Dutch Poisons Information Center (DPIC), University Medical Center Utrecht, P.O. Box 85.500, NL-3508, GA, Utrecht, The Netherlands; Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508, TD, Utrecht, The Netherlands.
Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508, TD, Utrecht, The Netherlands.
Toxicol In Vitro. 2017 Dec;45(Pt 1):60-71. doi: 10.1016/j.tiv.2017.05.010. Epub 2017 May 13.
The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements. We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine. We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC values on hSERT, possibly due to experimental differences. Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.
新精神活性物质（NPS）的流行和使用正在增加，目前存在超过 600 种 NPS。许多非法药物和 NPS 通过抑制和/或逆转单胺再摄取转运体（DAT、NET 和 SERT）来增加大脑单胺水平。这通常使用劳动密集型、放射性终点测量来研究。我们研究了一种新颖且创新的测定法的适用性，该测定法基于荧光单胺模拟底物。DAT、NET 或 SERT 表达的人胚肾（HEK293）细胞暴露于常见药物（可卡因、dl-苯丙胺或 MDMA）、NPS（4-氟苯丙胺、PMMA、α-PVP、5-APB、2C-B、25B-NBOMe、25I-NBOMe 或甲氧基苯丙胺）或抗抑郁药氟西汀。我们证明，这种基于荧光微孔板读取器的测定法可检测不同药物对不同转运体的抑制作用，并可区分药物。大多数 IC 值与放射性测定法的先前结果一致，并且在估计的人脑浓度范围内。然而，苯乙胺在 hSERT 上显示出更高的 IC 值，可能是由于实验差异。与放射性测定法相比，这种高通量荧光测定法不复杂，可以在生理条件下测量，不需要特定的设备，并且允许进行动力学测量，从而能够检测瞬态效应。因此，该测定法是研究非法药物和 NPS 对单胺再摄取转运体的影响的放射性测定法的良好替代品。
