Neurotoxicity and biomarkers of zinc oxide nanoparticles in main functional brain regions and dopaminergic neurons

Journal Pre-proof Neurotoxicity and biomarkers of zinc oxide nanoparticles in main functional brain regions and dopaminergic neurons

Huanliang Liu, Honglian Yang, Yanjun Fang, Kang Li, Lei Tian, Xiaohua Liu, Wei Zhang, Yizhe Tan, Wenqing Lai, Liping Bian, Bencheng Lin, Zhuge Xi PII:

S0048-9697(19)35804-8

DOI:

https://doi.org/10.1016/j.scitotenv.2019.135809

Reference:

STOTEN 135809

To appear in:

Science of the Total Environment

Received date:

26 September 2019

Revised date:

8 November 2019

Accepted date:

26 November 2019

Please cite this article as: H. Liu, H. Yang, Y. Fang, et al., Neurotoxicity and biomarkers of zinc oxide nanoparticles in main functional brain regions and dopaminergic neurons, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.135809

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© 2019 Published by Elsevier.

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Neurotoxicity and biomarkers of zinc oxide nanoparticles in main functional brain regions and dopaminergic neurons Huanliang Liua,b,#, Honglian Yanga,b#, Yanjun Fanga,b , Kang Lia,b, Lei Tiana,b, Xiaohua Liua,b, Wei Zhanga,b, Yizhe Tana,b, Wenqing Laia,b, Liping Biana,b, Bencheng Lin a,b, , Zhuge Xi a,b,

a

Tianjin Institute of Environmental and Operational Medicine, Tianjin 300050, China

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b

Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment &

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Food Safety, Tianjin 300050, China

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Abstract

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Manufactured zinc oxide nanoparticles (Nano-ZnO) are being used increasingly in many fields

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owing to their excellent physicochemical properties. Consequently, biosecurity has become a growing concern for human health and the environment. In the present study, Nano-ZnO

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neurotoxicity was investigated in vivo and in vitro. In vivo results showed that Nano-ZnO particles delivered through intranasal instillation were translocated to the brain, specifically deposited in the olfactory bulb, hippocampus, striatum, and cerebral cortex, and caused ultrastructural changes, oxidative damage, inflammatory responses, and histopathological damages there, which may be important for inducing Nano-ZnO neurotoxicity. Further in vitro studies on PC12 cell line illustrated that exposure to Nano-ZnO for 6 h affected cell morphology, decreased cell viability,

* Corresponding authors: Tel: +086 022 84655124; +086 022 84655324 E-mail addresses: [email protected] (Z. Xi); [email protected] (C Lin) # These authors contributed equally to this work.

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increased lactate dehydrogenase and oxidative stress activity levels, impaired mitochondrial function, and disturbed the cell cycle. In addition, Nano-ZnO could destroy neuronal structure by affecting cytoskeleton proteins (tubulin-α, tubulin-β and NF-H), resulting in the interruption of connection between nerve cells, which lead to nervous system function damage. Meanwhile, Nano-ZnO could induce neuronal repair and regeneration disorders by affecting the growth-related

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protein GAP-43 and delayed neurotoxicity by affecting the calcium/calcium-regulated kinase (CAMK2A/CAMK2B protein) signaling pathway.

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1. Introduction

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Regulatory proteins; Cytoskeleton proteins

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Keywords: Nano-ZnO; Oxidative stress activity; Inflammatory responses; Mitochondrial function;

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Since the rapid development of nanotechnology, the manufacture and use of nanoparticles

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have increased. Consequently, people are more likely to be exposed to nanoparticles occupationally or environmentally. Among the many types of nanomaterials used in production process and

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detection methods, nano-zinc oxide nanoparticles (Nano-ZnO) is one of the most mature and functional nanomaterial after carbon nanotubes. Due to its excellent performance in light, electricity, magnetism, sensitivity, antibacterial, and disinfection, Nano-ZnO has been widely used in the preparation of antibacterial, disinfection, deodorization, and anti-ultraviolet products, as well as in gas sensors, medicine, electronics, and other industries. The toxicological research on Nano-ZnO is far behind the speed of its application on account of the conventional view that zinc oxide is non-toxic. However, existing studies have found that Nano-ZnO is more toxic than most nano-metal oxides（Jeng and Swanson, 2006; Liu et al., 2013; Liu et al., 2015）. Due to its unique physical and chemical properties, such as small size and large surface area,

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nanoparticles have high surface reactivity and produce more active sites to participate in different biochemical reactions compared to normal particles. Therefore, nanoparticles have a strong ability to penetrate tissue and cells as well as a strong oxidation and catalytic ability. Recent research has shown that the central nervous system is a potential nanoparticle toxicity target. Han et al. (2011) suggested that the spatial learning and memory ability is attenuated by synaptic plasticity alteration

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in rats intraperitoneally injected with Nano-ZnO. Another study showed that after intravenous administration into ICR mice, radioactive zinc oxide nanoparticles exhibit a primary retention in the

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lung for the first hour and begin to be translocated to intestinal tract for fecal excretion at a later

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stage, indicating that Nano-ZnO does not stay in the body for a long time after entering the blood

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circulation (Chen et al., 2010). The effect of Nano-ZnO on voltage-dependent sodium and

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potassium currents and evoked action potentials has been studied in acutely isolated rat

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hippocampal CA3 pyramidal neurons and the results have shown that Nano-ZnO solution leads to Na+ influx and intracellular Na+ accumulation as well as K+ efflux plus cytoplasmic K+ loss. These

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may disturb the ionic homeostasis and the physiological functions of neurons (Zhao et al., 2009). The toxicity of five kinds of nano-metal oxides on mouse brain neuroma cell line Neuro-2A has also been studied and compared, suggesting that compared with Nano-Al2O3, Nano-TiO2, NanoFe3O4, and Nano-CrO3 having similar particle sizes, Nano-ZnO is the most toxic particle, which could cause mitochondrial function decrease, lactate dehydrogenase leakage increase, and cell apoptosis (Jeng and Swanson, 2006). It has also been found that after exposing to 15 μg/mL Nano-ZnO particles for 3 h, the activity of mouse neural stem cells (NSCs）was decreased, DNA was damaged, and cell apoptosis was induced (Deng et al., 2009). Till date, research on Nano-ZnO neurotoxicity has mainly focused on the toxic damage to

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hippocampus, cortex, and the cognitive function. However, the response of striatum and dopaminergic neurons to Nano-ZnO remains ill-defined especially as a companion of in vivo or in vitro study. Neurodegenerative diseases are often accompanied by damage to the main functional brain areas, such as hippocampus and striatum, which further damages the corresponding nerve function. Patients with neurodegenerative diseases are susceptible to environmental toxicants (Lozano and Kalia, 2015). According to literature survey, the concentration of nanoparticles

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released into the air can reach 0.15 μg/m3 during their productive process (Arora et al., 2008).

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In the present study, we investigated Nano-ZnO neurotoxicity in vivo and in vitro. The in vivo

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study was carried out to locate Nano-ZnO in the brain using transmission electron microscope

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(TEM) after intranasal instillation of Nano-ZnO, and to concomitantly evaluate the potential

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particle-induced oxidative stress, inflammatory response, ultrastructural changes, and pathological

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injury in main functional brain regions. Moreover, we conducted in vitro studies in the dopaminergic neuron PC12 cell line to evaluate the damage induced by Nano-ZnO and measured

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changes in cell viability, cell morphology, LDH (lactate dehydrogenase) leakage, oxidative stress level, mitochondrial function, and cell cycle. Furthermore, in order to explore the possible biological molecular mechanisms of Nano-ZnO-mediated cell proliferation toxicity and morphological or phenotypic changes, we measured the expression levels of six proteins related to neuronal development, neuronal signal transduction, and neuronal cytoskeleton, namely, growth associated

protein-43

(GAP-43),

calcium

(Ca2+)/calmodulindependent

kinase

type

II

(CAMK2A/CAMK2B), and cytoskeleton proteins [tubulin-alpha (α), tubulin-beta (β) and neurofilament (NF-H)]. This study clarified the neurotoxicity of Nano-ZnO intranasal exposure on distribution and

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potential particle-induced injuries in mainly functional brain areas in vivo and Nano-ZnO neurotoxicity on dopaminergic neurons in vitro; especially, six proteins were selected as biomarkers. Our findings are of important significance for environmental or occupational safety following inhalation exposure to nanoparticles. 2. Materials and Methods

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2.1. Particle preparation

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Manufactured Nano-ZnO particles were purchased from Shenzhen Nanuo Nanomaterials Corp.

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The particles were vortexed in physiological saline for 10 s of 10 times. For breaking down the

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agglomerates, the particles were sonicated ten times for 30 s after every 2 min at 4 °C. The

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Nano-ZnO particle sizes and shapes were measured using TEM (JEM-1230; Japan; Fig. 1A). The BET surface area analyzer (AUTOSORB-MP) was used to detect the Nano-ZnO particle specific

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surface area (SSA). The crystal structure of the Nano-ZnO particles were detected with X-ray

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diffraction (XRD; MiniFlex II; Rigaku; Fig. 1B). The zeta potential was measured by applying an electrical field in 1 mM NaCl through MilliQ water, based on Smoluchowski’s theory (Delsa™Nano C; Beckman Coulter) (Hanna et al., 2008). The composition and density were obtained from the supplier datasheets. 2.2. In vivo study 2.2.1. Animals and intranasal instillation Healthy male Wistar rats [6-week old, 180-220 g BW (body weigh)] were obtained from the Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). This work was approved by the Animal Experiments and Experimental Animal Welfare Committee of Peking Union Medical College, Institute of Radiation Medicine of Chinese Academy of Medical Sciences. 5

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A proof/certificate of approval is available upon request. All the rats received food and water ad libitum and were housed in wire cages at 22±2 °C temperature, 60±10% humidity, and 12:12 h light: dark cycle. All treatments were performed in a Grade II animal room without any air pollutants in the environment. A week after adjusting to the environment, the rats were divided into treatment and control

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groups randomly, with each group comprising 20 rats. According to the previously published protocols of our laboratory (Liu et al., 2013; Liu et al., 2015), the treatment and control group mice

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were intranasally instilled with 20 μg Nano-ZnO/g body weight and equivalent saline volume daily,

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respectively. In the preliminary experiment, we assessed oxidative stress level, immune

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inflammatory response, and pathological changes in rat brain tissue every 5 days. No positive

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results were found within 5 and 10 days. As positive results were found on the 15th day, we

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evaluated zinc content in brain tissue and Nano-ZnO location in brain tissue at the end of the experiment. Hence, in the follow-up experiment, we assessed them at 15 and 30 days.

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The translocation and distribution of Nano-ZnO, as well as the ultrastructural changes were estimated in crucial functional regions of rat brains following Nano-ZnO and saline intranasal instillation within 15 days. Later, the oxidative stress level and immune inflammatory response were evaluated in crucial functional regions of rat brains within 15 to 30 days, while the histological changes were evaluated in these regions in 30 days. In the first experiment, the animals were anesthetized with 40 mg/kg 1% sodium pentobarbital and 20 μL Nano-ZnO was instilled in the nostrils of five rats of the treatment group held in supine position. Physiological saline was instilled in the control group rats. The animals were anaesthetized with 40 mg 1% sodium pentobarbital/kg body weight 15 days post-instillation. The brain regions of interest like cerebral cortex, striatum,

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hippocampus, and olfactory bulb were isolated over ice from the brains of three rats to determine the zinc content by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500ce, Agilent Technology Co.; USA). The cerebral cortex, striatum, hippocampus, and olfactory bulb of the remaining two rats were used for TEM observation. In the second experiment (intranasal instillation once daily for 15 and 30 days, respectively; 6 rats per group at each time point), the four

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above-mentioned brain regions were homogenized to assay for oxidative damage and cytokine

histopathologically examined 30 days post-instillation.

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2.2.2. Determination of Zn content

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levels. Furthermore, the four above-mentioned brain regions of the remaining three rats were

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The zinc content in the cerebral cortex, striatum, hippocampus, and olfactory bulb was

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determined by ICP-MS method. Each tissue was individually placed in Teflon digestion vials,

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weighed, and digested in 2 ml ultrapure nitric acid (65% nitric acid; Merck; GER) for 4 h. For removing the remaining nitric acid, the vials were heated at 120 ℃, until the samples were

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completely digested and the solutions became clear (0% nitric acid). Eventually, the processed solutions were diluted to 3 mL with deionized water. The zinc content in the samples were analyzed using ICP-MS with 1 ng/mL scandium as the standard element. 2.2.3. Oxidative stress-related biomarker assay The four brain regions were weighed and processed with cold phosphate-buffered saline (PBS, w/v=1:9). The mixtures were homogenized at 4 ℃ using a high-throughput tissue grinder (TECHIN, Tianjin, China) and then centrifuged for 5 min at 14000× g and 4 ℃. The supernatants were collected and assayed for oxidative stress and immune inflammatory response biomarkers. The protein contents were determined using the Bradford method with bovine serum albumin (BSA) as

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the standard (Beyotime Biotech, China). The glutathione activity (GSH) and malondialdehyde level (MDA) were determined by colorimetry using commercial kits (Beyotime Biotech) according to the protocols, and the optical density was detected with a microplate reader (MD SpectraMax M5e, USA) at 412 and 532 nm, respectively. 2.2.4. Measurement of immune inflammatory responses

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Interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels in the above-mentioned brain region homogenates were detected using rat-specific double-antibody

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sandwich enzyme linked immunosorbent assay (ELISA) kits (Abcam, UK) as indicators for the

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immune inflammatory response within the central nervous system (CNS) after Nano-ZnO exposure.

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The assays were performed according to the manufacturer instructions. The optical densities were

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2.2.5. Ultrastructure examination

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measured by an MD SpectraMax M5e multiplate reader at 450 nm.

Fresh olfactory bulbs, hippocampus, striatum and cerebral cortex samples were soaked

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overnight in 2.5% glutaraldehyde at 4 ℃ to fix the tissues and cells and then sufficiently washed with PBS. The samples were then fixed with 1% osmium tetroxide, dehydrated with a series of concentration gradient ethanol, embedded in epoxyresin, and polymerized for 24 h at 37 ℃. Ultrathin sections (70 nm) were cut using an ultramicrotome (LKB-V, Sweden), stained with lead citrate and uranyl acetate, and observed by TEM. 2.2.6. Histopathological examination Rat hippocampus, striatum, and cerebral cortex exposed to Nano-ZnO were collected and immediately fixed in 4% paraformaldehyde for 5 days. All tissues were processed in an automated tissue processor and embedded in paraffin. Each tissue was cut into 5 μm sections. After

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hematoxylin-eosin (HE) staining, the sections were visualized using a microscope (Olympus BX53, Japan). 2.3. In vitro study 2.3.1. Cell culture and exposure to nanoparticles The PC12 cell line was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences in Shanghai. The cell line was grown in RPMI-1640 medium supplemented

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with 100 U/mL penicillin, 10% horse serum, 100 μg/mL streptomycin, 50 ng/mL nerve growth

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factor (NGF), and 5% fetal bovine serum, in a cell incubator with 5% CO2 at 37 ℃.

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Nano-ZnO suspensions were freshly prepared prior to cell exposure. Suspensions were diluted

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with medium to 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg/mL and immediately applied to the cells.

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Untreated cells served as control.

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2.3.2. Cell morphology

The cells were seeded into 96-well plates (2×104 cells/well), and cultured for 6 h in 8 and 16

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μg/mL particle suspensions in an incubator. Cell morphology was observed under an inverted microscope (Olympus BX51, Japan).

2.3.3. Cell viability assay and LDH measurement Cell viability was assessed using the CCK-8 assay kits (Beyotime Biotech). Cells were cultured in 96-well plates (3×104 cells/well) for 24 h and treated with different nanoparticle suspension concentrations for 6 and 12 h. After the treatment, 10 μL CCK-8 dye was added to each well, mixed thoroughly, and incubated for 2 h at 37 ℃. To prevent particles from interfering with the result analysis, the solution of each well was quantitatively transferred to an empty well of another plate as described by Zhang et al (2012). Afterwards, the absorbance was analyzed using a

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microplate reader (MD SpectraMax M5e, USA). LDH leakage is another objective cytotoxicity measurement based on membrane integrity, which was measured by a commercial LDH kit (Beyotime Biotech). Briefly, after treatment with Nano-ZnO particles for 6 h, the cell culture medium was centrifuged at 3000 rpm for 10 min. The supernatant was collected for the LDH activity measurement by measuring the optical density with

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an MD SpectraMax M5e at 490 nm. 2.3.4. Intracellular reactive oxygen species (ROS) and oxidative damage production

was

monitored

by

the

oxidation

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ROS

sensitive

fluorescent

dye

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2′7′-dichlorofluorescein diacetate (Beyotime Biotech). The 10 mM DCFH-DA stocking solution

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was diluted to a 10 μΜ working solution with serum-free RPMI-1640. Cells (1×106) stimulated

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with 4–20 μg/mL Nano-ZnO were incubated with 2 mL DCFH-DA working solution at 37 ℃ for 30

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min and the fluorescence intensity was measured by flow cytometer (BD FACSCalibur™, USA). The MDA, nitric oxide (NO), and superoxide dismutase (SOD) levels were measured by

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respective reagent kits (Beyotime Biotech). After 6 h of stimulation with different Nano-ZnO concentrations, PC12 cells were collected and lysed in lysis buffer (Beyotime Biotech) in ice for 5 min. The lysate was then centrifuged at 14000× g for 10 min at 4 ℃. The supernatants were collected to measure the intracellular MDA, NO, and SOD levels. 2.3.5. Measurement of mitochondrial membrane potentials and ATP level The mitochondrial membrane potentials were measured with a fluorescent lipid cationic compound

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanide

iodide

(JC-1,

Beyotime Biotech). After 6 h stimulation with 4–20 μg/mL Nano-ZnO, 6×105 cells were extracted and suspended in 0.5 mL medium. Then, 0.5 mL JC-1 working solution was added, mixed, and

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2.3.6. Measurement of cell cycle For cell cycle detection, cells were harvested, fixed, and permeabilized in 70% ice-cold ethanol

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and then stored at 4 ℃ for at least 24 h after exposure to 4–20 μg/mL Nano-ZnO for 6 h. The cells

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were resuspended in 1 mL lysis buffer (0.1% Triton X-100, 1 mg/mL RNase A, and 50 μg/mL PI)

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and incubated for 30 min at 37 ℃. The stained cell samples were analyzed using the flow cytometer.

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2.3.7. Western blot analysis

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After stimulating the PC12 cells with 4–20 μg/mL Nano-ZnO for 6 h, the cells were harvested, washed with ice-cold PBS, and lysed in ice-cold protein lysis buffer (Beyotime Biotech) with a

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protease inhibitor cocktail. The lysate was then centrifuged at 14000× g for 10 min at 4 ℃ to collect the supernatants. After quantifying the protein concentration by BCA protein assay kit (Beyotime Biotech), 30–50 μg total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck & Co. Inc; New Jersey, USA). After blocking for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST), the membranes were incubated with anti-GAP-43, -CAMK2A, -CAMK2B, -tubulin-α, -tubulin-β, and -NF-H antibodies (Protein Tech, USA) overnight at 4 ℃. The membranes were then washed with TBST thrice and incubated with horseradish peroxidase-conjugated secondary antibody (HuaAn Biotechnology Co. Ltd,

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Hangzhou, China) in TBST for 1 h at room temperature. After washing with TBST again, the blots were visualized with the enhanced chemiluminescence ECL Kit (Affinity Biosciences Co. Ltd, Cincinnati, USA) and the results were analyzed with a Gel-pro analyzer (Tanon Science & Technology Co. Ltd, Shanghai, China). 2.4. Statistical analyses

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Statistical analyses were performed using SPSS V 21.0. The data were presented as the mean ± standard deviation (SD). The results were carried out using ANOVA followed by the least

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significant difference (LSD) test (equal variances) or Dunnet’s T3 post hoc test (unequal variances).

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P < 0.05 was considered significant.

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3. Results

3.1. Structures and properties of Nano-ZnO

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As shown in the TEM image (Fig. 1A), the particles were nearly spherical and around 10–30

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nm in size. The XRD spectrum clearly shows that the crystalline structure of nanoparticles was hexagonal. Detailed structure and properties are listed in Table 1.

Fig. 1. Images of Nano-ZnO by (A) TEM and (B) XRD Table 1Characterization of Nano- ZnO

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Zeta potential Supplier

SSA

Crystalline

(g/cm3)

(m2/g)

structure

5.78

45

Hexagonal

Size (nm) (mV)

Nano-ZnO

Density

18.63

19.61±5.83

Shape

Composition

sphere

ZnO >99.9%

3.2. Zn content in each brain region The Zn contents in the olfactory bulb, hippocampus, striatum, and cerebral cortex were

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determined after an intranasal instillation of Nano-ZnO for 15 days, and the results are shown in Fig.

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2. The Zn content increased in all brain regions, however, there was no significant difference compared with the content in control group rat brain regions. While investigating the reasons, we

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observed that zinc is one of the essential trace elements in organisms and a neurotransmitter of

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excitatory synapses as well. It plays an important role in stress response and maintenance of central

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nervous CNS metabolism and has a relative high distribution in the brain. Therefore, the difference

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in the background value of zinc in rats may affect the results in this study. Hence, we combined the results with TEM results to explore whether nanoparticles were deposited in the above-mentioned

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brain regions and observe their ultrastructural changes.

Fig. 2. Zn content in regions of the rat brain (n = 3) 15 days after intranasal instillation with 20μg Nano-ZnO/g body weight. 3.3. Ultrastructural changes 13

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Fig. 3 shows ultrastructural micrographs of the olfactory bulb, hippocampus, striatum and cerebral cortex tissue after intranasally instilling Nano-ZnO for 15 days. TEM images of the olfactory bulb tissue from the Nano-ZnO-instilled rats revealed that the Golgi apparatus and mitochondria appeared markedly dilated and swelled, mitochondria matrix was vacuolated, the mitochondrial cristae was lysised , the number of rough endoplasmic reticula decreased, while that

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of the lysosomes increased; fine particles with high electron density were found scattered or clustered in the nerve fibers. TEM images of the hippocampus revealed that the number of

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lysosomes increased slightly, free ribosomes were relatively abundant, Golgi apparatus were

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somewhat dilated, mitochondria matrix was vacuolated partially, and more fine particles with high

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electron density were observed in cytoplasmic and intramedullary regions. TEM images of the

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striatum from the Nano-ZnO-instilled rats revealed that neuronal cell nucleus was oval, nuclear

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membrane was slightly twisted, the number of lysosomes increased, Golgi apparatus appeared markedly dilated, rough endoplasmic reticula shortened and decreased in number, mitochondrial

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matrix degraded, and fine particles with high electron density were found dispersed in the nerve fiber section. TEM images of the cerebral cortex indicated that Golgi apparatus in the cytoplasm was dilated and its structure was blurred, free ribosomes were relatively abundant, rough endoplasmic reticulum was slightly dilated, mitochondrial structure was blurred, and more fine particles with high electron density were observed in the cytoplasm and nerve fiber.

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Fig. 3. TEM images of the olfactory bulb, hippocampus, striatum and cerebral cortex tissue

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after intranasally instilling 20 μg Nano-ZnO/g body weight for 15 days.

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The CNS is susceptible to changes in its microenvironment. Therefore, trace amounts of

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extraneous material entering the brain microenvironment may result in changes in the balance of the

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internal milieu and cause damage to the CNS. Consequently, our above findings indicated that it

functional brain areas.

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was imperative to evaluate whether treatment with Nano-ZnO induced potential injuries in the four

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3.4. Oxidative damage and inflammatory responses in regions of the rat brain After 15 and 30 days of Nano-ZnO exposure, MDA concentration was markedly increased in the olfactory bulb (p<0.01), hippocampus (p<0.01), and striatum (p<0.01), as well as in the cortex (p<0.05) after 30 days of exposure compared to those in unexposed control (Fig. 4A). GSH activity was decreased in the olfactory bulb (p<0.01), hippocampus (p<0.05 in 15 days exposure group; p<0.01 in 30 days exposure group), and striatum (p<0.01) after 15 and 30 days of exposure to Nano-ZnO, and in the cerebral cortex after 30 days of exposure compared to those in unexposed control (Fig. 4B). Overall, Nano-ZnO elevated MDA concentration and reduced GSH activity in a time-dependent manner. In addition, TNF-α levels in the olfactory bulb (p<0.05 in 15days exposure

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group; p<0.01 in 30days exposure group), hippocampus (p<0.05 in 15days exposure group; p<0.01 in 30days exposure group), and striatum (p<0.01) significantly increased 15 and 30 days post-exposure compared to the levels in unexposed controls (Fig. 4C). IL-1β levels in the olfactory bulb, hippocampus, and striatum also significantly increased (p<0.01) 15 and 30 days post-exposure

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compared to the levels in unexposed controls (Fig. 4 D).

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Fig. 4. Levels of oxidative stress (MDA and GSH) and cytokine (TNF-α and IL-1β) in the olfactory

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bulb, striatum, cerebral cortex, and hippocampus of rats (n=6) intranasally instilled with 20 μg Nano-ZnO/g body weight 15 and 30 days post-exposure. (A) MDA; (B) GSH; (C) TNF-α; (D) IL-1β. *p < 0.05; **p < 0.01 versus the control group. 3.5. Histological changes of the hippocampus and striatum As shown in Fig. 5, the histological changes of the olfactory bulb ， cerebral cortex, hippocampus, and striatum revealed no observable damage in control group after treatment. In contrast, different degrees of disordered cell arrangement, cell degeneration (nuclear pyknosis, lysis or disappearance and fiber entanglement), and inflammatory cell infiltration were observed in CA1, CA3, and dentate gyrus regions of the hippocampus and cerebral cortex after intranasal exposure to Nano-ZnO, in which CA3 and dentate gyrus region damages were most serious. In addition, the 16

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striatum of the treatment group rats showed nuclear pyknosis, neurocyte edema, and inflammatory cell infiltration. Compared with the control group rats, the olfactory bulb contained disordered cell

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layers with some cells gathering and inflammatory cells infiltrating into the tissues.

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Fig. 5. The histological changes of the olfactory bulb, cerebral cortex, hippocampus, and striatum

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after intranasally instilling 20 μg Nano-ZnO/g body weight for 30 days (×200 magnification).

green arrow: inflammatory cells.

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3.6. Cell morphological changes

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Yellow arrow: neurons; black arrow: tangled fibers; red arrow: disordered arrangement of neurons;

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PC12 cells were undifferentiated, spherical in shape and did not produce neurite before NGF exposure (Fig. 6A). Following NGF exposure, PC12 cells differentiated into neuronal type cells, and neurites could be seen extending to the periphery to form a sparse network, as shown in Fig. 6B. Control PC12 cells were normal with long neurites, multiple intercellular neurite connecting points, multiple intercellular processes, and transparent cytoplasm. After treating with 8 and 16 μg/mL Nano-ZnO for 6 h, PC12 cells showed different degrees of deformation, including rounding and floating, full nuclear condensation, and formation of several intracellular vacuoles (Fig. 6C, D). Additionally, the number and length of cellular neurites were significantly reduced or shortened and intercellular neurite connecting points were decreased and even disappeared in some groups. The cellular transparency was visibly reduced. The distribution of nanoparticles had an effect on the 17

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growth and metabolism of cells, which resulting in viable cell number reduction.

Fig. 6. Morphological changes in PC12 cells after 6 h exposure to Nano-ZnO observed using

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inverted phase contrast microscopy (×400 magnification). (A.) Control cells without NGF; (B)

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Control cells with NGF; (C) Cell with 8 μg/mL Nano-ZnO; (D) Cell with 16 μg/mL Nano-ZnO

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3.7. Cytotoxicity of Nano-ZnO in PC12 cells

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NGF-stimulated differentiated PC12 cells demonstrate the typical morphological and

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functional characteristics of dopaminergic neurons and are extensively used as a paradigm in neurobiological research (Ishima et al., 2008). After treatment with 8–20 μg/mL Nano-ZnO for 6

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and 12 h, a dose-dependent significant reduction in cell viability was observed (p<0.05 in

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8–12μg/mL Nano-ZnO for 6 h, p<0.01 in 14–20μg/mL Nano-ZnO for 6 h; p<0.05 in 8–10μg/mL Nano-ZnO for 12 h, p<0.01 in 12–20μg/mL Nano-ZnO for 12 h) (Fig. 7A). After exposure to 16 and 20 μg/mL Nano-ZnO for 6 h, the LDH levels were significant increased (p<0.01) compared with those in controls (Fig. 7B).

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Fig. 7. The effects of Nano-ZnO on viability and LDH levels of PC12 cells. (A) Viability of PC12 cells after 6 and 12 h exposure to Nano-ZnO (n=6); (B) Leakage of LDH from PC12 cells after 6 h treatment with Nano-ZnO (n=6). *p < 0.05; **p < 0.01 versus control group. 3.8. Influence on levels of oxidative stress and mitochondrial function in PC12 cells To further explicate the injury process, cytotoxicity was determined according to the intracellular oxidative stress levels as detected by MDA and NO production, SOD inhibition, and

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ROS generation. As shown in Fig. 8A–D, after 6 h treatment with 8–20 μg/mL Nano-ZnO, the

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MDA and NO concentrations were increased significantly (p<0.01). Simultaneously, after treatment

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with 4–20 μg/mL Nano-ZnO, ROS generation were increased (p<0.01) and SOD level was

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decreased (p<0.01) significantly compared with controls. The results were in accordance with the

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results of in vivo studies, indicating that oxidative stress might be an important route for inducing

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Nano-ZnO cytotoxicity. Our findings revealed that Nano-ZnO has an effect on cell viability, however, whether the intracellular ROS increase affects cell organelles, such as the mitochondria,

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needs to be further investigated. The present study resulted in a decrease in mitochondrial membrane potential after treatment of PC12 cells with 4–20 μg/mL Nano-ZnO for 6 h (p<0.05 in 4 μg/mL Nano-ZnO; p<0.01 in 8–20 μg/mL Nano-ZnO) (Fig. 8E). As shown in Fig. 8F, after 6 h incubation of PC12 cells with 8–20 μg/mL Nano-ZnO, ATP levels was markedly decreased compared with the control (p<0.01).

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Fig. 8. Levels of oxidative stress (A. MDA; B. NO; C. SOD; D. ROS), membrane potentials (E)

3.9. Cell cycle assay

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group; ** p<0.01 versus control group.

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and ATP (F) of PC12 cell exposed to Nano-ZnO after 6 h exposure (n=3). * p<0.05 versus control

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The cell cycle distribution of PC12 cells incubated with 4–20 μg/mL Nano-ZnO for 6 h is

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shown in Fig. 9. An increase in the percentage of cells in G2 phase was found after treatment with all concentrations of Nano-ZnO (p<0.01). As the percentages of cells in the G1 phase reduced, cells in the G2 phase also accumulated. These data suggested that Nano-ZnO significantly inhibited cell proliferation as characterized by G2 cell cycle phase arrest.

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< 0.05; **p < 0.01 versus the control group.

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Fig. 9. Effects of Nano-ZnO on cell cycle progression of PC12 cells after 6 h treatment (n=3). *p

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3.10. Effects of Nano-ZnO on CAMKII, GAP-43, tubulin, and NF-H protein levels in PC12

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cells

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To further explore the mechanism of Nano-ZnO-induced neuronal damage, the expression levels of six proteins related to the neuronal cytoskeleton, development, and signal transduction of

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tubulin-α, tubulin-β, NF-H, CAMK2A, CAMK2B and GAP-43,

were detected. As shown in Fig.

10, after 6 h incubation, the tubulin-α level of PC12 cells treated with 12–20 μg/mL Nano-ZnO (p<0.05 in 12–16 μg/mL Nano-ZnO; p<0.01 in 20 μg/mL Nano-ZnO), tubulin-beta and CAMK2A levels of PC12 cells treated with 8–20 μg/mL Nano-ZnO (p<0.05 in 8 μg/mL Nano-ZnO; p<0.01 in 12–20 μg/mL Nano-ZnO), NF-H level of PC12 cells treated with 4–20 μg/mL Nano-ZnO (p<0.05 in 4 μg/mL Nano-ZnO; p<0.01 in 8–20 μg/mL Nano-ZnO), CAMK2B level of PC12 cells treated with 8–20 μg/mL Nano-ZnO (p<0.05 in 8–16 μg/mL Nano-ZnO; p<0.01 in 20 μg/mL Nano-ZnO), and GAP-43 level of PC12 cells treated with 4–20 μg/mL Nano-ZnO (p<0.01) significant decreased compared with that of the control group.

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Fig. 10. Effects of Nano-ZnO on CAMKII, GAP43, tubulin, and NF-H protein levels in PC12 cells

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after 6 h treatment (n=3). *p < 0.05; **p < 0.01 versus the control group.

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4. Discussion

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The TEM assay results showed that Nano-ZnO was adsorbed and deposited in the nasal

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mucosa of rats after intranasal instillation and then transported along the olfactory nerve to the CNS. Nanoparticle translocation and distribution in vivo are affected by many factors including particle

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size, surface charge, and surface hydrophilic and lipophilic characteristics (Islam et al., 2017; Iqbal

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et al., 2018). In addition, two factors can influence nanoparticle entrance to cells: physicochemical properties and the type of cell. Nanoparticles can cross cell membranes and enter cells with relative ease due to their small size and high surface activity. However, their ability to enter cells differ with the type of cells. After surface modifications, nanoparticles possess excellent biocompatibility, which makes it relatively easy for them to enter cells. Moreover, mammalian cell surface is negatively charged, therefore considering electrostatic interactions, positively charged nanoparticles can enter the cell easily than negatively charged nanoparticles. Various type of cells have different capability to intake nanoparticles, which directly affects the number of nanoparticles taken up into the cell. Microglial cells possess phagocytic ability, resulting in a strong ability to intake nanoparticles. In this study, the Nano-ZnO particle size was relatively small (<30 nm) and 22

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positively charged, such that these particles could be taken up easily by the tissue cells. When adsorbed and deposited on the nasal mucosa, Nano-ZnO could be taken up easily by the olfactory mucosa, then transported to the olfactory bulb tissue, and finally to the brain through the olfactory nerve. As the most representative product of lipid peroxidation within cells, MDA indicates lipid

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peroxidation rate and intensity (Christodoulou et al., 2017). GSH concentration reflects the antioxidant ability of the cell and body directly. Thus, our result suggested that tissues and cells

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were attacked by free radicals, decreasing antioxidant abilities such as free radical scavenging

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activity. Due to heavy oxygen demand, the brain appeared to be particularly susceptible to oxidative

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stress-mediated damage compared to other tissues. Many hypotheses describing the mechanisms

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that mediate degenerative loss of dopaminergic neurons in Parkinson's disease (PD), i.e., oxidative

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stress, mitochondrial dysfunction, inflammation, and immune dysregulation have been proposed previously. Among them, oxidative stress has been considered the initiating factor, whereas the

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others are consequences of a highly oxidized state in dopaminergic neurons. Persistent and serious oxidative stress can probably activate inflammation related genes and transcription factors to turn on the inflammatory process (Tarique et al., 2016). The oxidative stress-induced release of pro-inflammatory cytokines, including IL-1β and TNF-α, could activate endothelial cells, astrocytes and microglia, stimulating IL-6 and IL-8 expressions (Block et al., 2007). Cytokines activate pro-apoptotic pathways within the Dopaminergic neuron (DN) as well as stimulate microglia through

inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2)

induction. These events lead to toxic effects in the DN. As signals from damaged DA cells further recruit and stimulate microglia, the process will spiral out of control and into full-blown

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neurodegeneration (Gao and Hong, 2008; Whitton, 2007). It has been demonstrated that IL-1β, IL-6, and TNF-α levels in the striatum of PD brains are elevated (Whitton, 2007). Moreover, inflammation is considered to play an essential role in dopaminergic neuron death and be involved in the pathological process of certain neurodegenerative diseases (Joshi and Singh, 2018; Malú and Matthew, 2010). In this study, Nano-ZnO caused injuries to the CNS, specifically the hippocampus

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and striatum, which were possibly induced by the toxic effects associated with oxidative stress and the release of inflammatory mediators. Thus, our data suggested that Nano-ZnO exposure could

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increase neurodegenerative disorder risk.

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The nervous system is composed of a large number of neurons; however, there is no protoplasm

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connection between these neurons. The neurons establish intercellular interactions only by mutual

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neurite contacts to form the synapse, which allows the functions associated with processes like

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intercellular substance transportation, information transmission, and signal transduction. The results of the present study indicated that Nano-ZnO might injure the neurite and lead to a reduction in the

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number of neurite connecting points, which results in incapability of nerve cells to perform normal material transportation and information transfer. This phenomenon induced the functional damage observed in the nervous system, resulting in toxicity in the nervous system. As a cytoplasmic marker, LDH concentration in cell culture supernatant is a stable and sensitive indicator of cell membrane integrity and cell activity to evaluate the degree of damage to cells (Cerdán, 2006). In this study, LDH activity after exposure to 16 and 20 μg/mL Nano-ZnO was significantly higher than that in the control group. However, this was not exactly the same with the results obtained from the CCK-8 assay at the same dosage level and exposure time, which indicated that cytotoxicity differed significantly after exposure to 8–20 μg/mL Nano-ZnO for 6 h.

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Consequently, it is possible that the cytotoxicity primarily stems from the cellular internalization of Nano-ZnO rather than physical injury to the cell membrane. Previous studies have shown that nanoparticles can cross the cell membrane and be taken up by cells through non-phagocytic mechanisms (Hwang et al., 2016). Excessive ROS generation has been reported to be associated with various diseases (Mates et al., 1999; Perluigi et al., 2009). In the present study, ROS production was significant increased after

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exposure to 4 μg/mL nano-ZnO (Fig. 8D), demonstrating that ultra-low Nano-ZnO doses might

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cause neuron oxidative stress. Mitochondria possess a very important parameter, the mitochondrial

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membrane potential, which is connected to various cellular mechanisms and whose disturbance has

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severe consequences to the cell. The mitochondrial electron transport chain is the major ROS

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production site, and ROS accumulation may impair mitochondrial ATP production. ATP, as the most

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important energy molecule, plays an important role in various physiological and pathological cellular processes and changes in ATP levels can affect the function of many cells. A decrease in

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ATP level usually indicates impaired or decreased mitochondrial function. In conclusion, our experiments indicated mitochondrial functionality loss after Nano-ZnO treatment. ROS may cause DNA damage, which is characterized by single-stranded and double-stranded DNA breaks and base modifications, leading to cell cycle arrest or even mutations (Singh et al., 2009). These experiments suggested that Nano-ZnO significantly inhibited cell proliferation as characterized by G2 cell cycle phase arrest. In this study, cell morphological observation found that Nano-ZnO could destroy the neural processes of neurons, reduce the number of intercellular nodes, and hinder the formation of neural network structures. The nervous system is composed of a large number of neurons with no

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protoplasmic link between the neurons in the structure. These neurons can only function through synapses formed by the contact between neural processes. Cytoskeleton, a major component of the nervous system axons, is a protein–fiber–reticulum system within cells. It can maintain the normal morphological structure of cells and participate in intracellular substance transport, information transmission and other activities. Microtubules,

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microfilaments, and neurofilaments are important neurocytoskeletal protein components. The morphology of nerve cells is mainly supported by cytoskeleton. The normal expression of various

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cytoskeleton proteins provides materials for the synthesis, assembly, and extension of synapses,

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axons, and dendrites, enabling nerve cells to be interconnected through neurofilaments. The

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microtubule proteins are also related to cell movement and morphological differentiation. The

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combined action of all cytoskeleton proteins provides a structural basis for the normal physiological

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functioning of the nervous system (Ayaz et al., 2012; Kueh and Mitchison, 2009; Rama, Rao et al.,

will be impaired.

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2011; Wang and Zhu, 2018). Once the cytoskeleton is damaged, the function of the nervous system

In this study, we found that Nano-ZnO could damage nerve fibers, destroy neuronal structure, shorten neuritis, and reduce the number of nerve nodes through tubulin-α, tubulin-β, and NF-H injury, which hinder neural network structure formation and interrupt the connection between neural networks and nerve cells. Intercellular substance transport and information transmission were impeded, leading to impaired neurological function. GAP-43 is a protein closely related to cell and tissue development. Widely distributed in neurons, it is closely related to nerve fiber growth and development, axon regeneration, and synaptic function maintenance. It is also involved in neurotransmitter regulation and is thought to

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be an important endogenous determinant of neuron development and regeneration (Benowitz and Routtenberg, 1997). It can be used as a biomarker for synaptic and axonal growth during neurodevelopment. Our study found that Nano-ZnO could induce GAP-43 protein expression down-regulation, suggesting that Nano-ZnO might induce neuronal dysplasia during repair and regeneration, thus causing permanent nerve injury.

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CAMKII is a relatively abundant protein in nerve tissue regulated by Ca2+ and calmodulin. It plays an important role in a series of life processes, such as synapse formation, remodeling,

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regeneration (Mockett et al., 2011; Redondo et al., 2010), and apoptosis (Chen et al., 2011;

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Hamdani et al., 2013). The CAMKII protein has two subtypes, namely CAMK2A and CAMK2B,

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each of which has a distinct function. The main function of CAMK2A protein is to regulate signal

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transduction between synapses, while CAMK2B is related to the number of synapses and the

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morphology and disappearance of axons and dendrites (Hamdani et al., 2013; Matsuzaki et al., 2004; Mockett et al., 2011; Redondo et al., 2010). Our study found that Nano-ZnO could down-regulate

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calmodulin-dependent protein kinase CAMK2A/CAMK2B protein expression, suggesting that Nano-ZnO might induce delayed neurotoxicity through the calcium/calcium-regulated kinase signaling pathway. 5. Conclusion The present study illustrated that the olfactory neuronal pathway represented an important route for CNS exposure to Nano-ZnO. Intranasally instilledNano-ZnO could be transported to the CNS through the olfactory nerve pathway and caused ultrastructural changes, oxidative damage, inflammatory responses, and histopathological damages in crucial functional regions of the rat brain (olfactory bulb, striatum, hippocampus and cerebral cortex). In vitro studies of dopaminergic

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neurons indicated that PC12 cells exposed to Nano-ZnO decreased cell proliferation due to an arrest of cell cycle progression in the G2 phase compared to control cell proliferation. Nano-ZnO can induce mitochondrial function damage and cell phase arrest of dopaminergic neurons through oxidative stress mechanism, which might be important factors for inducing neuronal cytotoxicity. In addition, Nano-ZnO can also destroy neuronal structure by affecting cytoskeleton proteins

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(tubulin-α, tubulin-β, and NF-H), resulting in the interruption of connection between nerve cells, obstruction of material transport and information transmission between cells, and ultimately lead to

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nervous system function damage. Concurrently, Nano-ZnO can induce neuronal repair and

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regeneration disorders by affecting the growth-related protein GAP-43, which can cause permanent

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damage to the nervous system. Furthermore, Nano-ZnO may induce delayed neurotoxicity through

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the calcium/calcium-regulated kinase (CAMK2A/CAMK2B protein) signaling pathway. This study

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is the first to establish a striatum damage model and discover the biomarker for Nano-ZnO damage to dopaminergic neurons. The olfactory neuronal translocation pathway is possibly operative in

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humans as it has also been demonstrated in non-human primates (Bodian and Howe, 1941; Howe and Bodian,1941). In summary, these results demonstrate that Nano-ZnO exposure is a potential risk factor for neurodegenerative diseases and may particularly affect the hippocampus, striatum, and dopaminergic neurons negatively. Acknowledgements This work was supported by National Natural Science Foundation of China (21407178, 81773390), the National Key Research and Development Program of China (2016YFC0206900) and AWS16J004. We are grateful to the editor and the two anonymous reviewers for their helpful comments and suggestions.

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Table 1 Characterization of Nano- ZnO Zeta potential Supplier

Density

Crystalline

Size (nm) 3

(mV)

structure

5.78

45

Hexagonal

Composition

sphere

ZnO >99.9%

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19.61±5.83

(m /g)

Shape

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18.63

(g/cm )

2

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Nano-ZnO

SSA

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Graphical abstract

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Journal Pre-proof Highlights * Nano-ZnO can be transported via the olfactory nerve pathways into the brain tissue. * Nano-ZnO can deposit in olfactory bulb, hippocampus, striatum, and cerebral cortex. * Nano-ZnO can cause toxic damage to crucial functional sub-brain regions.

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* Nano-ZnO can damage neuronal mitochondrial function by oxidative stress mechanism. * Nano-ZnO can reduce the expression of CAMKII, GAP43, tubulin, and NF-H in neurons.

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