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Benzo[a]pyrene (B[a]P), a representative of polycyclic aromatic hydrocarbons (PAHs), is ubiquitously spread in the environment and showing deleterious impacts on male steroidogenesis, including testosterone synthesis disorder. However, the precise mechanisms involved in B[a]P-induced steroidogenesis perturbation remains obscure. In the present study, we integrated in vivo tests, transcriptome profiling, in vitro assays, and conjoint in silico toxicological approaches to delineate the detailed mechanisms. In mouse models, we observed that B[a]P administration remarkably inhibited testosterone synthesis accompanied by ultrastructural impairments of mitochondria and mitophagosome formation in mouse Leydig cells. Transcriptome profiling showed that B[a]P down-regulated the expression of Ndufa9, Ndufa6, Ndufa10, and Ndufa5 in mouse testes, which are identified as critical genes involved in the assembly and functionality of mitochondrial complex I. In the in vitro tests, the bioactive B[a]P metabolite BPDE induced perturbation of testosterone synthesis by NDUFA10-mediated mitochondrial impairment, which was further exacerbated by mitophagy in TM3 Leydig cells. The findings of in silico toxicological analyses were highly consistent with the experimental observations and further unveiled that B[a]P/BPDE-involved PPARα activation could serve as a molecular initiating event to trigger the decline in Ndufa10 expression and testosterone synthesis. Overall, we have shown the first evidence that mitochondrial compromise in Leydig cells is the extremely crucial target in B[a]P-induced steroidogenesis perturbation.

Sertoli cells play a pivotal role in the complex spermatogenesis process. This study aimed to investigate the effects of PM2.5 on Sertoli cells using the TM4 cell line and a real time whole-body PM2.5 exposure mouse model, and further explore the underlying mechanisms through the application of metabolomics and transcriptomics. The results in vivo and in vitro showed that PM2.5 reduced Sertoli cells number in seminiferous tubules and inhibited cell proliferation. PM2.5 exposure also induced Sertoli cell dysfunction by increasing androgen binding protein (ABP) concentration, reducing the blood-testis barrier (BTB)-related protein expression, and decreasing glycolysis capacity and lactate production. The results of transcriptomics, metabolomics, and integrative analysis of multi-omics in the TM4 Sertoli cells revealed the activation of xenobiotic metabolism, and the disturbance of glutathione and purine metabolism after PM2.5 exposure. Further tests verified the reduced GSH/GSSG ratio and the elevation of xanthine oxidase (XO) activity in the PM2.5-exposed TM4 cells, indicating that excessive reactive oxygen species (ROS) was generated via metabolic disorder caused by PM2.5. Moreover, the redox imbalance was proved by the increase in the mitochondrial ROS level, superoxide dismutase (SOD) and catalase (CAT) activity, as well as the activation of the Nrf2 antioxidative pathway. Further study found that the redox imbalance caused by PM2.5 induced DNA damage response and cell cycle arrest. Additionally, PM2.5 induced ferroptosis through iron overload and lipid peroxidation. Taken all together, our study provided new insights for understanding proliferation inhibition and dysfunction of TM4 Sertoli cells exposed to PM2.5 via metabolic disorder and redox imbalance-mediated DNA damage response and ferroptosis.