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Toxicogenomic Assessment of 6-OH-BDE47 Induced Developmental Toxicity in Chicken Embryo Ying Peng, Pu Xia, Junjiang Zhang, Daniel L. Villeneuve, Jiamin Zhang, Zhihao Wang, Si Wei, Hongxia Yu, and Xiaowei Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04467 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016
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Toxicogenomic Assessment of 6-OH-BDE47 Induced
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Developmental Toxicity in Chicken Embryo
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Ying Peng†#, Pu Xia†#, Junjiang Zhang†, Daniel L. Villeneuve‡, Jiamin Zhang†,
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Zhihao Wang†, Si Wei†, Hongxia Yu†, Xiaowei Zhang†*
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† State Key Laboratory of Pollution Control & Resource Reuse, School of the
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Environment, Nanjing University, Nanjing, 210023, PR China
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‡
United States Environmental Protection Agency, Mid-Continent Ecology Division,
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Duluth, MN, 55804, USA.
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# These authors contributed equally to this paper
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*Correspondence:
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Xiaowei Zhang, PhD, Prof
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School of the Environment
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Nanjing University
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Nanjing, 210089, China;
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Tel.: 86-25-89680623
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Fax: 86-25-89680623
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E-mail:
[email protected] 19
[email protected] 20 21
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Abstract:
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Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are analogs of PBDEs
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with hundreds of possible structures and are frequently detected in the environment.
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However, the in vivo evidence on the toxicity of OH-PBDEs is still very limited. Here
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the developmental toxicity of 6-OH-BDE47, a predominant congener of OH-PBDEs
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detected in the environment, in chicken embryos was assessed using a toxicogenomic
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approach. Fertilized chicken eggs were dosed via in ovo administration of 0.006 to
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0.474 nmol 6-OH-BDE47/g egg followed by 18-days incubation. Significant embryo
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lethality (LD50 = 1.940 nmol/g egg) and increased hepatic somatic index (HSI) were
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caused by 6-OH-BDE47 exposure. The functional enrichment of differentially
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expressed genes (DEGs) was associated with oxidative phosphorylation, generation of
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precursor metabolites and energy, and electron transport chain, which suggest that
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6-OH-BDE47 exposure may disrupt the embryo development by altering the function
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of energy production in mitochondria. Moreover, aryl hydrocarbon receptor (AhR)
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mediated responses including up-regulation of CYP1A4 was observed in the livers of
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embryos exposed to 6-OH-BDE47. Overall, this study confirmed the embryo lethality
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by 6-OH-BDE47 and further improved the mechanistic understanding on OH-PBDEs
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caused toxicity. Ecological risk assessment via application of both no observed effect
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level (NOEL) and the sensitive NOTEL (transcriptional NOEL) suggested that
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OH-PBDEs might cause ecological risk to wild birds.
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Introduction
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Hydroxylated
and
methoxylated
polybrominated
diphenyl
ethers
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(OH-/MeO-PBDEs) are analogs of PBDEs which have been used as flame retardants
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and are widely distributed in the environment.1 OH-/MeO-PBDEs are chemicals with
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hundreds of possible structures and many of them have been detected in abiotic
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matrices or biota including surface water, fish, birds, polar bear, marine mammals and
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human blood.2-6 Many in vitro studies have shown that some OH-PBDEs are more
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potent than their postulated precursor PBDEs and corresponding MeO-PBDEs for
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several toxicologically relevant molecular endpoints, including binding to steroid
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hormone receptors,7-10 toxicity to neural cells,11 cytotoxic effects,12 and alteration of
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DNA repair pathways.13 However, the in vivo evidence concerning the toxicity of
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OH-/MeO-PBDEs remains limited.
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6-OH-BDE47 was one of the predominant congeners of OH-PBDEs detected in
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biota.14-16 Our previous study using an in vitro AhR- luciferase reporter gene (LRG)
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test suggested that OH-/MeO-PBDEs can activate aryl hydrocarbon receptor (AhR) in
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both mammalian and avian species, and 6-OH-BDE47 was one of the most potent
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congeners.17,
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showed that this chemical can also modulate other molecular pathways in zebrafish19,
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molecular-level pathways were impacted following activation of AhR and potentially
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other targets, by 6-OH-BDE47 in the embryo development remains unclear.
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Although 6-OH-BDE47 is a known AhR activator,17,
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evidence
and demonstrated thyroid hormone activity in in vitro Yeast hybrid assays.21 How
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Toxicogenomic approaches which provide global view of molecular pathways that
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might be disturbed by the chemical exposure have been widely used to help elucidate
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molecular mechanisms of chemical toxicity. In particular, RNA-seq-based
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transcriptomics has increasingly been used to study gene transcript expression due to
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its much broader genome coverage and higher sample throughput compared with the
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previous microarray technologies.
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The primary goal of this study was to test the hypothesis that 6-OH-BDE47 could
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cause embryo toxicity in avian species. The developmental toxicity of 6-OH-BDE47
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in chicken (Gallus gallus) was tested by in ovo administration. The hepatic
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distribution of 6-OH-BDE47 was confirmed by instrumental analysis and the hepatic
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transcriptomic profile was evaluated to examine the underlying molecular response in
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6-OH-BDE47 induced embryo toxicity. Furthermore, we also assessed the potential
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ecological risk of 6-OH-BDE47 on wild birds.
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2. Materials & method
2.1 Chemicals and solutions
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6-MeO-BDE4, 6-OH-BDE47 and other 10 MeO-PBDEs, 10 OH-PBDEs were
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synthesized at City University of Hong Kong (purities > 98%).22 6-OH-BDE82,
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6-MeO-BDE-82, 6-MeO-BDE85, 6-MeO-BDE87 were purchased from AccuStandard
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(New Haven, CT, USA). 6-OH-BDE47 was dissolved in dimethyl sulfoxide (DMSO,
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Generay Biotech, Shanghai, China) to prepare stock solutions and then diluted in
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DMSO to the desired test concentrations when injected to the chicken embryo.
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2.2 Procedures of egg preparation, injection and incubation
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Fertilized chicken eggs were purchased from local farm with no potential dioxin
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exposure history in Nanjing, Jiangsu Province, China. In general, preparation of
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injection solutions and egg injection procedures follow methodology described in
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Powell et al. (1996)23 with minor modifications. Eggs were stored in a cooler for no
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longer than one week at 13-15 °C until 24 h before injection. Eggs were weighed to
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the nearest 0.1 g and then held to a bright light to detect subtle damage to the shell.
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Undamaged eggs with mean weights of 42.08 ± 3.62 g (± SD) had the center of their
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air cells marked with pencil to outline the injection site. A pipette with sterile
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aciculiform tip (Eppendoff, Germany) was inserted horizontally through the air cell
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after filing to thin the egg shell. Five doses (0.474, 0.158, 0.053, 0.018, 0.006 nmol
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6-OH-BDE47 /g egg) with DMSO solution (5 µL) were injected into chamber of
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chicken eggs (n=10 eggs per treatment). The air cell was chosen as the site of
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injection because of ease and speed of delivery of the chemical into the egg.24 The site
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of injection was then sealed using liquid paraffin wax. Controls included non-injected
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and solvent vehicle control eggs. Concentration of DMSO in final test solutions did
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not exceed 0.01%.
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The ten eggs from each treatment were incubated in an automatic digital display
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incubator (Wansheng, Nanjing, JS, China) until hatch or 18 d. After 18 d incubation,
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any eggs which had not hatched were opened and dead embryos were recorded.
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Pipped embryos were removed and euthanized by decapitation. For all embryos that
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pipped, the following measurements were recorded: embryo mass, liver mass, tarsus
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length, head plus bill length (head + bill: back of head to tip of bill). Liver samples
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were collected and quickly frozen with liquid nitrogen and four liver samples from
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each treatment group were picked at random for further analysis. Each liver sample
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was split with half of sample allocated for RNA extraction, subsequent RNA-seq and
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qRT-PCR analysis (n=4). The other half of each liver was allocated for chemical
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analysis and was stored at -80°C until extracted.
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2.3 Chemical Analysis
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Liver samples (approximately 0.3 g, ww) were mixed with anhydrous sodium
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sulfate (5 times of liver sample weight). A surrogate recovery standard was added
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(6-OH-BDE82 and 6-MeO-BDE85) and then samples were extracted with 50%
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dichlormethane (DCM)/hexane (HEX) using an accelerated solvent extraction system
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(Dionex ASE 200). The column extraction eluent was concentrated to 10 mL and 1
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mL (10% of the sample) was removed for gravimetric lipid determination, the
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remaining volume of extract was dissolved with HEX and then partitioned with
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potassium hydroxide (0.5M in 50% ethanol). The neutral fraction used for BDEs and
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MeO-BDEs analysis was treated with 2 mL concentrated sulfuric acid to remove
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lipids, then cleaned on a multilayer silica gel column (inner diameter 0.8 cm, packed
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with 8 cm neutral silica (3% deactivated, w/w) and 8 cm sulfuric acid silica (2:1
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w/w)) and eluted with 35 mL DCM: HEX (1:1, v/v). Finally, the extract was blown to
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near-dryness under a gentle stream of nitrogen, and 10 µL BDE-77 and
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6-MeO-BDE87 were added respectively as internal standards and made up to 100 µL
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prior to GC/MS analysis. The alkaline phase used for OH-BDE analysis was acidified
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to pH < 2 with hydrochloric acid , then extracted two times with HEX: MTBE (1:1,
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v/v, 6 mL and 3 mL, respectively) and dried by anhydrous sodium sulfate. Samples
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were derivatized with diazomethane overnight to methylate the hydroxyl group. After
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methylation, the samples were purified on a silica gel column (inner diameter 0.8 cm)
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packed with 8 cm neutral silica (3% deactivated, w/w) and 8 cm sulfuric acid silica
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(2:1 w/w) and eluted with 35 mL DCM: HEX (1:1, v/v). Each extract was blown to
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near dryness and reconstituted in 50 mL iso-octane. 6-MeO-BDE87 was added as an
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internal standard for GC/MS analysis. The analysis of MeO-PBDEs and BDEs was
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performed using a TSQ Quantum GC/MS (Thermo Scientific, USA) coupled with
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electron capture negative ionization (ECNI) source in the selected ion monitoring
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mode (SIM). Concentration and potential biotransformation of 10 OH-PBDEs, 10
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MeO-PBDEs were also determined. Detailed protocols for instrumental conditions
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and quality assurance and quality control (QA/QC) are provided in the Supporting
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Information.
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2.4 RNA Sequencing
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To analyze transcriptomic response in the liver of embryos after exposure to
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6-OH-BDE47, samples from the 0.01% DMSO vehicle treatment group, 0.018 nmol/g
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egg, 0.052 nmol/g egg, 0.158 nmol/g egg and 0.474 nmol/g egg treatment groups
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were used for RNA-seq. Total RNA of chicken livers was isolated by RNeasy mini
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kit, followed by digestion of genomic DNA (QIANGEN, GmbH, Hilden). Total RNA
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samples were stored at -80°C. The concentrations of total RNA were measured by
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Synergy H4 Hybrid Take3 reader (BioTek Instruments, Winooski, VT), followed by
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determination of the RNA quality using Agilent 2100 bioanalyzer (Agilent
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technologies, Santa Clara, CA, US). 16 total RNA samples, including 4 control
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samples and 12 treatment samples (N=4 for 0.018 nmol/g and 0.158 nmol/g,
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respectively; N=2 for 0.052 nmol/g and 0.474 nmol/g, respectively), were submit to
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the libraries construction using Dynabeads mRNA DIRECT Micro Kit (Life
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technologies, AS, Oslo, Norway) and Ion Total RNA-Seq Kit v2 (Life technologies,
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Austin). The prepared libraries were then submitted to high throughput sequencing on
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Ion Torrent Proton (Life technologies, Carlsbad, CA, USA) following manufacturer’s
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protocol.
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2.5 RNA-seq Data Analysis
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Raw sequences were mapped to Gallus gallus 4.75 (Ensembl Genes 8325) using the
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Torrent Mapping Alignment Program (TMAP). The aligned files were submitted to R
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package, Genomic Alignments (Bioconductor version 1.6.3)26 for gene expression
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analysis. Normalization of gene expression was performed to account for library
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preparation effects. The normalized expressions of each gene across control to
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treatment groups (measured concentrations) were submitted to analysis of
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differentially expressed genes (DEGs).
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Two approaches were used to identify DEGs according to the measured hepatic
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chemical concentrations. First, linear regression analysis on expression of genes was
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conducted using basic function ‘lm’ in R.27 The p-values of linear regression of each
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gene were submitted to multiple hypothesis test (Benjamini & Hochberg method) to
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get adjusted-p value. Significant linear regression genes (adjusted-p value
