Effect of pubertal BPA exposure on mammary stem cells

Perinatal exposure to bisphenol A (BPA) has been shown to cause aberrant mammary gland morphogenesis and mammary neoplastic transformation. Yet, the underlying mechanism is poorly understood. We tested the hypothesis that mammary glands exposed to BPA during a susceptible window may lead to its susceptibility to tumorigenesis through a stem-cell mediated mechanism. We exposed 21-day-old Balb/c mice to BPA by gavage (25 µg/kg/day) during puberty for 3 weeks, and a subset of animals were further challenged with one oral dose (30 mg/kg) of 7,12-dimethylbenz[a]anthracene (DMBA) at 2 months of age. Primary mammary cells were isolated at 6 weeks, and 2 and 4 months of age for mammary stem cell (MaSC) quantification and function analysis. Pubertal exposure to the low-dose BPA increased lateral branches and hyperplasia in adult mammary glands and caused an acute increase of MaSC in 6-week-old glands and a delayed increase of luminal progenitors in 4-month-old adult gland. Most importantly, pubertal BPA exposure altered the function of MaSC from different age groups, causing pre-neoplastic lesions in their regenerated glands similar to those induced by DMBA exposure, which indicates that MaSCs are susceptible to BPA-induced transformation. Deep sequencing analysis on MaSC-enriched mammospheres identified a set of aberrantly expressed genes associated with pre-neoplastic lesions in human breast cancer patients. Thus, our study for the first time shows that pubertal BPA exposure altered MaSC gene expression and function such that they induced early neoplastic transformation. Overall design: Four samples were analyzed (control, BPA, DMBA, BPA+DMBA), and for each treatment group, mammospheres from 5 animals were pooled for RNA extraction.

围产期暴露于双酚A（BPA）已被证实可引发异常乳腺形态发生与乳腺肿瘤性转化，但其潜在机制仍尚未明确。本研究验证了如下假说：在易感窗口期暴露于BPA的乳腺，可通过干细胞介导的机制增加其肿瘤发生易感性。我们于青春期对21日龄Balb/c小鼠进行为期3周的灌胃暴露，BPA剂量为25微克/千克/天；并于小鼠2月龄时，对其中一部分动物施加单次口服剂量30毫克/千克的7,12-二甲基苯并[a]蒽（DMBA）攻毒处理。分别于6周龄、2月龄及4月龄时分离原代乳腺细胞，用于乳腺干细胞（MaSC）的定量与功能分析。青春期暴露于低剂量BPA可提升成年小鼠乳腺的侧支分支与增生程度，在6周龄小鼠乳腺中引发MaSC的急性升高，并在4月龄成年小鼠乳腺中导致腔祖细胞的延迟升高。最为关键的是，青春期BPA暴露改变了不同年龄组MaSC的功能，使其再生腺体出现与DMBA暴露诱导相似的癌前病变，这表明MaSC易受BPA诱导的转化影响。对富集MaSC的乳腺球进行深度测序分析，鉴定出一组与人类乳腺癌患者癌前病变相关的异常表达基因。因此，本研究首次证实：青春期BPA暴露可改变MaSC的基因表达与功能，进而诱导早期肿瘤性转化。实验整体设计：共设置4组样本（对照组、BPA暴露组、DMBA暴露组、BPA+DMBA联合暴露组）；每个处理组混合5只动物的乳腺球用于RNA提取。