Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA.
Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA; Research Centre for Toxic Compounds in the Environment (RECETOX), Masaryk University, Kamenice 753/5, Brno, Czech Republic.
Neurotoxicology. 2021 Jul;85:173-185. doi: 10.1016/j.neuro.2021.05.011. Epub 2021 May 25.
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.
苯并吡唑类的氟虫腈是一种抑制中枢神经系统γ-氨基丁酸（GABA）离子型受体的杀虫剂。实验证据表明，氟虫腈作为一种神经毒素，与神经退行性疾病有关；然而，其神经毒性机制尚未完全阐明。本研究的目的是量化氟虫腈诱导多巴胺细胞神经毒性的机制。用氟虫腈（0.25 至 500 μM，取决于测定方法）处理大鼠原代永生化中脑多巴胺能细胞（N27）。我们测量了与线粒体生物能学、线粒体自噬、线粒体膜电位和 ATP 产生相关的终点，此外还辨别了农药对转录组的反应。氟虫腈在 24 小时暴露后在 500 μM 时降低细胞活力，并且在 6 和 12 小时时，250 和 500 μM 的氟虫腈使 caspase 3/7 活性显著增加。因此，在低于细胞毒性的浓度下评估了后续终点。我们在氟虫腈（0.25、2.5、25 或 50 μM）单次处理 24 小时后测量了 N27 细胞的氧化呼吸。与载体对照和 0.25 或 2.5 μM 氟虫腈处理相比，氧消耗率（OCR）在 0.25 或 2.5 μM 氟虫腈处理之间没有差异，但基础呼吸降低了约 40-60%，并且在 50 μM 时减少了寡霉素诱导的 ATP 产生。OCR 的降低据推测与线粒体自噬导致的线粒体质量下降有关。线粒体膜电位也对氟虫腈敏感，在 2.5 μM 及以上浓度时受到损害。为了进一步阐明与神经毒性相关的机制，我们在多巴胺细胞中用 25 μM 氟虫腈进行了转录组学研究。氟虫腈抑制了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。这项研究阐明了氟虫腈诱导的神经毒性的分子靶点，并通过多种证据支持氟虫腈作为多巴胺细胞中的线粒体毒物。这与帕金森病等神经退行性疾病有关，因为接触氟虫腈与啮齿动物中黑质纹状体多巴胺能神经元的进行性丧失有关。
