Zn stimulates the phospholipids (PL) biosynthesis via the pathways of

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Zn stimulates the phospholipids (PL) biosynthesis via the pathways of oxidative and endoplasmic reticulum (ER) stress in the intestine of freshwater teleost yellow catfish Kun Wu, Zhi Luo, Christer Hogstrand, Guanghui Chen, Chuan-Chuan Wei, and Dan-Dan Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02967 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Zn stimulates the phospholipids (PL) biosynthesis via the pathways of oxidative and

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endoplasmic reticulum (ER) stress in the intestine of freshwater teleost yellow catfish

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Kun Wua, Zhi Luoa,b*, Christer Hogstrandc, Guang-Hui Chena, Chuan-Chuan Weia,

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Dan-Dan Lia

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a

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Fishery College, Huazhong Agricultural University, Wuhan 430070, China

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b

Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture of P.R.C.,

Collaborative Innovation Center for Efficient and Health Production of Fisheries in

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Hunan Province, Changde 415000, China

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c

12

London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK

Diabetes and Nutritional Sciences Division, School of Medicine, King’s College

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ABSTRACT:

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The hypothesis of our study was that waterborne Zn exposure evoked phospholipids

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(PL) biosynthesis to compensate for the loss of membrane integrity, and the pathways

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of oxidative stress and ER stress mediated the Zn-evoked changes of PL biosynthesis.

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Thus, we conducted RNA sequencing RNA-seq to analyse the differences in the

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intestinal transcriptomes between the control and Zn-treated P. fulvidraco. 56-day Zn

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exposure increased the intestinal Zn accumulation, and mRNA levels of 816 genes

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were markedly up-regulated and 263 genes down-regulated. Many DEGs in pathways

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of PL biosynthesis and protein processing in ER were identified. Their expression

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profiles indicated that waterborne Zn exposure injured protein metabolism and

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induced PL biosynthesis, caused oxidative stress and ER stress, and activated the

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unfolded protein response. Then, using the primary enterocytes, we identified the

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mechanism of oxidative and ER stress mediating Zn-induced PL biosynthesis, and

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indicated that the activation of these pathways constituted adaptive mechanisms to

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reduce Zn toxicity. Our study demonstrated that Zn exposure via the water increased

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Zn accumulation and PL biosynthesis, and that oxidative stress and ER stress were

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interdependent and mediated the Zn-induced PL biosynthesis of the intestine in the

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freshwater teleost.

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GRAPHICAL ABSTRACT

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1. INTRODUCTION

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The pollution of aquatic ecosystems by metals is of concern due to their toxicity and

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bioaccumulation. Among these metals, zinc (Zn) is an essential trace element for

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many important biochemical processes in vertebrates including fish, but can be toxic

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above the threshold concentration in the body. Among aquatic organisms, fish are

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considered to be the ideal organism for pollution monitoring in aquatic ecosystems,1

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and unique among the vertebrates because they have two routes of metal acquisition,

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from the diet and from the water.2 In contrast to marine fish, freshwater fish do not

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drink considerable volumes of water for osmotic homeostasis, and waterborne Zn is

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absorbed mainly via the gill.3 The influence of waterborne Zn on gill has been well

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studied due to its direct involvement in Zn toxicity in freshwater fish.4 The intestine is

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also very sensitive to a wide range of stressors.5 In our pilot study, we found

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incidentally that waterborne Zn exposure increased Zn accumulation in the intestine

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of yellow catfish. We found this interesting and toxicologically relevant because

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increased Zn accumulation in the intestine could potentially influence the

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physiological functions of the intestine. However, whether and how waterborne Zn

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had effects on physiological functions of the intestine are not exactly known in

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freshwater teleost, and the underlying mechanisms of intestinal homeostatic

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regulation are not well understood.

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In fish, the failure to maintain Zn homeostasis results in the damage of cellular

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structure and intracellular membranes, and impedes a wide range of physiological

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processes. 6 Therefore, it is meaningful to emphasize that Zn exposure via water can

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increase intestinal Zn accumulation and in turn initiate the change of intestinal

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physiological functions. Phospholipids (PL) are the major lipid components of cellular

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and intracellular membranes.7,8 Despite the importance of PL in maintaining integrity

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and physiological state of membrane, no studies have addressed the effects of mineral

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elements (including Zn) on PL metabolism. Since excess Zn in the water can damage

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cellular structures, we hypothesize that waterborne Zn can affect PL biosynthesis in

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the intestine.

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The ER, an important organelle, is responsible for folding and assembly of

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proteins, PL synthesis and Ca2+ homeostasis. The changes in the ER increase the

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amounts of unfolded/misfolded proteins in the ER, evoking ER stress and increasing

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the concentration of Ca ion in the cytoplasm.9 To recover the ER homeostasis, cells

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activate the UPR, a pathway governed by three sensors of ER stress: PERK, IRE1 and

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ATF6 (their full names were shown in Abbreviation list in Supporting Information,

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SI).8 Together, they evoke gene transcription to increase folding capacity and

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down-regulate protein synthesis, and induce elimination of unfolded proteins through

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ER associated degradation (ERAD).10 At present, increasing evidence indicates that

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the UPR is involved in the regulation of lipid and PL biogenesis.11,12 However, the

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molecular mechanism linking the UPR to PL biogenesis has not been elucidated. In

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addition to ER stress, Zn also induces oxidative stress13,14 by accelerating the

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generation of ROS.15 Moreover, generation of ROS during oxidative stress directly or

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indirectly (or both) affects ER homeostasis and UPR activation.16,17 Therefore, we

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reasoned that this ROS-based model would be very helpful in illuminating the

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undefined mechanism linking ER stress to Zn-induced changes of PL metabolism.

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Here, we hypothesize that Zn exposure via the water affected PL metabolism,

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and that the pathways of oxidative stress and ER stress mediated the Zn-induced

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alterations of PL metabolism. Therefore, using RNA-seq technology, the present

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study ascertained the effects of waterborne Zn influencing physiological responses of

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the intestine in Pelteobagrus fulvidraco. Using primary yellow catfish enterocytes, the

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complex relationship between Zn and oxidative stress/UPR/PL metabolism was

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investigated. Yellow catfish, an omnivorous freshwater teleost, is widely cultured in

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several Asian countries and used as experimental animals. We reveal an

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unprecedented role for the ER stress and oxidative stress mediating Zn-induced

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changes of PL biosynthesis.

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2. MATERIALS AND METHODS

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The study consisted of three experiments. All experiments followed the ethical

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guidelines of Huazhong Agricultural University.

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2.1. Expt. 1: Experimental procedures and sample analysis

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2.1.1. Zn exposure and sampling

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The experimental procedures for waterborne Zn were described in our parallel

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study.9 Briefly, 144 healthy yellow catfish (mean body weight: 8.4 ± 0.4 g) were were

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randomly transferred to 6 fiberglass tanks (300 L water, 24 fish per tank). They were

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exposed to two nominal Zn concentrations: 0 (control, no extra Zn addition) and 7.69

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μM (0.5 mg/L, 5% of the 96-h LC5018), respectively, with triplicate tanks for each

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treatment. Zn was added in the form of ZnSO4·7H2O. Here, the Zn concentnration

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(0.5 mg/L) was lower than those in some contaminated waters in China, and also

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lower than these in water quality criteria II according to GB 3838-2002 of China (1.0

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mg Zn/l, GB3838-2002). All fish were fed twice daily with diets (Zn content: 17.45

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mg/kg diet). The actural Zn concentrations for two treatments were 0.007 ± 0.001 and

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0.514 ± 0.070 mg/l, respectively.19 The experiment continued for 56 days, and the

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entire intestine samples (including anterior, mid- and posterior intestine) were

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collected for the determination of contents of triglyceride (TG) and metal elements,

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enzymatic activity and mRNA expression.

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2.1.2. Measurements of metal, TG content and enzymatic activities

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The

intestine

Zn

content

was

measured

using

ICP-AES.19

Quality

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assurance/quality control (QA/QC) procedures included analysis of three method

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blanks (purified water), three certified reference tissues (DORM-2, LUTS-1 and

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DOLT-2) and two randomly selected duplicate samples.

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Activities of G6PD, FAS, 6PGD, ME and ICDH (their full names were listed in

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SI) were analyzed based on the methods described in our previous study.19 The TG

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content and activities of Cu, Zn-SOD were measured by using commercial kits

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(Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).

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2.2. Transcriptome analysis

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2.2.1. RNA isolation, library preparation and sequencing

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Three fish per tank were randomly selected for RNA isolation. Trizol reagent

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(Invitrogen, CA, USA) was used for total RNA isolation. In total, three fish from each

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tank and three replicates per treatment (six samples in total) were used for

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transcriptomic sequencing. The construction of cDNA library and sequencing

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(Illumina HiSeq 2000 platform, Illumina, CA, USA) were undertaken at the Beijing

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Genome Institute (BGI, Beijing, China) according to the methods described

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previously.19

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2.2.2. De novo assembly and annotation

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Before assembly, clean reads were obtained from raw data by filtering adaptor

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sequences and low-quality sequences. They were then de novo assembled into

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unigenes using Trinity.20 We annotated the transcripts over 200 bp using the BLASTx

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alignment (e-value < 10-5) between unigenes and databases, including NR

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(non-redundant protein sequence), non-redundant nucleotide (Nt), Swiss-Prot, KEGG

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(Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous

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Groups). Blast2GO was used to accomplish Gene Ontology (GO) annotation ,21 and

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WEGO program for further gene classification .22

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2.2.3. Differentially expressed genes (DEGs) and Q-PCR validation

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After obtaining de novo transcriptomic data, the BGISEQ-500 platform (BGI,

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Beijing, China) was used to quantify the expression levels of genes. 6 independent

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cDNA libraries (three equivalent quantities of RNA samples from the same tank were

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combined into one pool, and there were three replicate tanks and accordingly 3 cDNA

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libraries per treatment) were constructed for RNA-Seq quantification analysis (all

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reads have been submitted to the Sequence Read Archive at NCBI, Accession

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Number: SRP116984). The gene expression levels were calculated using the

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fragments per kilobase of transcript per million mapped reads (FPKM) method23 and

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DEGs were screened using Noiseq method24 with a value of log2fold-change > 1 and a

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diverge probability ≥ 0.8. All DEGs were mapped to terms in GO and the KEGG

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database. In the present study, twelve candidate genes were randomly selected for

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Q-PCR validation. 19 Primers are given in Table S1 (SI). A set of seven housekeeping

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genes (β-actin, b2m, elfa, gapdh, rpl7, hprt and tbp) were selected from the

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literature25 in order to test their transcription stability. The mRNA levels of β-actin

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and hprt were the most stable, based on the analysis of geNorm software25

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Accordingly, the gene expression levels were normalized to the geometric mean of the

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β-actin and hprt. The 2-ΔΔCt method26 was used to calculate the fold changes in

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relative expression to the control.

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2.3. Expt. 2: in vitro studies

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2.3.1. Enterocytes culture and treatment

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Enterocytes were isolated from yellow catfish, based on the published

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methods27,28. Four pathway inhibitors were used, and they are TPEN (Zn2+ chelator,

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Sigma, MO, USA), NAC (ROS inhibitor, Selleck, TX, USA), fatostatin (SREBP1

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inhibitor, Selleck, TX, USA) and STF-083010 (IRE1 inhibitor, Selleck, TX, USA).

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Ten groups were designed as follows: control, 40 μM Zn, 2 μM TPEN, 1mM NAC,

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10 μM fatostatin, 50 μM STF-083010, 40 μM Zn + 2 μM TPEN, 40 μM Zn + 1 mM

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NAC, 40 μM Zn + 10 μM fatostatin and 40 μM Zn + 50 μM STF-083010. The

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concentrations of Zn and inhibitors were selected according to our pilot trials and to

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other in vitro studies.9,29-31 Each treatment was performed in triplicates. The cells were

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collected at 48 h for following analysis.

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2.3.2. Cell viability, TG and PC content

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Cell

viability

was

evaluated

by

3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) assay method.19 Intracellular TG content was

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measured as described above. The amount of phosphatidylcholine (PC) was measured

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using a PC assay kit (Sigma, MO, USA).

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2.3.3. Zn2+ and Ca2+ measurements

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Two cell permeant probes, Newport Green DCF (Ex/Em = 505nm/535nm) and

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Fluo-4 (Ex/Em = 494nm/516nm) were used to measure intracellular Zn2+ and Ca2+

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concentrations, respectively9,32 Fluorescence was detected and quantified using laser

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scanning confocal microscopy (Leica, Wetzlar, German).

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2.3.4. Flow cytometry for the determination of apoptosis and ROS

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Apoptosis analysis was conducted with 5 μl Annexin V-FITC and 5μl propidium

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iodide (PI), accompanied with flow cytometry (Beckman, CA, USA) with ModFit 3.0

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LTTM software.

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ROS production was measured with 10 μM 2, 7-dichlorofluorescin diacetate

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(DCFH-DA).33 Fluorescence intensity was measured at the wavelengths of excitation

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at 490 nm and emission wavelength at 525 nm, respectively.34 For fluorescence

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intensity

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(2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α] pyrazin-3-one), a specific

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chemiluminescence probe for superoxide anion and singlet oxygen, the PBS-washed

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cells were incubated with 8 μM MCLA for 20 min. Fluorescence intensity was

measurement

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MCLA

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measured at the wavelengths of excitation at 430 nm and emission wavelength at 546

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nm, respectively.

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2.3.5. Enzymatic activity, MDA content and Q-PCR

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Activities of G6PD, FAS, 6PGD, ME and ICDH were analyzed following the

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methods in our previous study.19 The malondialdehyde (MDA) content and activities

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of total-SOD and Cu, Zn-SOD were measured by commercial kits (Nanjing Jian

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Cheng Bioengineering Institute, Nanjing, China). Soluble protein content of the

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homogenates was determined by the method of Bradford.35

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Q-PCR for the in vitro experiment was performed based on the methods

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mentioned above, andβ-actin and tbp were used as the control genes according to

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geNorm software.

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2.4. Expt. 3: Bioaccumulation of Zn in tissues

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Considering that dietary Zn might have the potential influence on the Zn uptake,

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we further performed an experiment to analyze the relative contribution of dietary Zn

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and waterborne Zn. 180 yellow catfish (initial mean weight: 10.8 ± 0.3 g) were

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randomly stocked in 12 tanks and there were 15 fish per tank. The yellow catfish were

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exposed to four treatments for 8 weeks, three replicates for each treatment. There

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were two waterborne Zn concentrations: 0 (no extra Zn addition in the water) and 7.69

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μM (0.5 mg/L, waterborne Zn addition group) waterborne Zn. For each waterborne

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Zn exposure, fish were fed 6.17 (dietary Zn deficiency group) or 17.45 mg/kg (dietary

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Zn addition group) Zn, respectively. The group of dietary Zn deficiency, TPEN (Zn

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chelator) was added at 0.01 g/kg to inhibit dietary Zn uptake. The feed composition

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and nutrient analysis of diets were shown in Table S6 (SI). The fish were fed diets at a

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ration of 4% of body weight twice a day. Fish were sampled on days 0, 14, 28, 42 and

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56, respectively. Gills, intestine and plasma were sampled for the determination of Zn

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content and mRNA expression of genes involved in Zn uptake and transport.

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2.5. Statistical analysis

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Statistical analysis was conducted with SPSS 19.0 software.19 Data were

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analyzed with one-way ANOVA and Student’s t-test where appropriate. Significant

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level was set at P < 0.05.

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3. RESULTS AND DISCUSSION

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3.1. Waterborne Zn had direct effects on Zn accumulation and PL metabolism of

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the intestine

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In the freshwater fish, Zn is assimilated both from waterborne and dietary phases.

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In the present study, Zn exposure via the water significantly increased Zn

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accumulation (Fig. 1A). 1079 unigenes were identified as DEGs by RNA-seq

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technology, including 816 up- and 263 down-regulated genes (Fig. S1, SI). The GO

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(Fig. S2, SI) and KEGG (Fig. S3, SI) database were further used to perform the

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pathway analysis (For detailed RNA-seq data and validation result, see SI, Table

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S2-S5, Fig. S4). The results indicated that waterborne Zn exposure impacted the

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physiological function of the intestine in many aspects and a series of genes and

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pathways may be of importance for Zn-induced physiological responses. Similar to

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the results in the in vivo study, intracellular Zn content of primary enterocytes were

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up-regulated after Zn incubation, and the changes were partly reversed by TPEN (Fig.

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S5, SI), which to some extent supports the argument. Overall, the present study

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indicated that waterborne Zn disrupted the Zn balance in the intestine, which thereby

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led to diverse physiological changes, similar to our study in yellow catfish

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hepatocytes.33 The present study found an interesting contradiction: Zn increased

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activities of enzymes involved in fatty acid (FA) synthesis (Fig. 1D) but reduced TG

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content (Fig. 1B) in the intestine. Considering that FA is not only incorporated into

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TG, but also for the synthesis of phosphatidic acid (PA), PL precursor, we

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accordingly analyzed the DEGs involved in PL biosynthesis pathway. The present

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study found that many genes, such as acc, fas, acsl, gpat, agpat, cds, ck, pap, ept1 and

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cept1, involved in FA and PL biosynthesis, were up-regulated by waterborne Zn

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exposure (Fig. 2). The activities of enzymes related to FA synthesis, such as 6pgd, me,

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icdh and fas, were significantly increased by waterborne Zn exposure (Fig. 1D). These

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observations indicated that waterborne Zn exposure induced PL biosynthesis.

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Similarly, several studies pointed out that the synthesis of PL was regulated in

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response to Zn status in yeast.36,37 Iwanyshyn et al.36 found that the activities of the

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CDP-diacylglycerol pathway enzymes were positively associated with the

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cytoplasmic Zn levels. GPAT and AGPAT were key enzymes for the initiation of

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PL synthesis.7 PC is the main PL and the precursor to SM, PS and PE.12 Some key

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enzymes: CK, CCT, CHPT1/CEPT1 and PEMT, played important roles in PC

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biosynthesis.12,38-40 Considering that waterborne Zn increased tissue MDA content

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(Fig. 1C) and PC is the main membrane PL in cells,12 it is plausible to speculate that

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increased PC synthesis compensated for the loss of membrane integrity induced by Zn

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exposure. Moreover, our study found that the expression of DGAT, a crucial enzyme

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catalyzing formation of TG from DG, was markedly decreased. Thus, it is reasonable

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to conclude that most of FA participated in the PL biosynthesis process, not in TG

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biosynthesis. To verify whether increased Zn content directly affected these changes

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in the intestine, Zn2+ chelator TPEN was used in the in vitro study. Our study

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indicated that TPEN significantly attenuated the Zn-induced increase of expression of

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accα, gpat, pap, cds, cct and ect (Fig. 3A), which in turn suppressed the Zn-induced

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accumulation of PC content and the reduction of TG content (Fig. 3B), as observed

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here.

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3.2. Waterborne Zn evoked ER stress and UPR

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Zn exposure influenced the protein metabolism and processing in ER (Fig. 4).

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For instance, the expression levels of uggt and bip were markedly elevated. UGGT

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and BiP are parts of the ER quality control system and preferentially recognizes the

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unfolded/misfolded proteins.10 Thus, the increased expression of uggt and bip

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probably reflected the increase of these unfolded/misfolded proteins in the ER. The

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current study also showed that mRNA levels of perk, atf6 and ire1 were significantly

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up-regulated (Fig. 4). It has been reported that under ER stress, aberrant proteins

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rapidly accumulates and accordingly induces the UPR in eukaryotic cells.10 Hence,

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these events suggested that waterborne Zn caused the UPR in the intestine of yellow

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catfish. One of the adaptive mechanisms that recover normal physiological state of ER

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is the upregulation of the folding capacity through induction of molecular

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chaperones.8 Here we found that expressions of chaperones, such as bip and cnx, were

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up-regulated, indicating the increased folding capacity in response to Zn exposure.

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Moreover, Zn increased the expression of many genes from ERAD and ubiquitin

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proteolysis, including the glycosylase ermani which catalyzes the important step in

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ERAD.41 The enhanced ERAD was conducive to degrade and remove excess

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unfolded/misfolded proteins in ER. It has been postulated that activation of UPR

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increases ERAD efficiency through the IRE1-XBP1 cascade.8,10 As expected,

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expression levels of ire1 and xbp1 were up-regulated here, which constituted a pivotal

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link between Zn-induced UPR and ERAD. Similarly, Sriburi et al.42,43 also reported

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that XBP-1 stimulated the expression of many genes in fibroblasts, including those

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encoding the protein translational machinery.

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3.3. The activation of IRE1-XBP1 pathway was involved in Zn-induced PL

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biosynthesis

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Many enzymes involved in PL biosynthesis, such as GPAT and AGPAT, are

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related with the ER.7,44 Studies also suggested that PL biosynthesis was induced in a

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response to ER stress in the yeast.45 Thus, we further explored whether Zn-induced

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occurrence of ER stress and UPR mediated PL biosynthesis. Here, we found that Zn

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exposure increased the expression of genes in ER stress and UPR, such as perk, atf6,

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ire1, bip and cnx (Fig. 3A), indicating their activation. TPEN markedly inhibited the

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Zn-induced up-regulation of mRNA abundance of grp94, crt, ire1α and perk, further

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indicating that Zn induced the occurrence of ER stress and UPR in vivo and in vitro.

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Similarly, van der Sanden et al.46 suggested that the UPR might provide a means to

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rapidly respond to an increased need for PL in Chinese hamster ovary cells. The

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present study indicated that STF-083010 (IRE1 inhibitor) significantly suppressed the

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Zn-induced up-regulation of mRNA expression of ire1α, xbp1, cds, cct and cept (Fig.

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5A). However, STF-083010 had no suppressed effect on Zn-induced increase of Zn

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content (Fig. S6, SI) and TG content (Fig. 5B). These findings suggested that CDS,

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CCT and CEPT, the key regulatory factors involved in the PC biosynthesis, were

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potential targets of IRE1-XBP1 ptahway, similar to the result by Sriburi et al.42 XBP1

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is also closely linked with PC synthesis.10 Enforced expression of XBP1 is sufficient

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to induce PC biosynthesis.42 Other studies indicated that the level and synthesis of

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CCTa protein, was up-regulated in XBP-1(S)-transduced fibroblasts, and accordingly

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elevated production of cytidine diphosphocholine (CDP-choline).43 The activation of

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ER stress and UPR by waterborne Zn exposure has been reported in the yellow catfish

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liver tissues.9 Other reports pointed out that during a physiological UPR, the

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IRE1-XBP1 pathway regulated synthesis of PL according to cellular needs for ER

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membrane components and its activation of IRE1/XBP1 branch of the UPR responded

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to increased demand for PL in B cells and fibroblasts.12,42 Thus, the XBP1 was

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activated, presumably to facilitate PL biosynthesis under the ER stress,42,43 which was

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essential for optimal cellular functions in response to Zn stimulation. In addition,

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unmitigated ER stress led to apoptosis.10,47,48 Similarly, the present study indicated

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that inhibition of IRE1 pathway during Zn incubation had a negetive effect on

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apoptosis (Fig. S7, SI) and cell viability (Fig. S8, SI). Thus, inhibition of IRE1

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hindered the remedy process of UPR, which likely injured the cells and initiated the

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apoptosis to eliminate unhealthy cells, as suggested by Sriburi et al.43. Therefore, all

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these observations above indicated that the UPR, especially the IRE1-XBP1 pathway,

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played a positive role in alleviating ER stress and maintaining normal functions of ER,

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at least partly via facilitating PC biosynthesis.

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3.4. SREBP1 mediated the regulation of Zn-induced PL biosynthesis

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SREBP1 is a transcription factor that regulate multiple genes involved in FA

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synthesis, including acc and fas.49 The present in vivo experiment found that Zn

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increased the srebp1 mRNA levels. Thus, srebp1 seemed to play key roles in

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Zn-induced alterations of PL metabolism.49-51 Using the SREBP1 inhibitor fatostatin

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(F), we found that, compared to the control, the PC content in the Zn-treated group

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was increased while the PC content in the Zn + F group was relatively stable (Fig. 6C),

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indicating that srebp1 was involved in Zn-induced PC biosynthesis. Compared to the

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control, activities of ME, G6PD and FAS, and mRNA levels of srebp1, accα, fas and

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all the tested genes involved in PC synthesis and PC content were enhanced by Zn

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incubation (Fig. 6A and 6B), further confirming the results of the transcriptome

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analysis. ACCα, G6PD and FAS are the crucial enzymes during FA biosynthesis.52

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SREBP1 inhibitor caused the reversion of Zn-induced increase of fas and accα mRNA

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expression (Fig. 6A), and increase of G6PD and FAS activities (Fig. 6B), thereby

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possibly impacting fatty acid synthesis. For genes involved in PL synthesis, compared

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to single Zn treatment, fatostatin pre-treatment suppressed the changes of gpat, pap

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and cds. Given the close correlation between the synthetic pathways of fatty acid and

373

PC, SREBP1 plays important roles in PC biosynthesis and SREBP1 activation may be

374

required for providing enough fatty acid to support PC biosynthesis.

375

3.5. The Zn-induced changes of physiological functions was partially attributable

376

to oxidative stress

377

To investigate how Zn exposure induced ER stress, we explored the change of 2+

378

Ca concentration in the enterocytes. However, we did not observe appreciable Ca2+

379

release (Fluo-4) after Zn addition (Fig. S9, SI). Considering that intracellular free

380

radicals, such as ROS, directly or indirectly affect ER homeostasis, and thereby lead

381

to UPR.16,17 We further determine the parameters involved in ROS generation. Here

382

we detected the increased fluorescence intensity of ROS (Fig. S10, SI), increased

383

activities of T-SOD and Cu, Zn-SOD, increased MDA content and increased MCLA

384

fluorescence intensity in the Zn-treated group (Fig. 7C), suggesting the accumulation

385

of intracellular ROS. Cu, Zn-SOD is one of the key free-radical scavengers.53 The

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upregulated Cu, Zn-SOD activity probably implied the production of cellular

387

superoxide anion radicals in yellow catfish intestines in a response to Zn exposure.

388

Similarly, previous studies also demonstrated that Zn stimulated the production of

389

ROS.14,54 In contrast, Zn incubation seemed to have no effect on ROS production in

390

yellow catfish hepatocytes.33 ROS augments ER stress.55,56 Our study indicated that

391

increasing Zn led to the generation of ROS and oxidative stress, and preincubation

392

with NAC prevented the Zn-evoked ROS generation, indicating that oxidative stress

393

was responsible for the Zn toxicity, in agreement with other studies.54 As a result, the

394

increased expression levels of bip, crt, ire1α and perk in the Zn-treated group was

395

partly reversed in the Zn + N group (Fig. 7A), indicating that Zn-induced ER stress

396

was, at least in part, attributable to generation of ROS. Meantime, NAC attenuated the

397

Zn-induced effects on PC content (Fig. 7B) and expression of genes involved in PL

398

biosynthesis (Fig. 7A), suggesting the involvement of ROS generation in Zn-induced

399

PL biosynthesis. High concentration of ROS can inflict direct damage to proteins and

400

lipids.16,57 In this study, Zn treatment significantly increased intracellular MDA

401

content. MDA has been widely used as a biomarker for lipid peroxidation (LPO).57

402

Taken together, Zn-induced ROS accumulation probably caused the peroxidation,

403

which further impaired proteins, lipids and membrane integrity. Other research also

404

suggests that Zn caused lipid peroxidation and loss of membrane.58,59 The demand for

405

membrane PL components would increase correspondingly and thereby caused a

406

series of adaptive mechanisms, including the activation of IRE1-XBP1 and SREBP1

407

pathways which initiated a cascade of biochemical events that enhanced supplies of

408

PL and alleviated stress. Thus, the present study provided the first evidence that

409

Zn-induced ROS and UPR signaling mutally mediated Zn-induced PL biosynthesis in

410

enterocytes.

411

3.6. Zn accumulation in tissues was dominated by waterborne uptake

412

Fish can uptake Zn from diet and from gill-plasma route. A potential problem is

413

that dietary Zn might have the potential influence on the intestinal Zn accumulation,

414

which might further influence the results. In order to clarify the effect, we analyzed

415

the effects of dietary and waterborne Zn exposure on Zn deposition (Fig. S11, SI). No

416

surprisingly, waterborne Zn exposure resulted in significant Zn accumulation in the

417

gill on day 56, similar to the study in rainbow trout.60 However, dietary Zn had no

418

significant effects on gill Zn accumulation. Plasma Zn concentration ranged from 10

419

to 25 μg/ml and increased significantly with time in waterborne Zn groups. Other

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420

studies also reported that waterborne Zn exposure increased Zn concentrations in the

421

plasma.61-63 Plasma Zn concentrations are maintained within narrow margins in the

422

fish61 and alterations may mean the occurrence of toxicity.63 Thus, it seemed that Zn

423

exposure via the water evoked the perturbations of plasma Zn and toxicity, whereas

424

the dietary exposure did not. Sappal et al.63 also found that chronic dietary Zn

425

exposure had no significant effect on plasma Zn content in rainbow trout. Similar to

426

the plasma, Zn exposure via the water increased the intestine Zn content much more

427

than dietary exposure did (Fig. S11 A, SI). Previous study in yellow catfish indicated

428

that only an extremely high dietary Zn concentration (213.84 mg/kg) had nearly the

429

same effect on tissues Zn accumulation as 0.35 mg/L waterborne Zn.64 In view of the

430

fact that dietary Zn content we used was much lower than 213.84 mg/kg and belongs

431

to the normal demand range for yellow catfish (dietary Zn requirement for yellow

432

catfish: 17.1-20.9 mg Zn/kg diet)65, our study found that the intestine Zn accumulation

433

came mainly from gill-plasma route. Furthermore, changes of dietary Zn

434

concentration did not result in changes of mRNA levels of znt1 and zip4 on day 56,

435

whereas waterborne Zn exposure increased znt1 expression and decreased zip4

436

expression compared to the group without Zn addition in the water (Fig. S12, SI),

437

indicating that waterborne Zn, but not dietary Zn, influenced mRNA expressions of

438

genes involved in Zn uptake and transport. Taken together, under the present

439

condition, our study strongly indicated that Zn accumulation and changes in the

440

physiological functions (PL biosynthesis) in the intestine were predominantly

441

influenced by waterborne Zn exposure. Our results are in agreement with the opinion

442

that Zn via the water is toxicologically more significant probably because Zn

443

permeates the gill more easily.63

444

In conclusion, for the first time, our data provide evidenced that Zn induced PL

445

biosynthesis by activating the pathways of oxidative stress and ER stress. Our study

446

offered new insights for elucidating the effects and mechanism of Zn toxicology,

447

which will be potential meaningful for incoming Zn risk assessments in aquatic

448

environment.

449

ASSOCIATED CONTENT

450

Supporting information

451

Table S1, primers used for Q-PCR analysis; Table S2, statistics of reads and assembly

452

quality; Table S3, summary of annotation and CDS results; Table S4, summary of

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453

sequencing data for each sample; Table S5, alignment statistics of reads align to

454

reference gene; Table S6, feed formulation and proximate analysis of experimental

455

diets ; Figures S1, the correlation between the gene expression profiles of control and

456

Zn-exposed groups; Figures S2, GO functional classification of DEGs; Figures S3,

457

KEGG functional classification of DEGs; Figures S4, comparison of mRNA levels

458

between RNA-seq and Q-PCR results; Figures S5, effect of Zn and/or TPEN on

459

intracellular Zn fluorescence; Figures S6, Effect of Zn and/or inhibitors on

460

intracellular Zn fluorescence; Figures S7, effect of Zn and/or STF-083010 (IRE1

461

inhibitor) on apoptosis; Figure S8, Effect of Zn and/or inhibitors on cell viability;

462

Figures S9, intracellular Ca fluorescence after Zn stimulation; Figures S10, effect of

463

Zn and/or NAC on intracellular ROS generation; Figure S11, effect of waterborne and

464

dietary Zn on tissues Zn accumulation; Figure S12, Effect of waterborne and dietary

465

Zn on mRNA levels.

466

AUTHOR INFORMATION

467

Corresponding Author

468

*Phone:

469

[email protected]; [email protected] (Z. Luo).

470

Notes

471

The authors declare no competing financial interest.

472

ACKNOWLEDGMENTS

473

The authors thank the staff of Aquatic Animal Nutrition and Feed Laboratory of

474

Huazhong Agricultural University for their assistance with excellent sample analysis.

475

This work was supported by the National Natural Science Foundation of China (Grant

476

Nos. 31422056, 31372547).

477

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deposition and metabolism in juvenile yellow catfish Pelteobagrus fulvidraco. Aquat.

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663 664

150-155.

665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681

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682 683

Fig. 1 Effect of waterborne Zn exposure on Zn (A), TG contents (B), MDA content

684

(C) and enzymatic activities (D) in the intestine of P. fulvidraco on day 56. Values are

685

means ± SEM (n= 3 replicate tanks, six fish were sampled for each tank). Asterisks (*)

686

indicate significant differences between control and Zn-exposed group.

687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704

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705 706

Fig. 2 Differentially expressed genes involved in phospholipid biosynthesis. The

707

up-regulated genes (Probability ≥ 0.8, and the absolute value of log2 (Ratio) ≥ 1) are

708

highlighted in red. The down-regulated genes (Probability ≥ 0.8, and the absolute

709

value of log2 (Ratio) ≥ 1) are highlighted in green. The non DEGs are highlighted in

710

grey. FA, fatty acid; PA, phosphatidic acid; DG, diacylglycerol; CL, cardiolipin; SM,

711

sphingomyelin;

712

phosphatidylethanolamine;

713

abbreviations of genes are shown in the Abbreviation List of the Supplementary file.

PC,

phosphatidylcholine; PS,

PI,

phosphatidylserine;

714 715 716 717 718 719 720 721 722

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phosphatidylinositol;

PE,

Etn,

The

ethanolamine.

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723 724

Fig. 3 Effects of Zn and/or TPEN (Zn2+ chelator) on mRNA levels (A), TG

725

accumulation (B) and PC content (C) in enterocytes of yellow catfish at 48 h. Values

726

are means ± SEM (n = 3). Bars that share different lowercase letters indicate

727

significant differences among groups (p < 0.05).

728 729 730 731 732

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Fig. 4 Differentially expressed genes involved in protein processing in ER. The

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up-regulated genes (Probability ≥ 0.8, and the absolute value of log2 (Ratio) ≥ 1) are

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highlighted in red. The down-regulated genes (Probability ≥ 0.8, and the absolute

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value of log2 (Ratio) ≥ 1) are highlighted in green. The non DEGs are highlighted in

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grey. G3M9: (Glc)3 (GlcNAc)2 (Man)9 (Asn)1; G1M9: (Glc)1 (GlcNAc)2 (Man)9

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(Asn)1; M9: (GlcNAc)2 (Man)9 (Asn)1; M8: (GlcNAc)2 (Man)8 (Asn)1; M5,

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(GlcNAc)2 (Man)5 (Asn)1; Ub, ubiquitin. The abbreviations of genes are shown in

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the Abbreviation List of the Supplementary file.

742 743 744 745 746 747 748 749 750 751 752 753 754 755 756

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Fig. 5 Effects of Zn and/or STF-083010 (IRE1 inhibitor) on mRNA levels of genes

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involved in UPR, lipogenesis and PL biosynthesis (A), TG accumulation and PC

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content (B) in enterocytes of yellow catfish at 48 h. Values are means ± SEM (n = 3).

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Bars that share different lowercase letters indicate significant differences among

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groups (p < 0.05).

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Fig. 6 Effects of Zn and/or fatostatin (SREBP1 inhibitor) on mRNA levels of genes

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involved in lipogenesis, lipolysis and PL biosynthesis (A), enzymatic activities (B),

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TG accumulation and PC content (C) in enterocytes of yellow catfish. Values are

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means ± SEM (n = 3). Bars that share different lowercase letters indicate significant

768 769

differences among groups (p < 0.05).

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Fig. 7 Effects of Zn and/or NAC (ROS inhibitor) on mRNA levels of genes involved

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in UPR, ER stress markers, lipogenesis and PL biosynthesis (A), TG accumulation

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and PC content (B), SOD activities, MDA content and MCLA fluorescent intensity (C)

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in enterocytes of yellow catfish. Values are means ± SEM (n = 3). Bars that share

776

different lowercase letters indicate significant differences among groups (p < 0.05).

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