Transport of the beta-O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1) - Requirement for glutathione or a non-sulfur-containing analog

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its me tabolite 4-(methylnitrosamino)-1(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In t he present study, we investigated the ATP-binding cassette (ABC) protein, M RP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesi cles prepared from transfected cells, we found that MRP1 transports [H-3]NN AL-O-glucuronide but is dependent on the presence of GSH (K-m 39 muM, V-max 48 pmol mg(-1) min(-1)). We also found that the sulfur atom in GSH was dis pensable because transport was supported by the GSH analog, gamma -glutamyl -alpha -aminobutyryl-glycine. Despite stimulation of NNAL-O-glueuronide tra nsport by GSH, there was no detectable reciprocal stimulation of [H-3]GSH t ransport. Moreover, whereas the MRP1 substrates leukotriene C-4 (LTC4) and 17 beta -estradiol 17 beta-(D-glucuronide) (E(2)17 betaG) inhibited GSH-dep endent uptake of [H-3]NNAL-O-glucuronide, only [H-3]LTC4 transport was inhi bited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were comp lex. A mutant form of MRP1, which transports LTC4 but not E(2)17 betaG, als o did not transport NNAL-O-glucuronide suggesting a commonality in the bind ing elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNA L-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhi bited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and thei r conjugated organic anion substrates, and extend the range of xenotoxins t ransported by MRP1 and MRP2 to include metabolites of known carcinogens inv olved in the etiology of lung and other cancers.