Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
Toxicol In Vitro. 2018 Feb;46:246-256. doi: 10.1016/j.tiv.2017.09.026. Epub 2017 Oct 10.
OATS/Oats are transmembrane proteins that transport a variety of drugs, environmental toxins and endogenous metabolites into the cell. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. In this study we specifically address Oat2 (Slc22a7) family. Conserved synteny analysis showed localization of zebrafish oat2 genes on two chromosomes, 11 and 17. All five zebrafish Oats were localized by live cell imaging in membranes of transiently transfected HEK293-T cells, and Oat2a, b, d, and e were confirmed using western blot analysis. Functional studies using the HEK293T cells overexpressing zebrafish Oats revealed two model fluorescent substrates of three Oats: Lucifer yellow for Oat2a and Oat2d (Km 122, and 49.7μM), and 6-carboxyfluorescein for Oat2b and Oat2d (Km 199.7, and 266.9μM). The initial screening of a series of diverse endo- and xenobiotics showed interaction with a number of compounds, including cGMP and diclofenac (IC50 27.74, and 19.14μM) with Oat2a; estrone-3-sulfate and diclofenac (IC50 30.96, and 12.6μM) with Oat2b; and fumarate and indomethacin (IC50 68.24, and 20.41μM) with Oat2d. This study provides the first comprehensive data set on Oat2 in zebrafish and offers an important basis for more detailed molecular and (eco)toxicological characterizations of these transporters.
OATs/Oats 是一种跨膜蛋白，可将多种药物、环境毒素和内源性代谢物转运到细胞内。斑马鱼（Danio rerio）有 7 种 OAT 同源物：Oat1、Oat2a-e 和 Oat3。在本研究中，我们特别关注 Oat2（Slc22a7）家族。保守的同线性分析表明，斑马鱼 oat2 基因定位于两条染色体 11 和 17 上。通过瞬时转染 HEK293-T 细胞的活细胞成像，定位了所有 5 种斑马鱼 Oat，并用 Western blot 分析证实了 Oat2a、b、d 和 e 的定位。使用过表达斑马鱼 Oat 的 HEK293T 细胞进行的功能研究表明，3 种 Oat 有 2 种模型荧光底物： Lucifer yellow 用于 Oat2a 和 Oat2d（Km 分别为 122 和 49.7μM），6-羧基荧光素用于 Oat2b 和 Oat2d（Km 分别为 199.7 和 266.9μM）。对一系列内源性和外源性化合物的初步筛选表明，与多种化合物相互作用，包括 cGMP 和双氯芬酸（Oat2a 的 IC50 分别为 27.74 和 19.14μM）；雌酮-3-硫酸盐和双氯芬酸（Oat2b 的 IC50 分别为 30.96 和 12.6μM）；以及富马酸和吲哚美辛（Oat2d 的 IC50 分别为 68.24 和 20.41μM）。本研究提供了斑马鱼 Oat2 的首个全面数据集，并为这些转运蛋白的更详细的分子和（生态）毒理学特征提供了重要基础。
