Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Environ Health Perspect
2005 Mar 01;1133:329-34. doi: 10.1289/ehp.7418.
Show Gene links
Show Anatomy links
Assessment of estrogenic endocrine-disrupting chemical actions in the brain using in vivo somatic gene transfer.
Trudeau VL
,
Turque N
,
Le Mével S
,
Alliot C
,
Gallant N
,
Coen L
,
Pakdel F
,
Demeneix B
.
???displayArticle.abstract???
Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpolebrain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brainventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.
Figure 2. Expression of GFP in adult goldfish brain. (A) Expression of GFP in the telencephalon (TEL) and optic tectum (OT) of freshly dissected intact brain. Note the high expression around the brain third ventricle (V3); bar = 100 μm. (B) Sagittal section (25 μm) through the telencephalon of a goldfish showing a highly branching neuron expressing GFP throughout. The third ventricle is to the right; bar = 5 μm. (C) Sagittal section (25 μm) through the telencephalon of a goldfish showing a neuron extending dorsolaterally. The cell body (not easily visualized) is toward the top left corner; bar = 5 μm.
Figure 3. Optimization of PEI-based gene transfer in the goldfish brain. (A) Comparison of the efficiencies of 22 kDa linear PEI used at different ratios of PEI amines to DNA anions. Animals were injected with CMV-LUC DNA (1 μg in 4 μL) complexed with 0 (n = 9), 3 (n = 10), 6 (n = 10), and 9 (n = 10) eq of PEI; brains were dissected at 48 hr postinjection; and luciferase activity (RLU/mg protein × 10–3; mean ± SEM) was determined. (B) Time course of expression of CMV-LUC in the goldfish brain. Animals were injected with CMV-LUC DNA (1 μg in 4 μL) complexed with 6 eq of PEI; brains were dissected at 2 hr (n = 10), 12 hr (n = 10), 24 hr (n = 10), 48 hr (n = 7), and 96 hr (n = 5) postinjection, and luciferase activity (RLU/mg protein × 10–3; mean ± SEM) was determined.
Figure 4. Effects of estrogenic chemicals on ERE-TK-LUC activity in the brains of male and female goldfish preexposed for 48 hr to E2 (10 nM; n = 14 males and 14 females), EE2 (10 nM; n = 8 males and 8 females), or ethanol vehicle (0.1 mL/L water; n = 8 males and 16 females). Data are presented as mean ± SEM.
*p < 0.05 compared with the male control values. levels
Abrahám,
Critical in vivo roles for classical estrogen receptors in rapid estrogen actions on intracellular signaling in mouse brain.
2004, Pubmed
Abrahám,
Critical in vivo roles for classical estrogen receptors in rapid estrogen actions on intracellular signaling in mouse brain.
2004,
Pubmed
Ackermann,
Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents.
2002,
Pubmed
Andersen,
Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals.
1999,
Pubmed
Arukwe,
Cellular and molecular responses to endocrine-modulators and the impact on fish reproduction.
2001,
Pubmed
Belfroid,
Occurrence of bisphenol A in surface water and uptake in fish: evaluation of field measurements.
2002,
Pubmed
Bosma,
Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic.
2001,
Pubmed
Bögi,
Functional genomics and sexual differentiation in amphibians.
2002,
Pubmed
,
Xenbase
Callard,
Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish.
2001,
Pubmed
Choi,
Molecular cloning of estrogen receptor alpha and expression pattern of estrogen receptor subtypes in male and female goldfish.
2003,
Pubmed
Coen,
A somatic gene transfer approach using recombinant fusion proteins to map muscle-motoneuron projections in Xenopus spinal cord.
1999,
Pubmed
,
Xenbase
Crump,
Octylphenol and UV-B radiation alter larval development and hypothalamic gene expression in the leopard frog (Rana pipiens).
2002,
Pubmed
Dellovade,
Thyroid hormone coadministration inhibits the estrogen-stimulated elevation of preproenkephalin mRNA in female rat hypothalamic neurons.
1999,
Pubmed
Feigelson,
Estrogens and breast cancer.
1996,
Pubmed
Graham,
Thoughts on tamoxifen resistant breast cancer. Are coregulators the answer or just a red herring?
2000,
Pubmed
Guillette,
Organization versus activation: the role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife.
1995,
Pubmed
Guissouma,
Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific.
1998,
Pubmed
Hess,
Estrogen in the adult male reproductive tract: a review.
2003,
Pubmed
Iwamuro,
Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larval Xenopus laevis.
2003,
Pubmed
,
Xenbase
Kim,
Topography of estrogen target cells in the forebrain of goldfish, Carassius auratus.
1978,
Pubmed
Kloas,
Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo.
1999,
Pubmed
,
Xenbase
Kolpin,
Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: a national reconnaissance.
2002,
Pubmed
Korach,
Update on animal models developed for analyses of estrogen receptor biological activity.
2003,
Pubmed
Le Guevel,
Assessment of oestrogenic potency of chemicals used as growth promoter by in-vitro methods.
2001,
Pubmed
Lee,
Determination of bisphenol A in sewage effluent and sludge by solid-phase and supercritical fluid extraction and gas chromatography/mass spectrometry.
2000,
Pubmed
Lemkine,
Polyethylenimines for in vivo gene delivery.
2001,
Pubmed
Levy,
Bisphenol A induces feminization in Xenopus laevis tadpoles.
2004,
Pubmed
,
Xenbase
Mackenzie,
Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds.
2003,
Pubmed
Matthews,
Ability of structurally diverse natural products and synthetic chemicals to induce gene expression mediated by estrogen receptors from various species.
2002,
Pubmed
,
Xenbase
McLachlan,
Environmental signaling: what embryos and evolution teach us about endocrine disrupting chemicals.
2001,
Pubmed
Menuet,
Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions.
2002,
Pubmed
Metcalfe,
Occurrence of neutral and acidic drugs in the effluents of Canadian sewage treatment plants.
2003,
Pubmed
Metcalfe,
Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias latipes).
2001,
Pubmed
Métivier,
Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter.
2003,
Pubmed
Métivier,
Synergism between ERalpha transactivation function 1 (AF-1) and AF-2 mediated by steroid receptor coactivator protein-1: requirement for the AF-1 alpha-helical core and for a direct interaction between the N- and C-terminal domains.
2001,
Pubmed
Navas,
Do gonadotrophin-releasing hormone neurons express estrogen receptors in the rainbow trout? A double immunohistochemical study.
1995,
Pubmed
Ouatas,
T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied by polyethylenimine based in vivo gene transfer.
1998,
Pubmed
,
Xenbase
Pasmanik,
Changes in brain aromatase and 5 alpha-reductase activities correlate significantly with seasonal reproductive cycles in goldfish (Carassius auratus).
1988,
Pubmed
Peter,
A stereotaxic atlas and technique for forebrain nuclei of the goldfish, Carassius auratus.
1975,
Pubmed
Petit,
Two complementary bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout estrogen receptor and trout hepatocyte cultures.
1997,
Pubmed
Pickford,
Assessing chronic toxicity of bisphenol A to larvae of the African clawed frog (Xenopus laevis) in a flow-through exposure system.
2003,
Pubmed
,
Xenbase
Robinson-Rechavi,
The nuclear receptor superfamily.
2003,
Pubmed
Rowe,
Autonomous regulation of muscle fibre fate during metamorphosis in Xenopus tropicalis.
2002,
Pubmed
,
Xenbase
Sanchez,
Diversity in the mechanisms of gene regulation by estrogen receptors.
2002,
Pubmed
Segner,
Potencies of estrogenic compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo.
2003,
Pubmed
Staples,
A review of the environmental fate, effects, and exposures of bisphenol A.
1998,
Pubmed
Sumpter,
Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment.
1995,
Pubmed
Ternes,
Behavior and occurrence of estrogens in municipal sewage treatment plants--I. Investigations in Germany, Canada and Brazil.
1999,
Pubmed
Trudeau,
Neuroendocrine regulation of gonadotrophin II release and gonadal growth in the goldfish, Carassius auratus.
1997,
Pubmed
Trudeau,
Octylphenol (OP) alters the expression of members of the amyloid protein family in the hypothalamus of the snapping turtle, Chelydra serpentina serpentina.
2002,
Pubmed
Tyler,
Endocrine disruption in wildlife: a critical review of the evidence.
1998,
Pubmed
Yamamoto,
Bisphenol A in hazardous waste landfill leachates.
2001,
Pubmed
Yin,
Occurrence and fate of hormone steroids in the environment.
2002,
Pubmed
Yoon,
Differential activation of wild-type and variant forms of estrogen receptor alpha by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements.
2001,
Pubmed
