Prenatal xenoestrogen exposure and mammary cancer

描述(申请人提供)：乳腺癌发病率的增加促使科学家考虑荷尔蒙活性环境化学物质的可能作用。然而，寻找成人环境暴露与乳腺癌发病率之间的相关性的流行病学研究主要是不确定的。然而，流行病学研究确实表明，胎儿雌激素水平的波动对成年后患乳腺癌的风险有长期影响。对小鼠的研究表明，产前暴露于低剂量的异种雌激素双酚A(BPA)会改变这些乳腺的发育。这些影响在停止接触后很久才显现出来。这些乳腺的末端芽和末端导管的数量增加特别相关，因为癌症起源于这些结构。我们推测，产前暴露于低剂量双酚A可能会增加患乳腺癌的风险，导致肿瘤结果的机制可能包括调节乳腺发育的基因的即兴表达。产前注射异种雌激素可能会改变雌激素反应基因的表达，进而影响乳腺发育计划中的下游基因，使这些动物容易患上癌症。为了验证这一假设，我们将使用一个大鼠模型，在青春期暴露于致癌物质亚硝基甲基尿素(NMU)会诱发乳腺癌。使用Wistar大鼠进行的一项初步研究表明，可以对该模型进行修改，以评估胎儿暴露于环境相关的异种雌激素水平的影响。它产生的数据与提出的假设一致。目的1：确定产前暴露双酚A导致乳腺癌发病率增加的水平。从妊娠第9天到出生，暴露于双酚A的Wistar大鼠的肿瘤发病率和潜伏期将被测量。在50天龄时，动物将被注射亚致癌剂量的NMU。目的2：验证产前双酚A暴露改变乳腺基因表达的假说。将从乳腺中提取总RNA，并利用DNA微阵列技术进行分析。在适当的情况下，还将使用激光捕获显微切割和/或RNA线性放大技术来获取样本。上调和下调的基因将通过QRT-PCR进行确认。细胞分布将通过原位杂交进行评估。需要分析的发育点是：1)孕期接触双酚A；2)出生后5天；3)青春期；4)导管发育完成后。目的：验证子宫内暴露于双酚A改变大鼠乳腺组织结构并导致肿瘤前表型表达的假说。这一目的将评估暴露于低剂量BPA是否会导致大鼠乳腺的特定形态变化，从而可能导致致癌倾向，例如在注射NMU时末端芽(TEB)和末端管(TD)的数量增加，TEB在青春期后的持续，以及在几个年龄点出现肿瘤前病变。此外，还将检查目标2中所研究时间点的乳腺组织结构。目标1、目标2和目标3紧密相连，因为它们寻求在生物组织的不同层面上研究同一现象。基因表达的改变可能意味着组织结构的后果，反之亦然。这项探索性研究是关于产前激素暴露与乳腺癌发生之间因果关系的可检验假说的产生的核心，并可能最终提供环境暴露与乳腺癌发病率之间的直接联系，这是研究人员在过去20年里一直在寻找的。此外，如果像怀疑的那样，低剂量的双酚A会增加乳腺癌的发病率，这项工作可能会对我们研究风险因素和进行流行病学研究的方式产生很大影响。它甚至可能影响有关预防的公共政策。

DESCRIPTION (provided by applicant): Increased breast cancer incidence has prompted scientists to consider the possible role of hormonally active environmental chemicals. However, epidemiological studies searching for correlations between adult environmental exposures and breast cancer incidence have been mainly inconclusive. Yet, epidemiological studies do suggest that fetal estrogen level fluctuations have long-term consequences on the risk of developing breast cancer as an adult. Studies in mice reveal that prenatal exposure to low doses of the xenoestrogen bisphenol A (BPA) alters the development of these mammary glands. These effects manifested long after exposure ceased. The increased number of terminal end buds and terminal ducts in these mammary glands is particularly relevant since carcinomas originate in these structures. We hypothesize that prenatal exposure to low doses of BPA may increase the risk of mammary cancer and that the mechanism responsible for the neoplastic outcome may include the extemporaneous expression of genes that mediate mammary gland development. Xenoestrogens administered prenatally may alter the expression of estrogen-responsive genes that would then affect downstream genes in the mammary development program and predispose these animals to cancer. To test this hypothesis we will use a rat model whereby exposure to the carcinogen nitroso-methyl-urea (NMU) at puberty induces mammary cancer. A pilot study performed using Wistar rats showed that this model could be modified to assess the effect of fetal exposure to environmentally relevant xenoestrogen levels. It produced data consistent with the proposed hypothesis. Aim 1: To determine the levels at which prenatal BPA exposure results in an increased incidence of mammary cancer. Tumor incidence and latency will be measured in Wistar rats exposed to BPA from gestational day 9 to birth. At 50 days of age, animals will be injected with a sub-carcinogenic dose of NMU. Aim 2: To test the hypothesis that prenatal BPA exposure alters gene expression in the mammary gland during both the period of BPA exposure and throughout life. Total RNA will be isolated from mammary glands and analyzed by DNA microarray technology. Where appropriate, laser-capture microdissection and/or RNA linear amplification techniques will also be used to obtain samples. Up- and down-regulated genes will be confirmed by QRTPCR. Cellular distribution will be assessed by in situ hybridization. Developmental points to be analyzed are: 1) during gestational BPA exposure; 2) 5 days postnatal; 3) peripubertal; and 4) after ductal development is completed. Aim 3: To test the hypothesis that in utero exposure to BPA alters the histoarchitecture of the rat mammary gland and results in the expression of pre-neoplastic phenotypes. This Aim will assess whether exposure to low doses of BPA results in specific morphological alterations in the rat mammary gland that may confer a propensity to carcinogenesis, such as an increase in the number of terminal end buds (TEBs) and terminal ducts (TDs) at the time of NMU administration), the persistence of TEBs beyond puberty, and the appearance of pre-neoplastic lesions at several age points. In addition, the histoarchitecture of the mammary gland at the time-points studied in Aim 2 will be examined. Aims 1, 2 and 3 are intimately linked, because they seek to study the same phenomenon at different levels of biological organization. Gene expression alterations may suggest histoarchitectural consequences and vice-versa. This exploratory research is central to the generation of testable hypotheses about cause-effect relationships linking prenatal hormonal exposure and mammary gland carcinogenesis and may finally provide the direct link between environmental exposures and incidence of breast cancer researchers have been looking for over the past 20 years. Moreover, if, as suspected, low doses of BPA increase the incidence of mammary cancer, this work may have a great impact on the way we study risk factors and conduct epidemiological studies. It may even influence public policy about prevention.