High-throughput transcriptomics screening of mixtures of BPA alternatives reveal additive effects in vitro

Humans are regularly exposed to mixtures of BPA alternative chemicals through their diets, consumer products and other sources. Like BPA, many alternatives perturb nuclear hormone receptors and are considered endocrine disrupting chemicals. We used high-throughput transcriptomics (HTTr) to evaluate the potency and potential modes of action of seven mixtures of BPA alternatives, along with their 12 individual components, in MCF-7 breast cancer cells. Our primary aim was to explore whether alternatives present in mixtures act additively. MCF-7 cells were exposed to chemicals (0.001 – 50 µM) for 48 h in serum-free conditions and TempO-Seq (BioSypder Inc.) was used to determine global and estrogen receptor alpha (ERa)-specific transcriptomic changes. Transcriptomic points of departure were derived using benchmark concentration (BMC) modelling. We first identified the concentration at which global transcriptional activity was robustly altered. Then, a novel ERa transcriptomic biomarker was used to identify ERa agonists and model a predicted ERa activation point of departure. Mixtures modelling was employed to predict potency of BPA alternatives and test for additive effects in vitro. Ingenuity Pathway Analysis (IPA; Qiagen) was used to identify upstream regulators and canonical pathways from genes fitting BMCs. All seven mixtures had additive effects across all transcriptomic points of departure modelled. BPAF was the most potent individual chemical tested overall, followed by BPA and BPC. The ERa transcriptomic biomarker classified all mixtures as ERa activators along with several individual BPA alternatives. Pathway analysis revealed that all mixtures and most individual components perturbed similar upstream regulators and pathways, suggesting common modes of action. These data support the use of HTTr as part of new approach methodologies to assess the toxicological potency of mixtures in vitro. Overall design: MCF-7 cells were exposed (0.001, 0.01, 0.1, 0.5, 1, 5, 10, 50 uM; 48hrs) to 7 different mixtures of BPA alternative chemicals and 12 individual chemicals (BPA, BPAF, 4,4'-BPF, BPC, BPS, P201, BPAP, 2,4'-BPS, 2,4'-BPF, BPB, BPZ, TMBPF), along with controls (solvent: 0.1% DMSO; E2: 0.1 nM, 1 nM). Mixtures 1 – 6 were equimolar: mix 1 (BPA + BPAF), mix 2 (mix 1 + BPC), mix 3 (mix 2 + 4,4'-BPF), mix 4 (mix 3 + BPAP), mix 5 (mix 4 + BPS), mix 6 (mix 5 + 2,4'-BPF + P201 + 2,4'-BPS). Mixture 7 (BPAF + 4,4'-BPF + BPA + BPB + BPZ + BPS + BPAP) was non equimolar and had a different exposure concentration range (0.66, 1.3, 2.6, 5.2, 10.5, 21 uM). All exposures were for 48 hours in serum-free conditions (DMEM + 2% BSA + 1% P/S).