Azole Fungicides Disturb Intracellular Ca2 in an Additive Manner in Dopaminergic PC12 Cells
Azole Fungicides Disturb Intracellular Ca2 in an Additive Manner in Dopaminergic PC12 Cells
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DOI：
10.1093/toxsci/kft119
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发表时间：
2013-08-01
影响因子：
3.8
通讯作者：
Westerink, Remco H. S.
中科院分区：
文献类型：
--
作者：
Heusinkveld, Harm J.;Molendijk, Jeffrey;Westerink, Remco H. S.
人类主要通过食物暴露于农药和其他化合物的复杂混合物。唑类杀菌剂是一种广谱抗真菌化合物，用于农业、人类和兽医学。抗真菌作用的机制依赖于抑制CYP 51，从而抑制真菌细胞生长。已知的唑类杀真菌剂对健康的不利影响主要与抑制真菌生长有关。此外，唑类杀真菌剂诱导的神经毒性也有报道，但其潜在机制尚不清楚。因此，我们研究了一组六唑类杀菌剂（抑霉唑、氟硅唑、氟康唑、戊唑醇、三唑酮和环丙唑醇）对细胞活力的影响，使用组合的阿利兰/CFDA-AM测定法，并使用H-2-DCFDA荧光测定法对氧化应激的影响。由于钙在神经元的存活和功能中起着关键作用，因此使用单细胞荧光显微镜研究了这六种唑类杀真菌剂以及唑类杀真菌剂的二元和四元混合物对多巴胺能PC 12细胞中细胞内钙浓度（[Ca-2]i）的影响，所述多巴胺能PC 12细胞装载有钙敏感性荧光染料Fura-2。仅观察到细胞活力和ROS产生的适度变化。然而，六个唑类杀真菌剂中有五个诱导电压门控钙通道（VGCC）的非特异性抑制，尽管效力不同。使用二元IC 20和四元IC 10混合物的实验表明，对VGCC的抑制作用是加和的。综合研究结果表明，通过抑制VGCC调节细胞内Ca-2是唑类杀真菌剂的一种新的作用模式。此外，唑类杀真菌剂的混合物显示出加和性，说明在人类风险评估中需要考虑混合物效应。
Humans are exposed to complex mixtures of pesticides and other compounds, mainly via food. Azole fungicides are broad spectrum antifungal compounds used in agriculture and in human and veterinary medicine. The mechanism of antifungal action relies on inhibition of CYP51, resulting in inhibition of fungal cell growth. Known adverse health effects of azole fungicides are mainly linked to CYP inhibition. Additionally, azole fungicideinduced neurotoxicity has been reported, though the underlying mechanism(s) are largely unknown. We therefore investigated the effects of a group of six azole fungicides (imazalil, flusilazole, fluconazole, tebuconazole, triadimefon, and cyproconazole) on cell viability using a combined alamar Blue/CFDA-AM assay and on oxidative stress using a H-2-DCFDA fluorescent assay. As calcium plays a pivotal role in neuronal survival and functioning, effects of these six azole fungicides and binary and quaternary mixtures of azole fungicides on the intracellular calcium concentration ([Ca-2]i) were investigated using single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive fluorescent dye Fura-2. Only modest changes in cell viability and ROS production were observed. However, five out of six azole fungicides induced a nonspecific inhibition of voltage-gated calcium channels (VGCCs), though with varying potency. Experiments using binary IC20 and quaternary IC10 mixtures indicated that the inhibitory effects on VGCCs are additive. The combined findings demonstrate modulation of intracellular Ca-2 via inhibition of VGCCs as a novel mode of action of azole fungicides. Furthermore, mixtures of azole fungicides display additivity, illustrating the need to take mixture effects into account in human risk assessment.