Characterizing properties of non-estrogenic substituted bisphenol analogs using high throughput microscopy and image analysis

Abstract

Animal studies have linked the estrogenic properties of bisphenol A (BPA) to adverse effects on the endocrine system. Because of concerns for similar effects in humans, there is a desire to replace BPA in consumer products, and a search for BPA replacements that lack endocrine-disrupting bioactivity is ongoing. We used multiple cell-based models, including an established multi-parametric, high throughput microscopy-based platform that incorporates engineered HeLa cell lines with visible ERα- or ERβ-regulated transcription loci, to discriminate the estrogen-like and androgen-like properties of previously uncharacterized substituted bisphenol derivatives and hydroquinone. As expected, BPA induced 70–80% of the estrogen-like activity via ERα and ERβ compared to E2 in the HeLa prolactin array cell line. 2,2’ BPA, Bisguaiacol F, CHDM 4-hydroxybuyl acrylate, hydroquinone, and TM modified variants of BPF showed very limited estrogen-like or androgen-like activity (< 10% of that observed with the control compounds). Interestingly, TM-BFP and CHDM 4-hydroxybuyl acrylate, but not their derivatives, demonstrated evidence of anti-estrogenic and anti-androgenic activity. Our findings indicate that Bisguaiacol F, TM-BFP-ER and TM-BPF-DGE demonstrate low potential for affecting estrogenic or androgenic endocrine activity. This suggest that the tested compounds could be suitable commercially viable alternatives to BPA.

Classification of estrogenic compounds by coupling high content analysis and machine learning algorithms

“…This cell model allows for the direct simultaneous visualization of compound-dependent effects on several aspects of the ER signaling pathway, including ER expression, nuclear translocation, chromatin binding, and chromatin remodeling (Fig 7). Importantly, we have previously observed qualitative and quantitative differences in ER pathway signaling endpoints in the cell model-based compound activity class (Fig 7B and 7C) [5,[7][8][9][10]. 384 multiwell plates seeded with cells were treated for 2 hrs with a single concentration (10 uM) of 45 reference compounds provided by the EPA along with 3 control treatments.…”

“…High throughput microscopy and high content analysis-based experiments were performed using the GFP-ER⍺:PRL-HeLa cell line model following the experimental methodology described previously [ 5 , 7 – 10 ]. This cell model allows for the direct simultaneous visualization of compound-dependent effects on several aspects of the ER signaling pathway, including ER expression, nuclear translocation, chromatin binding, and chromatin remodeling ( Fig 7 ).…”

“…The estrogenic potential of different chemicals can be measured using cell-based or cellfree in vitro assays by recording several facets of the ER mechanism of action (i.e., ligand binding, cell proliferation, gene expression, etc.) [5][6][7]. Previously, a high content/high throughput microscopy-based assay in HeLa cells, engineered to harbor a visible multicopy integration of the ER response element present within the prolactin promoter/enhancer, was developed to capture several mechanistic steps of the ER pathway by imaging [5,[7][8][9][10].…”

“…e ., ligand binding, cell proliferation, gene expression, etc .) [ 5 – 7 ]. Previously, a high content/high throughput microscopy-based assay in HeLa cells, engineered to harbor a visible multicopy integration of the ER response element present within the prolactin promoter/enhancer, was developed to capture several mechanistic steps of the ER pathway by imaging [ 5 , 7 – 10 ].…”

“…[ 5 – 7 ]. Previously, a high content/high throughput microscopy-based assay in HeLa cells, engineered to harbor a visible multicopy integration of the ER response element present within the prolactin promoter/enhancer, was developed to capture several mechanistic steps of the ER pathway by imaging [ 5 , 7 – 10 ]. Coupled with stable expression of GFP-ER⍺, this high content analysis-based approach allows the direct visualization of ligand-induced DNA binding by ER as a visually distinct nuclear spot/array when using standard fluorescent microscopy.…”