Department of Cell Toxicology , Helmholtz Centre for Environmental Research - UFZ , Permoserstraße 15 , 04318 Leipzig , Germany.
Institute for Biodiversity and Ecosystem Dynamics , University of Amsterdam , Science Park 904 , 1098 XH Amsterdam , Netherlands.
Chem Res Toxicol. 2018 Aug 20;31(8):646-657. doi: 10.1021/acs.chemrestox.8b00019. Epub 2018 Jul 16.
Cellular uptake kinetics are key for understanding time-dependent chemical exposure in in vitro cell assays. Slow cellular uptake kinetics in relation to the total exposure time can considerably reduce the biologically effective dose. In this study, fluorescence microscopy combined with automated image analysis was applied for time-resolved quantification of cellular uptake of 10 neutral, anionic, cationic, and zwitterionic fluorophores in two reporter gene assays. The chemical fluorescence in the medium remained relatively constant during the 24-h assay duration, emphasizing that the proteins and lipids in the fetal bovine serum (FBS) supplemented to the assay medium represent a large reservoir of reversibly bound chemicals with the potential to compensate for chemical depletion by cell uptake, growth, and sorption to well materials. Hence FBS plays a role in stabilizing the cellular dose in a similar way as polymer-based passive dosing, here we term this process as serum-mediated passive dosing (SMPD). Neutral chemicals accumulated in the cells up to 12 times faster than charged chemicals. Increasing medium FBS concentrations accelerated uptake due to FBS-facilitated transport but led to lower cellular concentrations as a result of increased sorption to medium proteins and lipids. In vitro cell exposure results from the interaction of several extra- and intracellular processes, leading to variable and time-dependent exposure between different chemicals and assay setups. The medium FBS plays a crucial role for the thermodynamic equilibria as well as for the cellular uptake kinetics, hence influencing exposure. However, quantification of cellular exposure by an area under the curve (AUC) analysis illustrated that, for the evaluated bioassay setup, current in vitro exposure models that assume instantaneous equilibrium between medium and cells still reflect a realistic exposure because the AUC was typically reduced less than 20% compared to the cellular dose that would result from instantaneous equilibrium.
细胞摄取动力学是理解体外细胞测定中时变化学暴露的关键。与总暴露时间相比，缓慢的细胞摄取动力学会大大降低生物有效剂量。在这项研究中，荧光显微镜结合自动图像分析用于在两个报告基因测定中时间分辨地定量测定 10 种中性、阴离子、阳离子和两性离子荧光染料的细胞摄取。在 24 小时测定过程中，介质中的化学荧光保持相对恒定，这强调了胎牛血清 (FBS) 中补充到测定介质中的蛋白质和脂质代表了大量可还原结合化学物质的储库，这些化学物质具有通过细胞摄取、生长和吸附到井材料来补偿化学物质消耗的潜力。因此，FBS 在稳定细胞剂量方面发挥作用的方式与基于聚合物的被动给药相似，我们在此将该过程称为血清介导的被动给药 (SMPD)。中性化学物质在细胞中的积累速度比带电化学物质快 12 倍。增加培养基中 FBS 的浓度会因 FBS 促进的运输而加速摄取，但由于更多地吸附到介质中的蛋白质和脂质，会导致细胞内浓度降低。体外细胞暴露是由几个细胞内外过程相互作用的结果，导致不同化学物质和测定设置之间的暴露具有可变性和时变特性。培养基 FBS 对热力学平衡以及细胞摄取动力学都起着至关重要的作用，因此会影响暴露。然而，通过曲线下面积 (AUC) 分析对细胞暴露进行定量表明，对于评估的生物测定设置，假设介质和细胞之间瞬间达到平衡的当前体外暴露模型仍然反映了真实的暴露，因为 AUC 通常比从瞬间平衡得出的细胞剂量降低不到 20%。
