The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 μM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 μM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 μM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 μM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 μM fipronil treatments, but there was a ∼40–60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 μM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 μM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 μM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson’s disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents.

苯吡唑氟虫腈是一种杀虫剂，可抑制中枢神经系统中的 γ-氨基丁酸 (GABA) 离子型受体。实验证据表明氟虫腈是一种神经毒素，它与神经退行性疾病有关；然而，神经毒性的机制尚未完全阐明。本研究的目的是量化氟虫腈诱导的多巴胺细胞神经毒性的机制。大鼠原代永生化中脑多巴胺能细胞 (N27) 用氟虫腈（0.25 至 500 μM，取决于试验）处理。除了辨别转录组对农药的反应外，我们还测量了与线粒体生物能量学、线粒体自噬、线粒体膜电位和 ATP 产生相关的端点。氟虫腈在 500 μM 暴露 24 小时后降低细胞活力，并且半胱天冬酶 3/7 活性在 250 和 500 μM 氟虫腈后 6 和 12 小时显着增加。因此，在低于细胞毒性的浓度下评估了随后的终点。我们在暴露于一剂 0.25、2.5、25 或 50 μM 氟虫腈 24 小时后测量了 N27 细胞的氧化呼吸。载体对照和 0.25 或 2.5 μM 氟虫腈处理之间的耗氧率 (OCR) 没有差异，但基础呼吸减少了 ~40-60%，并且在 50 μM 时寡霉素诱导的 ATP 产生减少。假设 OCR 的减少与线粒体自噬导致的线粒体质量降低有关。线粒体膜电位对氟虫腈也很敏感，在浓度为 2.5 μM 及以上时会受到影响。为了进一步阐明与神经毒性相关的机制，我们在用 25 μM 氟虫腈处理后的多巴胺细胞中进行了转录组学。氟虫腈抑制与线粒体相关的转录网络（损伤、去极化、通透性和裂变），这与其对线粒体膜电位的影响一致。改变的基因网络还包括与阿尔茨海默病、炎症性疾病、神经纤维变性和神经原纤维缠结相关的基因网络。本研究阐明了氟虫腈诱导的神经毒性的分子靶点，并通过多条证据支持氟虫腈在多巴胺细胞中充当线粒体毒物。