Faltermann Susanne, Prétôt René, Pernthaler Jakob, Fent Karl
University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland; University of Zürich, Institute of Plant Biology, Limnological Station, Seestrasse 187, 8802 Kilchberg, Switzerland.
University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
Aquat Toxicol. 2016 Feb;171:69-76. doi: 10.1016/j.aquatox.2015.11.016.
Microcystin-LR (MC-LR) and nodularin are hepatotoxins produced by several cyanobacterial species. Their toxicity is based on active cellular uptake and subsequent inhibition of protein phosphatases PP1/2A, leading to hyperphosphorylation and cell death. To date, uptake of MC-LR and nodularin in fish is poorly understood. Here, we investigated the role of the organic anion transporting polypeptide Oatp1d1 in zebrafish (drOatp1d1, Slco1d1) in cellular uptake in zebrafish. We stably transfected CHO and HEK293 cell lines expressing drOatp1d1. In both transfectants, uptake of MC-LR and nodularin was demonstrated by competitive inhibition of uptake with fluorescent substrate lucifer yellow. Direct uptake of MC-LR was demonstrated by immunostaining, and indirectly by the high cytotoxicity in stable transfectants. By means of a synthesized fluorescent labeled MC-LR derivative, direct uptake was further confirmed in HEK293 cells expressing drOatp1d1. Additionally, uptake and toxicity was investigated in the permanent zebrafish liver cell line ZFL. These cells had only a low relative abundance of drOatp1d1, drOatp2b1 and drOatp1f transcripts, which correlated with the lack of MC-LR induced cytotoxicity and transcriptional changes of genes indicative of endoplasmic reticulum stress, a known effect of this toxin. Our study demonstrates that drOatp1d1 functions as an uptake transporter for both MC-LR and nodularin in zebrafish.
微囊藻毒素-LR（MC-LR）和节球藻毒素是由几种蓝藻物种产生的肝毒素。它们的毒性基于细胞的主动摄取以及随后对蛋白磷酸酶PP1/2A的抑制，从而导致过度磷酸化和细胞死亡。迄今为止，鱼类对MC-LR和节球藻毒素的摄取情况仍知之甚少。在此，我们研究了有机阴离子转运多肽Oatp1d1在斑马鱼（drOatp1d1，Slco1d1）细胞摄取中的作用。我们稳定转染了表达drOatp1d1的CHO和HEK293细胞系。在这两种转染细胞中，通过荧光底物荧光素黄竞争性抑制摄取证明了MC-LR和节球藻毒素的摄取。通过免疫染色直接证明了MC-LR的摄取，并通过稳定转染细胞中的高细胞毒性间接证明了这一点。借助合成的荧光标记MC-LR衍生物，在表达drOatp1d1的HEK293细胞中进一步证实了直接摄取。此外，还在永久性斑马鱼肝细胞系ZFL中研究了摄取和毒性。这些细胞中drOatp1d1、drOatp2b1和drOatp1f转录本的相对丰度较低，这与缺乏MC-LR诱导的细胞毒性以及内质网应激相关基因的转录变化相关，内质网应激是这种毒素的已知效应。我们的研究表明，drOatp1d1在斑马鱼中作为MC-LR和节球藻毒素的摄取转运体发挥作用。
