Transgenic (cyp19a1b-GFP) zebrafish embryos as a tool for assessing combined effects of oestrogenic chemicals

Abstract

Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.

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

“…Results from these studies demonstrate that binary or multi-component mixtures of ER agonists generally act in an additive manner on the expression of ER-regulated genes in both brain and liver (Thorpe et al 2001, Rajapakse et al 2004, Lin and Janz 2006, Kortenkamp 2007, Petersen and Tollefsen 2011, Petersen et al 2013. In some cases, some deviations from additivity can be observed, especially with infra-additive effects, both in in vitro and in vivo experiments (Rajapakse et al 2004, Lin and Janz 2006, Petersen and Tollefsen 2011, Petersen et al 2013).…”

“…In some cases, some deviations from additivity can be observed, especially with infra-additive effects, both in in vitro and in vivo experiments (Rajapakse et al 2004, Lin and Janz 2006, Petersen and Tollefsen 2011, Petersen et al 2013). …”

“…The cyp19a1b gene is extremely sensitive to (xeno-)estrogens and this regulation is ER-dependent (Le Page et al 2008. In the past few years, we developed both in vitro and in vivo bioassays, based on zebrafish cyp19a1b gene, that were used in this study: i) a human glial cell culture (U251-MG) co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 and zfERβ2) and a luciferase gene under the control of the zebrafish cyp19a1b promoter (Le Page et al 2006), ii) a transgenic zebrafish (cyp19a1b-GFP) line expressing GFP under the control of the zebrafish cyp19a1b promoter which is suitable to detect estrogenicity of chemicals alone and in mixtures , Petersen et al 2013. These in vivo and in vitro bioassays were used to assess the estrogenic responses of EE2 and LNG in mixtures; responses that were modeled using the concentrationaddition (CA) prediction model.…”

Toxicity Effects of the Environmental Hormone 4-Tert-Octylphenol in Zebrafish (Danio Rerio)

“…Transgenic biosensor zebrafish embryos which express the green florescent protein (GFP) under the control of estrogen-inducible promoter had been developed for studying potential health effects of environmental estrogens [29]. Exposure of the transgenic biosensor of zebrafish to 4-t-OP induced GFP expressed demonstrating that 4-t-OP possesses ability to act as natural estrogen activity in zebrafish [30].…”

Expression Patterns of the chgH:rfp Transgene in Response to 17α-Ethinylestradiol (EE2) Exposure in Marine Medaka Oryzias dancena

“…A number of transgenic fish models incorporating fluorescence protein reporters have been established for detecting waterborne estrogenic compounds, in which estrogen-responsive promoters were designed to drive the expression of visible fluorescent protein in specific response to exposure to exogenous estrogenic chemicals (Chen et al, 2010;Petersen et al, 2013). As an in vivo model system, such a transgenebased approach to measure environmental estrogenicity is useful since it allows the simple and non-invasive detection of the response to estrogens in living fish (Kurauchi et al, 2008;Chen et al, 2010).…”