Titanium dioxide nanoparticles induce proteostasis disruption and autophagy in human trophoblast cells

Accepted Manuscript Titanium dioxide nanoparticles induce proteostasis disruption and autophagy in human trophoblast cells Yuqing Zhang, Bo Xu, Mengmeng Yao, Tianyu Dong, Zhilei Mao, Bo Hang, Xiumei Han, Zhongning Lin, Bian Qian, Min Li, Yankai Xia PII:

S0009-2797(18)30744-0

DOI:

10.1016/j.cbi.2018.09.015

Reference:

CBI 8416

To appear in:

Chemico-Biological Interactions

Received Date: 7 June 2018 Revised Date:

20 July 2018

Accepted Date: 24 September 2018

Please cite this article as: Y. Zhang, B. Xu, M. Yao, T. Dong, Z. Mao, B. Hang, X. Han, Z. Lin, B. Qian, M. Li, Y. Xia, Titanium dioxide nanoparticles induce proteostasis disruption and autophagy in human trophoblast cells, Chemico-Biological Interactions (2018), doi: https://doi.org/10.1016/j.cbi.2018.09.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Titanium dioxide nanoparticles induce proteostasis disruption and autophagy in

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human trophoblast cells

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Yuqing Zhanga,b1, Bo Xua,b1, Mengmeng Yaoc1, Tianyu Donga,b, Zhilei Maod, Bo

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Hange, Xiumei Hana,b, Zhongning Lin f, Bian Qiang, Min Lih*,Yankai Xiaa,b*

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a

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Public Health, Nanjing Medical University, Nanjing, 211166, China.

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Health, Nanjing Medical University, Nanjing, 211166, China.

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State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of

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Key Laboratory of Modern Toxicology of Ministry of Education, School of Public

Healthcare Management, International Business Center of Nanjing University,

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Nanjing, 211166, China.

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Medical University, Changzhou, 213003, China.

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e

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Laboratory, Berkeley, CA 94720, USA.

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f

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of Public Health, Xiamen University, Xiamen, P. R. China

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Disease Control and Prevention, Nanjing, 210009, China

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The Affiliated Changzhou Maternity and Child Health Care Hospital, Nanjing

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Biological Systems and Engineering Division, Lawrence Berkeley National

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State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School

Department of Toxicology and Function Assessment, Jiangsu Provincial Center for

Department of Anatomy, Nanjing Medical University, Nanjing, 211166, China.

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* To whom correspondence should be addressed:

These authors contributed equally to this work.

ACCEPTED MANUSCRIPT Dr. Yankai Xia and Dr. Min Li.

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State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of

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Public Health, Nanjing Medical University, No.101 Longmian Road, Nanjing, 211166,

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China.

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Phone: +86-25-86868425; E-mail: [email protected]

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Department of Anatomy, Nanjing Medical University, Nanjing, 211166, China.

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Phone: +86-25-86869106; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Titanium dioxide nanoparticles (TiO2 NPs) exist in many nano-products and concerns

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have been raised about their potential toxicity on human beings. One such issue is

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their potential effects on placental function, and the studies on this topic are limited

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and the mechanism remains unclear. Here we employed human trophoblast

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HTR-8/SVneo cells to investigate the effects of TiO2 NPs on trophoblast. Results

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showed that TiO2 NPs could enter cells and were mostly distributed in lysosomes,

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with some in the cytoplasm. TiO2 NPs and protein aggregation were found in both

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fetal bovine serum (FBS) in culture medium and cytoplasm of HTR-8/SVneo cells. In

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consistence with that, proteostasis of HTR-8/SVneo cells was significantly disrupted

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and endoplasmic reticulum (ER) stress related markers including PERK, IRE1-α were

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increased. After high speed centrifugation, the proteins PERK and IRE1-α were

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dramatically decreased in the highest TiO2 NPs treatment group, which indicated

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interactions between TiO2 NPs and these two proteins. Meanwhile, the protein

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expressions of LC3-II/LC3-I and P62, the autophagy biomarkers, were increased and

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the autophagy flux was not blocked. Cellular ROS stress increased and mitophagy

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related genes including PINK and Parkin increased along with the increased

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co-localization of LC3 and mitochondria. Taken together, these results indicated that

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TiO2 NPs interacted with intracellular proteins and activated ER stress and mitophagy

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in HTR-8/SVneo cells, which might do damage to placental function.

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Keywords:

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Titanium dioxide nanoparticles (TiO2 NPs); Trophoblast; Proteostasis; Autophagy;

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Mitophagy

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ACCEPTED MANUSCRIPT 1. Introduction

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Titanium dioxide nanoparticles (TiO2 NPs) are one type of mostly used nanoparticles,

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with foods, consumer products and household products being the most important

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sources of exposure [1, 2]. Previous research has shown that titanium materials are in

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many personal care products such as sunscreens and toothpastes by weight of 1

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to >10%, at least 36% of which are in the form of nanoparticles [1]. Due to their small

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size and biological activity, TiO2 NPs can get into human body and pass through

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biological barriers [3, 4].

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Previous studies have confirmed that TiO2 NPs could pass through placental barrier and do damage to the developing fetuses [5, 6]. Studies have shown that

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prenatal exposure to TiO2 NPs could impair lung development, decrease neurogenesis

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and damage memory of offspring [7-9]. Exposure to TiO2 NPs has adverse effects on

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mothers as well. Maternal exposure to TiO2 NPs could induce cytotoxicity in human

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amniotic fluid-derived cells as well as structural and functional abnormalities in the

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placenta on the maternal side [6, 10]. However, how TiO2 NPs impact placenta and

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the exact mechanisms of inducing placental dysfunction are not clear.

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Efforts have been made to explore the effects of nanomaterials on organisms.

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Interaction of nanoparticles with proteins in cell uptake and drug delivery [11-13] is

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one of the research foci. However, almost all the studies have focused on the

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nano-protein interactions in blood circulation and extracellular environment [11, 14].

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The effect of nanoparticles on proteins inside cells is rarely studied so far [15, 16].

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Trophoblast is a crucial tissue of highly active protein synthesis and secretion [17].

ACCEPTED MANUSCRIPT TiO2 NPs, which can enter trophoblast cells, have the ability of adsorbing proteins

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and forming “protein corona” on particle surface [18-21] and then might induce

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trophoblast dysfunction. In this study, we employed HTR-8/SVneo cells as a

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representative trophoblast cell model to investigate nanoparticle-protein interactions

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inside these cells and the potential mechanisms to induce trophoblast dysfunction

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upon exposure to TiO2 NPs.

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2. Materials and methods

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2.1. Characterization of TiO2 NPs

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TiO2 NPs (Sigma, USA) were suspended in either the same medium applied to cell

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culture, which was Roswell Park Memorial Institute (RPMI) -1640 medium (Gibco,

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USA) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum

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(FBS) (Gibco, USA), or in distilled and deionized water. Before detection,

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suspensions were sonicated in ice water for 30 min (100 W). The morphology was

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determined using transmission electron microscope (TEM, JEOL JEM 2011, Japan).

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Hydrodynamic diameter and surface zeta potential of TiO2 NPs were measured on

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Malvern Zetasizer Nano-ZS90 (Malvern, UK).

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2.2. Cell culture

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The trophoblast HTR-8/SVneo cell line was purchased from American Type Culture

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Collection (ATCC, Mamassas VA, USA) and incubated in complete RPMI-1640

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medium at 37oC, 5% CO2. After ultraviolet sterilization for 30 min, TiO2 NPs were

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suspended with complete RPMI-1640 medium as stock solution at the concentration

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ACCEPTED MANUSCRIPT of 1 mg/ml and sonicated as described above. The suspensions were diluted to

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different concentrations with complete culture medium (1, 10 and 100 µg/ml) before

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applied to cell culture. Another group without TiO2 NPs treatment was served as

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control group. 200 nM Rapamycin (Beyotime, China) or 10 µM chloroquine (Sigma,

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USA) was added to the culture medium with TiO2 NPs when detecting the autophagy

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flow.

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2.3. Cell viability

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HTR-8/SVneo cells were seeded 1.5x104 per well one day prior to the experiment in

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96-multiwell plates and cultured with 100 µl TiO2 NPs suspensions at the

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concentrations of 1, 10 and 100 µg/ml respectively. The results were compared with

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control group having no TiO2 NPs. After treatment for 24 h or 48 h, we used Cell

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Counting Kit 8 (CCK-8) (Dojindo, Japan) to determine cell viability following the

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reference manual. In brief, after incubated with CCK-8-solution for 1 h at 37oC, 100

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µl supernatant of was transferred to another 96-multiwell plate and absorbance was

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detected at 450 nm on UV/vis spectrometer (Ocean Optics, HR4000). This

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experiment was performed three times independently.

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2.4. Cellular uptake of TiO2 NPs

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After incubation with TiO2 NPs for 24 h, samples were pretreated as described before

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[22]. Briefly, through fixation, post-fixation and a series of dehydration,

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HTR-8/SVneo cells were embedded in Araldite and stained with lead citrate and

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uranyl acetate before TEM examination.

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HTR-8/SVneo cells were seeded in plates (Corning Costar, USA) with TiO2 NPs

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determined using a light microscope (ECLIPSE, TS100, Japan).

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2.5. Nano-protein negative staining assay

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TiO2 NPs were suspended with complete RPMI-1640 culture medium and sonicated

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in ice water for 30 min (100 W). Afterwards the suspension was negatively stained

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with phosphotungstic acid (1% in PBS) and then examined using TEM (JEOL JEM

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2011, Japan).

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After treatment with TiO2 NPs for 24 h, HTR-8/SVneo cells were washed with

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PBS to remove nanoparticles on the surface as far as possible to decrease potential

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disturbance from the extracellular nanoparticles. Cells were harvested and lysed for

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30 min on ice. Then cell lysate was differential-speed centrifuged with 1000 rpm for

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10 min to remove cell debris then the supernatant was centrifuged at 12000 rpm for

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30 min to collect nano-protein composite. Deposition was re-suspended in PBS and

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negatively stained with phosphotungstic acid (1% in PBS) and examined using TEM.

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2.6. Protein preparation and Western blotting assay

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After incubated with different concentrations of TiO2 NPs for 24 h, HTR-8/SVneo

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cells were lysed for 30 min in RIPA buffer (Beyotime, China) with 1%

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phenylmethanesulfonyl fluoride (PMSF) (Beyotime, China) as protease inhibitor.

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Lysates were centrifugalized at 1,000 rpm for 10 min at 4oC to collect supernatant.

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BCA Kit (Beyotime, China) was used to quantitatively analyze protein concentrations.

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80 µg of total protein of each group was electrophoresed in sodium dodecyl sulfate

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(SDS)-polyacrylamide gel electrophoresis (PAGE) gel and blotted in polyvinylidene

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ACCEPTED MANUSCRIPT fluoride (PVDF) membrane (Bio-Rad, USA). Membranes were blocked with 5% BSA

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(Sigma, USA) buffer for 1 h and respectively incubated with the following primary

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antibodies overnight at 4oC: rabbit anti-LC3 (1:1000), rabbit anti-PERK (1:1000),

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rabbit anti-phospho PERK (1:1000), rabbit anti-IRE1-α (1:1000), rabbit

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anti-SAPK/JNK (1:1000) and mouse anti-phospho SAPK/JNK (1:1000)(CST, USA),

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rabbit anti-P62 (Abcam, USA, 1:1000), mouse anti- GAPDH (Beyotime, China,

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1:1000). PVDF membranes were washed with TBST buffer (20 mM tris (pH7.6), 137

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mM NaCl, and 0.1% Tween 20) for 1 h, and then incubated with secondary antibodies

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including horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary

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antibody (Beyotime, China, 1:2000) and HRP-conjugated goat anti-mouse secondary

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antibody (Beyotime, China, 1:2000). Chemiluminescence was developed by the ECL

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Western blot detection kit (Millipore, USA) and bands were scanned in a Bio-Rad

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Imaging System. These blots were repeated three times independently.

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2.7. Quantitative real-time PCR assay

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Total RNAs were isolated with TRIZOL regent (Invitrogen, Carlsbad, CA) and the

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concentration was assessed with Nanodrop 2000 (Thermo Fisher Scientific, USA).

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Reverse transcription was carried out according to instructions of PrimerScript RT

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reagent Kit (TaKaRa, Japan). The products were stored at -200C until used. The

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reaction system was conducted following the instruction of SYBR Green mix kit

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(TaKaRa, Japan). Real-time PCR was performed on ABI7900 Fast Real-Time System

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(Applied Biosystems, USA) and the housekeeping gene gapdh was used as the

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internal reference. Expression levels of mRNAs were calculated using 2^-∆Ct

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ACCEPTED MANUSCRIPT (threshold cycle) method. All the primers were synthesized by Invitrogen (Invitrogen,

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Shanghai) and the detailed information was shown in Table S1. At least three

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separate experiments were carried out to assess expression levels of mRNAs.

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2.8. Immunofluorescence analysis

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After treated with TiO2 NPs on Confocal Dish (Cellvis, China) for 24 or 48 h, culture

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medium was replaced with fresh one containing 100 nM MitoTracker® Red CMXRos

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(Invitrogen, USA) to stain mitochondria for 30 min at 37oC. Cells were fixed with 4%

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paraformaldehyde for 30 min at room temperature. Following membrane permeation

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with 0.1% Triton X-100 (Beyotime, China) and blocking with 1% BSA, cells were

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incubated overnight at 4oC with rabbit anti-LC3 antibodies (CST, USA, 1:200) and

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followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary

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antibody (Beyotime, China, 1:200) for 4 h at room temperature. The nuclei were

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stained with 4', 6-diamidino-2-phenylindole (DAPI) (Beyotime, China, 1:1000) for 5

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min. Immunofluorescence photos were obtained by confocal microscope (Nikon,

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Japan).

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2.9. Derivative-quenched bovine serum albumin (DQ-Red BSA) assay

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DQ-Red BSA (Molecular Probes, USA) is a self-quenched lysosome degradation

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biomarker used to monitor enzyme activity of lysosomes. Cells were seeded onto

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Confocal Dish (Cellvis, China) and incubated with TiO2 NPs for 24 h. Then cells were

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further incubated with fresh RPMI-1640 culture medium containing 10 mg/mL

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DQ-Red BSA for 3 h at 37oC. Then nuclei were stained with Hoechst (Beyotime,

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China) and fluorescence was visualized by confocal microscopy (Nikon, Japan).

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ACCEPTED MANUSCRIPT 2.10. Reactive oxygen species (ROS) assay

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HTR cells were exposed to different concentrations of TiO2 NPs or control medium

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for 24 h. Cells were collected with trypsin (Beyotime, China) and incubated with

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fresh culture medium containing 5 µM dichlorofluorescin diacetate (DCFH-DA) for

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30 min at 37oC. Afterwards, cellular fluorescence intensity was measured by FACS

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Calibur Flow Cytometry (BD Biosciences, USA) immediately. This assay was carried

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out three times separately.

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2.11. Statistical analysis

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Results were expressed as mean ± standard error (S.E.) from at least three separate

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experiments for all analyses. Statistical significance was defined as two-tailed p<0.05.

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Comparisons between groups were conducted by ANOVA. Further comparison

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between control group and different treated groups were performed by the Dunnett’s

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multiple comparison test.

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3.

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3.1. Characteristics of TiO2 NPs

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The characteristics of TiO2 NPs were represented in Fig. 1. TEM images

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demonstrated that TiO2 NPs suspended in distilled and deionized water were nearly

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spherical, with the average diameter of 36.90 nm. The particle size distribution

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showed that TiO2 NPs slightly aggregated in complete culture medium and water,

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with hydrodynamic diameters of 73.29±5.75 nm and 65.48±1.63 nm, respectively.

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Zeta potential of TiO2 NPs in water was -8.59±0.37 mV, while in complete culture

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ACCEPTED MANUSCRIPT medium was -9.56±0.92 mV.

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Fig. 1. (A) TEM image of TiO2 NPs suspended in distilled and deionized water.

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Bar=100 nm. (B) Size distribution of TiO2 NPs in RPMI-1640 complete culture

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medium. (C) Characteristics of TiO2 NPs in water or complete culture medium.

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3.2. Effects of TiO2 NPs on cell viability and morphology

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Treatment with TiO2 NPs did not change cell viability even at a relatively high

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concentration of 100 µg/ml at both 24 h (Fig. 2A) and 48 h (Fig. 2B). The following

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analysis of this study were all conducted after treatment for 24 h. The treated cells

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colored yellowish-brown because of uptake of non-transparent nanoparticles. In the

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same time, some vacuoles were formed inside the exposed cells (Fig. 2C).

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Under TEM, we found TiO2 NPs inside the cells, and most of them were

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distributed in lysosomes, with some in the cytoplasm (Fig. 2D). We also observed that

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the autophagosomes were increased in the TiO2 NPs-treated group, which indicates

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autophagy activation (Fig. 2D).

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Fig. 2. (A)Viability of HTR-8/SVneo cells treated with various concentrations of TiO2

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NPs for 24 h. (B) Viability of HTR-8/SVneo cells treated with various concentrations

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of TiO2 NPs for 48 h. Results of A and B were expressed as mean ± standard error

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(S.E.) from three separate experiments. (C) Light microscope images with 100x

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magnification of control cells and TiO2 NPs treated cells (100 µg/ml) after treating for

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24 h. (D) TEM images of control and nanoparticle-treated groups after 24 h treatment.

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TiO2 NPs were mostly distributed in the lysosome, with some in the cytoplasm.

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Autophagosomes were indicated by red arrows.

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3.3. Nano-protein aggregation outside and inside HTR-8/SVneo cells after 24 h

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exposure

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To determine if TiO2 NPs adsorb proteins in culture medium, using TEM, we found

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protein corona around nanoparticles suspended in complete culture medium (Fig. 3A),

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while no protein corona was found around the ones in distilled and deionized water

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(Fig. 1A). We further negatively stained nanoparticles suspended in complete culture

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medium and found nano-protein aggregation (Fig. 3B). It is known that nanoparticles enter cells by endocytosis [23], and that enzymes

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in the endosome degrade them [24]. DQ-Red BSA is a fluorogenic substrate for

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proteases and bright red fluorescent fragments will be released after proteolysis in the

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lysosome. As TEM images showed, the brightness of red fluorescent fragments was

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weakened after TiO2 NPs treatment (Fig. 3C), indicating that the lysosome protease

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activity was decreased. This also suggests the structural disruption of lysosomes to

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certain extent and the release of nanoparticles in the cytoplasm.

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We used phosphotungstic acid to negatively stain TiO2 NPs in cell lysis precipitate after differential-speed centrifugation and found nano-protein aggregation (Fig. 3D).

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Fig. 3. (A) TEM image of TiO2 NPs suspended in complete culture medium. Bar=500

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nm. (B) Phosphotungstic acid-stained TEM image of nano-protein aggregation in

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complete culture medium. (C) DQ-Red BSA (bright red fluorescent fragments) was

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weakened after treated with TiO2 NPs (100 µg/ml) for 24 h, which indicated

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decreased proteolytic capacity in treated group. Bar=20 µm. (D) Phosphotungstic

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acid-stained

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centrifugation.

precipitant after differential-speed

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TEM image of cell lysis

3.4. TiO2 NPs exposure impairs proteostasis after 24 h

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Since TiO2 NPs could disrupt the structure of lysosomes and adsorb proteins in the

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cytoplasm, the protein homeostasis could also be disrupted after TiO2 NPs uptaken by

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the cells. We investigated whether there was endoplasmic reticulum stress (ER stress)

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after TiO2 NPs treatment, which is a consequence of proteostasis impairment. We

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measured mRNA expression of ER stress related-sensors, inositol-requiring enzyme

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1–alpha (IRE1-α) and protein kinase (PKR)-like ER kinase (PERK), and found that

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they were increased as the concentrations of TiO2 NPs increased (Fig. 4A). After

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treatment with different concentrations of TiO2 NPs, we collected and lysed cells to

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detect ER stress-related proteins PERK, IRE1-α and c-Jun N-terminal kinase (JNK).

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The results showed increased expression of several sensor proteins as shown in Fig.

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4B, indicating proteostasis impairment. To explore whether TiO2 NPs could absorb

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these proteins, we separated cell lysate with high-speed centrifugation and found the

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levels of PERK, IRE1-α were dramatically decreased at the highest concentration

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group while the reference protein GAPDH remained unchanged (Fig. 4C).

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Fig. 4. (A) ER stress-related sensors (IRE1-α and PERK) were detected by RT-PCR

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using gapdh as internal control. Values were expressed as mean ± standard error (S.E.)

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from three separate experiments. (B) Cell lysis was separated with low-speed

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centrifugation to obtain total protein. Protein expressions of stress-activated protein

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kinase (JNK) and ER stress-related proteins (PERK, IRE1-α) were detected by

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Western blot with GAPDH as internal control. (C) Two ER stress-related proteins

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(PERK, IRE1-α) were detected with Western blot after high-speed centrifugation,

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which showed dramatically decreased protein levels in the highest treatment group.

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Asterisk indicates significant difference when the values were compared to that of the

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control (*p < 0.05，**p<0.01, ***p<0.001).

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3.5. TiO2 NPs treatment for 24 h induces autophagy

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Autophagy is known to be a pro-survival pathway activated by certain stressors [25].

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As shown in Fig. 2C, our TEM images indicated that the number of autophagosomes

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components of autophagy. The expression of Beclin 1 and P62 were increased after

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TiO2 NPs treatment (Fig. 5A). After treating HTR-8/SVneo cells with different

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concentrations of TiO2 NPs for 24 h, the protein expressions of LC3-II/LC3-I and P62

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were increased significantly (Fig. 5B). Next, rapamycin (MTORC1 inhibitor to

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stimulate autophagy) and chloroquine (lysosomal fusion inhibitor to block autophagy)

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were added to culture medium with 100 µg/ml of TiO2 NPs to test whether the

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autophagic flux was altered. We found that the LC3-II/LC3-I protein ratios were all

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increased compared to the controls in TiO2 NP-treated groups, indicating that the

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autophagic flux was not blocked and autophagosomes were increased after TiO2 NPs

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treatment (Fig. 5C).

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Fig. 5. (A) mRNA expressions of two autophagy-related genes (Beclin1 and 312

SQSTM1/P62) were detected by RT-PCR after treated with TiO2 NPs for 24 h. Values 313

were expressed as mean ± standard error (S.E.) from three separate experiments. (B) 314

Western blot analysis of the expression of microtubule-associated light chain 3 (LC3)

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and Ubiquitin-Binding Protein P62 of different concentration groups with GAPDH as 316

loading control. (C) Western blot analysis of autophagic flux activation with 317

rapamycin (a MTORC1 inhibitor to stimulate autophagy) and chloroquine (a 318 319

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lysosomal fusion inhibitor) added to culture medium with TiO2 NPs. Asterisk indicates significant difference when the values were compared to that of the control 320

(*p < 0.05，**p<0.01, ***p<0.001).

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3.6. Clearance of damaged mitochondria after TiO2 NPs treatment for 24 h

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Proteostasis disruption may disturb the function of mitochondria. When mitochondria

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are impaired, PTEN induced putative kinase 1 (PINK1) accumulates in mitochondria,

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which recruits and activates proteins such as Parkin (PARK2). The latter is an

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ubiquitin ligase for the degradation of damaged mitochondria [26, 27]. We firstly

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investigated the oxidative stress the cells may undertake and found that the levels

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were higher as the concentrations of TiO2 NPs increased (Fig. 6A). The mRNA levels

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of PINK1 and Parkin were increased after TiO2 NPs treatment as well (Fig. 6B). We

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then used immunofluorescence and confocal microscope to confirm the

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co-localization of autophagosomes and mitochondria. After treating HTR cells with

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TiO2 NPs for 24 h, we found the activation of autophagy was increased and

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membrane potential of mitochondria was decreased (Fig. 6C). The overlap of these

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two kinds of fluorescent fragments was reduced compared to the control group (Fig.

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6C), suggesting that the damaged mitochondria were cleared up by the process of

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autophagy.

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Fig. 6. (A) Intracellular reactive oxygen species (ROS) formation measured by FCM. 339

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results in A and B were expressed as mean ± standard error (S.E.) from three separate 341

experiments. (C) Confocal microscopy to show co-localization of LC3 and 342

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4.

Discussion

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In this study, we explored the potential interaction between TiO2 NPs and intracellular

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proteins as well as the possible mechanisms on trophoblast cells. We treated HTR

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cells at a relative high concentration of TiO2 NPs at which concentration cell viability

ACCEPTED MANUSCRIPT was not decreased. We demonstrated that TiO2 NPs could enter HTR cells and mostly

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distributed in the lysosomes. The lysosomal degradation function was impaired,

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leading to some of the TiO2 NPs leaking into the cytoplasm and adsorbing the proteins

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in the cytoplasm, thus leading to proteostasis disturbance and ER stress. In the

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meantime, the mitochondria were impaired, leading to autophagy towards the

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damaged mitochondria.

Increasing evidence has shown that engineered nanoparticles could pass through

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placental barrier and damage growing fetuses [28-30]. Although lacking of human

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studies, TiO2 NPs has been reported to penetrate placenta and present in placental

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trophoblast and fetus in rodent models [6, 8, 31]. TiO2 NPs used in our study was

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spherical with hydrodynamic diameter of 73.29±5.75 nm, smaller than the

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nanoparticles mentioned in above studies, which suggests trophoblast exposure during

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pregnancy. In our study, cell viability after exposure for 24 h was not changed at the

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relative high concentration. Pervious studies have demonstrated nanoparticle toxicity

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without causing cell viability decrease [32, 33].

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In our study, nano-protein aggregation was found when particles were suspended

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in complete culture medium, which was in agreement with a previous study [34]. TiO2

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NPs are not the only nanoparticles exhibiting the ability of protein absorption.

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Nanoparticles like SiO2, carbon black, CdS and CeO have been reported to absorb

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proteins in culture medium or plasm [35-38]. In a study comparing protein absorption

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by several types of materials, TiO2, CeO2 and ZnO showed strong abilities [34]. The

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potential mechanism of strong protein absorption ability of TiO2 NPs might due to

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negative charges on surface [14] and positive charge on proteins. All of these could

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lead to nano-protein aggregation [18-21]. Nanoparticle-protein interaction might persist during all the process of

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bio-distribution. After taken by cells, whether there is direct interaction between

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nanoparticles and intracellular proteins is not clear. Nanoparticles often get into cells

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by endocytosis. Endosomes containing the ingested materials then fuse with

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lysosomes and form lysosome-endosome hybrids, which are rich of enzymes [39].

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Acid condition and enzyme in lysosomes could degrade nanoparticles and protein

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aggregation formed by FBS[24], whereas nanoparticle could interact with enzymes

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inside lysosomes and lead to dysfunction. In our study, we found decreased

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proteolytic capacity and structural disruption of lysosomes after TiO2 NPs treatment,

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which phenomenon has been reported present in some other nanoparticles as well [22,

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40, 41]. As the TEM images showed in this study, some of the nanoparticles were

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present in the cytoplasm where is full of proteins. The destiny of nanoparticles

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escaped to cytoplasm is not well studied. A previous study found that SiO2

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nanoparticles could enter HEp-2 cells and aggregate with proteins in cell nuclei,

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which impairs nuclear function [15]. By contrast, TiO2 NPs in our study did not

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appear in cell nuclei. Instead, they were present in cytoplasm after escaped from

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lysosomes and nano-protein aggregation was still existent. However, the composition

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of aggregated proteins was not clear, which might include proteins in FBS, enzymes

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in lysosomes as well as proteins in cytoplasm.

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To make out if TiO2 NPs absorbed proteins in cytoplasm and led to proteostasis disruption. We assessed the expression of ER stress related markers. Soluble secretory

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and transmembrane proteins are synthesized in membrane-bound ribosomes in the

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form of peptides and then folded in the endoplasmic reticulum (ER) and modified in

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Golgi [42]. Protein folding in ER is very sensitive to changes in the intracellular and

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extracellular environments [43], especially in cells like trophoblast cells with a high

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rate of protein synthesis. Many studies have established that nanoparticle exposure

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could lead to ER stress [33, 44, 45]. In this study, the expression levels of ER

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stress-related markers increased after TiO2 NPs treatment, which indicated

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proteostasis disruption inside cells. The potential possibility might be absorption of

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endoplasmic reticulum related proteins onto TiO2 NPs, which disturbed the function

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of endoplasmic reticulum. One of our previous studies has shown that TiO2 NPs could

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absorb Tau protein inside the HY5Y cells [22]. In this study, we found that the

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expression of IRE1-α and PERK showed a dramatic decrease in the HTR cells treated

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with highest concentration of TiO2 NPs. These two proteins are highly expressed in

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ER, where plenty of peptides are folded. TiO2 NPs might adhere to the surface of ER

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and absorb these two proteins. Proteins absorbed on TiO2 NPs maybe not specific and

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may lead to various disruptions inside cells. Although some of the protein

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identifications were determined, many of them are not.

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At the early stage of TiO2 NPs exposure, some pre-survival reaction might be

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activated to conserve energy and maintain cellular homeostasis, of which autophagy is

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an important one [46-48]. In autophagy process, Beclin1 encodes a protein mediating

ACCEPTED MANUSCRIPT vesicle-trafficking processes and regulating autophagy [49]. SQSTM1/P62 binds

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directly to LC3 to facilitate degradation of ubiquitinated proteins aggregated by

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autophagy [50]. LC3 is a widely used biomarker of autophagy and the ratio of

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LC3-II/LC3-I corresponds to the amount of autophagosomes [51]. In our study, gene

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expression of Beclin1 and P62 were increased as well as ratio of LC3-II/LC3-I protein

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expression increased after TiO2 NPs treatment. The accumulation of autophagosomes

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may be due to autophagy activation or blockage of autophagy flux [52, 53].

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Rapamycin (a MTORC1 inhibitor to stimulate autophagy) and chloroquine (a

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lysosomal fusion inhibitor) are commonly used to detect whether there is blockage in

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autophagy flux [51]. Our results showed that the ratio of LC3-II/LC3-I was both

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increased after adding rapamycin or chloroquine to culture medium with TiO2 NPs

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when compared to the control, indicating that there was no autophagic flux blockage

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after TiO2 NPs treatment. Previous studies have indicated that autophagy in

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trophoblast might lead to miscarriage, preeclampsia, intrauterine growth restriction

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(IUGR) and other disorders in both mothers and fetuses [54-56]. Although cell

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viability was not changed by comparing with the control group in our study,

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autophagy might induce cell dysfunction after long time treatment.

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Most of the proteins in mitochondria are synthesized in the free ribosomes in

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cytoplasm and imported through the double membrane of the mitochondria in their

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unfolded state [57]. Inadequate protein transportation into mitochondria may lead to

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mitochondrial dysfunction. In addition, studies have shown that ions or molecules

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released after lysosomal degradation of nanoparticles could damage mitochondria and

ACCEPTED MANUSCRIPT induce high levels of ROS [24]. In our study, TiO2 NPs in the cell might absorb

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proteins critical to mitochondrial function and cause molecules to be released from

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lysosomes, either of which could damage the function of mitochondria. In the current

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study, ROS levels were increased in the treated groups and the increased level of ROS

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might in turn damage mitochondria and generate more ROS by the defective ones[58].

442

Besides, mitochondrial membrane potential was decreased in the treatment group,

443

which suggested imbalance between mitochondrial outer membrane and inter

444

membrane [59]. All these suggests the damage of mitochondria. However, the exact

445

mechanism of mitochondrial damage was not clear in our study. It has been reported

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that, under stress, mitophagy is initiated after accumulation of PINK and

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phosphorylated Parkin on the outer mitochondrial membranes [60]. The expression

448

levels of PINK1 and Parkin were indeed increased in our study, suggesting that there

449

was a mitophagy activation. In addition, immunofluorescence and confocal

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microscope analysis confirmed that mitochondria were damaged and autophagy was

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activated to clean up the damaged ones.

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In conclusion, we revealed that exposure to TiO2 NPs could disrupt the structure

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of lysosomes and led to the leakage of these particles into the cytoplasm. As a result,

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they absorbed cellular proteins in the cytoplasm which may account for the

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proteostasis disruption. In addition, we observed that, mitochondria were damaged

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and autophagy was activated upon exposure to TiO2 NPs. These findings provide

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insight into the toxicological mechanisms of TiO2 NPs effects on human health. A

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pictorial presentation of the potential mechanisms is outlined in Fig. 7. Certainly more

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ACCEPTED MANUSCRIPT studies are warranted to further identify which proteins may interact with TiO2 NPs

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and the more mechanisms of cellular dysfunction induced by these nanoparticles.

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Fig. 7. Potential mechanisms of proteostasis disruption and autophagy in human

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trophoblast cells caused by TiO2 NPs treatment. TiO2 NPs and proteins in FBS are

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aggregated in culture medium. Then particles are uptaken by endocytosis. Endosomes

465

containing the ingested materials fuse with lysosomes and form lysosome-endosome

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hybrids. The lysosomal degradation function is impaired, leading to some of the TiO2

467

NPs leaking into the cytoplasm and adsorbing the proteins in the cytoplasm, thus

468

leading to ER stress. Ions or molecules released after lysosomal disruption damage

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mitochondria. Proteostasis disturbance inside cells might lead to mitochondrial

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damage as well. All of these activate mitophagy to clear the impaired mitochondria.

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References

475 476 477

[1]

A. Weir, P. Westerhoff, L. Fabricius, K. Hristovski, N. von Goetz, Titanium dioxide nanoparticles in food and personal care products, Environ Sci Technol 46 (2012) 2242-2250.

ACCEPTED MANUSCRIPT

[6]

[7]

[8]

[9]

RI PT

SC

[5]

M AN U

[4]

TE D

[3]

L. Sang, Y. Zhao, C. Burda, TiO2 nanoparticles as functional building blocks, Chem Rev 114 (2014) 9283-9318. P.J. Borm, D. Robbins, S. Haubold, T. Kuhlbusch, H. Fissan, K. Donaldson, R. Schins, V. Stone, W. Kreyling, J. Lademann, J. Krutmann, D. Warheit, E. Oberdorster, The potential risks of nanomaterials: a review carried out for ECETOC, Part Fibre Toxicol 3 (2006) 11. A. Peters, B. Veronesi, L. Calderon-Garciduenas, P. Gehr, L.C. Chen, M. Geiser, W. Reed, B. Rothen-Rutishauser, S. Schurch, H. Schulz, Translocation and potential neurological effects of fine and ultrafine particles a critical update, Part Fibre Toxicol 3 (2006) 13. M. Umezawa, H. Tainaka, N. Kawashima, M. Shimizu, K. Takeda, Effect of fetal exposure to titanium dioxide nanoparticle on brain development - brain region information, J Toxicol Sci 37 (2012) 1247-1252. K. Yamashita, Y. Yoshioka, K. Higashisaka, K. Mimura, Y. Morishita, M. Nozaki, T. Yoshida, T. Ogura, H. Nabeshi, K. Nagano, Y. Abe, H. Kamada, Y. Monobe, T. Imazawa, H. Aoshima, K. Shishido, Y. Kawai, T. Mayumi, S. Tsunoda, N. Itoh, T. Yoshikawa, I. Yanagihara, S. Saito, Y. Tsutsumi, Silica and titanium dioxide nanoparticles cause pregnancy complications in mice, Nat Nanotechnol 6 (2011) 321-328. A. Ebrahimzadeh Bideskan, A. Mohammadipour, A. Fazel, H. Haghir, H. Rafatpanah, M. Hosseini, A. Rajabzadeh, Maternal exposure to titanium dioxide nanoparticles during pregnancy and lactation alters offspring hippocampal mRNA BAX and Bcl-2 levels, induces apoptosis and decreases neurogenesis, Exp Toxicol Pathol 69 (2017) 329-337. A. Mohammadipour, A. Fazel, H. Haghir, F. Motejaded, H. Rafatpanah, H. Zabihi, M. Hosseini, A.E. Bideskan, Maternal exposure to titanium dioxide nanoparticles during pregnancy; impaired memory and decreased hippocampal cell proliferation in rat offspring, Environ Toxicol Pharmacol 37 (2014) 617-625. E. Paul, M.L. Franco-Montoya, E. Paineau, B. Angeletti, S. Vibhushan, A. Ridoux, A. Tiendrebeogo, M. Salome, B. Hesse, D. Vantelon, J. Rose, F. Canoui-Poitrine, J. Boczkowski, S. Lanone, C. Delacourt, J.C. Pairon, Pulmonary exposure to metallic nanomaterials during pregnancy irreversibly impairs lung development of the offspring, Nanotoxicology 11 (2017) 484-495. M.S. Acar, Z.B. Bulut, A. Ates, B. Nami, N. Kocak, B. Yildiz, Titanium dioxide nanoparticles induce cytotoxicity and reduce mitotic index in human amniotic fluid-derived cells, Hum Exp Toxicol 34 (2015) 74-82. C.D. Walkey, J.B. Olsen, H. Guo, A. Emili, W.C. Chan, Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake, J Am Chem Soc 134 (2012) 2139-2147. D. Brambilla, R. Verpillot, B. Le Droumaguet, J. Nicolas, M. Taverna, J. Kona, B. Lettiero, S.H. Hashemi, L. De Kimpe, M. Canovi, M. Gobbi, V. Nicolas, W. Scheper, S.M. Moghimi, I. Tvaroska, P. Couvreur, K. Andrieux, PEGylated

EP

[2]

AC C

478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521

[10]

[11]

[12]

ACCEPTED MANUSCRIPT

[16]

[17] [18] [19]

[20]

RI PT

EP

[21]

SC

[15]

M AN U

[14]

TE D

[13]

nanoparticles bind to and alter amyloid-beta peptide conformation: toward engineering of functional nanomedicines for Alzheimer's disease, ACS Nano 6 (2012) 5897-5908. K. Ulbrich, K. Hola, V. Subr, A. Bakandritsos, J. Tucek, R. Zboril, Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies, Chem Rev 116 (2016) 5338-5431. Z. Lin, N.A. Monteiro-Riviere, J.E. Riviere, Pharmacokinetics of metallic nanoparticles, Wiley Interdiscip Rev Nanomed Nanobiotechnol 7 (2015) 189-217. M. Chen, A. von Mikecz, Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles, Exp Cell Res 305 (2005) 51-62. X. Yan, J. Li, Q. Liu, H. Peng, A. Popowich, Z. Wang, X.F. Li, p-Azidophenylarsenoxide: An Arsenical "Bait" for the In Situ Capture and Identification of Cellular Arsenic-Binding Proteins, 55 (2016) 14051-14056. G. Riquelme, Review: Placental syncytiotrophoblast membranes--domains, subdomains and microdomains, Placenta 32 Suppl 2 (2011) S196-202. N. Duran, C.P. Silveira, M. Duran, D.S. Martinez, Silver nanoparticle protein corona and toxicity: a mini-review, J Nanobiotechnology 13 (2015) 55. Z. Ma, J. Bai, X. Jiang, Monitoring of the Enzymatic Degradation of Protein Corona and Evaluating the Accompanying Cytotoxicity of Nanoparticles, ACS Appl Mater Interfaces 7 (2015) 17614-17622. M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K.A. Dawson, Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts, Proc Natl Acad Sci U S A 105 (2008) 14265-14270. S. Tenzer, D. Docter, J. Kuharev, A. Musyanovych, V. Fetz, R. Hecht, F. Schlenk, D. Fischer, K. Kiouptsi, C. Reinhardt, K. Landfester, H. Schild, M. Maskos, S.K. Knauer, R.H. Stauber, Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology, Nat Nanotechnol 8 (2013) 772-781. Z. Mao, B. Xu, X. Ji, K. Zhou, X. Zhang, M. Chen, X. Han, Q. Tang, X. Wang, Y. Xia, Titanium dioxide nanoparticles alter cellular morphology via disturbing the microtubule dynamics, Nanoscale 7 (2015) 8466-8475. S. Zhang, H. Gao, G. Bao, Physical Principles of Nanoparticle Cellular Endocytosis, ACS Nano 9 (2015) 8655-8671. J. Deng, C. Gao, Recent advances in interactions of designed nanoparticles and cells with respect to cellular uptake, intracellular fate, degradation and cytotoxicity, Nanotechnology 27 (2016) 412002. P. Ravanan, I.F. Srikumar, P. Talwar, Autophagy: The spotlight for cellular stress responses, Life Sci (2017). J.J. Lemasters, Variants of mitochondrial autophagy: Types 1 and 2 mitophagy and micromitophagy (Type 3), Redox Biol 2 (2014) 749-754.

AC C

522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

[22]

[23] [24]

[25] [26]

ACCEPTED MANUSCRIPT

[32]

[33]

[34]

[35]

[36]

[37]

RI PT

[31]

SC

[30]

M AN U

[29]

TE D

[28]

A. Eiyama, K. Okamoto, PINK1/Parkin-mediated mitophagy in mammalian cells, Curr Opin Cell Biol 33 (2015) 95-101. S. Sweeney, A. Adamcakova-Dodd, P.S. Thorne, J.G. Assouline, Multifunctional nanoparticles for real-time evaluation of toxicity during fetal development, PLoS One 13 (2018) e0192474. S.J. Hawkins, L.A. Crompton, A. Sood, M. Saunders, Nanoparticle-induced neuronal toxicity across placental barriers is mediated by autophagy and dependent on astrocytes, 13 (2018) 427-433. B. Zhang, L. Tan, Y. Yu, B. Wang, Z. Chen, J. Han, M. Li, J. Chen, T. Xiao, B.K. Ambati, L. Cai, Q. Yang, N.R. Nayak, J. Zhang, X. Fan, Placenta-specific drug delivery by trophoblast-targeted nanoparticles in mice, Theranostics 8 (2018) 2765-2781. F. Hong, Y. Zhou, X. Zhao, L. Sheng, L. Wang, Maternal exposure to nanosized titanium dioxide suppresses embryonic development in mice, Int J Nanomedicine 12 (2017) 6197-6204. X. Ji, B. Xu, M. Yao, Z. Mao, Y. Zhang, G. Xu, Q. Tang, X. Wang, Y. Xia, Graphene oxide quantum dots disrupt autophagic flux by inhibiting lysosome activity in GC-2 and TM4 cell lines, Toxicology 374 (2016) 10-17. R. Chen, L. Huo, X. Shi, R. Bai, Z. Zhang, Y. Zhao, Y. Chang, C. Chen, Endoplasmic reticulum stress induced by zinc oxide nanoparticles is an earlier biomarker for nanotoxicological evaluation, ACS Nano 8 (2014) 2562-2574. M. Horie, K. Nishio, K. Fujita, S. Endoh, A. Miyauchi, Y. Saito, H. Iwahashi, K. Yamamoto, H. Murayama, H. Nakano, N. Nanashima, E. Niki, Y. Yoshida, Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells, Chem Res Toxicol 22 (2009) 543-553. H. Ruh, B. Kuhl, G. Brenner-Weiss, C. Hopf, S. Diabate, C. Weiss, Identification of serum proteins bound to industrial nanomaterials, Toxicol Lett 208 (2012) 41-50. E. Casals, T. Pfaller, A. Duschl, G.J. Oostingh, V.F. Puntes, Hardening of the nanoparticle-protein corona in metal (Au, Ag) and oxide (Fe3O4, CoO, and CeO2) nanoparticles, Small 7 (2011) 3479-3486. E. Casals, T. Pfaller, A. Duschl, G.J. Oostingh, V. Puntes, Time evolution of the nanoparticle protein corona, ACS Nano 4 (2010) 3623-3632. P. Roach, D. Farrar, C.C. Perry, Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry, J Am Chem Soc 128 (2006) 3939-3945. S.T. Stern, P.P. Adiseshaiah, R.M. Crist, Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity, Part Fibre Toxicol 9 (2012) 20. Y. Zhu, J.W. Eaton, C. Li, Titanium dioxide (TiO2) nanoparticles preferentially induce cell death in transformed cells in a Bak/Bax-independent fashion, PLoS One 7 (2012) e50607. S. Behzadi, V. Serpooshan, W. Tao, M.A. Hamaly, M.Y. Alkawareek, E.C. Dreaden, D. Brown, A.M. Alkilany, O.C. Farokhzad, M. Mahmoudi, Cellular

EP

[27]

AC C

566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609

[38]

[39]

[40]

[41]

ACCEPTED MANUSCRIPT

[46]

[47] [48]

[49] [50]

[51]

RI PT

SC

[45]

M AN U

[44]

TE D

[43]

EP

[42]

uptake of nanoparticles: journey inside the cell, Chem Soc Rev 46 (2017) 4218-4244. E.M. Sontag, R.S. Samant, J. Frydman, Mechanisms and Functions of Spatial Protein Quality Control, Annu Rev Biochem 86 (2017) 97-122. P. Walter, D. Ron, The unfolded protein response: from stress pathway to homeostatic regulation, Science 334 (2011) 1081-1086. Q. Yang, Y. Wang, Q. Yang, Y. Gao, X. Duan, Q. Fu, C. Chu, X. Pan, X. Cui, Y. Sun, Cuprous oxide nanoparticles trigger ER stress-induced apoptosis by regulating copper trafficking and overcoming resistance to sunitinib therapy in renal cancer, Biomaterials 146 (2017) 72-85. L. Huo, R. Chen, L. Zhao, X. Shi, R. Bai, D. Long, F. Chen, Y. Zhao, Y.Z. Chang, C. Chen, Silver nanoparticles activate endoplasmic reticulum stress signaling pathway in cell and mouse models: The role in toxicity evaluation, Biomaterials 61 (2015) 307-315. J.S. Nowak, D. Mehn, P. Nativo, C.P. Garcia, S. Gioria, I. Ojea-Jimenez, D. Gilliland, F. Rossi, Silica nanoparticle uptake induces survival mechanism in A549 cells by the activation of autophagy but not apoptosis, Toxicol Lett 224 (2014) 84-92. M.N. Moore, J.I. Allen, P.J. Somerfield, Autophagy: role in surviving environmental stress, Mar Environ Res 62 Suppl (2006) S420-425. M.N. Moore, Autophagy as a second level protective process in conferring resistance to environmentally-induced oxidative stress, Autophagy 4 (2008) 254-256. Y. Cao, D.J. Klionsky, Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein, Cell Res 17 (2007) 839-849. S. Pankiv, T.H. Clausen, T. Lamark, A. Brech, J.A. Bruun, H. Outzen, A. Overvatn, G. Bjorkoy, T. Johansen, p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy, J Biol Chem 282 (2007) 24131-24145. K. Peynshaert, B.B. Manshian, F. Joris, K. Braeckmans, S.C. De Smedt, J. Demeester, S.J. Soenen, Exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticle-induced autophagy in biomedical research, Chem Rev 114 (2014) 7581-7609. A. Lau, Y. Zheng, S. Tao, H. Wang, S.A. Whitman, E. White, D.D. Zhang, Arsenic inhibits autophagic flux, activating the Nrf2-Keap1 pathway in a p62-dependent manner, Mol Cell Biol 33 (2013) 2436-2446. X.J. Zhang, S. Chen, K.X. Huang, W.D. Le, Why should autophagic flux be assessed?, Acta Pharmacol Sin 34 (2013) 595-599. L. Avagliano, L. Terraneo, E. Virgili, C. Martinelli, P. Doi, M. Samaja, G.P. Bulfamante, A.M. Marconi, Autophagy in Normal and Abnormal Early Human Pregnancies, Reprod Sci 22 (2015) 838-844. T.H. Hung, S.F. Chen, L.M. Lo, M.J. Li, Y.L. Yeh, T.T. Hsieh, Increased autophagy in placentas of intrauterine growth-restricted pregnancies, PLoS One 7 (2012) e40957.

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[52]

[53] [54]

[55]

ACCEPTED MANUSCRIPT

[57] [58] [59]

[60]

S. Saito, A. Nakashima, A review of the mechanism for poor placentation in early-onset preeclampsia: the role of autophagy in trophoblast invasion and vascular remodeling, J Reprod Immunol 101-102 (2014) 80-88. L. Ellenrieder, H. Rampelt, T. Becker, Connection of Protein Transport and Organelle Contact Sites in Mitochondria, J Mol Biol 429 (2017) 2148-2160. R.J. Youle, A.M. van der Bliek, Mitochondrial fission, fusion, and stress, Science 337 (2012) 1062-1065. D.P. Evenson, Z. Darzynkiewicz, M.R. Melamed, Simultaneous measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell motility, J Histochem Cytochem 30 (1982) 279-280. S.M. Jin, R.J. Youle, The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria, Autophagy 9 (2013) 1750-1757.

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ACCEPTED MANUSCRIPT Acknowledgements

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This work was supported by the National Key R&D Program of China

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(2016YFC1000207), the National Natural Science Foundation of China (81630085,

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81602884, 81502832), Qing Lan Project of Jiangsu Province, Six Talent Peaks Project

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of Jiangsu Province (JY-052), Second Level of Training Object of Jiangsu Province

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"333"

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(BK20161585).

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Competing interests

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The authors declare that they have no competing interests.

and

the Natural Science Foundation of Jiangsu Province

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ACCEPTED MANUSCRIPT Highlights: - TiO2 NPs and intracellular protein aggregation was found in HTR-8/SVneo cells - ER stress and mitophagy was activated after TiO2 NPs treatment

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- Interaction between TiO2 NPs and proteins like PERK and IRE1-α was suggested