Inhibition of the efflux of glutathione S-conjugates by plant polyphenols

The formation of dinitrophenylglutathione (DNP-SG) in human colon adenocarcinoma cells was identified and quantified by an HPLC-UV method, following exposure to 1-chloro-2,4,dinitrobenzene (CDNB) at 10 degrees for 40 min. The rate of efflux of DNP-SG at 37 degrees likewise, was measured by monitoring the DNP-SG content in the extracellular medium. Among the polyphenols examined for their action on DNP-SG export, butein was the most potent inhibitor with an IC50 value of 15 mu M. The others, in order of decreasing potencies, were quercetin, tannic acid, 2'-hydroxychalcone, 2-hydroxychalcone and morin, all of which have re,, values in the micromolar range. These polyphenols did not affect the ATP or the glutathione content of the cells. Mg2+-ATPase extracted from the plasma membrane of the cells was activated by DNP-SG in a concentration-dependent manner, and the reaction showed saturation kinetics with K-m and V-max values of 110 mu M and 12.3 nmol/min/mg protein, respectively. However, the six polyphenols mentioned above had negligible effects on the Mg2+-ATPase activity, suggesting that this was probably not the target of their inhibitory action. Probenecid, p-trifluoromethoxy-phenylhydrazone (FCCP) and chlorambucil also showed varying degrees of inhibition of the export of DNP-SG. Copyright (C) 1996 Elsevier Science Inc.