Dynamics of Glutathione Conjugation and Conjugate Efflux in Detoxification of the Carcinogen, 4-Nitroquinoline 1-Oxide: Contributions of Glutathione, GlutathioneS-Transferase, and MRP1

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Abstract
4-Nitroquinoline 1-oxide (NQO) is a reactive electrophile with potent cytotoxic as well as genotoxic activities. NQO forms a conjugate, QO-SG, with glutathione, which greatly reduces its chemical reactivity. Previous studies demonstrated that glutathione S-transferase (GST) P1a-1a and multidrug resistance protein (MRP) 1/2 act in synergy to confer resistance to both cyto- and genotoxicities of NQO, whereas protection afforded by GSTP1a-1a or MRP alone was much less. To better understand the role of glutathione, GSTP1a-1a, and MRP1 in NQO detoxification, we have characterized the kinetics and cofactor requirements of MRP1-mediated transport of QO-SG and NQO. Additionally, using recombinant GSTP1a-1a and physiological conditions, we have examined the enzymatic and nonenzymatic formation of QO-SG. Results show that MRP1 supports efficient transport of QO-SG with a Km of 9.5 μM and a Vmax comparable to other good MRP1 substrates. Glutathione or its S-methyl analogue enhanced the rate of 3H-QO-SG transport, whereas QO-SG inhibited the rate of 3H-glutathione transport. These data favor a mechanism for glutathione-enhanced, MRP1-mediated QO-SG transport that does not involve cotransport of glutathione. NQO was not transported by MRP1 either alone or in the presence of S-methyl glutathione. Transport of 3H-NQO was observed in the presence of glutathione, but uptake into MRP1-containing vesicles was entirely attributable to its conjugate, QO-SG, formed nonenzymatically. While the nonenzymatic rate was readily measurable, enzyme catalysis was overwhelmingly dominant in the presence of GSTP1a-1a (rate enhancement factor, (kcat/Km)/k2, ∼3 × 106). We conclude that MRP1 supports detoxification of NQO via efficient, glutathione-stimulated efflux of QO-SG. While nonenzymatic QO-SG formation and MRP1-mediated conjugate efflux result in low-level protection from cyto- and genotoxicities, this protection is greatly enhanced by coexpression of GSTP1-1 with MRP1. This result emphasizes the quantitative importance of enzyme-catalyzed conjugate formation, a crucial determinant of high-level, MRP-dependent protection of cells from NQO toxicity.
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