Active Pharmaceutical Ingredient Uptake by Zebrafish (Danio rerio) Oct2 (slc22a2) Transporter Expressed in Xenopus laevis Oocytes

Abstract

Uptake of active pharmaceutical ingredients (APIs) across the gill epithelium of fish is via either a passive or facilitated transport process, with the latter being more important at the lower concentrations more readily observed in the environment. The solute carrier (SLC) 22A family, which includes the organic cation transporter OCT2 (SLC22A2), has been shown in mammals to transport several endogenous chemicals and APIs. Zebrafish oct2 was expressed in Xenopus oocytes and the uptake of ranitidine, propranolol, and tetraethylammonium characterized. Uptake of ranitidine and propranolol was time- and concentration-dependent with a k_m and V_max for ranitidine of 246 µM and 45 pmol/(oocyte × min) and for propranolol of 409 µM and 190 pmol/(oocyte × min), respectively. Uptake of tetraethylammonium (TEA) was inhibited by propranolol, amantadine, and cimetidine, known to be human OCT2 substrates, but not quinidine or ranitidine. At external media pH 7 and 8 propranolol uptake was 100-fold greater than at pH 6; pH did not affect ranitidine or TEA uptake. It is likely that cation uptake is driven by the electrochemical gradient across the oocyte. Uptake kinetics parameters, such as those derived in the present study, coupled with knowledge of transporter localization and abundance and API metabolism, can help derive pharmacokinetic models. Environ Toxicol Chem 2022;41:2993-2998. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Keywords: Absorption; emerging pollutants; in vitro toxicology; pharmaceuticals.

Figure 1

Time‐dependent accumulation of radiolabelled ranitidine, propranolol, and tetraethylammonium into Xenopus oocytes expressing zebrafish OCT2 (black squares) or sham injected controls (white circles). Values represent the average ± SEM of between seven and 15 oocytes.

Figure 2

The uptake rate of 200 µM radiolabelled tetraethylammonium into Xenopus oocytes expressing zebrafish OCT2 in the absence (control) and presence of 2 mM amantadine, cimetidine, propranolol, quinidine, and ranitidine. Values represent the average ± SEM of between seven and 15 oocytes. The columns with the same letter are not significantly different from each other (one‐way analysis of variance followed by post hoc Tukey's test, p < 0.05).

Figure 3

Dose‐dependent uptake rate of radiolabelled ranitidine and propranolol into Xenopus oocytes expressing zebrafish OCT2. Values represent the average ± SEM of uptake in seven to 15 OCT2 injected oocytes adjusted for the uptake in sham injected oocytes at each concentration. Michaelis–Menten parameters: ranitidine k _m = 246.1 µM, V _max = 44.6 pmol/(oocyte × min); propranolol k _m = 408.9 µM, V _max = 190 pmol/(oocyte × min).

Figure 4

The uptake rate of radiolabelled ranitidine, propranolol, and tetraethylammonium into Xenopus oocytes expressing zebrafish Oct2 (grey bars) or sham injected controls (white bars) at pH 6, 7, and 8. Values represent the average ± SEM of between seven and 15 oocytes. Asterisks indicate a significant difference between sham and Oct2 injected oocytes (Student's t‐test, p < 0.05) and A above the columns indicates a difference in the uptake rate in the Oct2 injected oocytes for propranolol at pH 7 and 8 compared with pH 6 (one‐way analysis of variance post hoc Tukey's test, p < 0.05).