Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures.

Endocrine effects of the tapeworm Ligula intestinalis in its teleost host, the roach ( Rutilus rutilus).

The effects of parasite infection by the cestode Ligula intestinalis on the reproductive function and endocrine system of wild roach Rutilus rutilus were evaluated. Gonad maturation, plasma vitellogenin, plasma steroid concentrations (i.e. progesterone, 11-keto-testosterone and 17-beta-estradiol) and brain aromatase activity were investigated in relation with parasitization. A low prevalence (8%) of ligulosed roach and a moderate impact of parasitization (mean parasitization index of 8.8%) were found in the studied population. Inhibition of gonad maturation generally resulted from infestation but 5% of the ligulosed roach nevertheless reached maturity. Main sex steroid plasma content was depleted in both genders. Male 11-keto-testosterone, female 17-beta-estradiol and progesterone plasma concentrations of both genders were, respectively, 27, 5 and 3 times lower in ligulosed fish when compared to their non-infected counterparts. Progesterone levels were negatively correlated with the parasitization index in females. Brain aromatase activity of infected roach was reduced to 50% of that of the non-infected fish. These results demonstrate significant negative effects on the reproductive function of wild roach infected by the tapeworm L. intestinalis collected from a site with low contamination.

Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio).

Many chemicals in the aquatic environment are able to adversely affect in vitro brain and ovarian aromatase expression/activity. However, it remains to be determined if these substances elicit in vivo effect in fish. With the view to further understanding possible effects of endocrine disrupting chemicals (EDCs) on aromatase function, we first developed methods to measure brain and ovarian aromatase expression/activity in a model species, the zebrafish, and assessed the effect of estradiol (E2) and androstatrienedione (ATD), a steroidal aromatase inhibitor. We showed that CYP19b gene was predominantly expressed in the brain whereas in the ovary CYP19a mRNA level was predominant. Moreover, aromatase activities (AA) were higher in brain than in ovary. In adult zebrafish, E2 treatment had no effect on aromatase expression/activity in brain, whereas at larval stage, E2 strongly triggered CYP19b expression. In the ovaries, E2 led to a complete inhibition of both CYP19a expression and AA. Exposure to ATD led to a total inhibition of both brain and ovarian AA but had no effect on CYP19 transcripts abundance. Together, these results provide relevant knowledge concerning the characterization of aromatase in the zebrafish, and reinforce the idea that brain and ovarian aromatase are promising markers of EDCs in fish and deserve further in vivo studies.