RU-521

Trihexyl phosphate (THP), a widely detected organophosphate flame retardant, poses emerging concerns due to its potential male reproductive toxicity, though mechanistic insights remain limited. Here, we delineate a downstream cascade linking THP exposure to testosterone suppression through mitochondrial dysfunction and cytosolic DNA-sensing pathways in adult Leydig cells (LCs). In vivo, oral THP exposure (0, 12.5, 25, 50 mg/kg/day, 14 days) in adult male C57BL/6 mice reduced serum testosterone without altering LH/FSH levels, accompanied by downregulated steroidogenic genes/proteins (NR5A1, SCARB1, STAR) at low-medium doses and diminished CYP11A1 + LC populations at the highest dose. In vitro, THP impaired MA-10 LC viability and steroidogenesis. Mechanistically, THP disrupted PINK1/Parkin-mediated mitophagy, increased mitochondrial ROS, and triggered mtDNA leakage, thereby activating the cGAS-STING-TBK1-IRF3 axis. This was accompanied by enhanced NLRP3 inflammasome signaling and pyroptotic responses. Pharmacological and genetic interventions supported pathway dependence: urolithin A restored mitochondrial homeostasis and attenuated cGAS-STING activation, while RU.521 and STING knockdown preserved testosterone output by suppressing NLRP3-associated signaling. Structural and transcriptomic analyses further supported STING-NLRP3 association and linked elevated cGAS-STING activity with mitophagy suppression and pyroptosis in LCs. Our findings provide evidence that THP disrupts testosterone synthesis via a mitophagy-mtDNA-cGAS-STING-pyroptosis axis, highlighting mitophagy enhancement and cGAS-STING inhibition as potential intervention strategies. The primary chemical initiating event and direct molecular target(s) of THP remain to be defined.

Vitiligo is a chronic depigmentary disorder initiated by oxidative stress, which activates inflammatory signaling and innate immunity, ultimately leading to melanocyte destruction. Although melanocyte defects have been widely studied, dermal fibroblasts-the predominant stromal regulators of cutaneous immunity-remain insufficiently characterized in vitiligo pathogenesis. Here, we demonstrate that subtoxic oxidative stress in normal human dermal fibroblasts (NHDFs) induces a VDAC1-dependent, non-apoptotic release of mitochondrial DNA (mtDNA), thereby linking redox imbalance to immune activation. Low-dose hydrogen peroxide preserved mitochondrial morphology while promoting VDAC1 oligomerization, forming pores that enabled selective mtDNA efflux from structurally intact mitochondria. The released mtDNA activated the cGAS-STING pathway and the NLRP3 inflammasome, driving the expression of IL-1β, IL-6, ICAM-1, and Occludin-a pattern consistent with a senescence-associated secretory phenotype. Pharmacological interventions using ethidium bromide, RU.521, VBIT-4, and exogenous mtDNA established mtDNA release as an upstream event in fibroblast innate immune activation. Notably, inhibiting VDAC1 oligomerization with VBIT-4 effectively prevented mtDNA leakage, attenuated fibroblast senescence and inflammatory signaling, and restored epidermal repigmentation in a vitiligo mouse model. These findings identify dermal fibroblasts as active sensors and amplifiers of oxidative stress via the VDAC1-mtDNA-cGAS-STING axis and highlight VDAC1 oligomerization as a promising therapeutic target.