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Interaction between zebrafish Oct1 and endo- and xenobiotics

Mihaljević, Ivan; Popović, Marta; Žaja, Roko; Maraković, Nikola; Šinko, Goran; Smital, Tvrtko (2017) Interaction between zebrafish Oct1 and endo- and xenobiotics. Aquatic Toxicology, 187 . pp. 18-28. ISSN 0166-445X

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Playing as key elements in absorption, distribution, metabolism and excretion (ADME) processes of numerous endo- and xenobiotics, human organic cation transporters (OCTs) have been in focus of the toxicological research for more than a decade. These membrane transporters belong to SLC22A family within the SLC (Solute carrier) protein superfamily and in human are present with three co-orthologs, organic cation transporter 1, 2 and 3 (OCT1, OCT2, and OCT3). There are two Oct orthologs in zebrafish, Oct1 and Oct2, however their structural and functional properties, together with their relevance in zebrafish toxicological processes are still unexplored. In this study we performed functional characterization of zebrafish Oct1, using transient and stable heterologous expression systems and model fluorescent substrates as the basis for interaction studies with endo- and xenobiotics. We also performed basic topology analysis and homology modeling in order to determine the structure and membrane localization of Oct1. Finally, we performed toxicological study using MTT assay in order to see toxic effect of identified interactors in human embryonic kidney cells (HEK293T) stably expressing Oct1. Results showed twelve transmembrane alpha helices of Oct1, forming active region with more than one active site. Interaction studies revealed numerous interactors, which inhibited the Oct1 dependent uptake of fluorescent substrates. We identified five new fluorescent substrates: ASP+ (Km = 25.97 µM), rhodamine 123 (Km = 103.7 nM), berberine (Km = 3.96 µM), DAPI (Km = 780 nM) and ethidium bromide (Km = 96.93 nM). Interactors ranged from physiological compounds, with emphasis on steroid hormones, to different classes of xenobiotics, with IC50 values in nanomolar for e.g., pyrimethamine and prazosin, to millimolar range for cimetidine. Finally, using the MTT assay we identified toxic effect of determined interactors, but also confirmed these interactors as Oct1 substrates. Apart from berberine, oxaliplatin and MMP+ are identified as as new substrates of zebrafish Oct1. The described novel insights on functional properties of zebrafish Oct1 provide important basis for more detailed molecular characterization of this transporter.

Item Type: Article
Uncontrolled Keywords: organic cation transporters (OCTs) ; Oct1 ; zebrafish ; functional characterization ; transient and stable transfection ; novel fluorescent substrates ; interaction with xenobiotics
Subjects: NATURAL SCIENCES > Biology > Biochemistry and Molecular Biology
NATURAL SCIENCES > Interdisciplinary Natural Sciences > Environmental Science
Divisions: Division for Marine and Enviromental Research
Project titleProject leaderProject codeProject type
Identifikacija i funkcionalna karakterizacija (eko)toksikološki važnih polispecifičnih membranskih transportnih proteina u zebrici (Danio rerio)-TRANS-ZEBRATOXTvrtko SmitalIP-2013-11-4806HRZZ
Depositing User: Marta Popović
Date Deposited: 25 Oct 2018 14:14
DOI: 10.1016/j.aquatox.2017.03.012

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