High-Content Image-Based Single-Cell Phenotypic Analysis for the Testicular Toxicity Prediction Induced by Bisphenol A and Its Analogs Bisphenol S, Bisphenol AF, and Tetrabromobisphenol A in a Three-Dimensional Testicular Cell Co-culture Model

Abstract

Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest.

Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels

“…An intriguing observation was the higher proportion of cells showing signs of DNA damage ( Figure 1F–H ). Previous data from a testicular coculture model indicated that BPA had no effect on the proportion of cells with signs of DNA damage when cultured for up to 72 h and at BPA concentrations up to ( Yin et al. 2020 ).…”

“…2016 ; Sahu et al. 2020 ; Yin et al. 2020 ), we also examined the proportion of cells staining positive for , a marker of DNA damage ( Rogakou et al.…”

Image-based profiling for drug discovery: due for a machine-learning upgrade?

“…Image-based profiling identified rosmarinic acid as a candidate with cardioprotective effects against the toxicity of doxorubicin 91 . This approach also revealed the toxic effects of bisphenol A and its analogues on a testicular cell co-culture model by simultaneously measuring multiple adverse end points such as changes to nuclear morphology and cytoskeletal structure 92 .…”

Genotoxic Mechanism of Action of TBBPA, TBBPS and Selected Bromophenols in Human Peripheral Blood Mononuclear Cells

“…Yin et al. ( 24 ) determined an early DNA damage response marker γ-H2AX to assess the genotoxicity of TBBPA in mouse testicular cell co-culture model. They observed that after 24 h of incubation, TBBPA at 15 µM (8.1 µg/mL) caused significant increase of the number of γ-H2AX positive cells.…”

“…For instance, earlier studies have shown no genotoxic effects of TBBPA that could have been associated with the use of very low doses of this compound ( 19 – 21 ). However, more recent studies have indicated genotoxic potential of TBBPA in spermatozoa of mice ( 22 ), blood cells of spotted snake ( Channa punctatus ) ( 23 ) and mouse testicular cell co-culture model ( 24 ). 2,4,6-TBP was not genotoxic in in vitro bacterial tests ( 25 , 26 ); however, it caused chromosomal aberrations (with and without metabolic activation) in in vitro tests on Chinese hamster cells ( 27 ).…”