title: Interaction between the zebrafish (Danio rerio) organic cation transporter 1 (Oct1) and endo- and xenobiotics.
abstract: Organic cation transporters (OCTs) serve as uptake transporters of numerous endo- and xenobiotics. They have been in the focus of medical toxicological research for more than a decade due to their key role in absorption, distribution, metabolism and excretion due to their expression on basolateral membranes of various barrier tissues. OCTs belong to the SLC22A family within the SLC (Solute carrier) protein superfamily, with three co-orthologs identified in humans (OCT1, 2 and 3), and two Oct orthologs in zebrafish (Oct1 and Oct2). The structural and functional properties of zebrafish Octs, along with their toxicological relevance, have still not been explored. In this study, we performed a functional characterization of zebrafish Oct1 using transient and stable heterologous expression systems and model fluorescent substrates as the basis for interaction studies with a wide range of endo- and xenobiotics. We also conducted a basic topology analysis and homology modeling to determine the structure and membrane localization of Oct1. Finally, we performed an MTT assay to evaluate the toxic effects of the seven interactors identified - oxaliplatin, cisplatin, berberine, MPP+, prazosin, paraquat and mitoxantrone - in human embryonic kidney cells (HEK293T) stably expressing zebrafish Oct1 (HEK293T-drOct1 cells). Our results show that the zebrafish Oct1 structure consists of 12 transmembrane alpha helices, which form the active region with more than one active site. Five new fluorescent substrates of Oct1 were identified: ASP+ (Km=26μM), rhodamine 123 (Km=103.7nM), berberine (Km=3.96μM), DAPI (Km=780nM), and ethidium bromide (Km=97nM). Interaction studies revealed numerous interactors that inhibited the Oct1-dependent uptake of fluorescent substrates. The identified interactors ranged from physiological compounds (mainly steroid hormones) to different classes of xenobiotics, with IC50values in nanomolar (e.g., pyrimethamine and prazosin) to millimolar range (e.g., cimetidine). Cytotoxicity experiments with HEK293T-drOct1 cells enabled us to identify berberine, oxaliplatin and MPP+ as substrates of Oct1. The data presented in this study provide the first insights into the functional properties of zebrafish Oct1 and offer an important basis for more detailed molecular and ecotoxicological characterizations of this transporter.
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000, Zagreb, Croatia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Mihaljević
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000, Zagreb, Croatia; Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, United Kingdom.
authors.forename: Marta
authors.initials: M
authors.lastname: Popović
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000, Zagreb, Croatia.
authors.forename: Roko
authors.initials: R
authors.lastname: Žaja
authors.affiliation: Biochemistry and Organic Analytical Chemistry Unit, Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 1000 Zagreb, Croatia.
authors.forename: Nikola
authors.initials: N
authors.lastname: Maraković
authors.affiliation: Biochemistry and Organic Analytical Chemistry Unit, Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 1000 Zagreb, Croatia.
authors.forename: Goran
authors.initials: G
authors.lastname: Šinko
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000, Zagreb, Croatia. Electronic address: smital@irb.hr.
authors.forename: Tvrtko
authors.initials: T
authors.lastname: Smital
mesh_terms: D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008297:Male; D009928:Organ Specificity; D027702:Organic Cation Transporter 1 / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014018:Tissue Distribution; D014162:Transfection; D014874:Water Pollutants, Chemical / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity*; D015262:Xenobiotics / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity*; D015027:Zebrafish / Q000378:metabolism*
chemical_list: D027702:Organic Cation Transporter 1; D014874:Water Pollutants, Chemical; D015262:Xenobiotics
keywords: Functional characterization; Interaction with xenobiotics; Novel fluorescent substrates; Organic cation transporter 1 (Oct1); Transient and stable transfection; Zebrafish