Reproductive toxicity potential of Fluazifop-butyl: In vitro, network toxicology, and molecular docking insights

Fluazifop-butyl is an aryloxyphenoxypropionate herbicide. Despite restrictions on its use in many countries, traces of its residues continue to be detected in the environment. Studies have shown that fluazifop-butyl can lead to reproductive and developmental toxicity in non-target species. This preliminary research explores the toxic effects of fluazifop-butyl exposure using an in vitro model, specifically Leydig (TM3) and Sertoli (TM4) cells, to better understand its potential effects on male reproductive health. The IC₅₀ of fluazifop-butyl was determined to be 0.940 mM for TM3 cells after 48 h of exposure, while the IC₅₀ for TM4 cells could not be established. Apoptosis/necrosis induction, reactive oxygen species (ROS) generation, reduced glutathione (GSH) levels, and genotoxicity were assessed in response to exposures ranging from 0 to 0.750 mM in both cell lines. Fluazifop-butyl resulted in higher ROS and lower GSH levels in TM3 cells, whereas in TM4 cells, ROS increased regardless of dose, and GSH levels increased in a dose-dependent manner. Apoptosis assessment revealed a dose-dependent increase in both early and late apoptotic cells in TM3, while higher concentrations led to a rise in late apoptosis in TM4 cells. Genotoxicity results showed more than a 4-fold increase in tail intensity in TM3 and more than a 5-fold increase in TM4 cells. Furthermore, a network toxicology and molecular docking approach was employed to investigate the reproductive toxicity of fluazifop-butyl, specifically its mechanism of testicular damage. Key targets identified include the androgen receptor, fibroblast growth factor receptor 3, and tumor necrosis factor-α, which mediate fluazifop-butyl's toxic effects.