Publication Date
2-1-2022
Journal
Environmental Health Perspectives
DOI
10.1289/EHP9297
PMID
35167326
PMCID
PMC8846386
PubMedCentral® Posted Date
2-15-2022
PubMedCentral® Full Text Version
Post-Print
Published Open-Access
yes
Keywords
Endocrine Disruptors, In Situ Hybridization, Fluorescence, Quality Control, Receptors, Estrogen, Single-Cell Analysis
Abstract
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
METHODS: We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with
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
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.
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