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Environ Health Perspect
2005 Mar 01;1133:329-34. doi: 10.1289/ehp.7418.
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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.
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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
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