title: Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.
abstract: 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.
authors: INERIS, Unité d'écotoxicologie in vitro et in vivo, Verneuil-en-Halatte, France. Electronic address: nathalie.hinfray@ineris.fr.
authors: N
authors: N
authors: Hinfray
authors: INERIS, Unité Modèles pour l'Ecotoxicologie et la Toxicologie, Verneuil-en-Halatte, France.
authors: C
authors: C
authors: Tebby
authors: INERIS, Unité d'écotoxicologie in vitro et in vivo, Verneuil-en-Halatte, France.
authors: C
authors: C
authors: Garoche
authors: INERIS, Unité d'écotoxicologie in vitro et in vivo, Verneuil-en-Halatte, France.
authors: B
authors: B
authors: Piccini
authors: IRSET, équipe NEED, Université de Rennes 1, Rennes, France.
authors: G
authors: G
authors: Bourgine
authors: INERIS, Unité d'écotoxicologie in vitro et in vivo, Verneuil-en-Halatte, France.
authors: S
authors: S
authors: Aït-Aïssa
authors: IRSET, équipe NEED, Université de Rennes 1, Rennes, France.
authors: O
authors: O
authors: Kah
authors: IRSET, Inserm U1085, équipe TREC, Université de Rennes 1, Rennes, France.
authors: F
authors: F
authors: Pakdel
authors: INERIS, Unité d'écotoxicologie in vitro et in vivo, Verneuil-en-Halatte, France.
authors: F
authors: F
authors: Brion
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001141:Aromatase / Q000235:genetics*; D001681:Biological Assay; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D004347:Drug Interactions; D004625:Embryo, Nonmammalian; D047628:Estrogen Receptor alpha / Q000235:genetics; D047629:Estrogen Receptor beta / Q000235:genetics; D004967:Estrogens / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000494:pharmacology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016912:Levonorgestrel / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011372:Progestins / Q000494:pharmacology*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; D011372:Progestins; D029961:Zebrafish Proteins; C000594790:estrogen receptor 2b, zebrafish; D049452:Green Fluorescent Proteins; D004997:Ethinyl Estradiol; D016912:Levonorgestrel; D008156:Luciferases; D001141:Aromatase; C440728:Cyp19a1b protein, zebrafish
keywords: Brain aromatase; Ethinylestradiol; Levonorgestrel; Mixture; Transgenic zebrafish; U251-MG cells