Florasulam impairs Leydig cell function by blocking autophagic flux and triggering PERK-eIF2α-ATF4-CHOP-mediated ER...

Environmental herbicides are increasingly recognized as contributors to male reproductive decline, yet the cellular mechanisms underlying their toxicity remain poorly defined. Here we show that Florasulam, a widely used triazolopyrimidine sulfonanilide herbicide, directly disrupts Leydig cell homeostasis and steroidogenic function. Florasulam reduces TM3 Leydig cell viability and promotes apoptosis, accompanied by decreased expression of the anti-apoptotic protein Bcl-2 and the steroidogenic enzyme HSD3B2. Transcriptomic profiling reveals early suppression of autophagy-related pathways together with enrichment of endoplasmic reticulum (ER) stress signatures. Consistent with these findings, Florasulam inhibits autophagic flux, marked by p62 accumulation, reduced LC3-II levels, and diminished autophagosome formation, and simultaneously activates the PERK-eIF2α-ATF4-CHOP signaling cascade indicative of unresolved ER stress. Pharmacological restoration of autophagy with Rapamycin or Torin1 attenuates ER stress and significantly reduces apoptosis. These results identify a critical autophagy-ER stress axis underlying Florasulam-induced Leydig cell injury and provide mechanistic insight into the reproductive risks associated with herbicide exposure.