Abstract

Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using HEK293 cells overexpressing sulfotransferase 1A3 (SULT1A3). Two mono-sulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by cell lysate followed the substrate inhibition kinetics (V_max = 0.66 nmol/min/mg, K_m = 12.9 μM and K_si= 58.1 μM for H-3'-S; V_max = 0.29 nmol/min/mg, K_m = 14.8 μM and K_si= 49.1 μM for H-7-S). The pan-MRP inhibitor MK-571 at 20 uM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the BCRP-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knock-down of MRP4 led to a substantial reduction (> 47.1%, p < 0.01) in sulfate excretion. Further, H-3'-S and H-7-S were good substrates for transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in the various organs/tissues (including the liver and kidney), MRP4 should be a determining factor to elimination and body distribution of hesperetin sulfates.