Prenatal xenoestrogen exposure and mammary cancer

描述（由申请人提供）：乳腺癌发病率的增加促使科学家考虑激素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相关性的流行病学研究基本上没有结论。然而，流行病学研究确实表明，胎儿雌激素水平波动会对成年后患乳腺癌的风险产生长期影响。对小鼠的研究表明，产前接触低剂量的异雌激素双酚 A (BPA) 会改变这些乳腺的发育。这些影响在停止接触后很长时间才显现出来。这些乳腺中末端芽和末端导管数量的增加尤其重要，因为癌起源于这些结构。我们假设产前接触低剂量的 BPA 可能会增加患乳腺癌的风险，并且导致肿瘤结果的机制可能包括介导乳腺发育的基因的临时表达。产前施用的异种雌激素可能会改变雌激素反应基因的表达，从而影响乳腺发育程序中的下游基因，并使这些动物易患癌症。为了验证这一假设，我们将使用大鼠模型，在青春期接触致癌物质亚硝基甲基脲（NMU）会诱发乳腺癌。使用 Wistar 大鼠进行的一项试点研究表明，可以修改该模型来评估胎儿暴露于环境相关异雌激素水平的影响。它产生的数据与所提出的假设一致。目标 1：确定产前 BPA 暴露导致乳腺癌发病率增加的水平。将测量从妊娠第 9 天到出生期间暴露于 BPA 的 Wistar 大鼠的肿瘤发生率和潜伏期。 50 天龄时，动物将被注射亚致癌剂量的 NMU。目标 2：检验以下假设：产前 BPA 暴露会改变 BPA 暴露期间和一生中乳腺的基因表达。将从乳腺中分离出总 RNA，并通过 DNA 微阵列技术进行分析。在适当的情况下，激光捕获显微切割和/或 RNA 线性扩增技术也将用于获取样本。上调和下调基因将通过 QRTPCR 进行确认。将通过原位杂交评估细胞分布。需要分析的发育点是： 1) 妊娠期 BPA 暴露期间； 2）产后5天； 3）青春期前后； 4) 导管发育完成后。目标 3：检验子宫内接触 BPA 会改变大鼠乳腺的组织结构并导致肿瘤前表型表达的假设。该目标将评估暴露于低剂量的 BPA 是否会导致大鼠乳腺发生特定的形态变化，从而可能赋予致癌倾向，例如 NMU 给药时末端芽 (TEB) 和末端导管 (TD) 数量的增加、TEB 在青春期后的持续存在以及在几个年龄点出现肿瘤前病变。此外，还将检查目标 2 中研究的时间点的乳腺组织结构。目标 1、2 和 3 密切相关，因为它们寻求在生物组织的不同层次上研究相同的现象。基因表达的改变可能表明组织结构的后果，反之亦然。这项探索性研究对于生成有关产前激素暴露与乳腺癌发生之间因果关系的可检验假设至关重要，并可能最终提供研究人员过去 20 年来一直在寻找的环境暴露与乳腺癌发病率之间的直接联系。此外，如果正如所怀疑的那样，低剂量的 BPA 会增加乳腺癌的发病率，这项工作可能会对我们研究危险因素和进行流行病学研究的方式产生巨大影响。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.