Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3–Overexpressing Human Embryonic Kidney 293 Cells

Abstract

Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the 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 human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-39-O-sulfate (H-39-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 the cell lysate followed the substrate inhibition kinetics (V max = 0.66 nmol/min per mg, K m = 12.9 mM, and K si = 58.1 mM for H-39-S; V max = 0.29 nmol/min per mg, K m = 14.8 mM, and K si = 49.1 mM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 mM essentially abolished cellular excretion of both H-39-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P < 0.01) in sulfate excretion. Further, H-39-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 various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

Arylsulfatase B Mediates the Sulfonation-Transport Interplay in Human Embryonic Kidney 293 Cells Overexpressing Sulfotransferase 1A3

“…SULT1A3-overexpressing HEK293 cells (called SULT293 cells) had been established and were characterized in our laboratory (Li et al, 2015;Sun et al, 2015a).…”

“…At each time point (0.5, 1, 1.5, and 2 hours), a 200-ml aliquot of incubation medium was sampled and immediately replenished with the same volume of dosing solution. The samples were subjected to UPLC analyses to determine the sulfate concentrations, as described previously (Sun et al, 2015a). After sampling at the last time point (2 hours), the cells were collected and disrupted to measure the intracellular amounts of sulfate conjugates.…”

“…Sulfate excretion experiments were performed following our published procedures (Li et al, 2015;Sun et al, 2015a). The cells were incubated with a 2-ml dosing solution, namely, the Hanks' balanced salt solution buffer containing hesperetin (10 or 40 mM) at 37°C.…”

“…After a 2-hour incubation time, the reaction was stopped by adding 50 ml of ice-cold acetonitrile. The samples were centrifuged at 18,000g for 15 minutes, and the supernatant was analyzed by UPLC (Sun et al, 2015a). The hydrolysis activity of the ARSC enzyme was derived by subtracting the hydrolysis rate of control lysate from that of the ARSC lysate.…”

“…In our previous studies, we have shown that MRP4 is primarily involved in the efflux transport of flavonoid sulfates [including hesperetin 7-O-sulfate (H7S) and hesperetin 39-O-sulfate (H39S)] in SULT1A3-overexpressing human embryonic kidney (HEK) 293 cells (also called SULT293 cells) (Li et al, 2015;Sun et al, 2015a). In the present study, we examined the sulfonation-transport interplay (or SULT1A3-MRP4 interplay) in SULT293 cells using hesperetin as a model compound.…”