In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals. In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals

Since initial regulatory action in 2010 in Canada, bisphenol A (BPA) has been progressively replaced by structurally related alternative chemicals. Unfortunately, many of these chemicals are data-poor, limiting toxicological risk assessment. We used high-throughput transcriptomics to evaluate potential hazards and compare potencies of BPA and 15 BPA alternative chemicals in cultured breast cancer cells. MCF-7 cells were exposed to BPA and 15 alternative chemicals (0.0005 – 100 µM) for 48 hrs. TempO-Seq sequencing (BioSpyder Inc.) was used to examine general toxicological effects and estrogen receptor alpha (ERα)-associated transcriptional changes. Benchmark concentration (BMC) analysis was conducted to identify two global transcriptomic points of departure (tPODs): (a) the lowest pathway median gene BMC and (b) the 25th lowest rank-ordered gene BMC. ERα activation was evaluated using a published transcriptomic biomarker and an ERα-specific tPOD was derived. Genes fitting BMC models were subjected to upstream regulator and canonical pathway analysis in Ingenuity Pathway Analysis. Biomarker analysis identified BPA and eight alternative chemicals as ERα active. Global and ERα tPODs produced highly similar potency rankings with BPAF as the most potent chemical tested, followed by BPA, BPC, 4,4’-BPF, BPAP, BPS, BADGE, 2,4’-BPF, Pergafast201®, D-8, 2,4’-BPS, TGSA, and BPS-MAE. Further, BPA and transcriptionally active alternative chemicals enriched similar gene sets associated with increased cell division and cancer-related processes. These data provide support for future read-across applications of transcriptomic profiling for risk assessment of data-poor chemicals and suggest that several BPA alternative chemicals may cause hazards at similar concentrations to BPA. Overall design: A total of 768 samples were sequenced for this toxicogenomics study on 16 bisphenol or bisphenol alternative chemicals, including technical controls (reference RNA; media only; cells with no treatment). The solvent controls were comprised of 0.1% DMSO. 10 concentrations of each bisphenol chemical were tested in quaduplicate, with a blocking design to avoid confounding across batches. Two reference chemicals, estradiol and dexamethasone, were also included in this study.

自加拿大2010年首次出台监管举措以来，双酚A（bisphenol A, BPA）正逐步被结构相关的替代化学品所取代。遗憾的是，其中多数替代化学品存在数据匮乏的问题，极大限制了毒理学风险评估的开展。
本研究采用高通量转录组学技术，在体外培养的乳腺癌细胞中评估双酚A及其15种替代化学品的潜在危害，并比较各化学品的毒理学效力。将MCF-7细胞置于双酚A及15种替代化学品（浓度区间0.0005–100 µM）中孵育48小时。采用TempO-Seq测序技术（BioSpyder公司）检测通用毒理学效应及雌激素受体α（estrogen receptor alpha, ERα）相关的转录组变化。
开展基准浓度（benchmark concentration, BMC）分析，以识别两类全局转录组效应起点（transcriptomic points of departure, tPODs）：(a) 最低通路中位基因基准浓度；(b) 按秩排列的第25位最低基因基准浓度。采用已发表的转录组学生物标志物评估雌激素受体α的激活活性，并推导得到雌激素受体α特异性的转录组效应起点。将符合基准浓度模型的基因导入Ingenuity通路分析（Ingenuity Pathway Analysis, IPA），开展上游调控因子及经典通路富集分析。
生物标志物分析结果显示，双酚A及8种替代化学品呈现雌激素受体α激活活性。全局转录组效应起点与雌激素受体α特异性转录组效应起点得到的效力排序高度一致，其中双酚AF（BPAF）为本次测试中效力最强的化学品，依次为双酚A、BPC、4,4’-双酚F（4,4’-BPF）、BPAP、双酚S（BPS）、BADGE、2,4’-双酚F（2,4’-BPF）、Pergafast201®、D-8、2,4’-双酚S（2,4’-BPS）、TGSA以及双酚S-MAE（BPS-MAE）。进一步分析显示，双酚A及具有转录激活活性的替代化学品，可富集得到与细胞增殖增强及癌症相关进程高度相似的基因集。
本研究数据为后续采用转录组谱分析开展数据匮乏化学品风险评估的交叉参照应用提供了理论依据，并提示多款双酚A替代化学品可能在与双酚A相近的暴露浓度下引发毒理学危害。
整体实验设计：本毒理基因组学研究共对16种双酚类或双酚类替代化学品的768个样本开展测序，其中包括技术对照（参考RNA、仅培养基、未处理细胞）。溶剂对照为0.1%二甲基亚砜（dimethyl sulfoxide, DMSO）。每种双酚类化学品设置10个浓度梯度，每个浓度设置4个复孔，并采用区组设计以避免批次间混杂效应。本研究同时纳入两种参考化学品：雌二醇与地塞米松。