Dynamics of glutathione conjugation and conjugate efflux in detoxification of the carcinogen, 4-nitroquinoline 1-oxide: contributions of glutathione, glutathione S-transferase, and MRP1.
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 K(m) of 9.5 microM and a V(max) comparable to other good MRP1 substrates. Glutathione or its S-methyl analogue enhanced the rate of (3)H-QO-SG transport, whereas QO-SG inhibited the rate of (3)H-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 (3)H-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, (k(cat)/K(m))/k(2), approximately 3 x 10(6)). 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.
Peklak-Scott
Christina
C
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Townsend
Alan J
AJ
Morrow
Charles S
CS
D015112:4-Nitroquinoline-1-oxide / Q000378:metabolism* / Q000633:toxicity; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity; D002384:Catalysis; D045744:Cell Line, Tumor; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008658:Inactivation, Metabolic; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D022161:Transport Vesicles / Q000737:chemistry / Q000378:metabolism
D015112:4-Nitroquinoline-1-oxide; D002273:Carcinogens; D005978:Glutathione; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D007527:Isoenzymes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C496556:GSTP1 protein, human; C421109:multidrug resistance-associated protein 1