Gene set analysis of microarray data

The heat map of Figure 3.1 depicts the results of the Gene Set Analysis (GSA) and shows enrichment of affected genes for KEGG pathways and GO- and InterPro terms (data shown for EC20 only). The GSA provides

a broad overview of the transcriptome response of fish embryos to the reference compounds EE2 (0.8 mg/l) and flutamide (1.4 mg/l) compared to the selected estrogenic and anti-androgenic test compounds bisphenol A (8.5 mg/l), genistein (2.4 mg/l), propanil (1.1 mg/l), methylparaben (24.4 mg/l), linuron (1.3 mg/l) and prochloraz (2.0 mg/l). The first two lanes (or columns) on the left show the complete set of significantly af- fected pathways (p-value <0.1), according to the affected KEGG-, GO- and InterPro- categories, for EE2 and flutamide; the categories are ranked by their odds ratios. The reference compound flutamide provoked a rel- ative weak transcriptome response (17 affected pathways) compared to EE2, which affected a considerably larger number of pathways (60 path- ways). For the non-reference compounds, the heat map shows only those

3.4 Results 47

affected pathways, which were also affected by either of the reference com- pounds, i.e. the gene sets of the intersections. Overall, the non-reference compounds affected more pathways (see Annex Tables C-1-C-8). Genis- tein caused the strongest alterations in gene expression with 711 affected pathways, followed by linuron, prochloraz, BPA and propanil with 251, 228, 196 and 182 pathways, respectively, and methylparaben showed the weakest response, with 101 regulated pathways.

Figure 3.1: Estrogenic and anti-androgenic pathways in 48hpf zebrafish embryos. Overview of affected pathways after exposure to 17α-ethinylestradiol (EE2), flutamide and comparison to bisphenol A (BPA), propanil, genistein, methylparaben, linuron and prochloraz. All significantly altered (p-value <0.1) and therefore overrepresented KEGG-, GO- and InterPro-terms for EE2 and flutamide are shown in the left two lanes (data were obtained from gene set analysis processed at the Babelomics 4 platform (http:www.fatigo.org). For the other substances, only the equivalent pathways and their regulation are shown. The color illustrates the odds ratios of the pathways, reflecting the probability for pathway- associated genes to be overrepresented in the identified part of the list; yellow fields indicate pathways containing upregulated genes, whereas blue color indicates downregulated pathways (3.3.6).

3.4 Results 49

Reference compounds A distinctive transcriptome pattern was retrieved for the model estrogen EE2 and the model anti-androgen flutamide (Fig- ure 3.1), clearly emphasizing that the modes of action of EE2 and flutamide are different in the fish embryos. Apart from one InterPro term, no similar- ities were found between the transcriptome responses of the two reference compounds. Figure 3.2 provides a summarized view of the affected GO and InterPro annotations as well as of the relationships between the single categories for EE2 (Figure 3.2a) and flutamide (Figure 3.2b), respectively. In the EE2 condition, growth related pathways, such as regulation of cell growth and insulin-like growth factor binding were strongly upregulated, showing the highest odds ratios (4.5 and 4.8, respectively) and signifi- cance values (0.003 and 0.002). Highly significant was also the induction of transcription related pathways, including biosynthesis, DNA binding and genes associated with homeodomain-like family. Further, genes asso- ciated with early steroid synthesis, e.g. isoprenoid synthesis, and steroid metabolism, e.g., sulfotransferase activity, were upregulated. Response to xenobiotic stimulus was another upregulated GO process, with the estro- genic biomarker aromatase b and vitellogenin 1 among the affected genes. Fewer pathways were downregulated by EE2. However, several GO terms related to protein metabolism were downregulated, indicating a decreased protein-turnover after EE2 exposure. Compared to EE2, the gene response to flutamide was less diverse and affected fewer pathways and molecular processes. The flutamide data suggest hormone activity, which is sup- ported by the induction of the related molecular functions “receptor bind- ing” and “neurotransmitter receptor activity” (Figure 3.2b). Further, in- duction of genes associated with the zona pellucida sperm-binding protein (IPR001507) (see Figure 3.1) implied an endocrine response in flutamide exposed fish embryos.

proteo- lysis bio- synthesis response to xenobiotic

stimulus metabolismDNA isoprenoid biosynthesis protein metabo- lism camera- type eye morpho- genesis protein catabolism ossification response to DNA damage stimulus regulation of cell growth sequence-specific DNA binding transciption factor activity ATP binding protein binding nucleo- side binding nucleic acid binding sequence- specific DNA binding chemokine receptor activity insulin-like growth factor binding Insulin-like growth factor- binding protein growth factor binding Fos transforming protein chemokine binding DNA binding sulfo- transferase activity Homeo- domain- like nucleus cell cortex membrane coat Molecular Function Biological Process Cellular Component A purine nucleotide metabolsim amine metabilsm cellular amino acid metabolsim receptor binding hormone activity hydrolase activity, acting on acid anhydrides, catalyzing transmenbrane movement of substances neuro- transmitter receptor activity

Biological Process Molecular Function

B

Figure 3.2: Visualization of estrogenic and anti-androgenic mode of actions in zebrafish em- bryos after 48h exposure to 17α-ethinylestradiol (EE2) and flutamide. The figure shows the main affected GO categories (biological processes, molecular functions and cellular components, depicted in rectangu- lar shapes) and InterPro-terms (hexagonal shapes) to EE2 (A) and flutamide (B), as summarized by REVIGO (http://revigo.irb.hr/revigo.jsp). Blue and yellow colored nodes depict downregulated and upregulated pathways, respectively, with the node size reflecting the value of the odds ratio. The node opacity reflects the corresponding adjusted p-value (opacity increases with increasing p-values). The width of the line between the nodes increases with the degree of similarity between the terms (determined by the SimRel semantic similarity measurement algorithm in REVIGO). Visualization of GO and Inter- Pro terms was performed with Cytoscape. Terms, which were sub-grouped into a functionally related category, do not appear in the graph, but are listed in tables 3.4 and 3.5 in the Appendix (3.3.6).

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Non-reference compounds Figure 3.1 shows also those affected pathways of the non-reference substance, which were concordantly affected by either EE2 or flutamide. The entire gene expression profile of each individual sub- stance is provided in the Annex (C-1-C-8). The heat map demonstrates that all tested non-reference substances regulated endocrine pathways. The numbers of affected estrogenic and anti-androgenic pathways as well as the commonalities with each reference substance, however, differ between the substances. After calculating the ratio between the sum of estrogenic and anti-androgenic pathways (regulated by a test compound) and the sum of all pathways regulated by EE2 and flutamide (all pathways, which are presented in Figure 3.1, were included in the calculation), the broad- est endocrine responses were found for genistein, linuron and prochloraz. They showed respectively 69%, 30% and 43% of pathway regulation agree- ment with EE2 and flutamide. BPA and propanil affected fewer estrogenic and anti-androgenic pathways (29% and 20%). Methylparaben showed the weakest endocrine response with only 12% of pathways shared with the reference compounds. The proportion of alignment of affected categories between the test compounds and EE2 or flutamide is depicted in Figure 3.3. Based on the number of concordantly affected pathways, a categorization as predominantly estrogenic or anti-androgenic was achievable for BPA (es- trogenic) as well as for propanil, linuron and prochloraz (anti-androgenic). Most of the BPA affected endocrine pathways were in agreement with the ones regulated by EE2. However, the anti-androgenic responses of linuron and especially prochloraz were ambiguous as they shared many pathways with EE2, implying other endocrine mechanisms of actions in addition to the anti-androgenic activity. For methylparaben, all pathways were regu- lated contrary to EE2, suggesting a predominantly anti-estrogenic activ- ity. Genistein affected proportionally more anti-androgenic than estrogenic pathways. However, the large overlap with flutamide and EE2, suggested a dual or multiple mode of action for genistein. Therefore, genistein can be considered as an anti-androgenic as well as an estrogenic acting compound.

Figure 3.3: Pathway intersections between bisphenol A (BPA), genistein, propanil, linuron or prochlo- raz and the reference compounds 17α -ethinylestradiol (EE2) and flutamide. For each substance, the per- centage reflects the ratio of the number of equally and differently regulated GO-, KEGG- and InterPro- pathways, compared to the number of all pathways affected by the reference compounds. Functionally closely related pathways (i.e. direct parent/child GO-and InterPro relationships) were merged prior to the calculation of the ratios (3.3.7). The pathways included in the calculation are listed in Tables 3.6 and 3.7.

Estrogenic and anti-androgenic transcriptome patterns When comparing the expression profiles of the estrogenic substances EE2, BPA and genis- tein, an intersection of commonly affected pathways became apparent. All substances induced the GO category “response to xenobiotic stimulus”, which comprises also the estrogenic biomarker genes cyp19a1b and vtg1. The upregulation of these genes has been confirmed by qPCR for EE2, BPA and genistein (data not shown for EE2 and BPA; for genistein, the validation has been published in Schiller et al. [2013]). Further, all sub- stances regulated terpenoid backbone pathway genes, a KEGG analogue to the GO biological process “isoprenoid synthesis”. The regulation for BPA, however, was opposite to EE2 and genistein. Moreover, growth related categories, such as “regulation of growth” and “growth factor binding” were induced by the estrogens. Among the corresponding gene sets, regu- lation affected genes encoding for proteins of the IGFBP superfamily, such as ctgf (connective tissue growth factor ) and igfbp1 (insulin-like growth factor binding protein 1 ), which were upregulated by BPA, genistein and EE2. The regulation of hox genes was also identified for the estrogenic

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compounds, with EE2 and BPA showing up- and genistein downregulation of these genes. Lastly, our data suggest some alterations of the energy metabolism since an enrichment of downregulated genes of the ATP bind- ing category was found for all estrogenic substances.

The expression profiles of propanil, linuron, prochloraz and genistein showed commonalities with the anti-androgen flutamide. Like flutamide, linuron and prochloraz upregulated genes associated with neurotransmit- ter receptor activity, which included several neuropeptide y receptor iso- forms (npy1r, npy4r, npy7r ) as well as kisspeptin receptors (kiss1b for prochloraz and flutamide and kiss1ra for linuron). In addition, prochloraz and flutamide induced hormone activity related genes, such as gnrh (go- nadotropin releasing hormone) (flutamide and genistein), lhbeta 1 (luteiniz- ing hormone beta 1 ) (flutamide) and fshb (follicle stimulating hormone beta (prochloraz), and linuron and prochloraz induced genes associated with the zona pellucida sperm-binding protein domain. Propanil shared affected pathways with flutamide, which are related to amino acid and purine metabolism as well as hydrolase activity.

Validation of microarray results For the validation of the microarray re- sults, gene expression analysis of selected genes for all tested compounds was performed by quantitative real-time PCR (qPCR). For genistein, the validation has previously been shown and described in Schiller et al. [2013]. For all other substances, genes were selected for validation, which showed an adjusted p-value below 0.05 in the microarray significance analysis and, if possible, appeared in the gene sets retrieved by GSA. QPCR was also applied to evaluate a concentration-dependency of established estrogenic marker genes after exposure to EE2. The qPCR validation confirmed the microarray results for the selected genes in terms of significant up- or down- regulation. On the other hand, a concentration-dependency could not be established for the expression of esr1, esr2a, vtg1 and cyp19a1b, verifying the suitability of the EC10 and EC20 concentration levels for the analysis

The corresponding data are available from the supplementary material in Annex 3.A and 3.A-3.