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Food Safety and Toxicology

Arsenic Induces Autophagy in Developing Mouse Cerebellum: Involvement of Blood-Brain Barrier’s Tight Junction Proteins and/or PI3K/Akt/mTOR Signaling Pathway Ram Kumar Manthari, Chiranjeevi Tikka, Mehdi Mohammad Ommati, Ruiyan Niu, Zilong Sun, Jinming Wang, Jianhai Zhang, and Jundong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02654 • Publication Date (Web): 22 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Journal of Agricultural and Food Chemistry

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Arsenic Induces Autophagy in Developing Mouse Cerebellum: Involvement of Blood-

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Brain Barrier’s Tight Junction Proteins and/or PI3K/Akt/mTOR Signaling Pathway

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Ram Kumar Manthari§, Chiranjeevi Tikka§, Mohammad Mehdi Ommati§,ψ, Ruiyan Niu§,

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Zilong Sun§, Jinming Wang§, Jianhai Zhang§, Jundong Wang§*

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§

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Medicine, College of Animal Science and Veterinary Medicine, Shanxi Agricultural

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University, Taigu, Shanxi-030801, China

Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary

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ψ

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65186, Iran

Department of Animal Science, College of Agriculture, Shiraz University, Shiraz 71441-

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*Corresponding author: Prof. Jundong Wang, Shanxi Key Laboratory of Ecological

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Animal Science and Environmental Veterinary Medicine, College of Animal Science and

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Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi-030801, China; Mobile

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No: +86 13603546490; Tel. No: +86-354-6288206; Fax: +86-354-6222942 E-mail ID:

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[email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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This study was designed to determine whether Blood-Brain Barrier’s (BBB) Tight Junction

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Proteins (TJs) and/or PI3K/Akt/mTOR Signaling Pathway are involved during arsenic (As)

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induced autophagy in developing mice cerebellum after being exposed to different As

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concentrations [0, 0.15mg, 1.5mg and 15mg As(III)/L] during gestational and lactational

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periods. The dosage was continued to the pups till postnatal day (PND) 42. Studies conducted

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at different developmental age points like PND21, PND28, PND35 and PND42 showed that

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exposure to As lead to a significant decrease in the mRNA expression levels of TJs

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(Occludin, Claudin, ZO-1 and ZO-2), PI3K, Akt, mTOR, and p62 with a concomitant

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increase in Beclin1, LC3I, LC3II, Atg5 and Atg12. Also, As significantly downregulated the

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occludin and mTOR protein expression levels with a concomitant upregulation of Beclin 1,

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LC3 and Atg12 in all the developmental age points. However, no significant alterations were

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observed in low and medium dose exposed groups of PND42. Histopathological analysis

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revealed the irregular arrangement of purkinje cell layer in the As exposed mice. Ultra

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structural analysis by transmission electron microscopy (TEM) revealed the occurrence of

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autophagosomes and vacuolated axons in the cerebellum of the mice exposed to high dose As

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at PND21 and 42 respectively. Finally, we conclude that developmental As exposure

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significantly altered TJ proteins resulting an increase in BBB permeability, facilitating As to

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cross and induces autophagy which might be partly by inhibition of PI3K/Akt/mTOR

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signaling pathway in an age-dependent manner, i.e., PND21 mice were found to be more

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vulnerable to As-induced neurotoxicity which could be due to the immature BBB that allows

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As to cross through it. However, the effect was not significant in PND42, which could be due

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to the developed BBB.

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KEYWORDS: Arsenic, Autophagy, Blood-brain barrier, Cerebellum, Postnatal day


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INTRODUCTION

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Arsenic (As), a metalloid element, is a major global environmental pollutant and most

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of the human populations are exposed to it through different pathways like diet, inhalation

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and or dermal contact, which may lead to the morphological and functional perturbations of

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different targeted organs like brain, liver, kidney, skin etc.1,2

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As-contaminated agricultural soil is one of the important exposure sources that affect

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human health through the food supply both quantitatively and qualitatively. Agricultural

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lands with increased As levels can reduce the soil effectiveness with a decrease in the yield of

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crops, and increases the chances of toxic effects to human health.3,4 As in soils is not only due

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to natural sources (weathering and erosion of rocks), but also different anthropogenic

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activities like emissions from industries, fertilizers and pesticides usage, atmospheric

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deposition and sewage irrigation.5-8 These enhanced human activities may lead to a

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significant increase in As levels on local, regional, and global scales.9,10 It was reported that

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industrial processes accounted for 58.2% of total annual input of As in agricultural soil, and

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the usage of wide-scale fertilizers was identified as second big contributor to the soil As

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levels.11

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It was reported that As exposure through occupational and non-occupational exposure

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sources results in various neuronal perturbations leading to permanent neuronal damage.12-15

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Moreover, increasing literature reported that a self-degradative process, autophagy, involves

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in the initiation and enhancement of As-induced neurotoxicity.16,17 Although As exists in

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three different forms (elemental: 0, trivalent: As III and pentavalent: As V), in terms of

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toxicity, As (III) is reported as more active due to its ability to bind with the protein

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sulfhydryl groups and disrupts their activity.18-20 Hence, arsenic trioxide (As2O3) has been

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used in this study.

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As is known to cross BBB and hence brain forms the first target for its toxicity causing

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oxidative stress surge rendering the brain towards free radicals [reactive oxygen species

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(ROS) and reactive nitrogen species (RNS)] leading to apoptosis of neural cells.21,22 BBB

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functioning is highly critical for the central nervous system (CNS) homeostasis.23 BBB

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actively transfers nutrients and metabolites between blood and the brain vice versa, also

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restricts the entry of different immune cells and plasma contents into the brain. Hence, the

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degree of a toxicant’s toxicity depends in part on the permeability of the BBB to that

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particular toxin.24 Surrounded the apical pole, endothelial cell sheets of brain microvessels

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forms a semi-permeable barrier with an circumferential seal called tight junctions (TJs). TJ

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components dislocation might be related to the alterations in BBB permeability, which plays

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a key role in the TJs structural integrity maintenance and barrier’s permselectivity.25,26 Hence,

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now-a-days, research has been carried out extensively on TJs at the morphological,

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functional, and molecular levels.

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During metabolic activation processes, As activates brain to generate ROS, which

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disrupts oxidant/antioxidant balancing system leading to an increased oxidative damage.27,28

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Recent literature has reported that ROS initiates autophagosomes formation by inhibiting

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mammalian target of rapamycin (mTOR), and enhances autophagy acting as cellular

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signaling molecules.29 Autophagy, an important cellular mechanism that sequesters

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cytoplasmic materials like endoplasmic reticulum, mitochondria and Golgi complex into

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double-membrane vesicle like structures called autophagosomes, and then finally translocated

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to lysosomes for recycling or degradation.30,31

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Exposure to heavy metals including As in different organisms could lead to

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autophagy.32 Autophagy acts as a self-defensive mechanism which is processed by a variety

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of proteins like phosphatidylinositol 3-kinase (PI3K), protein kinase Akt, the mammalian

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target of rapamycin complex (mTORC), various autophagy-related genes (ATGs),


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microtubule associated protein light chains 3 (LC3), and Beclin1. These proteins are well

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recognised as the biomarkers to evaluate autophagy mechanisms in different studies. While,

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Beclin1 is considered as the biomarker for the occurrence of autophagy; Atg12-Atg5 and

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LC3 are regarded as the biomarkers for the autophagosomes.

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Increasing evidences reported that the PI3K/Akt/mTOR pathway plays a prominent role

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in the autophagy regulation.33-35 PI3K/Akt/mTOR signalling pathway is commonly

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hyperactivated during different cancers, inhibited by different toxic chemicals and often

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confers a poor prognosis.36-39 As it is known that As cross BBB which is under-developed

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during developmental period and produce neurotoxic effects,40-42 we have chosen four

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different developmental age points like PND (postnatal day) 21, PND28, PND35 and PND42

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to evaluate whether As induces autophagy by inhibiting PI3K/Akt/mTOR pathway. And

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moreover, different regions of brain may respond differently to toxic chemicals, hence, the

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present study was designed with an aim to examine the As-induced autophagy in cerebellum.

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Moreover, till today, no study reports the involvement of BBB tight junction proteins during

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As-induced autophagy in developing mice. Hence, we also tried to investigate the role of

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BBB tight junction proteins in As-induced developmental autophagy. In this study, we

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hypothesize that As treatment can influence the BBB integrity at the level of the TJs and

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contribute to the development of autophagy in cerebellum of the mouse.

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With respect to the United States Environmental Protection Agency, the maximum

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contaminant level for As in drinking water is 10ppb.43 However, in most of the regional wells

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and ground water, the As levels were greater than 10 ppb, and even 5,000 ppb.44 Moreover,

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Agency for Toxic Substances and Disease Registry (ATSDR), reported that human beings are

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10–100 times more vulnerable to As(III). And Reilly et al. (2014) reported that the

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approximated mean every day As intake is 50.6 µg/day for adult females.45 Keeping in view



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of this, in the present study, the average daily drink of the maternal mice which is about

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10mL/day; resulted in 1.5, 15 and 150 µg of As intake per day per mouse.

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MATERIALS AND METHODS

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Animals and treatments

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Sixty-8-weeks old, healthy female Kunming mice were obtained from Experimental

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Animal Centre, Academy of Military Medical Sciences, China. Before the experiment

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initiation, all the mice were maintained in plastic cages having wooden shavings, in

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temperature-controlled rooms with controlled lighting (12 h light: 12 h dark) for

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acclimatization to the environment. Deionized water and commercial rodent pellets were

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freely provided throughout the experimental period. All animal procedures were done in

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compliance with the regulations and guidelines of International Ethics Committee on Animal

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Welfare, Shanxi Agricultural University, China.

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After two-weeks of acclimatization, the mice were randomly divided into four groups

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(15 per group): control group (drinking deionized water) and three As treated groups

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[concentration of As(III) in deionized water: 0.15 mg As2O3/L, 1.5 mg As2O3/L, 15 mg

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As2O3/L]. Maternal mice on an average daily drink about 10 mL/day, which resulted in 1.5

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µg, 15 µg and 150 µg As intake per mouse per day in low, medium and high groups

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respectively. The doses were chosen based on recent studies from our laboratory.46 The day

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vaginal plug was observed, considered as embryonic day 0 (E0), and then the pregnant

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females were housed separately in a plastic cage until offspring were born. Then all the

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maternal mice were treated with deionized water and different As dosages with respect to

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their treatment group throughout the whole lactation period. Once, the lactation period is

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over, the dosage was continued to the pups till postnatal day 42 (PND42).


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Twelve offspring males from each group were sacrificed using cervical dislocation

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according to animal ethical standards at different developmental age points like PND21,

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PND28, PND35 and PND42. Cerebellum was removed, then frozen quickly in liquid

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nitrogen, and stored at −80°C for total RNA extraction and western blotting analysis. Two

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samples from control and high dose treated groups of PND21 and 42 were fixed in 2.5%

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glutaraldehyde solution for the transmission electron microscope (TEM) analysis.

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Tissue preparation for TEM analysis

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Post 2 hrs fixation at room temperature (RT), control and high dose treated samples of

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PND21 and PND42 were washed with phosphate buffer, and then fixed in osmium tetroxide

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for 2 hrs at RT, followed by 10 min pre-staining in acetate-barbitone. After dehydration

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through graded ethanol series, the samples were embedded in Spurr’s resin. Sections were

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prepared and then stained with uranyl acetate and lead citrate. Finally, the ultra structure of

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purkinje neurons in cerebellum was observed in JEM-1400 (JEOL Ltd., Tokyo, Japan) TEM.

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Organ coefficient

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The cerebellum from every group was carefully separated and weighed. The percentage

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of organ coefficient was calculated by the formulae: Organ coefficient = [Organ wet weight

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(g)/Body weight (g)] X 100.

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Histopathological assessment

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The cerebellum was fixed in 10% formalin, and then samples were rinsed by running

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water, dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin. Then, they

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were cut into 5-µm sections with a rotary microtome and then eight histological sections were

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stained with hematoxylin and alcoholic eosin. Finally, the histological alterations were



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observed and photographed using an Olympus BX50 (Olympus, Japan) photomicroscope. An

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experienced pathologist who was blind to experiment performed the histopathological

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analyses.

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Total RNA extraction and real-time PCR (RT-PCR)

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Total RNA was extracted using the Trizol Reagent following the manufacturer’s

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instructions. RNA quality and quantity were estimated by NanoDrop ND-1000

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Spectrophotometer (Nano-Drop, USA) and agarose gel electrophoresis respectively. The

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reverse transcription assays were executed with 500ng total RNA in 10µl reaction mixture by

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following the protocol of PrimeScript® RT Master Mix. The list of primers was given in

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Table 1. RT-PCR was performed by using the SYBR Premix Ex TaqTM II QRT-PCR kit on

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the Mx3000PTM RT-PCR system (Stratagene, USA). The RT-PCR conditions were

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maintained as follows: 95°C for 10 s, 40 cycles of 95°C for 5 s, 61°C for 15 s and 72°C for 6

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s. Finally, 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s. All experiments were done in

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triplicate. The 2-∆∆Ct method was used to evaluate the relative expression levels of genes.

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Western blotting analysis

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The total protein from the cerebellum was extracted by RIPA lysis buffer (100:1

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contained PMSF, Sigma Chemical, St. Louis, MO, USA), and centrifuged at 12,000 g for 10

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min at 4°C. Bovine serum albumin as the standard, the protein concentration of each sample

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was determined by BCA protein assay kit (Beyotime Institute of Biotechnology, Ltd.,

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Shanghai, China). 50µg of protein was loaded and resolved by electrophoresis on 6% or 8%

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or 15% SDS polyacrylamide gels based on the molecular weight of the target protein. Then,

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the target stripe was transferred onto a nitrocellulose (NC) membrane and run for 60 min at

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35 V. Blots were blocked with 5% non-fat dried milk in Tris-buffered saline with 0.05 %


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Tween 20 (TBST) for 2 h at RT. Then the membrane was incubated with β-actin (1:5000,

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Santa Cruz Biotechnology, Inc., USA), mTOR (1:6000, Abcam, Shanghai, China), Beclin

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(1:1000, Santa Cruz Biotechnology, Inc., USA), LC3 (1:1000, Proteintech, Ltd, Wuhan,

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China), Atg12 (1:700, Santa Cruz Biotechnology, Inc., USA), Occludin (1:2500, Proteintech,

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Ltd, Wuhan, China) in primary antibody dilution buffer (Solarbio Life Sciences, Beijing,

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China) overnight at 4°C. After 3 washes (10 min each) with PBST, the membrane was

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incubated in m-IgGκ BP-HRP (1:4000, Santa Cruz Biotechnology, Inc., USA) or HRP-

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conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1:5000, Proteintech, Ltd, Wuhan,

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China) in TBST buffer at RT for 2 h. Following 3 washes with PBST, the target protein

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bands were visualized with enhanced chemiluminescent (super ECL, KeyGEN BioTECH,

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Beijing, China). Optical density was calculated using the Alpha View software (Version:

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3.2.2.0) on the FluorChem Q system (AlphaInnotech, CA, USA).

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Statistical analysis

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Experimental data was shown as the mean ± SEM and analyzed by GraphPad Prism 5

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software (GraphPad Software Inc., San Diego, USA). Differences among the experimental

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and control groups were calculated using one-way analysis of variance (ANOVA) followed

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by a Tukey's Multiple Comparison test. The value of p < 0.05 was considered statistically

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significant.

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RESULTS

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Influence of As on cerebellum organ coefficient

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Influence of As exposure on the cerebellum organ coefficient was shown in Fig 1.

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Exposure to low dose As significantly increased the cerebellum organ coefficient compared



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to the control group at PND42 (p < 0.05). However, no remarkable changes were observed

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between As treated and their respective controls of all the other developmental age periods.

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Effect of As exposure on mRNA expression levels of BBB tight junction proteins

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In this present study, we have investigated the mRNA expression levels of BBB TJ

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proteins including occludin, cladudin, ZO-1 and ZO-2. In all the developmental age points

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(PND21, 28, 35 and 42) studied, compared with their respective control groups, exposure to

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low, medium and high As doses, significantly decreased the mRNA expression levels of

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Occludin (Fig. 2A-2D), Claudin (Fig. 2E-2H), ZO-1 (Fig. 3A-3D), and ZO-2 (Fig. 3E-3H)

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expression levels in cerebellum. However, no significant changes were observed in case of

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Occludin in medium dose exposed group of PND42 animals; Claudin in low dose exposed

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groups of both PND35 and 42 animals; ZO-1 in medium and low dose exposed groups of

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PND35 and 42 respectively; and ZO-2 in low dose exposed groups of PND21 and 42, and

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high dose exposed group of PND28.

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Effect of As exposure on protein expression level of Occludin

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In this present study, along with the mRNA expression levels, we have evaluated the

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protein expression levels of occludin, which play an important role in the BBB integrity. In

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all the developmental age points (PND21, 28, 35 and 42) studied, compared with their

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respective control groups, exposure to low, medium and high As doses, significantly

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decreased the protein expression levels of Occludin (Fig. 4A-4D) in cerebellum. However, no

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significant changes were observed in case of medium dose exposed group of PND42.

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Effect of As exposure on mRNA expression levels of mTOR and its regulated genes


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In this present study, we have investigated the mRNA expression levels of mTOR and

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its regulated genes including PI3K, Akt, AMPK and PDK1. In all the developmental age

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points (PND21, 28, 35 and 42) studied, compared with their respective control groups,

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exposure to low, medium and high As doses, significantly decreased the mRNA expression

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levels of PI3K (Fig. 5A-5D), Akt (Fig. 5E-5H), mTOR (Fig. 5I-5L), PDK1 (Fig. 6A-6D),

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with concomitant increase in AMPK (Fig. 6E-6H) expression levels. However, no significant

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changes were observed in case of PI3K and Akt in medium and high dose exposed groups

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respectively of PND28 animals; PDK1 and Akt in medium dose exposed group of PND35

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animals; PI3K and Akt in low dose exposed group and AMPK in both low and medium dose

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exposed groups of PND42 animals.

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Effect of As exposure on mRNA expression levels of autophagy pathway related genes

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We then evaluated the mRNA expression levels of autophagy pathway related genes

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including Beclin (Fig. 7A-7D), LC3-I (Fig. 7E-7H), LC3-II (Fig. 7I-7L), p62 (Fig. 8A-8D)

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Atg5 (Fig. 8E-8H) and Atg12 (Fig. 8I-8L). In all the developmental age points (PND21, 28,

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35 and 42) studied, compared with their respective control groups, exposure to low, medium

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and high As doses, significantly increased the mRNA expression levels of all the autophagy

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pathway related genes. However, no significant changes were observed in case of LC3-II in

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low and high dose exposed groups, Atg5 in low and medium exposed groups of PND21;

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LC3-I and Atg5 in low and medium dose exposed group, LC3-II in low dose exposed group

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of PND28; LC3-II in low dose exposed group and p62 in medium dose exposed group of

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PND35; Beclin, p62 and Atg5 in low dose exposed group, LC3-I and Atg12 in low and

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medium dose exposed groups of PND42 animals.

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Effect of As exposure on protein expression levels of mTOR and autophagy markers



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In this present study, along with the mRNA expression levels, we have evaluated the

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protein expression levels for mTOR and some autophagy pathway related markers like

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Beclin1, LC3 and Atg12. In all the developmental age points (PND21, 28, 35 and 42) studied,

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compared with their respective control groups, exposure to low, medium and high As doses,

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significantly decreased the protein expression levels of mTOR (Fig. 9A-9E) with concomitant

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increase in Beclin (Fig. 9F-9J), LC3 (Fig. 10A-10E) and Atg12 (Fig. 10F-10J) expression

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levels. However, no significant changes were observed in case of LC3 in low dose exposed

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group of PND21 and PND28; Beclin and LC3 in low dose exposed group of PND35, Beclin

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in low dose exposed group, Atg12 and LC3 in low and medium dose exposed groups of

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PND42 animals.

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Histological alterations in cerebellum

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Examination of control group sections revealed the normal histological structure of the

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cerebellum, which was arranged in three successive strata, outer molecular layer, middle

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Purkinje layer and inner granular layer. The molecular layer had scanty population of

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neurons. The Purkinje cell layer was constituted with a single line of large pyriform somata

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of Purkinje neurons with pale nuclei and prominent nucleoli. In case of granular layer, a large

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number of neurons with rounded dark nuclei and scanty cytoplasm, fibers and small

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capillaries were observed. In total, most of the Purkinje cells were disc or pear in shape with

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cytoplasm distributed evenly surrounding the nucleus in the representative lobules of

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cerebella of the control animals (Fig. 11: PND21 and 28; Fig. 12: PND35 and 42).

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However, in case of As exposed groups of all age groups, structural alterations were

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highly exhibited in the Purkinje cell layer. Loss of neurons in purkinje layer was observed in

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PND21 animals. Sections of all age groups revealed shrunken Purkinje cell bodies which are

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irregular with deeply stained cytoplasm and hardly identified nuclei. Purkinje cells deposited


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in multiple layers were noticed in medium and high dose exposed groups of all the

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developmental age points. Also, pyknotic cells with deeply stained purkinje cells were

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observed (Fig. 11: PND21 and 28; Fig. 12: PND35 and 42).

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Ultrastructural abnormalities in cerebellum

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Further to explore histopathology in cerebellum, and to characterize the exact

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autophagy pathway status, we have chosen ultrastructural analysis, because this is the golden

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standard method for autophagy analysis.47 In both PND21 (Fig. 13A and 13B) and 42 (Fig.

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14A) control groups, no signs of autophagy were detected. Moreover, clear outlined nucleus

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with endoplasmic reticulum, golgi bodies, and mitochondria were observed. In high dose As

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exposed mice of PND21 (Fig. 13C and 13D), we observed axons with single layered

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autophagosome-like structures, with mitochondria and other electron dense cellular

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components engulfed in them. Copious alteration of axon myelin sheaths in the high dose

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exposed animals of PND42 (Fig. 14B) was evident. The defective myelin sheaths had

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damaged myelin-sheath layers with a frayed lamellae structure and unusual myelin

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protrusions into cortical neurons. And moreover, we detected axons containing the vacuoles

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that are embedded in myelin-sheath.

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DISCUSSION

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Heavy metals induced toxicity and related neurological diseases have become a most

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important problem of the developing countries.48 As-induces toxicity through agricultural

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soil, food and ground water which may lead to the occurrence of heavy metal

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intoxication.15,49 The capability of As to cross BBB makes the brain as the first target and

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more sensitive towards its toxicity.50 In order to better understand the developmental

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neurotoxicity of As and to confirm the toxicity exerted by a prenatal exposure, we evaluated



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the effects of As on BBB TJ proteins, autophagy and its relation with PI3K/Akt/mTOR

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signalling pathway in cerebellum of mice exposed to As during developmental periods.

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BBB plays a major role not only in maintaining the brain homeostasis but also several

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neural functions and contributes to the pathological alterations related with a wide variety of

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neurological disorders including tumours.51-53 The interconnections in the endothelial cells

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through TJ complexes restricts the paracellular entry of molecules. Increasing evidences

330

suggests that TJs proteins like Occludin, Claudin, ZO-1, and ZO-2 plays a major role in the

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BBB regulation during pathological damages and various neurodegenerative disorders.54

332

In this study, As treatment significantly decreased the mRNA expression levels of TJ

333

proteins like occludin, claudin, ZO-1 and ZO-2 in cerebellum of all the developmental age

334

periods which may lead to an increase in the BBB permeability. In corroboration with our

335

current results, many other recent studies reported an increased TJ permeability with

336

decreased protein expression of TJ members upon exposure to different toxic chemicals like

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As, Cd, Hg.55-57 A recent study conducted by Song et al. (2014) reported a significant

338

decrease in the occludin and ZO-1 in rat brain endothelial cells that are exposed to Pb.58

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Autophagy, an important degradation or recycling mechanism, protects neurons from

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toxic protein accumulation and nonadaptive organelles through fast clearance.59 However,

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increased autophagy can harm cells and tissues and results in the occurrence of pathological

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disturbances.60 Also, hyper-activation of autophagy might lead to cell death in the spine.61,62

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Just as two sides of a coin, recent studies reports the dual role of autophagy in cancer, i.e.,

344

protecting cell survival or contributing to cell death.63,64

345

Recent reports have proved that various signaling molecules, like MAPKs, mTOR, and

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class III PI3K, have been correlated to autophagy.38,65,66 Our results revealed that As

347

exposure triggered the inhibition of mTOR and its regulated genes like PI3K, PDK1, Akt

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with a concomitant increase in AMPK in all the developmental periods. Autophagy can be


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negatively regulated by PI3K/Akt signaling pathway by phosphorylating mTOR.38,65 This

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might explain our current results regarding the down-regulation of PI3K and Akt, with the

351

increase in autophagy levels. Previous investigations by Wang et al. (2017) reported that As

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activates the PI3K/Akt/mTOR pathway frequently in the cells under survival stress-induced

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autophagy.35 In agreement with our results, previous study by Chung and Chang, (2003) has

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reported that As exposure lead mTOR inhibition.67 And moreover, ROS generated as a result

355

of As intoxication could stimulate autophagy through inhibiting mTOR.68

356

Beclin1, LC3 and Atg12 are important indicators to detect autophagy. Beclin1, an

357

important autophagy protein is involved in various biological activities. Beclin1 binds to

358

class III PI3K (Vsp34) and other proteins, forming a complex which is necessary for

359

autophagosome initiation. In this study, the mRNA and protein expression levels of Beclin1,

360

LC3 and Atg12 were up-regulated by As exposure in all the developmental age points. In

361

agreement with our data, previous studies have reported that As induced autophagy with an

362

increased levels of Beclin1 and LC3 with a concomitant decrease in p62 in the brain tissues

363

of Gallus gallus.69 The possible explanation for the enhanced Atg12, LC3 levels might be due

364

to the increased conversion from LC3-I to LC3-II and the activation of Atg12-Atg5-Atg16

365

complex and LC3 embedded on the membrane. However, western blot analysis of LC3

366

protein revealed only single band was observed i.e., LC3-II rather than two bands (LC3-I and

367

LC3-II). This might be due to the increased levels in the conversion of LC3-I to LC3-II

368

during the formation of autophagosomes, which is the key step in the process of autophagy.

369

And moreover, LC3-II remains bound to the autophagolysosome membrane, the fusion

370

product of autophagic vacuole and lysosome.70 Moreover, Wang et al. (2015) reported

371

autophagy in the cortical neurons exposed to Cd as evaluated by the autophagosomes

372

occurrence and LC3-I to LC3-II conversion.71 From the results, we observed that As

373

activated autophagy as evidenced by increased levels of Beclin1 and LC3. This explains the



Page 16 of 48

374

important role of autophagy in inhibiting oxidative damage induced by heavy metals like

375

As.72

376

p62, a regulator of autophagy, was used as a marker of autophagic flux.73 Increased

377

levels of p62, enwraps the dysfunction of autophagic flux, can be involved in the cancer

378

initiation and progression.74 Results of present investigation revealed the downregulation of

379

p62 levels upon exposure to As in all the developmental periods which implies autophagy

380

dysregulation in As exposed mice. Similar results were reported by Liu et al. (2017) in L-02

381

cells that are subjected to acute or chronic As treatment.73

382

Ultrastructural analysis of cerebellum by TEM revealed the crunch of autophagosomes

383

in the control groups of PND21 and 42. This could be due to the high capability of

384

constitutive macroautophagy in control tissues with most potent vesicular trafficking in

385

maintaining balance between occurrence and clearance of autophagosomes.75,76 However,

386

Moreira et al. (2010) suggested substitute explication stating that neurons usually keep up

387

low levels of autophagosome biosynthesis.77 However, As-induced autophagy can be

388

evidenced by the increased number of autophagic vesicles that are observed in the ultra

389

structure of As exposed tissues. This increase in autophagosomes might be due to decreased

390

autophagosomes degradation.76,78 Formation of autophagosomes generally happens in the

391

reconciling response to stress situations, like malnourishment, toxic metal insult, decreased

392

insulin signaling, and mTOR inactivation. In a study conducted by Nixon et al. (2005)

393

reported a massive autophagosomes accumulation within neuritic processes and synaptic

394

terminals in the Alzheimer’s disease brain.79

395

Among the four different developmental age periods studied, PND21 mice were found

396

to be more vulnerable to the As-induced neurotoxicity. This age dependent toxicity could be

397

due to the As transfer across BBB during developmental stages. Moreover, it is reported that

398

chances of metal accumulation in immature animal’s brain is more than adult’s brain which


Page 17 of 48


399

could be due to the underdeveloped or immature BBB.41,80,81 However, the effect was not

400

significant in PND42, which could be due to the developed BBB.

401

In summary, we conclude that developmental exposure to As significantly altered TJ

402

proteins resulting an increase in BBB permeability. Thus, the leaky BBB in cerebellum may

403

facilitate the transfer of As and induces autophagy which might be partly by inhibition of

404

PI3K/Akt/mTOR signaling pathway in an age-dependent manner, i.e., among the four

405

developmental age periods, PND21 animals were found to be more vulnerable to the As-

406

induced neurotoxicity than the other three age periods.

407 408

ABBREVIATIONS USED

409

AMPK, AMP-activated protein kinase; As, Arsenic; As2O3, Arsenic trioxide; Atg,

410

Autophagy-related gene; BBB, Blood-brain barrier; LC3, microtubule associated protein light

411

chains 3; mTOR, mammalian target of rapamycin; PDK1, 3‐Phosphoinositide‐Dependent

412

Kinase 1; PI3K, phosphatidylinositol 3-kinase; PND, Postnatal Day; TEM, Transmission

413

electron microscope.

414 415

ACKNOWLEDGEMENT

416

This work is supported by China National Natural Science Foundation (Grant No. 31672623

417

and 31372497).

418 419

CONFLICT OF INTEREST

420

The authors declare that there are no conflicts of interest.

421 422 423



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FIGURE CAPTIONS

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Figure 1. Effect of As on cerebellum organ coefficient in different developmental age

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periods (A-PND21, B-PND28, C-PND35 and D-PND42) of mice following gestational

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and lactational As exposure continued till PND42. Data represents the mean ± SEM (n

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= 6). Asterisk (*) indicates significant difference compared to the control group (*p

Involvement of Blood-Brain Barrier's Tight Junction ... - ACS Publications

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