Hierarchical clustering analysis of differentially expressed genes and biochemical evidences.

<p>(A) Transcriptional profiles of estrogen receptor genes (i.e. <i>erα</i>, <i>erβ</i>, and <i>erγ</i>) in the BPS- or BaP-exposed liver tissues of <i>P</i>. <i>olivaceus</i>; (B) the effect of BPS and BaP injections on the plasma 17β-estradiol (E2) concentration in the liver tissues of <i>P</i>. <i>olivaceus</i>. E2 concentration is expressed as pg/mL. Each value is an average of five biological replicates, and data are shown as means ± S.D; (C) the effect of BPS and BaP injections on the plasma vitellogenin (VTG) concentration in the liver tissues of <i>P</i>. <i>olivaceus</i>. VTG concentration is expressed as μg/mL. Each value is an average of five biological replicates, and data are shown as means ± S.D; (D) transcriptional expressions of genes associated with the innate immunity in the BPS- or BaP-exposed liver tissues of <i>P</i>. <i>olivaceus</i>. (E) the effect of BPS and BaP injections on the plasma complement activity in the liver tissues of <i>P</i>. <i>olivaceus</i>. The activity is expressed as U/mL. Each value is an average of five biological replicates, and data are shown as means ± S.D. The asterisk symbol (*) indicates statistical significance (<i>P</i> < 0.05) compared to the control values.</p

Differentially expressed mRNAs in BPS/control [fold change (log 2) >10].

<p>Differentially expressed mRNAs in BPS/control [fold change (log 2) >10].</p

Differentially expressed mRNAs in BaP/control [fold change (log 2) >10].

<p>Differentially expressed mRNAs in BaP/control [fold change (log 2) >10].</p

Comparative analysis of distinctive transcriptome profiles with biochemical evidence in bisphenol S- and benzo[<i>a</i>]pyrene-exposed liver tissues of the olive flounder <i>Paralichthys olivaceus</i>

<div><p>Flounder is a promising model species for environmental monitoring of coastal regions. To assess the usefulness of liver transcriptome profiling, juvenile olive flounder <i>Paralichthys olivaceus</i> were exposed to two pollutants, bisphenol S (BPS) and benzo[<i>a</i>]pyrene (BaP), which have different chemical characteristics and have distinct modes of metabolic action in teleost. Six hours after intraperitoneal injection with BPS (50 mg/kg bw) or BaP (20 mg/kg bw), liver transcriptomes were analyzed using the Illumina Hiseq 3000 platform. Interestingly, the transcriptome was highly sensitive and was distinctively expressed in response to each chemical. The primary effect of BPS was significantly increased transcription of egg process and vitellogenesis related genes, including vitellogenins (<i>vtg1</i>, <i>vtg2</i>), zona pellucida sperm-binding proteins (<i>zp3</i>, <i>zp4</i>), and estrogen receptors (<i>erα</i>, <i>erβ</i>), with increases in plasma 17β-estradiol (E2) and vitellogenin (VTG) concentrations. Following BaP treatment, detoxification- and biotransformation-related genes such as <i>cyp1a1</i> and UDP-glucuronosyltransferase (<i>ugt1a1</i>) were significantly increased, with an increase in EROD activity. In both transcriptomes, mRNA expression of genes involved in antioxidant defense systems was increased, while genes involved in innate immunity were decreased upon BPS or BaP exposure with a decrease in complement activity. This study provides useful insight into the chemical-specific hepatic transcriptional response of <i>P</i>. <i>olivaceus</i> and suggests a basis for further studies examining biomarker application of liver transcriptomes for environmental pollution.</p></div

Comparison of KEGG pathways with mapped genes.

<p>(A) “Protein procession in endoplasmic reticulum” and (B) Benzo[<i>a</i>]pyrene metabolism in “Metabolism of xenobiotics by cytochrome P450” in the BPS- or BaP-exposed liver tissues of <i>P</i>. <i>olivaceus</i>. The orange color denotes the gene mapped from the BPS-exposed liver transcriptome to each KEGG pathway. The blue-green color denotes the gene mapped from the BaP-exposed liver transcriptomes to each KEGG pathway. Notably, no gene was solely mapped from the BPS-exposed liver transcriptome to a KEGG pathway. (C) The hepatic EROD activity in the liver tissue of BPS- or BaP-injected olive flounder. Data are presented as the mean ± standard deviation (S.D.). Significant differences from the control value are indicated by an asterisk (*) on the data bar (<i>P</i> < 0.05).</p

qPCR validation results on the mRNA expressions of 15 randomly selected genes.

<p>(A) The mRNA expressions of 15 genes were selected from the RNA-seq data (<i>P</i> < 0.05). (B) Validation of the mRNA expression patterns of the selected 15 genes by qPCR. Abbreviations of the gene names are as follows: Major facilitator superfamily domain contating 2A, <i>mfsd2a</i>; Vitellogenin 1, <i>vtg1</i>; Zona pellucida sperm-binding protein 3, <i>zp3</i>; Zona pellucida sperm-binding protein 4, <i>zp4</i>; Estrogen receptor alpha, <i>erα</i>; Cytochrome P450 1A1, <i>cyp1a1</i>; Glutathione S-transferase zeta 1, <i>gstz</i>; Catalase, <i>cat</i>; Hepcidin antimicrobial peptide, <i>hamp</i>; Calreticulin 3, <i>calr3</i>; Complement C9, <i>c9</i>; Complement H, <i>ch</i>; Calnexin, <i>cnx</i>; <i>lectin</i>; Selenoprotein F, <i>selenof</i>.</p

Summary of the assembly statistic information.

<p>Summary of the assembly statistic information.</p

Comparison of transcriptional expression patterns of whole libraries and analysis of chemical-specifically expressed genes.

<p>(A) Transcriptional pattern analysis of each library (i.e., two control, three BPS-exposed, and three BaP-exposed liver tissues) by employing heat map and hierarchical clustering; (B) PCA plot analysis of transcriptional profile of each library. Each sample is depicted with a different color. (C) Number of statistically significant transcripts (i.e., over 2 fold; <i>P</i> < 0.05) in <i>P</i>. <i>olivaceus</i> liver tissues exposed to BPS or BaP; (D) the number of uniquely or commonly up- or downregulated transcripts in the <i>P</i>. <i>olivaceus</i> liver tissues exposed to BPS or BaP. Detailed list of the commonly modulated genes is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196425#pone.0196425.s006" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196425#pone.0196425.s007" target="_blank">S6</a> Tables.</p

Schematic summary of unique and common transcriptional responses in the BPS- and BaP-exposed liver tissues of <i>P</i>. <i>olivaceus</i>.

<p>Red arrow means increased transcriptional metabolism and green arrow represents decreased transcriptional metabolism.</p

Functional classification of differentially expressed genes.

<p>Comparison of Gene Ontology (GO) in terms of (A) biological process, (B) cellular components, and (C) molecular function that were enriched in the BPS- or BaP-exposed liver tissues of <i>P</i>. <i>olivaceus</i>. Composition of each GO term is represented as a percentage. For detailed information see in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196425#pone.0196425.s004" target="_blank">S3 Table</a>.</p