BDE-47 and male infertility: integrated network toxicology, single-cell transcriptomics, and in vitro evidence of GSTM1 downregulation and ferroptosis-related molecular remodeling in Leydig cells.

BACKGROUND: BDE-47 is a widely detected polybrominated diphenyl ether with potential male reproductive toxicity, but the key testicular cell populations and molecular basis remain unclear.

METHODS: We integrated network toxicology, single-cell transcriptomic data from BDE-47-exposed mouse testes and human idiopathic non-obstructive azoospermia (NOA), and bulk transcriptomic data. High-dimensional weighted gene co-expression network analysis, exploratory metacell-based feature prioritization with SHapley Additive exPlanations analysis, ferroptosis-driver gene signature scoring, exploratory immune deconvolution, molecular docking, 100-ns molecular dynamics simulation, and TM3 cell experiments were performed.

RESULTS: We identified 113 shared candidate genes derived from BDE-47-related and NOA-associated gene sets, mainly enriched in xenobiotic metabolism, glutathione metabolism, oxidative stress, ferroptosis, and reproductive endocrine pathways. Mouse and human single-cell analyses localized the computational candidate signature to Leydig cells as one of the enriched somatic cell populations. GSTM1 was selected as an exploratory candidate based on multialgorithm screening, relative SHAP contribution, expression consistency across evidence layers, and redox-detoxification biological plausibility; it was expressed at lower levels in human NOA Leydig cells and bulk testicular tissue. At the cell level, lower GSTM1 expression was associated with a higher ferroptosis-driver gene signature score. Molecular simulations suggested potential structural compatibility between BDE-47 and GSTM1. In TM3 cells, BDE-47 reduced cellular metabolic activity and GSTM1 protein expression, decreased Gpx4 and Slc7a11 mRNA expression, and increased Acsl4 mRNA expression; ferrostatin-1 partially attenuated these transcriptional changes.

CONCLUSIONS: BDE-47-related molecular evidence and human NOA-associated evidence showed molecular convergence around GSTM1 and ferroptosis-related molecular features in a Leydig-cell context. In TM3 cells, BDE-47 exposure was accompanied by GSTM1 downregulation and ferroptosis-related transcriptional remodeling. These observations are hypothesis-generating and do not establish direct binding, GSTM1-mediated effects, ferroptotic cell death, or a causal relationship between BDE-47 exposure and human NOA.