Evaluation of a Direct Cellular Assay for NQO1 in the Presence of Phytochemicals

Abstract

NAD(P)H:quinone oxidoreductase NQO1 is a phase ll enzyme that catalyzes the linked intracellular conversion of NADPH2 to NADP and duroquinone (DQ) to hydroduroquinone (DQH2) in cells. There are different methods to determine NQO1 activity. The classic NQO1 enzyme assay is the usual method for measuring NQO1 activity in cell lysates. We chose to determine the intact-cell activity to investigate the effect of the four compounds Quercetin (Q), Epigallocatechin-3-gallate, (EGCG), indole-3-carbinol (I3C), and Sulforaphane (SFN) in stimulating NQO1 activity. In brief, DQ-mediated reduction of the cell-membrane-imperative secondary electron acceptor, ferricyanide, was used to quantify intact-cell NQO1 activity. This approach involves adding quinone duroquinone to the cells and then measuring the appearance rate of the two-electron reduction product, durohydroquinone, by its ferricyanide reduction. In conclusion, I3C and SFN did not interfere with the enzymatic reaction. In contrast, Q and EGCG can interfere with the enzymatic reaction of NQO1 because Q and EGCG possess quinone structures, unlike I3C and SFN, which do not have the same shape.