Deciphering the mechanisms of Cupferron reproductive toxicity: insights from in vitro assays, network toxicology, and molecular docking

Cupferron, widely used in industrial and analytical contexts, has been proposed as a potential nitric oxide (NO) donor; however, its effects on the male reproductive system remain unclear. We assessed toxicity in TM3 (Leydig) and TM4 (Sertoli) mouse cells. Cytotoxicity (6 to 0.0035 mg/ml) was measured by MTT/NRU; genotoxicity by comet assay; oxidative stress markers (MDA, 8-OHdG, GSH, SOD) and testosterone by ELISA; cell death and ROS by flow cytometry; and gene expression by RT-qPCR. MTT IC50 values were 0.131 mg/ml (TM3) and 0.219 mg/ml (TM4). At 0.125 mg/ml, comet assay revealed markedly increased DNA damage, ≥ 16-fold (P ≤ 0.05) in both TM3 and TM4 cells. In TM4, MDA and 8-OHdG rose ≥ 1.3-fold, while SOD activity increased in both TM3 (1.2-fold) and TM4 (1.5-fold) cells (P ≤ 0.05). Annexin V/PI analysis indicated increased necrosis without significant changes in apoptosis. Testosterone levels were unaffected at all doses. RT-qPCR showed upregulation of SOD1, HMOX1, GSTA1, GPX1 antioxidant genes in both TM3 and TM4 (P ≤ 0.05). Network toxicology highlighted NOS1, NOS3, and PTGS2 as putative targets, supported by docking indicating high affinity and substrate-like poses, implicating modulation of oxidative/inflammatory pathways. ADMETLab 3.0 predicted genotoxic, hepatotoxic, and carcinogenic risks. Overall, Cupferron induces oxidative stress, DNA damage, necrosis, and antioxidant gene activation in Leydig and Sertoli cells, supporting potential male reproductive toxicity and the need for comprehensive in vivo and mechanistic in vitro studies.