Chemical structures of compounds used in this study. (A) α-Methyl aromatic amino acids. (B) Iodinated aromatic amino acids. (C) Fluorinated aromatic amino acids. Groups I, II, and III represent compounds with a hydroxyl group at the para-, meta-, and ortho-positions of the benzene ring, respectively.

Transport of FAMT by OAT1. (A) Concentration-dependent inhibition of [¹⁴C]PAH uptake by FAMT in OAT1 cells. Uptake of [¹⁴C]PAH (1 μM) was measured in the presence of various concentrations of FAMT. Uptake values were fitted to inhibition curves. IC₅₀ of FAMT on OAT1-mediated [¹⁴C]PAH uptake was 453.1 ± 14.3 μM (n = 4). (B) Concentration dependence of FAMT-induced [¹⁴C]PAH efflux in OAT1 cells. The efflux of preloaded [¹⁴C]PAH from OAT1 cells was measured for 1 minute in the presence or absence of extracellular FAMT. The FAMT-induced efflux at each concentration of FAMT was obtained as described in Materials and Methods and plotted against the concentration of FAMT. The curve fit to Michaelis-Menten equation. The inset shows Eadie-Hofstee plot [ordinate: V (% radioactivity per minute); abscissa: V (% radioactivity per minute)/[FAMT] (micromolar)], which was used to determine kinetic parameters. K_m of 690.8 ± 18.9 μM and V_max of 38.1% ± 7.3% radioactivity/min were obtained (n = 4). (C) Concentration dependence of [¹⁴C]FAMT uptake mediated by OAT1. OAT1-mediated uptake of [¹⁴C]FAMT at each concentration was measured for 1 minute. Uptake rates were fit to Michaelis-Menten curve. Inset shows Eadie-Hofstee plot [ordinate: V (picomole per milligram protein per minute]; abscissa: V [picomole per milligram protein per minute)/[FAMT] (micromolar)]. K_m of 171.9 ± 16.6 μM and V_max of 3391 ± 27.3 pmol/mg per minute were obtained (n = 4).

Comparison of IMT and 4-I-m-Tyr in interaction with OAT1. (A) Concentration-dependent inhibition of [¹⁴C]PAH uptake by IMT and 4-I-m-Tyr in OAT1 cells. Uptake of [¹⁴C]PAH (1 μM) was measured in the presence of varied concentrations of IMT or 4-I-m-Tyr. Uptake values were fitted to inhibition curves with IC₅₀ listed in Table 1. (B) [¹⁴C]PAH efflux mediated by OAT1 induced by IMT and 4-I-m-Tyr. The efflux of preloaded [¹⁴C]PAH from OAT1 cells was measured for 1 minute in the presence or absence (−) of extracellular compounds at the indicated concentration. The radioactivity released from the cells was expressed as the percentage of total preloaded radioactivity. *P < 0.05; **P < 0.01; ***P < 0.001 vs. (−). Data are expressed as means ± S.D. (n = 4).

Effect of removal of the α-methyl, hydroxyl, or halogen group from IMT and FAMT. The concentration-dependent inhibition of [¹⁴C]PAH uptake (A) and the induction of OAT1-mediated [¹⁴C]PAH efflux (B) were compared among IMT and the compounds (3-IT, 3-I-AMP, and AMT) in which the α-methyl, hydroxyl, or halogen group was removed from IMT. The effect of removal of the α-methyl, hydroxyl, and halogen groups was also examined for FAMT in the comparison with 3-FT, 3-F-AMP, and AMT: the inhibition of [¹⁴C]PAH uptake (C); the induction of [¹⁴C]PAH efflux (D). The inhibition and efflux experiments were performed as described in the legend to Fig. 3. Uptake values were fitted to inhibition curves except 3-F-AMP and 3-I-AMP, which were difficult to fit (A and C). AMT in (C), same as that in (A), is shown as comparison. In (B and D), *P < 0.05; **P < 0.01; ***P < 0.001 vs. (−). Data are expressed as means ± S.D. (n = 4).

Effect of fluoro group position in para-tyrosine, meta-tyrosine, and ortho-tyrosine. The effect of fluoro group position was examined with a hydroxyl group fixed at position 4 (para-position), position 3 (meta-position), or position 2 (ortho-position) of the benzene ring. The concentration-dependent inhibition of [¹⁴C]PAH uptake (A, C, and E) and the induction of OAT1-mediated [¹⁴C]PAH efflux (B, D, and F) were compared between the compounds at indicated concentrations. The inhibition and efflux experiments were performed as described in the legend to Fig. 3. In (A, C, and E), the uptake values were fitted to inhibition curves, except 2-FT, which was difficult to fit. 3-FT in (A), same as that in Fig. 4C, is shown for comparison. In (B, D, and F), *P < 0.05; **P < 0.01; ***P < 0.001 vs. (−). Data are expressed as means ± S.D. (n = 4).

Comparison of fluoro and iodo groups in the interaction with OAT1. The contribution of fluoro and iodo groups in the interaction with OAT1 was compared between the compound with fluoro group and that with iodo group at the same position: 2-FT and 2-IT (A); 3-FT and 3-IT (B); 4-F-m-Tyr and 4-I-m-Tyr (A). The inhibition of [¹⁴C]PAH uptake (A, C, and E) and the induction of OAT1-mediated [¹⁴C]PAH efflux (B, D, and F) were examined as described in the legend to Fig. 3. In (A, C, and E), the uptake values were fitted to inhibition curves, whereas the efflux values are fitted to Michaelis-Menten curves in (B, D, and F). Blue: compounds with a fluoro group; black: compounds with an iodo group. In (A, C, and E), the compounds, except 2-IT, were presented in the other figures and shown for comparison (2-FT from Fig. 5; 3-FT and 3-IT from Fig 4; 4-F-m-Tyr from Fig 5; 4-I-m-Tyr from Fig. 3). Data represent means ± S.D., n = 4.

Interaction of 2-FAMT with OAT1. 2-FAMT, which has a fluoro group at position 2, and FAMT, with a fluoro group at position 3, were compared in the inhibition of [¹⁴C]PAH uptake (A) and the induction of OAT1-mediated [¹⁴C]PAH efflux at 100, 300, 1000 μM. (B). 2-FAMT exhibited less inhibition on OAT1-mediated [¹⁴C]PAH uptake than FAMT (A). Similarly, 2-FAMT induced less [¹⁴C]PAH efflux mediated by OAT1 compared with FAMT (B). The inhibition and efflux experiments were performed as described in the legend to Fig. 3. FAMT in (A), same as that in Fig. 4C, is shown for comparison. In (B), *P < 0.05; **P < 0.01; ***P < 0.001 vs. (−). Data are expressed as means ± S.D. (n = 4).