Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) that activate the aryl hydrocarbon receptor (Ahr) pathway, and endocrine disruptors acting through the estrogen receptor pathway are among environmental pollutants of major concern. In this work, we exposed Atlantic cod (Gadus morhua) precision-cut liver slices (PCLS) to BaP (10 nM and 1000 nM), ethynylestradiol (EE2) (10 nM and 1000 nM), and equimolar mixtures of BaP and EE2 (10 nM and 1000 nM) for 48 h, and performed RNA-Seq based transcriptome mapping followed by systematic bioinformatics analyses. Our gene expression analysis showed that several genes were differentially expressed in response to BaP and EE2 treatments in PCLS. Strong up-regulation of genes coding for the cytochrome P450 1a (Cyp1a) enzyme and the Ahr repressor (Ahrrb) was observed in BaP treated PCLS. EE2 treatment of liver slices strongly up-regulated genes coding for precursors of vitellogenin (Vtg) and eggshell zona pellucida (Zp) proteins. As expected, pathway enrichment and network analysis showed that the Ahr and estrogen receptor pathways are among the top affected by BaP and EE2 treatments, respectively. Interestingly, two genes coding for fibroblast growth factor 3 (Fgf3) and fibroblast growth factor 4 (Fgf4) were up-regulated by EE2 in this study. To our knowledge, the fgf3 and fgf4 genes have not previously been described in relation to estrogen signaling in fish liver, and these results suggest the modulation of the FGF signaling pathway by estrogens in fish. The signature expression profiles of top differentially expressed genes in response to the single compound (BaP or EE2) treatment were generally maintained in the expression responses to the equimolar binary mixtures. However, in the mixture-treated groups, BaP appeared to have anti-estrogenic effects as observed by lower number of differentially expressed putative EE2 responsive genes. Our in-depth quantitative analysis of changes in liver transcriptome in response to BaP and EE2, using PCLS tissue culture provides further mechanistic insights into effects of the compounds. Moreover, the analyses demonstrate the usefulness of PCLS in cod for omics experiments.

激活芳烃受体（Ahr）途径的多环芳烃（如苯并[ a ] re（BaP））和通过雌激素受体途径起作用的内分泌干扰物是主要关注的环境污染物。在这项工作中，我们暴露了大西洋鳕鱼（Gadus morhua）将肝脏切片（PCLS）精确切成BaP（10 nM和1000 nM），乙炔雌二醇（EE2）（10 nM和1000 nM）以及BaP和EE2（10 nM和1000 nM）的等摩尔混合物，持续48 h，以及进行了基于RNA-Seq的转录组定位，然后进行了系统的生物信息学分析。我们的基因表达分析表明，在PCLS中，响应BaP和EE2处理，几个基因差异表达。在BaP处理的PCLS中观察到了编码细胞色素P450 1a（Cyp1a）酶和Ahr阻遏物（Ahrrb）的基因的强烈上调。EE2处理肝脏切片会强烈上调编码卵黄蛋白原（Vtg）和蛋壳透明带（Zp）蛋白前体的基因。不出所料，途径富集和网络分析表明，Ahr和雌激素受体途径是受BaP和EE2治疗影响最大的途径，分别。有趣的是，在这项研究中，两个编码成纤维细胞生长因子3（Fgf3）和成纤维细胞生长因子4（Fgf4）的基因被EE2上调。据我们所知，fgf3和fgf4以前没有关于鱼肝中雌激素信号转导的基因的描述，这些结果表明鱼体内雌激素对FGF信号通路的调节。通常在对等摩尔二元混合物的表达响应中维持响应于单一化合物（BaP或EE2）处理的顶部差异表达基因的签名表达谱。但是，在混合物治疗组中，BaP似乎具有抗雌激素作用，这是通过较少数量的差异表达的推定EE2反应基因所观察到的。我们使用PCLS组织培养技术对BaP和EE2响应的肝转录组变化进行了深入的定量分析，从而进一步了解了这些化合物的作用机理。此外，分析证明了鳕鱼中PCLS在组学实验中的有用性。