DataSheet_1_Analysis by Metabolomics and Transcriptomics for the Energy Metabolism Disorder and the Aryl Hydrocarbon Receptor Activation in Male Reproduction of Mice and GC-2spd Cells Exposed to PM2.5.docx

Fine particulate matter (PM2.5)-induced male reproductive toxicity arouses global public health concerns. However, the mechanisms of toxicity remain unclear. This study aimed to further investigate toxicity pathways by exposure to PM2.5in vitro and in vivo through the application of metabolomics and transcriptomics. In vitro, spermatocyte-derived GC-2spd cells were treated with 0, 25, 50, 100 μg/mL PM2.5 for 48 h. In vivo, the real-world exposure of PM2.5 for mouse was established. Forty-five male C57BL/6 mice were exposed to filtered air, unfiltered air, and concentrated ambient PM2.5 in Tangshan of China for 8 weeks, respectively. The results in vitro and in vivo showed that PM2.5 exposure inhibited GC-2spd cell proliferation and reduced sperm motility. Mitochondrial damage was observed after PM2.5 treatment. Increased Humanin and MOTS-c levels and decreased mitochondrial respiratory indicated that mitochondrial function was disturbed. Furthermore, nontargeted metabolomics analysis revealed that PM2.5 exposure could disturb the citrate cycle (TCA cycle) and reduce amino acids and nucleotide synthesis. Mechanically, the aryl hydrocarbon receptor (AhR) pathway was activated after exposure to PM2.5, with a significant increase in CYP1A1 expression. Further studies showed that PM2.5 exposure significantly increased both intracellular and mitochondrial reactive oxygen species (ROS) and activated NRF2 antioxidative pathway. With the RNA-sequencing technique, the differentially expressed genes induced by PM2.5 exposure were mainly enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, of which Cyp1a1 was the most significantly changed gene. Our findings demonstrated that PM2.5 exposure could induce spermatocyte damage and energy metabolism disorder. The activation of the aryl hydrocarbon receptor might be involved in the mechanism of male reproductive toxicity.

细颗粒物（PM2.5）引发的雄性生殖毒性已引发全球公共卫生领域的广泛关注，但其具体毒性机制尚未完全阐明。本研究旨在通过代谢组学与转录组学技术，开展PM2.5体内外暴露实验，深入探究其毒性通路。体外实验中，以浓度分别为0、25、50、100 μg/mL的PM2.5处理生精细胞来源的GC-2spd细胞48小时；体内实验方面，本研究构建了小鼠PM2.5真实暴露模型：将45只雄性C57BL/6小鼠分别置于中国唐山地区，暴露于过滤空气、未过滤空气以及浓缩环境PM2.5中，持续8周。体内外实验结果均显示，PM2.5暴露可抑制GC-2spd细胞增殖，并降低精子活力；PM2.5处理后可观察到线粒体损伤，人素（Humanin）与线粒体肽MOTS-c水平升高，同时线粒体呼吸功能下降，提示线粒体功能出现紊乱。此外，非靶向代谢组学分析结果显示，PM2.5暴露可干扰三羧酸循环（TCA循环），并抑制氨基酸与核苷酸的合成。从机制层面而言，芳香烃受体（aryl hydrocarbon receptor, AhR）通路在PM2.5暴露后被激活，CYP1A1的表达水平显著上调。进一步研究发现，PM2.5暴露可显著升高细胞内与线粒体内的活性氧（reactive oxygen species, ROS）水平，并激活NRF2抗氧化通路。通过RNA测序技术分析发现，PM2.5暴露诱导的差异表达基因主要富集于细胞色素P450介导的外源性物质代谢通路，其中Cyp1a1是表达变化最为显著的基因。本研究结果表明，PM2.5暴露可诱导生精细胞损伤与能量代谢紊乱，芳香烃受体的激活可能参与了雄性生殖毒性的发生机制。