engleski

Interaction of zebrafish (Danio rerio) organic anion transporter 2d (Oat2d) with xeno- and endobiotics

Organic anion transporters (OATs/Oats) are polyspecific membrane uptake transporters that mediate entrance of compounds into the cell. Consequently, they are key determinants of toxicological response to various xenobiotics. Despite their physiological importance and role in cellular detoxification, the knowledge about uptake transporters in non-mammalian species is scarce. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. Mammalian OAT2 was characterized in human, rat and mouse, and was shown to play an important role in uptake and distribution of physiological compounds, as well as anionic toxins and drugs. As OAT2 orthologs have been poorly studied in non-mammalian species, the goal of our study was to determine phylogenetic relationships, tissue distribution and substrate specificity of zebrafish Oat2d. Phylogenetic analysis of OAT/Oat genes confirmed similarities among mammalian and zebrafish Oat transporters. Zebrafish Oat2d highly expressed in intestine and moderately expressed in testes and brain. Western blot analysis revealed protein band of 60 kDa, and immunocytochemistry showed its correct localization in the plasma membrane. Functional studies using HEK293T cells overexpressing zebrafish Oat2d revealed two model fluorescent substrates of Oat2d: lucifer yellow (LY, Km = 56.36 µM) and 6- carboxyfluorescein (Km = 210.1 µM). The initial screening with various endo- and xenobiotics showed significant inhibition of Oat2d mediated uptake of LY by endogenous compounds (α- ketoglutarate, fumarate, bilirubin and deoxycholic acid) and exogenous compounds (p- aminohippurate, perfluorooctanesulfonic acid, perfluorooctanoic acid, furosemid, chlorpyrifos, tetracycline and diclofenac). Selected potent inhibitors, fumarate and indomethacin, showed dose dependent inhibition of Oat2d transport (IC50 = 68.24 μM and 20.41 μM, respectively).

Membrane Proteins ; Phylogeny ; Tissue Expression ; Cell Localization ; Functional Analysis

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