Tetramethyl bisphenol a inhibits leydig cell function in late puberty by inducing ferroptosis

Tetramethyl bisphenol A (TMBPA) is a commonly used bisphenol analog, used as a fire retardant. However, whether it inhibits the function of Leydig cells in late puberty remains unclear. In this study, 35-day-old male Sprague-Dawley rats were gavaged with 0, 10, 100, and 200 mg/kg body weight TMBPA for 21 days. TMBPA significantly reduced serum testosterone levels at 10 mg/kg and higher doses without altering serum luteinizing hormone and follicle-stimulating hormone levels. TMBPA significantly increased serum iron concentraion while reducing the ratio of serum glutathione (GSH) and GSH/GSSG (oxidized glutathione disulfide). In addition, TMBPA significantly increased testicular iron amount at 10 mg/kg and higher doses and malondialdehyde level at 200 mg/kg. TMBPA down-regulated the expression of Leydig cell genes, including Nr5a1, Star, Scarb1, Insl3, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd11b1, and their proteins. In addition, TMBPA markedly down-regulated the expression of genes in the ferroptosis pathway (Tp53, Slc7a11, Sod1, Sod2, Cat, Sqstm1, Keap1, and Hmox1). TMBPA significantly reduced the levels of ferroptosis pathway proteins (TP53, SLC7A11, GPX4, SQSTM1, KEAP1, NRF2, and HMOX1) in Leydig cells in vivo. Immature and adult Leydig cell culture in vitro also showed that TMBPA significantly reduced testosterone concentrations in the medium, which can be reversed by a ferroptosis inhibitor. After 24 h of culture in primary Leydig cells at 10 and 50 μM, TMBPA significantly induced reactive oxygen species and lowered the mitochondrial membrane potential. TMBPA also altered protein levels in the ferroptosis pathway in Leydig cells in vitro. In conclusion, TMBPA directly inhibits the activity of rat Leydig cell steroidogenic enzymes and induces the ferroptosis of Leydig cells, thereby inhibiting the testosterone synthesis of Leydig cells in the late puberty. [Display omitted] TMBPA downregulates Leydig cell gene expression and proteins, leading to lower level of testosterone (T) resulting from its induction of ferroptosis by ROS generation via TP53-SLC7A11-GPX4 and SQSTM1-KEAP1-NRF2-HMOX1 pathway. •TMBPA inhibits testosterone biosynthesis in the late puberty after in vivo exposure.•TMBPA induces ferroptosis via GPX4 pathway after in vivo exposure.•TMBPA induces ROS production both in vivo and in vitro.•TMBPA inhibits testosterone production in primary Leydig cells.•TMBPA induces ferroptosis in primary Leydig cells.