Integrated network toxicology, transcriptomics and metabolomics to elucidate mechanism of di(2-ethylhexyl) phthalate inducing reproductive toxicity in laying hens.

Di(2-ethylhexyl) phthalate (DEHP) is ubiquitous in agricultural ecosystems due to the widespread use of plastic mulch films and plasticized agrochemicals, posing a significant threat to poultry health. This study investigates DEHP's reproductive toxicity and molecular mechanisms in laying hens. Hy-Line brown hens were divided into control and 150 mg/kg DEHP-exposed groups for 15 weeks. Production performance, egg quality and ovary oviduct histomorphology were assessed combined with transcriptomics, metabolomics, network toxicology and molecular docking. The results showed that DEHP reduced feed intake, altered yolk color, thinned eggshells and induced ovarian follicular atresia and oviduct uterine epithelial atrophy. Integrated omics showed disrupted ovarian pathways like neuroactive ligand-receptor interactions, glutathione and glycerophospholipid metabolism plus key metabolite-gene pairs such as CMPK2 and aspartic acid. Uterine transcriptomics revealed abnormal extracellular matrix, ion transport and eggshell mineralization genes. Network toxicology identified 10 core targets including Bcl2, Casp3 and PIK3CA with DEHP metabolite mono(2-ethylhexyl) phthalate (MEHP) stably binding them via docking. Integrated analysis of multi-omics and network toxicology, along with western blot results, further confirmed that the apoptosis and lysosomal pathways were activated in the ovaries and oviduct uterus of laying hens. Protein-protein interaction analysis positioned Bcl2 as a potential regulator linking these pathways. In conclusion, DEHP exposure is associated with reproductive toxicity in laying hens. Future studies should employ multi-dose designs and validate multi-omics-derived mechanisms with targeted functional experiments to strengthen causal inference.