Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
Aquat Toxicol. 2018 Dec;205:193-203. doi: 10.1016/j.aquatox.2018.10.016. Epub 2018 Oct 25.
Zebrafish Mate7 belongs to solute carrier protein superfamily and specifically to subfamily of multidrug and toxin extruders. It is co-orthologous to mammalian Mates, and is ubiquitously expressed in zebrafish tissues with the highest expression in kidney. It has been shown to interact with both endogenous (steroid hormones) and xenobiotic compounds (pharmaceuticals), implying a role in efflux of toxic compounds. The objective of our study was to analyse interaction of environmental contaminants with zebrafish Mate7 using a newly developed high throughput screening (HTS) Mate7 assay. A full-length zebrafish mate7 sequence was obtained from zebrafish cDNA originating from male kidney, and a stable expression of Mate7 in genetically engineered HEK293 Flp-In cells was achieved. Stable Mate7 transfectants were then used for development and optimization of a new HTS cellular uptake protocol, with DAPI and ASP + as model fluorescent substrates. The developed assay was used for identifying zebrafish Mate 7 interactors and discerning the type of interaction. A series of 89 diverse environmental contaminants, including industrial chemicals, pesticides, and pharmaceuticals, was tested and highly effective Mate7 interactors were identified in all of the aforementioned groups. Some of the inhibitors identified could be of environmental concern because they may potentially impair Mate7 efflux function, lowering the fish defence capacity against environmental contaminants, or interfering with transport of yet unidentified physiological substrates. In addition, we found significant differences between zebrafish Mate7 and mammalian Mates' substrate preferences, a finding that should be taken into consideration when using zebrafish as a model organism in toxicokinetic studies.
斑马鱼 Mate7 属于溶质载体蛋白超家族，特别是多药和毒素外排器亚家族。它与哺乳动物的 Mate 是共直系同源物，在斑马鱼组织中广泛表达，在肾脏中表达最高。已证明它与内源性（甾体激素）和外源性（药物）化合物相互作用，暗示其在有毒化合物的外排中起作用。我们的研究目的是使用新开发的高通量筛选（HTS）Mate7 测定法分析环境污染物与斑马鱼 Mate7 的相互作用。从雄性肾脏来源的斑马鱼 cDNA 中获得全长斑马鱼 mate7 序列，并在遗传工程 HEK293 Flp-In 细胞中实现了 Mate7 的稳定表达。然后，使用稳定的 Mate7 转染细胞来开发和优化新的 HTS 细胞摄取方案，以 DAPI 和 ASP ⁇ 为模型荧光底物。开发的测定法用于鉴定斑马鱼 Mate7 相互作用体并辨别相互作用的类型。测试了一系列 89 种不同的环境污染物，包括工业化学品、农药和药物，并在上述所有组中鉴定出高效的 Mate7 相互作用体。鉴定出的一些抑制剂可能引起环境关注，因为它们可能潜在地损害 Mate7 外排功能，降低鱼类对环境污染物的防御能力，或干扰尚未鉴定的生理底物的运输。此外，我们发现斑马鱼 Mate7 和哺乳动物 Mate 之间的底物偏好存在显著差异，在将斑马鱼用作毒代动力学研究中的模型生物时，应考虑到这一点。
