An integrated experimental and computational systems biology analysis reveals molecular mechanisms underlying reproductive toxicity induced by manganese and atrazine.

Atrazine (ATZ) and manganese (Mn) are individually known to influence reproductive health, but their combined impact on the male reproductive function remains unknown. Here, we investigate the combined reproductive effects of ATZ and Mn in male Wistar rats using biochemical analyses and an in silico approach. Rats (n = 6 per group), including control, ATZ (10 mg/kg), Mn (10 mg/kg), and combined low (2.5 + 2.5 mg/kg) and high dose (10 + 10 mg/kg), were treated for 28 days, respectively. Assessments include testicular function, reproductive hormones, sperm quality, oxidative and inflammatory response indices, and DNA fragmentation. An in silico approach to atrazine and manganese toxicities involved protein-protein interaction networks, KEGG pathway enrichment, and ADMET ATZ-profiling. Co-exposure significantly (p > 0.05) reduced biomarkers of testicular function and sperm quality, while increasing sperm abnormalities, oxidative stress, apoptosis, and pro-inflammatory cytokines. Computational analysis identified key hub proteins, including CASP3, TNF, TP53, IL6, NFE2L2, and HIF1A, as central mediators of the observed toxicity. Enrichment analyses highlighted activation of the TNF signalling and apoptosis pathways, which are associated with disruption of redox homeostasis and inflammatory responses. Docking analysis indicates that ATZ shows the strongest predicted interactions with NFE2L2, as supported by molecular dynamics simulations that establish its stability, suggesting that ATZ promotes oxidative stress and inflammation. In conclusion, combined exposure to ATZ and Mn induces reproductive toxicity via oxidative stress, inflammation, and apoptosis. A systems-level analysis identified critical molecular targets and signalling pathways, providing mechanistic insight into the synergistic effects of environmental toxicant-induced reproductive disturbances, particularly following co-exposure to ATZ and Mn in a rat model.