ABSTRACT:The involvement of the canalicular multidrug resistance protein 2 (Mrp2) in the hepatobiliary excretion of acetaminophen (APAP)-glutathione (GSH) conjugate and its derivatives was investigated using transport-deficient (TR Acetaminophen (APAP 1 ) is biotransformed through phase I and phase II reactions into negatively charged, hydrophilic metabolites that are eliminated in urine and bile. Cytochrome P450 converts APAP to the highly reactive electrophile N-acetyl-p-benzoquinoneimine (Dahlin et al., 1984), which produces liver injury unless it is neutralized by conjugation with glutathione (GSH) (Mitchell et al., 1973). The resulting conjugate (APAP-GSH) can be sequentially metabolized to APAP-cysteinylglycine (APAP-CG), -cysteine (APAP-CYS), and -mercapturate (N-acetylated L-cysteine) (APAP-NAC). By contrast, conjugation with glucuronic acid and sulfate produces APAP-glucuronide (APAP-GLU) and APAP-sulfate (APAP-SUL), respectively. In several species, a large fraction of APAP-GSH undergoes biliary excretion in its original form and the rest is excreted in urine and bile as a mixture of thiol-containing derivatives, whereas the majority of APAP-GLU and APAP-SUL is excreted in urine (Gregus et al., 1988).Measuring the amount of APAP metabolites in urine and/or bile is considered a useful in vivo method for examining the effects of other chemicals on APAP metabolism, because changes in urinary and/or biliary excretion of APAP conjugates correlate well with changes in APAP biotransformation produced by xenobiotics (Jollow et al., 1974;Madhu et al., 1989;Liu et al., 1993). This approach assumes that membrane-associated transport systems for APAP metabolites in hepatocytes are not affected by xenobiotic treatment. With the recent identification and characterization of several apical and basolateral transporters (Muller and Jansen, 1997), it is now clear that the hepatic levels of some transport systems can be modulated by prototypical microsomal inducers such as pregnenolone 16␣-carbonitrile (Salphati and Benet, 1998), dexamethasone (Courtois et al., 1999), and phenobarbital (Ogawa et al., 2000). Therefore, these types of APAP disposition studies should be interpreted with respect to both xenobiotic inducibility of hepatic transporters and molecular mechanisms for the hepatic excretion of APAP and its metabolites.We recently showed that the biliary concentration of APAP-GSH, APAP-NAC, and APAP-GLU, but not that of APAP itself, APAP-SUL or APAP-CG/CYS, was significantly decreased by coadministration of the nonmetabolizable organic anion indocyanine green (ICG) in male CD-1 mice (Chen et al., 2000). These findings suggest that several APAP conjugates share hepatobiliary transport systems with ICG.A primary active transporter on the canalicular domain of hepatocytes, known as multidrug resistance protein 2 (Mrp2), mediates the hepatobiliary transport of a wide range of organic anions, including GSH-S-conjugates (e.g., leukotriene C4 and 2,4-dinitrophenyl-S-GSH), oxidized GSH (GSSG), glucuronide conjugates (e.g., ...