The role of small GTPases in bisphenol AF-induced multinucleation in comparison with dibutyl phthalate in the male germ cells

by Selleckchem | Jun 22 2023

The goal of this study is to examine bisphenol AF (BPAF)-induced multinucleation (MNC) in comparison with dibutyl phthalate (DBP), known to induce MNC in mouse gonocytes in vivo. We performed image-based single-cell high content analysis (HCA) in the mouse spermatogonia C18-4 cells treated with various concentrations of BPAF and DBP. BPAF as low as 5 µM was cytotoxic and resulted in 40% cell death of the C18-4 cells after 72 h. HCA revealed that 5 µM of BPAF significantly increased the number of MNC by an average of 3.6-fold. DBP did not induce MNC in the doses we tested. Cytokinesis is tightly regulated by various small GTPase-signaling pathways. We, therefore, tested 5 selective GTPase inhibitors and found that Y27632, a ROCK inhibitor, reduced the BPAF-induced MNC by nearly 30%. Inhibition of Cdc42 by ML141 conversely increased the number of BPAF-induced MNC. We performed a hierarchical cluster analysis of the HCA data and demonstrated that the cytoskeletal disruption by BPAF was reversely modified by Y27632. We found that mRNA expression of genes regulating Rho and Rac GTPase activities, p190RhoGap and MgcRacGap, was altered in BPAF-treated C18-4 cells in a time-dependent manner. Multinucleated gonocytes are often indicators of disease pathologies. Our results provided the first evidence of mechanisms of the dual toxicity by BPAF to male germ cells, which induces chromosome endoreplication without the coordinated cytokinetic cellular components. The unique genotoxic mechanism of forming multinucleated germ cells suggests a novel mode of action in the male repro-toxicity concern over the increasingly ubiquitous presence of BPA analogs.

Comments:

The aim of this study was to investigate the effects of bisphenol AF (BPAF) and dibutyl phthalate (DBP) on multinucleation (MNC) in mouse spermatogonia C18-4 cells. The researchers conducted image-based single-cell high content analysis (HCA) on C18-4 cells treated with different concentrations of BPAF and DBP.

The study found that BPAF at concentrations as low as 5 µM exhibited cytotoxicity, resulting in 40% cell death after 72 hours of treatment. HCA analysis revealed that 5 µM of BPAF significantly increased the number of MNC by an average of 3.6-fold. In contrast, DBP did not induce MNC under the tested doses.

Since cytokinesis, the process of cell division, is regulated by various small GTPase-signaling pathways, the researchers examined the effects of selective GTPase inhibitors on BPAF-induced MNC. They found that Y27632, an inhibitor of the ROCK pathway, reduced the number of BPAF-induced MNC by approximately 30%. Conversely, inhibition of Cdc42 by ML141 increased the number of BPAF-induced MNC.

To further analyze the HCA data, the researchers performed hierarchical cluster analysis and demonstrated that the cytoskeletal disruption caused by BPAF was reversed by Y27632 treatment. Additionally, they observed time-dependent alterations in the mRNA expression of genes involved in regulating Rho and Rac GTPase activities, namely p190RhoGap and MgcRacGap, in BPAF-treated C18-4 cells.

Multinucleated gonocytes, as observed in this study, are often associated with pathological conditions. The findings of this research provide the first evidence of the mechanisms underlying the dual toxicity of BPAF on male germ cells. BPAF induces chromosome endoreplication without proper cytokinetic cellular components, leading to the formation of multinucleated germ cells. This unique genotoxic mechanism suggests a novel mode of action contributing to the male reproductive toxicity associated with the widespread presence of BPA analogs.

In summary, this study highlights the cytotoxic effects of BPAF on mouse spermatogonia cells, its ability to induce multinucleation, and the involvement of small GTPase-signaling pathways in this process. The findings contribute to the understanding of the reproductive toxicity associated with BPA analogs and emphasize the need for further research in this area.