Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule

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Abstract
The positively charged fluorescent dyes ethidium (Et+) and propidium (Pr2+) are widely used as DNA and necrosis markers. Et+ is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (≤500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et+ accumulated predominantly in the S2/S3 segments of the PT, but not Pr2+. In cells stably overexpressing human OCTs (hOCTs), Et+ uptake was observed with K m values of 0.8 ± 0.2 μM (hOCT1), 1.7 ± 0.5 μM (hOCT2), and 2.0 ± 0.5 μM (hOCT3), whereas Pr2+ was not transported. Accumulation of Et+ was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP+), cimetidine, and tetraethylammonium (TEA+). For hOCT1 and hOCT2, the IC50 values for MPP+, TEA+, and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et+ uptake by MPP+ and cimetidine was shown to be competitive. Et+ also inhibited transport of 0.1 μM [3H]MPP+ by all hOCT isoforms with IC50 values between 0.4 and 1.3 μM, and the inhibition of hOCT1-mediated uptake of MPP+ by Et+ was competitive. In Oct1/2−/− mice, Et+ uptake in the PT was almost abolished. The data demonstrate that Et+ is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et+ and their strong expression in various organs, strict safety guidelines for Et+ handling should be reinforced.

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