For this gene expression study we chose a sub-toxic dose of 90 µM offering an optimum compromise between viability and biological effect.

Fig. 1 Characterization of ZnO-NPs. STEM images of ZnO-NP-FBS (a) and ZnO-NPsilane (b) taken on a Hitachi S5500.


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Fig. 2 Cell viability after a 24 hour incubation with zinc acetate (), ZnO-NP-FBS (), or ZnO-NP-silane (). The arrow shows the concentration (90 µM or 7.3 µg/mL) chosen for the gene expression studies.

Indeed, at higher but still sub-toxic concentrations (150 µM), gene expression of usual internal normalizers showed too much variability, a phenomenon which is explained by the proliferating effect of zinc at this dose. 31 In order to capture early responses triggered by zinc, we also reduced the exposition to 6 hours, a condition where no cell death was yet measurable, even at concentrations as high as 300 µM (data not shown). The responses of HepG2 cells to a 6 h incubation with 90 µM zinc, brought either by zinc acetate, ZnO-NP-FBS or ZnO-NPsilane were studied by quantitative RT-PCR. For oxidative stress, we chose the classical genes CAT (the catalase), SOD1 (the cytoplasmic Cu, Zn super-oxide dismutase), SOD2 (the mitochondrial Mn super-oxide dismutase) and GCLM, the regulating subunit of the glutamate-cysteine ligase which is the first rate-limiting enzyme of glutathione (GSH) synthesis. We also added HMOX1, the inducible isoform of heme oxygenase which can act as an antioxidative protein and was found to be induced by ZnO-NP stress in colon and skinderived cell lines32-33 and by CuO-NP exposure in both macrophages34 and HepG2.25 For zinc homeostasis35 we chose MTF1, the metal regulatory element-binding transcription factor; Met1X, representing the metallothionein gene family; as well as various zinc transporters: ZnT1, the plasma membrane zinc exporter, ZnT7, involved in zinc storage in the Golgi apparatus and Zip1 the main plasma membrane zinc importer. For iron homeostasis we looked at HAMP, the gene encoding hepcidin, a cysteine-rich protein which controls iron homeostasis.36 Finally, we added the chaperone HSPA6, member of the HSP 70 family which responds to various stresses and has been shown to respond to CuO-NPs in HepG2 cells;25 and EGR1, an early response transcription factor which is

induced by a broad spectrum of extracellular signals, including growth factors, cytokines and environmental stress. EGR1 is known to be a tumour suppressor that plays a role in cell growth, differentiation and cell survival.37 For all treatments, the highest induction was observed for Met1X, which was up-regulated around 50-fold (Fig. 3). Metallothioneins, ZnT1 and ZnT7 gene expressions are known to be activated via MTF1.38 MTF1 functions as a cellular zinc sensor, which in the presence of excess zinc migrates to the nucleus and activates genes involved in zinc homeostasis, as well as in protection again metal toxicity, via binding to MREs (metal-response elements). Metallothioneins are cysteine-rich proteins which function as cytoplasmic soft-metal chelators that bind zinc with high affinity.39 They are primarily considered as an important defence mechanism against metal poisoning. 40 We also found a 4-fold increase in the expression of ZnT1 which is involved in exporting excess zinc but no change in ZnT7 which is involved in zinc storage, nor in Zip1 which is involved in zinc entry. These results are in agreement with previous work of Cousins et al.41 in THP1 cells treated with 40 µM ZnSO4 showing a 3-fold increase in ZnT1 expression and no significant variation for ZnT7 and Zip1. We found no evidence in the literature of regulation of Zip1 via MTF1. In HEK293 cells, it was shown that Zip1 is not regulated at the transcript level but rather posttranslationally by altering the protein subcellular distribution. 42 Finally, there was no increase in the expression of MTF1 showing that zinc excess induces the translocation of MTF1, and does not require de novo synthesis under our conditions. Although zinc homeostasis is linked to other metal homeostasis, such as that of copper and iron, Hepcidin, a hormone involved in iron metabolism and itself regulated via MTF1 8,43 also showed no variation in expression in our conditions. On the oxidative stress side, we found no modification of the major enzymes dedicated to detoxification of ROS. CAT, SOD1 and SOD2 are expressed constitutively and we can postulate that cells are able to cope with a “moderate” oxidative stress without requiring additional induction of the corresponding genes. However we showed 3.5-4.5 fold inductions for HMOX1 and GCLM which are classified as phase 2 enzymes in term of antioxidant response, and are regulated by the transcription factor Nrf2 via ARE, the antioxidant response element.44 Interestingly, GCLM was also described to be regulated by MTF1.45 In light of these results, it remains unclear if 90 µM zinc induces an oxidative stress or solely a metal homeostasis disruption. As such, metal and oxidative stress responses are tightly imbricated. On the one hand, GSH which is present in millimolar concentrations in cells, binds large amounts of zinc in vitro and on the other hand metallothioneins can function as antioxidants.1,5 HMOX1 cleaves the heme ring to form biliverdin and then bilirubin, known to chelate metals.46 Moreover, although zinc in biology is redox inert, in contrast to copper or iron, zinc excess can indirectly influence the redox balance. 1 The chaperone HSPA6 was slightly induced in particular with ZnO-NP-silane. Chaperones of the HSP70 family are activated by several heavy metals such as Cd2+ and Zn2+ and the heat shock element (HSE) has been proposed to mediate the responses to metal ions via heat shock factor 1 (HSF1) activation.47

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Fig. 3 Quantitative PCR analysis of mRNA expression in HepG2 cells after a 6 hour incubation with Zn acetate, ZnO -NP-FBS or ZnO-NP-silane at 90 µM (Note the 10-fold change in scale of the right panel). Results are presented as relative expression changes after normalizing with HPRT and 36B4 mRNA. Each value represents the mean of relative expression +SEM from three independent experiments. *: p