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Uptake of TiO2 Nanoparticles into C. elegans Neurons Negatively Affects Axonal Growth and Worm Locomotion Behavior Chun-Chih Hu, Gong-Her Wu, Tzu-En Hua, Oliver I. Wagner, and Ta-Jen Yen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18818 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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ACS Applied Materials & Interfaces

Uptake of TiO 2 Nanoparticles into C. elegans Neurons Negatively Affects Axonal Growth and Worm Locomotion Behavior

Chun-Chih Hu†, Gong-Her Wu‡, Tzu-En Hua‡, Oliver I. Wagner*,‡, and Ta-Jen Yen*,†

†

‡

Department of Materials Science and Engineering

Department of Life Science and Institute of Molecular & Cellular Biology National Tsing Hua University, Hsinchu 30013, Taiwan

*Corresponding authors: Ta-Jen Yen, PhD, Professor, Department of Materials Science and Engineering Oliver I. Wagner, PhD, Associate Professor, Department of Life Science National Tsing Hua University 101, Sec. 2, Kuang-Fu Road Hsinchu 30013 Taiwan R. O. C. Emails: [email protected], [email protected] (to whom correspondence should be addressed)

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Abstract We employ the model organism Caenorhabditis elegans to effectively study the toxicology of anatase and rutile phase titanium dioxide (TiO 2 ) nanoparticles (NPs). The experimental results show that the nematode C. elegans can take up fluorescein isothiocyanate (FITC)-labeled TiO 2 NPs and that both anatase and rutile TiO 2 NPs can be detected in the cytoplasm of cultured primary neurons imaged by transmission electron microscopy (TEM). After TiO 2 NPs exposure, these neurons also grow shorter axons, which may be related to the detected impeded worm locomotion behavior. Furthermore, anatase TiO 2 NPs did not affect the worm’s body length; however, we determined that a concentration of 500 µg/ml anatase TiO 2 NPs reduced a worm population by 50% within 72 hours. Notably, rutile TiO 2 NPs negatively affect both body size and worm population. Worms unable to enter the L4 larval stage explain a severe reduction in the worm population at TiO 2 NPs LC 50 /3d. To obtain a better understanding of the cellular mechanisms involved in TiO 2 NPs intoxication, DNA microarray assays were employed to determine changes in gene expression in the presence or absence of TiO 2 NPs exposure. Our data reveal three genes (with significant changes in expression levels) were related to metal binding or metal detoxification (mtl-2, C45B2.2 and nhr-247), six genes involved in fertility and reproduction (mtl-2, F26F2.3, ZK970.7, clec-70,

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K08C9.7 and C38C3.7), four genes involved in worm growth and body morphogenesis (mtl-2, F26F2.3, C38C3.7 and nhr-247), and five genes involved in neuronal function (C41G6.13, C45B2.2, srr-6, K08C9.7 and C38C3.7).

Keywords: Titanium dioxide nanoparticles (TiO 2 NPs), anatase, rutile, DNA microarray chip, primary C. elegans neurons

1. INTRODUCTION Engineered metal oxide nanoparticles (NPs) have attracted considerable attention in nanotechnology applications due to their unique size-dependent optical and electrical properties, high reactivity, a relatively large surface area, and other factors. Among many metal oxides known, titanium dioxide (TiO 2 ) has been recognized as a promising material in nanotechnology and has been applied in a variety of applications, such as in photocatalysis 1, solar cells 2, sensors, dielectric ceramics, medical devices, human implants, pigments, and cosmetics 3-8. TiO 2 can be employed in such a broad range of applications due to its wide bandgap, abundance, largely varying crystalline conformations, distinctive morphology, and nano-range size. In nature, TiO 2 occurs in three mineral forms, namely, rutile, anatase, and the rare form brookite.

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In addition to the numerous aforementioned technological applications, several studies on the potential environmental toxicity of TiO 2 NPs have recently been carried out due to increasing attention on eco-awareness. Specifically, the effective use of C. elegans in these studies has been widely demonstrated 9-12. The importance using C. elegans for toxicology studies stems from its small size (easy to maintain on agar plates), short life-cycle (approximately 2.5 days) 13, and fully sequenced genome 14, which are all advantageous for genetic studies. Furthermore, changes in worm locomotion behavioral assays can be related to altered motor neuron function, which is important in understanding neurological diseases such as Amyotrophic Lateral Sclerosis (ALS) and Parkinson’s diseases 15-17. In the biomedical field, the antibacterial features of anatase are receiving increasing attention 18. Another positive aspects of TiO 2 is that coating glass surfaces with TiO 2 NPs (or TiO 2 nanotubes) visibly improves cell growth and differentiation 19-23. The biocompatibility as well as the toxicity of TiO 2 NPs have been widely demonstrated in C. elegans with TiO 2 NPs concentrations ranging from 1 pg/ml 24-25 to 100 µg/ml 26-28 under both, dark conditions 25, 27 as well as under UV illumination (to enhance its photocatalytic effetcs) 26. Critically, various studies have concluded that the toxicity of TiO 2 NPs results from the production of cell-damaging reactive oxygen species (ROS) in C. elegans 24-26, 28-30. Toxicity

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effects of low TiO 2 NPs concentrations (i.e., ≤ 100 µg/ml) under light have been extensively studied 26-28; however, the effects of high TiO 2 NPs concentrations (i.e., 500 µg/ml) in the dark are not explored. More researches would provide deeper insights into the toxic effects of TiO 2 NPs in cases of, e.g., accidental uncoating of TiO 2 layers (corrosion effects) on implant surfaces 31. Specifically, whether TiO 2 NPs can be directly taken up by neurons and whether this would lead to pathological phenotypes remains unknown 32. Notably, metal intoxication has been implicated in Parkinson’s disease (resulting in movement disorders), and C. elegans has been extensively used to study these effects 17, 33-34. Thus, the focus of this study was to a) understand the toxic effects of elevated TiO 2 NPs concentrations on C. elegans, b) determine whether C. elegans neurons are able to take up these TiO 2 NPs into their cytoplasm, c) understand the concomitant effects on neuronal growth, and d) understand the underlying cellular mechanism causing the observed pathological phenotypes. Critically, primary neurons can be easily isolated from C. elegans embryos and cultured for several days at room temperature without the need to adjust the CO 2 atmosphere. Additionally, to understand the relationship between TiO 2 NPs-induced phenotypes and changes in underlying cellular mechanisms, commercially available DNA microarray

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biochips can be used. Employing this assay, changes in the expression of 22,548 genes in C. elegans can be detected when comparing control animals with TiO 2 NPs-treated animals.

2. EXPERIMENTAL SECTION 2.1. Characterization of TiO 2 NPs. Both anatase and rutile phase TiO 2 NPs were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The morphology (shapes and sizes) of TiO 2 NPs was characterized by a field-emission scanning electron microscope (FE-SEM, JSM-7000F), operating at 10 kV. The phase of TiO 2 NPs was confirmed by a X-ray diffractometer (Simatsu, XRD 6000, Japan) operating at scanning angles ranging from 10° to 90° at a scan rate of 4°/min (at 30 kV and 20 mA). The elemental composition of the TiO 2 NPs was investigated by using an energy dispersive X-ray spectroscope (EDS elemental analysis, Horiba, EX220, Japan) which was attached to the aforementioned FE-SEM. Dynamic light scattering (DLS) measurements and zeta potential were used to investigate the agglomeration/aggregation characteristics of TiO 2 NPs and FITC-APTMS-TiO 2 NPs in S-medium. 2.2. C. elegans Maintenance and TiO 2 Exposure. C. elegans wild type (N2) strains were maintained on NGM (nematode growth medium) agar plates seeded with (uracil auxotroph) E. coli OP50 (serving as a food source) at 20°C according to standard methods 13. 6


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Synchronized worms were exposed to either anatase or rutile TiO 2 NPs prepared in S-medium (worm liquid growth medium) 35 in the dark. To guarantee dark conditions, 15 ml falcon tubes containing worms immersed in S-medium were wrapped with aluminum foil. Due to the aggregation propensity of TiO 2 NPs, S-medium (containing TiO 2 NPs) was ultrasonicated prior to the addition of worms. To understand whether TiO 2 NPs exert toxicity effects on OP50 bacteria (eventually killing them) we determined OP50 growth curves (OD600 value) in the presence or absence of TiO 2 NPs (Figure S1). As a result, TiO 2 NPs under dark conditions (with reduced phototoxicity) do not kill OP50 bacteria. 2.3. TiO 2 NPs Labeling with FITC-APTMS. To determine whether worms can take up TiO 2 NPs, the TiO 2 NPs were labeled with fluorescein isothiocyanate modified with 3-aminopropyltrimethoxysilane (FITC-APTMS) based on a protocol by Sui et al. 36. Here, APTMS acts to chemically link FITC to TiO 2 NPs. In detail, 4 mg of FITC was dissolved in 2 ml of anhydrous ethanol and 20 μl of APTMS. After stirring at room temperature for 4 hours (in the dark) the solution is then mixed with TiO 2 NPs (ratio TiO 2 NPs:APTMS = 1:0.1). After further 12 hours of stirring at room temperature in the dark, the modified TiO 2 NPs were centrifuged for 10 min at 15,000 rpm (3 times) to remove excess APTMS. Particle sizes of FITC-APTMS-TiO 2 NPs were measured using an FE-SEM (JSM-7000F) operating

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at 10 kV and vary only slightly from unlabeled TiO 2 NPs (Table 1). Worms were incubated with the modified TiO 2 NPs using concentrations of 100 µg/ml for 12 hours (in S-medium in the dark) prior to imaging using confocal spinning disk microscopy (Olympus IX81 microscope with a DSU Nipkow spinning disk unit connected to an Andor iXon DV897 EMCCD camera). 2.4. Population Assay, Body Length Measurements, and Locomotion Analysis. Changes in the worm population after exposure to TiO 2 NPs were assessed by counting the number of offspring worms under a dissecting microscope. In addition, we determined the percentage of L4 worms in the worm population to better evaluate the effects of TiO 2 NPs on worm development. Note that the worm’s life cycle (approximately 2.5 days from egg hatching to the adult animal) is indicated by subsequent larval stages from L1 (after egg hatching) to L2, L3 and L4 (following the adult stage). The L4 stage can be easily distinguished from the L1, L2 and L3 stages because these worms exhibit a characteristic white triangular mark near their vulva. In both experiments, the worms were synchronized by the bleaching method, in which adult hermaphrodite worms are exposed to a “bleach solution” (that contains 7 ml ddH 2 O, 1 ml 5 M NaOH and 2 ml 6-12% NaOCl) that disintegrates the cuticle and allows for the collection of eggs. Sucrose, density gradient centrifugation was

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utilized to separate dead worm bodies and eggs. Eggs were collected by micropipette at the appropriate sucrose layer and subsequently counted via a cell counter. These eggs are then incubated with either anatase or rutile TiO 2 NPs for 72 hours in S-medium prior to calculating the number of offspring worms. The same worms were also used to measure body length as well as thrashing frequencies. Body length was determined by analyzing bright field images of the worm population using the open source imaging software NIH ImageJ (http://rsb.info.nih.gov/ij/). Here, we first straightened digital images of (typically) bended worms using the ImageJ plugin ‘Straighten’ and then used the ‘Line Tool’ to measure the worm’s body length. Changes in locomotion behavior (thrashing assay) were assessed by counting the worms’ body bends per minute in liquid S-medium. Note that the TiO 2 NPs incubation time for experiments was 42 hours, a time at which worms are presumed to have successfully entered the L4 larval stage. Each experiment noted above was independently repeated 3-5 times for each used TiO 2 NPs concentration. 2.5. Isolation and Culturing of Primary C. elegans Neurons. Primary C. elegans neurons were isolated and cultured based on protocols by Christensen et al. 37 and Strange et al. 38. Specifically, the neurons were cultured from worms expressing the pan-neuronal marker pUNC-104::UNC-104::mRFP 39 to improve the visualization of neuronal structures

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(soma, axons and dendrites) under a confocal microscope. In short, eggs from these worms were collected from gravid hermaphrodites using the above described bleaching method. These eggs were then incubated with the enzyme chitinase (Sigma) (1 U/ml egg solution) that allows for dissolving the compact egg shell. The exposed cells were subsequently plated on lectin-coated glass-bottom petri dishes using Leibovitz’s L-15 medium (containing 10% FBS and antibiotics) mixed with TiO 2 NPs in dark conditions. After 3 days, neurons were fully developed and could be imaged and analyzed. To measure axonal lengths, we first straightened the digital images of axons using the ImageJ plugin ‘Straighten’ and then used the ‘Line Tool’ to measure the lengths. Each experiment was independently repeated 3-5 times for each TiO 2 NPs concentration used. Note that in another experiment, cells collected from dissolved eggs were incubated with FITC-TiO 2 NPs instead of unlabeled TiO 2 NPs for the visualization of NP uptake into neurons and the subsequent analysis of colocalization events. 2.6. TEM Observation of Primary C. elegans Neurons Treated with TiO 2 NPs. To obtain a deeper understanding of neuronal TiO 2 NPs uptake, the primary C. elegans neurons (prepared and cultured as noted above) were imaged by using transmission electron microcopy (TEM). Here, cultured neurons were further processed by fixing with 2.5%

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glutaraldehyde and 4% paraformaldehyde prior to embedding in Spurrs’ resin. Ultramicrotomy yielded 50- to 100-nm-thick slices of resin-embedded neurons, which were then imaged using a Hitachi HT7700 TEM operating at 100 kV. Details on TEM operation are provided in the Supporting Information. 2.7. DNA Microarray Method. DNA microarray analysis was employed by using the ‘Affymetrix C. elegans gene chip’ (Thermo Fisher Scientific Inc., Santa Clara, CA, USA) covering 22,548 transcripts. In short, total RNA was isolated from synchronized worms grown in liquid culture (exposed to 500 µg/ml anatase TiO 2 NPs) using TRIZOL-based standard protocols 40. The quality of RNA was monitored using optical density measurements and an OD 260/280 ratio value of >1.9 was regarded as adequate quality. Subsequent fluorescent labeling was performed using the Genispehere 3DNA Array350 kit (Genisphere Inc., Hatfiled, PA, USA). The gene chip was hybridized to cDNA overnight, washed stringently to remove nonspecifically bound probe, and then poststained with fluorescent dendrimers. After posthybridization a GenePix scanner and GenePix Pro 4.0 image analysis software (Molecular Devices, Sunnycale CA, USA) was used to aquire fluorescence intensities. We considered hybridization spots as positive if signal intensity was twice (or more) the background in at least one channel of half of the replicates. Per-chip normalization

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was performed by dividing the expressed genes by the median of two housekeeping control genes, β-tubulin and cyclophilin. For data mining and statistical analysis, we used Rosetta Resolver System (Rosetta Biosoftware, Seattle, WA, USA) 41 as well as Transcriptome Analysis Console (TAC) Software 42 (Affymetrix, Waltham, MA, USA). To understand the cellular functions of the genes, the Wormbase database (wormbase.org) 43 was utilized. The database contains all known and annotated C. elegans genes 43 and can be employed to understand their differential expression in different tissues . All microarray experiments were carried out at the NHRI Microarray Core Laboratory (Zhunan, Taiwan) which houses an Affymetrix GeneChip system consisting of a GeneChip Hybridization oven 640, GeneChipFluidics Station 450, and GeneChipScanner 3000 (Affymetrix, Waltham, MA, USA). 2.8. Statistical Analysis. Statistical analysis was conducted using Student’s t-test (employing Microsoft Office Excel’s ‘Data Analysis’ tool). Three independent experiments were performed for each experimental condition. Data are represented as mean ± STD obtained from at least three independent experiments. P values are represented as *p

C. elegans Neurons - ACS Publications - American Chemical

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