摘要：Background:
Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses.
Objective:
We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen
receptor-
α
(ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner.
Methods:
We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with
∼
100
toxicants, over a large number of biological and technical replicates.
Results:
We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence
in situ hybridization (RNA FISH).
Discussion:
We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants.
https://doi.org/10.1289/EHP9297
