Animals and exposure
X. laevis adults were obtained from Nasco (Fort Atkinson, WI, USA). To alleviate suffering, animals were maintained humanely in accordance with the guidelines of the American Society for Testing and Materials [23]. Adult frogs (2 years old) were exposed to the environmentally relevant concentrations of 0.06 and 0.3 μg/L cis-BF (Sigma, St. Louis, MO, USA). Stock solutions were prepared with alcohol, and the alcohol concentrations were identical for all treatment groups. Each treatment consisted of 27 male frogs that were randomly assigned to three replicates. Experiments were carried out at the same photoperiod (12 h light: 12 h dark) and temperature (22°C). During the 3 months of exposure, the exposure solutions were replaced daily to maintain the concentration of cis-BF. After exposure, the frogs were anesthetized in MS-222 and frozen in liquid nitrogen for subsequent analysis. All studies were approved by the Experimental Animal Ethics Committee of Zhejiang University.
Extraction and quantification of cis-BF
The cis-BF in the exposure solutions and intestines was extracted and detected according to a previous study with slight modifications [24]. For exposure solutions, 200 mL of exposure solution was mixed with sodium chloride (5.0 g) and dichloromethane (200 mL) in a 500 mL separatory funnel. The mixture was shaken for 5 min and left at room temperature for 5 min to separate the organic and aqueous phases. Then, the organic phase was collected, and the extraction procedure was repeated once again. The combined organic phase was evaporated to near dryness on a rotatory evaporator at 40°C. Frog intestine contents (1 g) were homogenized and transferred into a 50 mL polypropylene tube preloaded with 0.25 g NaCl and 1 g MgSO4. Then, 5 mL acetonitrile was added to extract cis-BF, followed by sonication for 20 min. The mixture was centrifuged at 4500 rpm for 10 min, and the organic phase was transferred into a glass flask. The extraction procedure was repeated once again, the organic phase was combined and subsequently evaporated almost to dryness on a rotatory evaporator at 40°C. The residues from the exposure solution and tissues were reconstituted with 1 mL acetonitrile and transferred into a 1.5 mL polypropylene tube that contained 200 mg PSA. After vortexing and centrifugation (12000 g, 10 min), the supernatant was filtered with a 0.22-μm polytetrafluoroethylene filter and transferred into vials for quantitative analysis.
Oil red O staining
Fresh hepatic tissues were fixed in 4% (v/v) formaldehyde in phosphate buffer for 24 hours. The hepatic tissues were transferred into 15% and 30% sucrose solutions in turn at 4°C for dehydration. After dehydration, the hepatic tissues were embedded in optimal cutting temperature medium and subsequently cut into 8-μm-thick sections. The sections were stained with Oil Red O solution (Servicebio, Wuhan, China) for 10 min in the dark. The background differentiation procedure was performed in 60% isopropanol (Servicebio, Wuhan, China). Then, the sections were washed in water for 10 s and stained with hematoxylin. Glycerin gelatin (Servicebio, Wuhan, China) was used to seal the slices. The slices were observed and photographed on a light microscope (Olympus, Tokyo, Japan).
16S rRNA gene sequencing
Total genomic DNA was extracted from the intestinal contents of frogs using the PowerSoil® DNA isolation kit according to the manufacturer’s instructions. Extracted DNA was amplified to target the V3-V4 regions of the 16S rRNA gene of bacteria using the primer pair 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT). The amplicons were assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and sequenced on an Ion S5TM XL platform. To obtain high-quality clean reads, the raw reads were filtered on Cutadapt (Martin M., 2011). In addition, chimera sequences were removed with the UCHIME algorithm (UCHIME Algorithm). Uparse software (Uparse v7.0.1001) was used to choose operational taxonomic units (OTUs) with a threshold of 97% sequence similarity. A representative sequence from each OTU was selected for taxonomic annotation using the Silva Database (Version 132). The taxonomic information of the 16S rRNA gene sequences was obtained at different classification levels, including the phylum, class, order, family and genus levels. Alpha diversity (observed species, Chao1, Shannon, Simpson, ACE, and good coverage indices) and beta diversity were calculated with QIIME (Version 1.7.0). Linear discriminant analysis (LDA) effect size (LEfSe) was performed using the LEfSe tool.
Metabolomics analysis
The intestinal contents (100 mg) were ground with liquid nitrogen, and the homogenate was resuspended in prechilled 80% methanol (-20°C). After vortexing, the samples were incubated for 1 h at -20°C and then centrifuged at 14000 g for 20 min at 4°C. The supernatants were transferred to a new tube, dried in a vacuum concentrator, and then reconstituted in 60% methanol for LC-MS/MS analysis. LC-MS/MS analysis was performed on a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Chromatographic separation was performed using a Hyperil Gold column (100×2.1 mm, 1.9 μm). For the positive polarity mode (pos), the eluents were 0.1% FA in water (A) and methanol (B). For the negative polarity mode (neg), the eluents were 5 mM ammonium acetate (A, pH 9.0) and methanol (B). The solvent gradient was programmed as follows: 98% A, 1.5 min; 98-0% A, 12.0 min; 0% A, 14.0 min; 0-98% A, 14.1 min; 98% A, 17 min. The flow rate was 0.2 mL/min. The other optimized parameters were as follows: spray voltage, 3.2 kV; capillary temperature, 320°C; sheath gas flow rate, 35 arb; and aux gas flow rate, 10 arb. The raw data were processed with Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) for peak alignment, peak picking, and quantitation for each metabolite. Then, the molecular features were matched with the mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases.
Transcriptomics analysis
The mRNA-Seq experiment was completed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Total RNA was isolated from frog liver using TRIzol reagent (Takara Bio Inc., Kusatsu, Japan). The RNA purity and integrity were assessed by using the A260/A280 ratio and agarose–formaldehyde gel electrophoresis, and the RNA concentration was also measured. A total amount of 3 μg of RNA was used for RNA sample preparation, and the sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). The quality of the sequencing library was assessed on the Agilent Bioanalyzer 2100 system. Then, the library was sequenced on an Illumina HiSeq platform to generate 125 bp/150 bp paired-end reads. Reads containing adapters and poly-N and low-quality reads were removed to obtain clean reads. FeatureCounts v1.5.0-p3 was employed to calculate the clean read numbers mapped to each gene, and the FPKM value of each gene was also calculated based on the length of the gene and the read counts mapped to this gene. The DESeq2 R package (1.16.1) was used to assess the differential expression analysis results, and genes with an adjusted p-value < 0.05 were considered differentially expressed. The statistical enrichment of differentially expressed genes in KEGG pathways was performed using the clusterProfiler R package.
Quantitative real-time PCR (qPCR) validation
Frog liver tissues (n = 3 replicates; each replicate contained three tissues) were homogenized with RNAiso plus (Takara Bio Inc, Kusatsu, Japan) to extract the total RNA. The total RNA contents were measured on an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The quality of the extracted total RNA was also examined via the 260/280 nm ratios and 1% agarose gel electrophoresis. The Prime Script TM RT reagent Kit (Takara Bio Inc, Kusatsu, Japan) was used to synthesize the cDNA. The real-time PCR was carried out on an ABI 7300 system (Applied Biosystems, CA, USA) using the SYBR Green kits (Takara Bio Inc, Kusatsu, Japan). Specific primers of the target genes were identified by the NCBI Primer-BLAST and showed in the SI (Table S1). The transcriptional stability of the reference gene was accessed using the GeNorm, and finally the 18s was chosen as the housekeeping gene. The gene transcription levels were calculated using 2−ΔΔCt method.
Statistics
Data were presented as the mean ± standard error (SEM). SPSS 20.0 (SPSS, Chicago, IL, USA) was used to perform statistical analyses, and the comparison between the exposure groups and control group was performed using one-way analysis of variance (ANOVA) followed by Tukey’s test. A p < 0.05 was were considered to be significant.