Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA’s reactive metabolite MBP and the development of valvular-cardiovascular disease states.

The bulbo-ventricular canal (BVC) and the atrio-ventricular canal (AVC), which are precursors to the BV and AV valves respectively, can be distinguished at 36 hours post fertilisation (hpf) (Mehta et al., 2008) and the sinus venosus valve is distinguishable by 48 hpf (Grimes et al., 2006). Initial valvulogenesis (formation of the endocardial rings) is completed shortly after 96 hpf (Hu et al., 2000;Grimes et al., 2006) and is described in detail for the atrio-ventricular valve (Hove et al., 2003;Bartman et al., 2004;Vermot et al., 2009;Staudt and Stainier, 2012;Chen et al., 2013), but not the bulboventricular valve or sinus venosus valve. By 48 hpf, the AVC endocardium thickens to form the endocardial ring, and by 55 hpf this consists of a single\ layer of polarized cuboidal endocardial cells that stain strongly for alcama (Scherz et al., 2008). Cuboidal cell formation and alcama expression is dependent on troponin T type 2a (tnnt2a) (Bartman et al., 2004;Beis et al., 2005). The cuboidal endocardial cell layer subsequently proliferates, folds and extends into the extracellular matrix to form the superior valve leaflet by 85 hpf, and the inferior leaflet by 102 hpf (Scherz et al., 2008). The valve leaflets consist of two layers of cells, with those in the layer closest to the AVC remaining cuboidal and those in the layer closest to the myocardium developing a rounded shape (Scherz et al., 2008). Heart valve formation and remodelling (e.g. AV valve transitioning from two to four leaflets) is completed by 35 dpf (Beis et al., 2005, Sarmah et al., 2016). At the a molecular level, initial formation of the AVC coincides with localised expression of bmp4, versican (cspg2) and tgfb in the AVC myocardium (Walsh and Stainier, 2001;Beis et al., 2005;Chen et al., 2013) and expression of notch1b, calcineurin (ppp3ca, b, c and ppp3r1, 2) (Beis et al., 2005); has2 (Hurelstone et al., 2003) and prss23 (Chen et al., 2013) in the AVC endocardium. Key genes (e.g. notch1b and bmp4) in gene networks regulating endocardial cell proliferation and valve morphogenesis (post 48 hpf) have been linked to Wnt/β-catenin signalling (Walsh and Stainier, 2001;Hurlestone et al., 2003), TGF-β, ErbB/Neuregulin and prostaglandin signalling (Scherz et al., 2008) and to pkd2/Hdac5/Klf signalling (Vermot et al., 2009). Data represent the range in water quality measurements for solvent controls, low and high-level exposure treatments in each study. MBP 4-methyl-2,4-bis(4hydroxyphenyl)pent-1ene

Analytical method
Analysis of water and tissue samples was performed by Liquid Chromatography and Mass Spectrometry (LC-MS).
Both analytes were separated using a linear gradient of (A) aqueous phase and (B) organic solvent with initial conditions shown in the table below. Solvent B increased to 100% in 4.5 min and this was maintained for 1 min, before returning to the initial condition. The flow rate was 500µL/min. Temperature of autosampler was set at 8˚C, while column was kept at a room temperature. Mass spectrometry was performed using a TSQ Vantage triple quadrupole mass spectrometer. The mass spectrometer was equipped with a heated electrospray (HESI II) source (ThermoFisher Scientific, Hemel Hempstead, UK). The HESI probe was operating in both negative and positive mode; an ion-spray voltage of -4.0 kV for both analytes. The heated capillary temperature was set at 275 °C and the vaporizer temperature was 60 °C. Nitrogen was employed as a sheath and auxiliary gas at a pressure of 60 and 2 arbitrary units, respectively.
The argon CID gas was used at a pressure of 1.5 mTorr and the optimum collision energy (CE) for each transition was selected. Quantification of the target compounds was performed by monitoring two characteristic multiple reaction monitoring (MRM) transitions (  Data are presented as the mean ± 95% confidence interval. # Whole body concentration was calculated based on a wet weight of 1200 µg for a single zebrafish larvae at 5 days post fertilization (dpf) (Hu et al., 2000). § Heart concentration was calculated based on a ventricle weight of 10% of the whole body weight at 5 dpf (Hu et al., 2000).
¥ Bio-concentration factor (BCF) was calculated as measured tissue concentration / measured aqueous exposure concentration.
Aqueous exposure concentrations were relatively stable of over time. During the longest period between the static renewal of exposure solutions (i.e. 0-5 days), maximum reductions in aqueous exposure concentrations were: 34% for BPA (from 108% to 74% of nominal for the 100 g/L exposure); 29% for MBP (from 96% to 67% of nominal for the 2.5 g/L exposure).   Relative mean fluorescence intensity of the ERE:GFP reporter was quantified relative to the solvent control at 5 and 15 days post fertilization (dpf). Data are presented as the mean ± 95% confidence interval.   In the low-level BPA exposure (100 µg/L) 131 genes were differentially expressed at 5 dpf and only 1 gene apolipoprotein Da, duplicate 2 (apoda.2 -associated with GO:0006810 ~transport) at 15 dpf (SI Table S8 .xls). At 5 dpf there was significant enrichment of genes / ontologies associated with transport: GO:0006810 ~transport, GO:0006811 ~ion transport, GO:0055085 ~transmembrane transport, GO:0035879 ~plasma membrane lactate transport. Cell signalling pathways were also enriched GO:0007219 ~Notch signalling pathway, dre04630:Jak-STAT signalling pathway, dre04060:Cytokinecytokine receptor interaction, dre04080:Neuroactive ligand-receptor interaction (SI Table S9a .xls).
High-level MBP exposure (25 µg/L) resulted in differential expression (predominantly downregulation) of 127 genes at 5 dpf. One of these genes: elastin microfibril interfacer 3 (emilin3) was consistent with the low-level MBP exposure treatment at 5 dpf, and 2 genes: activated leukocyte cell adhesion molecule b (alcamb) and transforming growth factor, beta-induced (tgfbi) were consistent with that seen for the high-level BPA exposure treatment (SI Table S8 .xls). There were two distinct gene groups with significantly enriched ontologies for i) 'Cellular and extracellular matrix interactions' including via KEGG pathways dre04510:Focal adhesion, dre04512:ECM-receptor interaction, and the process GO:0060536 ~cartilage morphogenesis; ii) 'Filamentous protein synthesis and activity' including GO:0045095 ~keratin filament, GO:0005882 ~intermediate filament, GO:0005198 ~structural molecule activity (Table 9d .xls). The transcriptomic effects of high-level MBP exposure at 15 dpf could not be established due to problems encountered in sample processing (PCR amplification of libraries). Tables S8 to S12.xls' for the following tables .xls Tables   Table S8: Differentially expressed genes in BPA and MBP exposure treatments (continued)
Distal enhancer or promoter elements (up to 100 kB) are also involved in regulating the expression of many estrogen receptor target genes, often through looping or other higher order chromatin structures (reviewed in Dietz and Carroll, 2008;Liu and Cheung, 2014;Magnani and Lupien, 2014). This highlights the difficulties in pinning down the regulation of individual genes by estrogen receptors and the benefit of wider scanning of flanking regions for sets of genes to evaluate enrichment of TFBS motifs for ER-related transcription factor binding. We elected to scan 50 kB up-and down-stream of our differentially expressed genes, since the mean intergenic region in zebrafish is 97 kB, with a standard deviation of 164 kB (Hu et al., 2015). Therefore scanning 100 kB would have a high risk of overlapping genes.     Sequence data were generated from hearts pooled from ~30 individuals from each of 4 separate aquaria (nominally n=4 experimental replicates) per exposure treatment. Enriched pathways were identified using Enrichr and referenced to the Reactome database (2016). Pathways highlighted in red boxes are calcific aortic valve disease (CAVD) biomarkers.

Figure S9: Enriched Transcription Factor Binding Site motifs 5 kB up-and down-stream of differentially expressed genes for BPA and MBP exposure treatments
Enriched transcription factor binding site motifs were identified using Analysis of Motif Enrichment (AME) in MEME suite 5.0.2. Enrichment is inversely proportional to adjusted p-value. Transcription factor binding site motifs associated with estrogen receptor signalling, included: estrogen receptor 2 (esr2); specificity proteins constituting ERE tethering factors (sp1, sp3, sp4); pioneer factors facilitating ERE binding (foxa1, nfkb2, pbx1, runx1); CAMP responsive element binding proteins (creb1, creb5).