Assessing the Impact of Metabolic Competence on Estrogenic Chemical Bioactivity in High-Throughput Profiling Assays
High-throughput profiling assays such as high-throughput phenotypic profiling (HTPP) with Cell Painting and high-throughput transcriptomics (HTTr) with TempO-SeqTM are new approach methods that have been used to characterize the bioactivity and potential hazards of chemicals. To better account for potential in vivo hazards in screening, we previously coupled both assays to an in vitro metabolism platform, Alginate Immobilization of Metabolic Enzymes (AIME). In this study, we expanded upon our previous results in estrogenic reference chemicals by screening an additional 40 chemicals in VM7Luc4E2 breast cells anticipated to have varied activity and/or shifts in activity with metabolism in an estrogen receptor transactivation assay (ERTA) using the AIME platform. Our results demonstrate how HTTr could detect ER activity and the influence of metabolism on estrogenicity through the activation of ER high-confidence (ERHC) gene signatures and metabolism-induced shifts in ERHC signature enrichment. Additionally, HTPP could detect cases where metabolism impacted chemical cytotoxicity and detect generation of metabolites that were bioactive in a different way than their respective parent compounds when comparing the profiles of active features between AIME conditions. Notably, our findings highlighted examples of chemicals that had very different phenotypic and gene expression profiles between metabolic conditions that would not be observed in traditional chemical screening in most cell models. This study exhibited how high-throughput profiling assays could distinguish changes in bioactivity with metabolic competence using the AIME platform, which will ultimately better inform next generation risk assessment by providing more comprehensive hazard characterizations.