Benzyl butyl phthalate promotes ferroptosis in Sertoli cells via disrupting ceruloplasmin-mediated iron balance

Abstract

Widespread environmental contamination with benzyl butyl phthalate (BBP) has raised concerns due to its high potential for bioaccumulation and male reproductive toxicity. However, the mechanisms underlying BBP-induced male reproductive damage remain unclear. As the adjacent Sertoli cell-formed blood-testis barrier (BTB) creates a privileged niche for spermatogenesis and may serve as the first target of reproductive toxicants, we mainly focused on the detrimental effect of BBP on Sertoli cells and the BTB in this study. C57BL/6 mice were administered BBP via oral gavage at doses ranging from 0 to 400 mg/kg/day for 60 consecutive days. A comprehensive investigation was performed to estimate testicular BBP levels, sperm parameters, histological alterations, functional permeability of the Sertoli cell–based BTB, and ferroptosis in mice. Isolated Sertoli cells were further used to explore and validate the role of ferroptosis in BBP-induced BTB disruption. The results showed that permeation of BBP into the testis induced reduction in sperm quantity and quality, accompanied by fractured BTB ultrastructure, compromised permeable ‘fence’ functions of BTB, decreased expressions of tight junction proteins (TJP1 and OCLN) and paracellular transepithelial electrical resistance (TER) of Sertoli cells. Moreover, BBP exposure significantly increased intracellular iron content, promoted lipid peroxidation, and activated ferroptosis in the testis of mice and primary Sertoli cells, which was involved in BBP-induced disruption of BTB integrity and function as confirmed by the ferroptosis inhibitors. In mechanism, BBP specifically downregulated the intracellular iron exporter ceruloplasmin (CP) level to inhibit Fe²⁺ export and the oxidization of Fe²⁺ into less toxic Fe³⁺, thus exacerbating ferroptosis in Sertoli cells. Overexpression of CP significantly suppressed ferroptosis and alleviated BBP-induced BTB disruption. These findings reveal the role of CP-mediated iron homeostasis in regulating Sertoli cell ferroptosis and BTB function, providing new insights into the mechanisms of BBP-related reproductive toxicity.