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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\u2009\u00b5M was cytotoxic and resulted in 40% cell death of the C18-4 cells after 72\u2009h. HCA revealed that 5\u2009\u00b5M 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.<\/jats:p>","DOI":"10.1093\/toxsci\/kfad005","type":"journal-article","created":{"date-parts":[[2023,1,10]],"date-time":"2023-01-10T13:36:45Z","timestamp":1673357805000},"page":"43-58","source":"Crossref","is-referenced-by-count":6,"title":["The role of small GTPases in bisphenol AF-induced multinucleation in comparison with dibutyl phthalate in the male germ cells"],"prefix":"10.1093","volume":"192","author":[{"given":"Chelin","family":"Hu","sequence":"first","affiliation":[{"name":"University of New Mexico , Albuquerque, New Mexico 87131, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zoey Hsuan","family":"Hsiao","sequence":"additional","affiliation":[{"name":"ReproTox Biotech LLC , Albuquerque, New Mexico 87131, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lei","family":"Yin","sequence":"additional","affiliation":[{"name":"ReproTox Biotech LLC , Albuquerque, New Mexico 87131, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5523-9583","authenticated-orcid":false,"given":"Xiaozhong","family":"Yu","sequence":"additional","affiliation":[{"name":"University of New Mexico , Albuquerque, New Mexico 87131, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2023,1,10]]},"reference":[{"key":"2023032007570798600_","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1071\/RD20150","article-title":"Di-n-butyl phthalate diminishes testicular steroidogenesis by blocking the hypothalamic-pituitary-testicular axis: Relationship with germ cell apoptosis in Japanese quail","volume":"33","author":"Alam","year":"2021","journal-title":"Reprod. 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