Brain and gonadal aromatases as molecular and biochemical targets of endocrine disrupters in a model species, the zebrafish (Danio

rerio)
by Nathalie HiNfray (1), Ksenia CHesHeNKo (2), slim ait-aissa (1), olivier Palluel (1), Jean-Marc PorCHer (1), farzad PaKdel (3), rik i.l. eggeN (2), olivier KaH (3) & franois BrioN (1)
ABsTrAcT. - this study was undertaken to characterise the effect of endocrine disrupting chemicals (edCs) on aromatases in a model fish species, the zebrafish. We first developed methods to measure brain and gonadal aromatases at mRNA, protein and enzymatic levels and characterised the baseline expression in adult and larvae control fish. We further determined that (xeno)-estrogens induced cyp19b gene expression in the brain while they inhibited cyp19a expression in the ovary. tCdd, an aryl hydrocarbon receptor agonist, presented anti-estrogenic effects on the estradiol-dependent induction of cyp19b gene expression. androstatrienedione, a steroidal aromatase inhibitor and clotrimazole, a fungicide known to inhibit aromatase activity in vitro, were able to perturb aromatase expression in vivo, but in a more complex fashion than expected based on in vitro data. Our work showed that fish aromatases are molecular and biochemical targets of EDCs belonging to diverse chemical classes (xeno-estrogens, dioxins, pesticides). overall, we provide new and relevant data on the effects and mechanisms of action of EDCs in fish, which raises the question of the physiological consequences of such aromatase perturbation for the organism. Key words. - Aromatase - Zebrafish - Brain - Gonads - Endocrine disrupting chemicals.

Introduction to date, most studies on endocrine disrupting chemicals (edCs) have focused on compounds that interact with sex steroids receptors, particularly the estrogen receptors (ers). However, edCs can affect the endocrine system through various pathways and mechanisms of action. the biosynthesis of steroid hormones, particularly the steps catalysed by cytochrome P450-dependent enzymes, represents a target for edCs action. in vertebrates, an essential sex-related enzyme is aromatase (P450aro). aromatase is an enzymatic complex which catalyzes the final rate-limiting step in the conversion of androgens into estrogens. in most teleosts, including model species such as zebrafish (Danio rerio), aromatase is encoded by two structurally distinct genes, cyp19a and cyp19b. Cyp19 genes are key players in sexual differentiation and reproduction. Brain aromatase is also suggested to have a role in neuronal development and adult neurogenesis. With the aim to broaden our knowledge on the effects of EDCs on fish development and reproduction, we investigated the effects of several classes of chemical compounds that contaminate the aquatic environment, on P450aro expression and activities in the zebrafish. our goals were (i) to define the nature and the mode of action of compounds that can perturb aromatase and (ii) to determine whether aromatase could be use as a marker of endocrine disruption to assess the edCs potency of chemicals. in this article, we review our recent studies on the effects of edCs on aromatase in fish.

Methods different methodologies were developed to measure brain and gonadal aromatases at mrNa, protein and enzymatic levels in the zebrafish. Cyp19a and cyp19b mrNa levels were measured by specific branched DNA assays as described by Hinfray et al. (2006a). Brain aromatase (aroB) protein expression was studied using a specific zf-aroB polyclonal antibody (Menuet et al., 2005) by immunohistochemistry and elisa methods. Brain and gonadal aromatases enzymatic activities (aa) were measured by the tritiated water assay according to Hinfray et al. (2006a). results and discussion Expression of aromatase under physiological conditions table i summarises the expression patterns of aromatase measured at the gene, protein and enzymatic levels in the brain and gonads of adult zebrafish. our study confirmed that cyp19b is predominantly expressed in the brain. in the ovary cyp19a mrNa level was predominant while in the testis cyp19b gene was predominantly expressed compared
table i. - Characterisation of aromatase expression / activities in adult zebrafish. nd : not detected.

(1) [INERIS, Unit dvaluation des risques cotoxicologiques, France. [nathalie.hinfray@ineris.fr] (2) EAWAG, Switzerland. (3) uMr CNrs 6026, rennes, france. Cybium 2008, 32(2) suppl. : 34-36.

Hinfray et al. to cyp19a (sawyer et al., 2006). at the enzymatic level, brain aa was higher than that in the ovary while no aa was detected in the testis. Using specific zf-AroB antibody, brain aromatase was localised in radial glial cells. the functional significance of the high aromatase activity in these cells in the brain of fish is not entirely understood but it has recently been shown that aromatase-expressing radial glial cells in the forebrain of teleosts are progenitor cells capable of generating new neurons (Pellegrini et al., 2007). altogether these data demonstrate that brain and gonads are major sites for aromatase expression and that zebrafish brain is characterized by a high capacity to synthesise estrogens. Effect of EDCs on aromatase Effects of (xeno)-estrogens Xeno-estrogens such as ethynilestradiol (ee2) or nonylphenol (NP) are well-known contaminants of the aquatic environment and are found at concentrations up to 830 ng/l (2.8 nM) and 180 g/l, respectively. they are characterised by their ability to induce er-dependent gene, such as vitellogenin expression in the liver of oviparous vertebrates (Brion et al., 2004). In the brain of fish, it was demonstrated that estradiol (E2) induction of the zebrafish cyp19b gene is mediated by ERs and requires the synergistic action of estrogens responsive element (ere) and 1/2 ere in the promoter region of this gene (Menuet et al., 2005). to assess whether xeno-estrogens could modulate brain aromatase, zebrafish were exposed during 7 days to EE2 (10 nM) or NP (20, 100, and 500 g/l) at different life-stage of development. We showed that EE2 and NP weakly induced cyp19b gene expression in the brain of adult males (about a 2-fold change for the higher concentrations) but not in females. in contrast, at larval stage, these compounds strongly triggered cyp19b expression suggesting a higher sensitivity of larvae compared to adults. ee2 and NP have already been shown to be able to induce cyp19b gene expression in juvenile salmon (Meucci and Arukwe, 2006) and in zebrafish larvae (Kazeto et al., 2004) at similar concentrations. in our study, induction of the cyp19b gene in larvae led to a de novo synthesis of aroB in radial glial cells. Moreover, aroB induction was blocked by the pure er antagonist, iCi 182,780 (0.5 M), confirming that the cyp19b induction requires functional ERs. This study demonstrated that EE2 and NP mimic the action of the natural estrogen e2 in the brain by up-regulating cyp19b expression at the transcriptional level. The effect of E2 on the ovarian aromatase was quite different. at larval stage, cyp19a was not affected by exposure to e2 whereas it was strongly downregulated in ovary of adult females leading to aa inhibition (Hinfray et al., 2006a). interestingly, both ee2 and NP led to downregulation of the ovarian cyp19a expression, confirming the negative effect of NP on cyp19a gene expression in the ovary of
Cybium 2008, 32(2) suppl.

Effects of EDCs on aromatase in fish

adult zebrafish (Alberti et al., 2005). However, the precise mode of action of (xeno)-estrogens is not yet understood and further studies are needed to clarify this point. Effects of dioxins aryl hydrocarbon receptor (ahr) ligands such as dioxin or polycyclic aromatic hydrocarbons are ubiquitous contaminants of the environment and negatively affect fish reproduction. several putative ahr responsive elements or dioxin responsive elements (dre) were predicted in the cyp19a and cyp19b gene promoters of zebrafish and their presence indicates the potential of these genes to be regulated by ahr. However, in vivo exposure of zebrafish larvae to the prototypic ahr ligand, tCdd, had effect neither on cyp19b nor cyp19a gene expression (Cheshenko et al., 2007). on the other hand, co-exposure of larvae to 10nM e2 and 0.1 nM TCDD led to a significant down-regulation of E2-induction of cyp19b expression. anti-estrogenic effect of tCdd was also observed at the protein level by immunochemistry (Cheshenko et al., 2007). We further showed that TCDD action was independent of the dre present in the promoter region of the cyp19b gene, but was dependent of a cross-talk between er and ahr (Cheshenko et al., 2007). thus, our results showed that tCdd down-regulates the estrogeninduced transcriptional activity of the cyp19b gene and demonstrated for the first time the anti-estrogenic effect of an ahr agonist on an e2-regulated gene within a glial cell context. altogether these results point out the possible disruption of neuroendocrine functions of estrogens as one of the endocrine disrupting effects of this compound. Effect of aromatase inhibitors (AI) as mentioned above, xeno-estrogens and dioxine-like compounds are able to up- or down-regulate the transcriptional activity of the cyp19b gene by interacting with er or ahr, respectively. it is advisable to consider that several environmental substances such as imidazole pesticides are able to inhibit in vitro aromatisation of androgens into estrogens by interacting directly with the aromatase enzymatic complex (Hinfray et al., 2006b). among them, clotrimazole, a fungicide used as pharmaceutical and found in the aquatic environment, is one of the most potent ai in vitro. Our first aim was thus to assess its in vivo effects on aromatase. for that purpose, a 7 day-exposure of adult female zebrafish to nominal concentrations of clotrimazole (4, 20, 100 and 500 g/l) was performed and brain and ovarian aromatase expression and activity were measured. the steroidal ai androstatrienedione (atd) was used as reference. atd inhibited aa in both brain and gonads without affecting cyp19 genes expression (Hinfray et al., 2006a). in contrast, the in vivo effects of clotrimazole were more complex. in brain, an inverted-u dose-response curve was observed on cyp19b mrNa level, aroB protein synthesis as well as aa (tab. ii). in ovary, cyp19a mrNa levels were induced at all
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Effects of EDCs on aromatase in fish table ii. - effect of clotrimazole on aromatase expression and activity in brain and ovary of adult zebrafish after a 7 days exposure.: induction, : inhibition, =: no difference compared to control. Numbers in brackets represent the factors of induction or inhibition. AA: aromatase activity.

Hinfray et al.
Acknowledgements. - this study was supported by grants BCrddrC17-02, BCrd-drC08-03, and PNrPe-5000194 of the french Ministry of ecology and sustainable development.

references

concentrations tested while aa was induced at low and inhibited at high concentrations (tab. ii). although the precise modes of action of clotrimazole on brain and ovarian aromatase expression are still unknown, this study provides evidence that this compound is able to perturb aromatase in vivo in fish. Such effect are more complex than those previously envisioned after in vitro experiments. it should be pointed out that the concentration of clotrimazole used in this study are high compared to those found in the environment (up to 33 ng/l) and it remains to be determined whether this compounds can affect aromatase at environmentally relevant concentrations. conclusions our work clearly shows that brain and gonadal aromatases are molecular and biochemical targets of edCs in fish. A wide range of compounds (xeno-estrogens, dioxins, pesticides) have been shown to modulate aromatase expression and/or activities both in vitro and in vivo by acting at the transcriptional level or by modulating aa. Moreover, these substances induced tissue, sex, and life stage specific responses. given the complex effects of edCs on aromatase, the use of different methods to measure aromatase at the gene, protein and enzymatic level should be recommended to fully characterise chemical impact on this biomarker. finally, our studies provide new and relevant data on the effects and mechanism of action of known edCs in fish. given the physiological importance of estrogens for fish development and reproduction, the question of the physiological and reproductive consequences of aromatase perturbations for the organisms is raised.

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