Developmental toxicity of environmental chemicals

环境化学品的发育毒性

• 批准号: 6542479
• 负责人: FREDERICK S VOM SAAL
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-04 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542479
• 关键词: androgen receptor; biological signal transduction; chemicals; developmental genetics; diethylstilbestrol; dihydrotestosterone; environmental toxicology; enzyme activity; epidermal growth factor; estrogen receptors; hazardous substances; histogenesis; immunocytochemistry; in situ hybridization; insulinlike growth factor; laboratory mouse; messenger RNA; phenols; polymerase chain reaction; prostate; receptor binding; receptor expression; seminal vesicles; testosterone 5 alpha reductase; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The goal of the proposed research is to investigate mechanisms mediating disruption by bisphenol A (BPA) of cellular signalling systems during prostate and seminal vesicle development. BPA leaches from plastic resulting in significant fetal exposure in humans. Exposure of fetal mice to low doses of BPA results in a permanent increase in prostate gland number, overall size and androgen receptors (AR), while a permanent decrease in seminal vesicle size occurs; preliminary evidence suggests this is due to a decrease in the enzyme 5a-reductase, required for dihydrotestosterone (DHT) formation. Our first hypothesis is that the effects of low, environmentally relevant doses of BPA (and low doses of diethylstilbestrol, DES, as a positive control) in the fetal mouse prostate occur via binding to estrogen receptor alpha (ERa), induction of EGF and, subsequently, also IGF-1 gene activity (and synthesis of these growth factors), leading to a permanent increase in AR gene expression and AR protein. Our second hypothesis is that much higher doses of DES, but not BPA, will result in the opposite outcome and interfere with prostate development via competing with DHT for binding to AR. In the seminal vesicles, we predict that there will be a dose-dependent inhibition of EGF and IGF-1, resulting in a permanent dose-related down-regulation of 5a-reductase activity. To test these hypotheses our first specific aim is to conduct in vivo studies in which pregnant CD-1 mice are administered environmentally relevant doses of BPA (and also DES). Our second specific aim is to conduct in vivo studies with high doses of BPA and DES, up to the maximum tolerated dose. Our third specific aim involves removing the fetal urogenital sinus and Wolffian ducts for studies in primary culture to answer specific mechanistic questions by administering very low through sub-lethal doses of DES and BPA. We will also determine whether EGF and IGF-l mimic effects of DES and BPA, and if administering antibodies to these proteins inhibits effects. The fourth specific aim is to determine whether high doses of DES, but not BPA, compete with DHT for binding to AR, thus producing an antiandrogenic effect. In these studies the prostate and seminal vesicles will be examined on gestation day 18, postnatal day 3 and in adult offspring. We will initially focus on AR, ERa, ERB, 5a-reductase, EGF and IGF-1, and measure both mRNA levels by RT-PCR and protein levels by western blot analysis. Organ morphology will be determined by 3-D computer reconstruction from histological sections, coupled with in situ hybridization and immunocytochemistry for the above mRNAs and corresponding proteins, including markers for cell types, proliferation and apoptosis. 5a-reductase activity will be determined by radiometric assay.

描述（由申请人提供）：拟议研究的目标是研究前列腺和精囊发育期间双酚A（BPA）介导细胞信号系统中断的机制。BPA从塑料中浸出，导致人类胎儿大量接触。胎鼠暴露于低剂量BPA会导致前列腺数量、总体大小和雄激素受体（AR）永久性增加，而精囊大小则永久性减少;初步证据表明，这是由于双氢睾酮（DHT）形成所需的5 α-还原酶减少所致。我们的第一个假设是，低剂量、环境相关剂量的BPA（和低剂量的己烯雌酚，DES，作为阳性对照）对胎鼠前列腺的影响是通过与雌激素受体α（ER α）结合、诱导EGF以及随后的IGF-1基因活性（以及这些生长因子的合成）而发生的，从而导致AR基因表达和AR蛋白的永久性增加。我们的第二个假设是，更高剂量的DES，而不是BPA，将导致相反的结果，并通过与DHT竞争结合AR来干扰前列腺发育。在精囊，我们预测，将有一个剂量依赖性的抑制EGF和IGF-1，导致永久剂量相关的下调5 α-还原酶活性。为了验证这些假设，我们的第一个具体目标是进行体内研究，其中怀孕的CD-1小鼠给予环境相关剂量的BPA（以及DES）。我们的第二个具体目标是进行高剂量BPA和DES的体内研究，达到最大耐受剂量。我们的第三个具体目标是通过给予非常低至亚致死剂量的DES和BPA，切除胎儿尿生殖窦和Wolffian管进行原代培养研究，以回答具体的机制问题。我们还将确定EGF和IGF-1是否模拟DES和BPA的作用，以及是否给予这些蛋白质的抗体抑制作用。第四个具体目标是确定高剂量的DES，而不是BPA，是否与DHT竞争结合AR，从而产生抗雄激素作用。在这些研究中，将在妊娠第18天、出生后第3天和成年后代中检查前列腺和精囊。我们将首先关注AR，ERa，ERB，5 α-还原酶，EGF和IGF-1，并通过RT-PCR和蛋白质印迹分析测量mRNA水平。器官形态将通过组织学切片的3-D计算机重建，结合上述mRNA和相应蛋白质的原位杂交和免疫细胞化学，包括细胞类型、增殖和凋亡的标志物来确定。5a-还原酶活性将通过放射性测定法测定。