Tetrachlorobisphenol A inhibits steroidogenesis in pubertal rat Leydig cells through disruption of copper homeostasis.

Tetrachlorobisphenol A (TCBPA) is a widely used flame retardant with potential endocrine-disrupting properties. This study investigated the effects of TCBPA on steroidogenesis in pubertal rat Leydig cells, focusing on copper homeostasis and on whether the cuproptosis pathway is engaged. Male Sprague-Dawley rats (35 days old) were administered TCBPA (0, 10, 100, and 200 mg/kg/day) for 21 days. TCBPA exposure (100 and 200 mg/kg) significantly reduced serum and testicular testosterone levels without affecting testis weight or Leydig cell numbers. TCBPA downregulated steroidogenic genes (Lhcgr, Scarb1, Star, Cyp11a1, Insl3, and Hsd11b1) and their corresponding proteins, while increasing Leydig cell proliferation. TCBPA elevated testicular copper levels and altered expression of cuproptosis-related proteins, upregulating FDX1 and LIAS while downregulating ATP7B. In vitro studies confirmed that TCBPA induced reactive oxygen species generation, reduced mitochondrial membrane potential, and triggered copper accumulation in primary Leydig cells. Co-treatment with the copper chelator tetrathiomolybdate attenuated these changes, with a significant TCBPA by tetrathiomolybdate interaction for six of eight endpoints, indicating dependence on copper availability. At concentrations that did not affect cell viability, two molecular features of cuproptosis were tested directly: the iron-sulfur cluster protein SDHB was not significantly reduced, and DLAT oligomerisation was not detected, although it was readily produced by the positive control elesclomol-CuCl2. These findings indicate that TCBPA impairs steroidogenesis in rat Leydig cells through suppression of steroidogenic enzymes and copper-dependent mitochondrial dysfunction, without engaging cuproptosis, providing a basis for evaluating the reproductive risk of halogenated bisphenols.