Microcystins (MCs) are the most studied cyanotoxins. The uptake of MCs in cells and tissues of mammals and fish species is mostly mediated by organic anion-transporting polypeptides (OATPs in humans and rodents; Oatps in other species), and the Oatp1d1 appears to be a major transporter for MCs in fish. In this study, six MC congeners of varying physicochemical properties (MC-LR, -RR, -YR, -LW, -LF, -LA) were tested by measuring their effect on the uptake of model Oatp1d1 fluorescent substrate Lucifer yellow (LY) in HEK293T cells transiently or stably overexpressing zebrafish Oatp1d1. MC-LW and -LF showed the strongest interaction resulting in an almost complete inhibition of LY transport with IC₅₀ values of 0.21 and 0.26 μM, while congeners -LR, -YR and -LA showed lower inhibitory effects. To discern between Oatp1d1 substrates and inhibitors, results were complemented by Michaelis–Menten kinetics and chemical analytical determinations of MCs uptake, along with molecular docking studies performed using the developed zebrafish Oatp1d1 homology model. Our study showed that Oatp1d1-mediated transport of MCs could be largely dependent on their basic physicochemical properties, with log P_OW being the most obvious determinant. Finally, apart from determination of the chemical composition of cynobacterial blooms, a reliable risk assessment should take into account the interaction of identified MC congeners with Oatp1d1 as their primary transporter, and herewith we demonstrated that such a comprehensive approach could be based on the use of highly specific in vitro models, accompanied by chemical assessment and in silico molecular docking studies.

微囊藻毒素 (MCs) 是研究最多的蓝藻毒素。哺乳动物和鱼类细胞和组织中 MCs 的摄取主要由有机阴离子转运多肽（人类和啮齿动物中的 OATPs；其他物种中的 Oatps）介导，Oatp1d1 似乎是鱼类中 MCs 的主要转运蛋白。在这项研究中，通过测量它们对模型 Oatp1d1 荧光底物路西法黄 (LY) 吸收的影响，测试了六种具有不同物理化学性质的 MC 同系物（MC-LR、-RR、-YR、-LW、-LF、-LA）在 HEK293T 细胞中瞬时或稳定地过表达斑马鱼 Oatp1d1。MC-LW 和-LF 表现出最强的相互作用，导致几乎完全抑制 LY 转运与 IC ₅₀值分别为 0.21 和 0.26 μM，而同源物 -LR、-YR 和 -LA 显示出较低的抑制作用。为了区分 Oatp1d1 底物和抑制剂，结果补充了 Michaelis-Menten 动力学和 MCs 摄取的化学分析测定，以及使用开发的斑马鱼 Oatp1d1 同源模型进行的分子对接研究。我们的研究表明，Oatp1d1 介导的 MCs 转运在很大程度上取决于它们的基本物理化学性质，log P _OW是最明显的决定因素。最后，除了确定蓝藻水华的化学成分外，可靠的风险评估应考虑已识别的 MC 同源物与 Oatp1d1 作为其主要转运蛋白的相互作用，因此我们证明了这种综合方法可以基于使用高度特异性的体外模型，伴随着化学评估和计算机分子对接研究。