Modulation of Human Multidrug Resistance Proteins by Reactive Quinone-based Glutathione Conjugates
Slot, Andrew Johannes
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The multidrug resistance protein 1 (MRP1) and MRP2 mediate the ATP-dependent cellular efflux of a diverse set of organic molecules including many glutathione (GSH) conjugates. In the present study, a series of GSH conjugated quinones (some of which have been shown to be toxic) were investigated for their ability to interact with human MRP1 and MRP2 using membrane vesicles enriched for these transporters.Several structurally and biologically distinct classes of endogenous (e.g. estradiol) and exogenous (e.g. hydroquinone (HQ),N-methyl-α-methyldopamine (N-Me-α-MeDA), and caffeic acid (CA)) GSH conjugated metabolites inhibited both MRP1- and MRP2-mediated vesicular transport of the prototypic MRPsubstrate (17β-estradiol-17β-D-glucuronide (E217βG)). The relative inhibitory potencies of the metabolites with respect to MRP1 versus MRP2 differed by approximately 10-fold, with the exception of4-hydroxy-2-(glutathion-S-yl)-17β-estradiol (4-OH-2-GS-E2), which differed by 300-fold. The catechol estradiols, N-Me-α-MeDA, and CA metabolites competitively inhibited MRP1-mediated E217βG transport and thus, are potential substrates for this transporter. The estradiol metabolites 2-hydroxy-1-(glutathion-S-yl)-17β-estradiol(2-OH-1-GS-E2) and 4-OH-2-GS-E2were bothsubsequently shown to besubstrates of MRP1 and MRP2by means ofa substrate depletion assay and a vesicular accumulation assay using HPLC with electrochemical detection. Transport of 2-OH-1-GS-E2 by MRP1 was inhibited by several MRP1 substrates (e.g. E217βG, leukotriene C4, and GSH disulfide) and modulators (e.g. MK571 and S-decyl-GSH). The chemically reactiveGSH-conjugated HQ metabolites also inhibited MRP1-mediated vesicular transport of E217βGwith IC50values ranging from 3 – 30 μM. To determine whether these compounds might modify MRP1, MRP1-enriched membrane vesicles were incubated with 2,5-(glutathion-S-yl)-hydroquinone (2,5-GS-HQ) and then immunoblotted with a MRP1-specific monoclonal antibody.An apparentreduction in the electrophoretic mobility of immuno-reactive bands relative to untreated vesicles was observed. However, subsequent limited trypsin digests did not show any differences in the digestion profiles between 2,5-GS-HQ-treated and control vesicles, suggesting no significant differences in protein structure with respect to accessible trypsin cleavage sites. Further experiments are needed to demonstrateMRP1 adduction by 2,5-GS-HQ.In conclusion, the data presented here are the first to show that reactive GSH-conjugated catechol and quinone metabolites can be substrates and modulators of MRP1 and MRP2 in vitro.