BPA: Cellular and epigenetic effects on the urethra

描述(申请人提供)：梗阻性排尿障碍(CVD)是男性衰老过程中的一种常见疾病，虽然不太常见，但也发生在女性。拟议的实验涉及出生前和出生后(寿命)暴露于双酚A(BPA)对雄性和雌性SD大鼠OVD发展的影响，在国家毒理学研究中心(NCTR)饲养并按GLP方案使用不同剂量的BPA治疗。对雄性和雌性大鼠和小鼠的初步研究表明，尿道腺发育和尿路畸形的变化是由雌激素化学物质引起的，包括双酚A，但低剂量和高剂量的具体效果不同。初步研究还表明，产前暴露于BPA会导致成年后暴露于外源雌激素的雄性小鼠发生OVD。假设胎儿暴露于低剂量的BPA会使雄性和雌性SD大鼠在衰老过程中易患OVD，这是由于出生后持续暴露于相同剂量的BPA所致。在目标1中，研究人员将检测BPA对男性和女性胎儿(怀孕第21天)尿道腺结构(使用计算机辅助连续切片重建)以及上皮和基质功能的影响，使用激光捕获显微解剖(LCM)结合下一代测序(NGS)检测DNA的基因表达和表观遗传学变化，以及免疫组织化学(IHC)确定特定的细胞类型、增殖和凋亡。在目标2中，将从患OVD的成年雄性和雌性大鼠或非疾病对照组特定年龄的大鼠身上收集尿路。研究人员将使用与胎儿相同的结果(组织病理学、免疫组织化学、LCM)检查尿道腺和间质的结构和功能，捕获上皮和间质细胞，使用NGS分析mRNA和基因组DNA，以确定转录图谱和基因组DNA的全基因组甲基化图谱。对暴露于不同剂量双酚A的胎儿和成人尿路组织进行详细的形态和功能分析相结合，具有很高的创新性。

DESCRIPTION (provided by applicant): Obstructive voiding disorder (CVD) is a common disease during aging in men, and while less common, it also occurs in women. The proposed experiments concern the role of prenatal and postnatal (lifespan) exposure to bisphenol A (BPA) on the development of OVD in both male and female SD rats reared and treated with different doses of BPA under a GLP protocol at the National Center for Toxicological Research (NCTR). Preliminary studies with both male and female rats and mice show that changes in periurethral gland development and malformations of the urethra are caused by estrogenic chemicals, including BPA, but specific effects are different at low and high doses. Preliminary studies also show that prenatal exposure to BPA results in OVD in male mice exposed to exogenous estrogen during adulthood. The hypothesis is that fetal exposure to low doses of BPA will predispose both male and female SD rats to development of OVD during aging as a result of continued exposure to the same dose of BPA during postnatal life. In aim 1 the investigators will examine the effects of BPA on male and female fetal (gestation day 21) periurethral gland structure (using computer-assisted reconstruction of serial sections) and function of epithelium and stroma, using laser capture microdissection (LCM) coupled with next generation sequencing (NGS) for examination of gene expression and epigenetic changes in DNA, as well as immunohistochemistry (IHC) to identify specific cell types, proliferation and apoptosis. In aim 2 the urethra will be collected from adult male and female rats that develop OVD or at specific ages in non-diseased controls. The investigators will examine periurethral gland and stroma structure and function using the same outcomes described for fetuses (histopathology, immunohistochemistry, LCM) to capture epithelial and stromal cells for analysis of mRNA and genomic DNA using NGS to determine transcriptomal profiles and to determine genome-wide methylation profiles of genomic DNA. The combination of detailed morphological and functional analyses of fetal and adult urethral tissues exposed to different doses of BPA is highly innovative.