"Methods

All experimental protocols were approved by the School of the Environment, Nanjing University, and the methods were carried out in accordance with the approved guidelines.

Fish Care and Exposure.

Five-month-old adult zebrafish (Danio rerio, AB strain; sex ratio, 1:1) were purchased from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Before the exposure period, the fish were cultured for more than 1 week in tap water (aerated and basked for two days for dechlorination before use, with oxygen saturation exceeding 80%) and allowed to acclimate to the following conditions28: 12h:12h light/dark regime, 26± 1 °C, pH 7.4–8.1 and a daily feeding (9:00 am) with dry flake food at a maximum of 2% body weight. Before exposure experiments were initiated, the 96h-LC50 of TPhP to adult zebrafish was determined following the guidance of OECD No. 20329 (details are shown in Supplementary information, Section S2). The 96h-LC50 of TPhP to adult zebrafish was found to be 1.026mg/L; thus, the experimental concentrations of TPhP were set at 0.050 and 0.300 mg/L. The concentration 0.050 mg/L was close to the maximum concentration of TPhP that has been reported in surface water. In total, 270 fish were exposed to related concentrations of TPhP (99+%, purchased from Sigma–Aldrich, USA) with a solvent control (0.1% dimethyl sulfoxide [DMSO], chromatographic grade, purchased from Merck, Germany) in 10 L glass tanks. Each tank contained 30 fish (15 males and 15 females), with 3 parallel tanks per treatment group. The exposure experiments were carried out with a semi-static protocol. Half of the solution in each tank was renewed daily. Other conditions were the same as in the above-mentioned acclimation period. The stability of the TPhP concentrations was confirmed using solid phase extraction and high-performance liquid chromatography (details are shown in Supplementary Information, Section S1). The exposure period lasted for 7 days. The fish were fed daily except on the sampling day. When the fish were sampled, they were first anesthetized in ice-cold water. The exposure experiment was performed twice (540 fish were used), once for metabolomic, transcriptomic and histopathological tests and once for blood and RT-qPCR (reverse transcription-polymerase chain reaction) tests.

Histopathological Observation.

After a one-week exposure period, the livers of zebrafish (10 fish for each concentration group) were dissected and fixed in 10% formalin at 4 °C for 24h. Subsequently, the fixed liver tissues were dehydrated in gradient ethanol, hyalinized in xylene, and embedded in paraffin wax at 56 °C. Then, the paraffin blocks were sectioned at 4-μm thickness. The sections were collected on glass slides and stained with hematoxylin and eosin (H&E) using an H&E Staining Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s recommendations (standard H&E staining protocol). Histologic lesions were observed using an optical microscope equipped with a digital camera.

Metabolomic Analysis.

After a one-week exposure period, the zebrafish were anesthetized on ice, and their livers were dissected and used for extraction immediately. The extraction was carried out using a methanol/chloroform/water system at 4 °C as described by C.Y. Lin et al. with minor modifications30. In detail, approximately 50mg frozen liver tissue (dissected from 12 fish) was homogenized using an electric tissue homogenizer in 400μL methanol (chromatographic grade, Merck, Germany) and 85 μL ultrapure water (UPW) in a glass tube. The homogenizer was washed with 75μL UPW; this fluid was also collected in the tube. Then, another 200μL UPW and 400μL chloroform (chromatographic grade, Merck, Germany) were added after the tube was vortexed for 60 seconds. Next, the tube was centrifuged at 2000 g at 4 °C for 5min, and 500 μL methanol/water (upper) and 350 μL chloroform (lower) were separately transferred to new glass tubes followed by lyophilization or vacuum drying, respectively. Polar and nonpolar residues were redissolved using 400 μL UPW+ 100 μL D2O (99.9 atom% D, containing 0.05wt% TSP, Sigma–Aldrich, USA) or 500 μL chloroform-d (99.8 atom% D, containing 0.03% v/v TMS, Sigma–Aldrich, USA) separately before NMR analysis. Six parallel samples (12 fish were sacrificed for each sample) were prepared for each treatment/control group. 1 H NMR spectra of all samples were acquired using a Bruker AV600 MHz spectrometer (Bruker Co., Germany) operating at 600.17 MHz and 298.5 K. Spectra of the aqueous samples were acquired using a Carr-Purcell-Meiboom-Gill, 1D acquisition with presaturation (CPMGPR1D) pulse program with 32 scans (FIDs) collected into 65536 data points with Fourier transformation. For the chloroform phase samples, spectra were acquired using a zg pulse program with 16 scans (FIDs) collected into 32768 data points with Fourier transformation. NMR spectra were processed (phase correction, baseline correction and ppm shift correction using trisilylpropionic acid [TSP] or tetramethylsilane [TMS]) and analyzed (GSD, Global Spectral Deconvolution) using MestReNova v6.1.0–6224 software. Metabolite resonances were identified using both Chenomx NMR Suite 8.02 and published literature31–33. NMR spectra and assigned metabolites are presented in Figure S1, Figure S2 and Table S1. Integrations of water resonance (4.70–5.15ppm in spectra of aqueous samples) and methanol resonance (3.34–3.38ppm in spectra of aqueous samples and 3.30–3.40ppm in spectra of chloroform phase samples) were excluded. Following the removal of these regions, the metabolites were then normalized to the total integrated spectral area (0.65–9.37 ppm for aqueous samples and 0.60–7.00ppm for chloroform phase samples). Then, the data set was log transformed and Pareto scaled (mean-centered and divided by the square root of the standard deviation of each variable) before statistical analysis. Significantly changed metabolites (SCMs) were identified by t-test and partial least squares-discriminant analysis (PLS-DA) using an online tool for metabolomics data analysis, MetaboAnalyst 3.034.

Transcriptomic Analysis.

The livers of 4 zebrafish were dissected and used for total RNA extraction. Total RNA extraction was performed using TRIzol reagent (Life Technologies). Two parallel total RNA samples were prepared for each treatment group. Then, the transcriptomic analysis was conducted by BGI Tech. (Shenzhen, China) using BGI RNA-Seq (Quantification) analysis (http://bgi-international.com/services/genomics/rna-seq-quantification/#tab-id-1). In detail, the total RNA samples were first treated with DNase I to degrade any possible DNA contamination. Then, the mRNA was enriched by using oligo(dT) magnetic beads (for eukaryotes). The mRNA was mixed with the fragmentation buffer and fragmented into short fragments (approximately 200bp). Then, the first strand of cDNA was synthesized by using random hexamer primers. Buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double-stranded cDNA was purified with magnetic beads. End repair and 3′-end single nucleotide A (adenine) addition were then performed. Finally, sequencing adaptors were ligated to the fragments. The fragments were enriched by PCR amplification. During the quality control step, an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were used to qualify and quantify the sample library. The library products were used for sequencing via an Illumina HiSeqTM 2000 or other sequencer when necessary. Then, the raw data were cleaned by removing low-quality reads as well as reads with adaptor sequences and reads containing unknown bases more than 10%. Clean reads were mapped to reference sequences and/or references. No more than 2 mismatches were allowed in the alignment. Expression levels of the assigned genes were calculated by using the reads per kb per million reads (RPKM) method35. Then, differentially expressed genes (DEGs) between TPhP-treated and control groups were screened using the NOIseq method33. DEGs were judged by the threshold “false discovery rate (FDR) ≤ 0.001 and the absolute value of log2Ratio ≥1”. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs compared with the whole genome background.

Blood Tests.

After the fish were exposed, fish blood samples were collected by tail cutting, as described previously by Chen et al.36. The tail of each fish was cut off using sterilized spring scissors to expose the caudal vein. Then, blood was collected via pipette (20 μL, Eppendorf, Germany) and transferred into anticoagulant tubes (200 μL, treated with heparin at a final concentration of 20U/mL blood). Approximately 15 μL blood was collected from each fish; blood from 10 fish (5 males and 5 females) was pooled to provide an adequate plasma volume for subsequent measurements. The collected blood samples were centrifuged at 2000 g at 4 °C for 5min, and the upper layer (plasma, approximately 50 μL) was collected and stored at −20 °C until use. Three parallel samples were prepared for each treatment group. The glucose, pyruvate, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) contents in the plasma were measured using a glucose assay kit, pyruvate assay kit, TG assay kit, TC assay kit, HDL-C assay kit, and LDL-C assay kit, respectively. All these assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

RT-qPCR.

The expression levels of 6 DEGs that were identified in the transcriptomic analysis were measured by RT-qPCR to verify the reliability of the transcriptomic analysis. Three parallel total RNA samples (4 fish each sample) were prepared for each group with the above-described method. The primer sequences are shown in Table S4. RT-qPCR experiments were performed using a RevertAid First Strand cDNA Synthesis Kit and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, USA) on a Bio-Rad CFX Connect Real-Time PCR Detection System.

Statistical Analysis.

In the metabolomic analysis, statistical analysis was performed using an online tool, MetaboAnalyst 3.0. SCMs were identified by p< 0.05 in t-tests and by a variable influence on projection (VIP) score of >1 in the PLS-DA analysis by comparing the metabolomic profiles between control and TPhP-treated groups. In the transcriptomic analysis, statistical analysis was also conducted by BGI Tech following their standard method of RNA-Seq analysis. For blood tests, the data are expressed as the means± standard deviation (SD); the blood test data were analyzed with SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Significant differences between different treatment groups were identified on the basis of p< 0.05 using one-way ANOVA followed by post hoc test based on least significant difference (LSD)."

Male and Female AB Strain Zebrafish