Transport of the β-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 metabolite 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 the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (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 vesicles prepared from transfected cells, we found that MRP1 transports [H-3]NNAL-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 dispensable because transport was supported by the GSH analog, gamma -glutamyl-alpha -aminobutyryl-glycine. Despite stimulation of NNAL-O-glueuronide transport by GSH, there was no detectable reciprocal stimulation of [H-3]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C-4 (LTC4) and 17 beta -estradiol 17 beta-(D-glucuronide) (E(2)17 betaG) inhibited GSH-dependent uptake of [H-3]NNAL-O-glucuronide, only [H-3]LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC4 but not E(2)17 betaG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.