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

Toxicol Sci. 2020 Feb 1;173(2):313-335. doi: 10.1093/toxsci/kfz233.


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.

Keywords: in vitro co-culture model; bisphenol A; bisphenol AF; bisphenol S; high-content image; machine learning; single-cell analysis; testicular toxicity; tetrabromobisphenol A.

MeSH terms

  • Animals
  • Benzhydryl Compounds / toxicity*
  • Cell Culture Techniques
  • Cell Survival / drug effects
  • Cells, Cultured
  • Coculture Techniques
  • DNA Damage / drug effects
  • Machine Learning
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Phenols / toxicity*
  • Phenotype
  • Polybrominated Biphenyls / toxicity*
  • Single-Cell Analysis*
  • Spermatogonia / drug effects
  • Sulfones / toxicity*
  • Testis / drug effects*
  • Testis / ultrastructure*


  • Benzhydryl Compounds
  • Phenols
  • Polybrominated Biphenyls
  • Sulfones
  • bis(4-hydroxyphenyl)sulfone
  • tetrabromobisphenol A
  • bisphenol A
  • 4,4'-hexafluorisopropylidene diphenol