Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3 Ϫ/Ϫ and Abcc4 Ϫ/Ϫ mice using a cassette dosing approach. In the livers of Abcc3 Ϫ/Ϫ and Abcc4 Ϫ/Ϫ mice, the basolateral excretory clearance of acetaminophen sulfate was reduced ϳ20 and ϳ20%, 4-methylumbelliferyl sulfate was reduced ϳ50 and ϳ65%, and harmol sulfate was decreased ϳ30 and ϳ45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by ϳ96, ϳ85, and ϳ40%, respectively, in the livers of Abcc3 Ϫ/Ϫ mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.Phase II metabolites formed in the liver, such as sulfate and glucuronide conjugates, are excreted into sinusoidal blood across the basolateral membrane or are extruded from the liver by transport across the canalicular (apical) membrane into bile. The molecular determinants of canalicular excretion have been characterized extensively (Keppler et al., 1997;Suzuki et al., 2003), and it is now known that MRP2 (ABCC2) and breast cancer resistance protein (ABCG2) mediate the biliary excretion of many phase II conjugates. In contrast to canalicular excretion, hepatic basolateral efflux mechanisms are poorly understood.Recently, two members of the MRP family, MRP3 and MRP4 Lee et al., 1998), have been implicated in hepatic basolateral excretion. MRP3 is expressed at low levels on the hepatic basolateral membrane in humans and rats, but it is susceptible to striking induction in cholestasis (Hirohashi et al., 1998;Konig et al., 1999). The substrate selectivity of MRP3 includes phase II metabolites, such as glucuronide and glutathione conjugates, as well as bile acids (Hirohashi et al., 1999(Hirohashi et al., , 2000Zeng et al., 1999). Mrp2-deficient rats, and to a lesser extent Mrp2 knockout mice, have increased hepatic expression of Mrp3 (Hirohashi

Human immunodeficiency virus-infected patients on antiretroviral drug therapy frequently experience hepatotoxicity, the underlying mechanism of which is poorly understood. Hepatotoxicity from other compounds such as bosentan and troglitazone has been attributed, in part, to inhibition of hepatocyte bile acid excretion. This work tested the hypothesis that antiretroviral drugs modulate hepatic bile acid transport. Ritonavir (28 M ϭ 2.1 and 6.4 M in human and rat, respectively), saquinavir (IC 50 ϭ 6.7 and 20 M, respectively), and efavirenz (IC 50 ϭ 43 and 97 M, respectively). Nevirapine (75 M) had no effect on bile acid transport in any model system. In conclusion, ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibited both the hepatic uptake and biliary excretion of taurocholate.

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(Ϫ/Ϫ), Abcg2(Ϫ/Ϫ), and Abcb1a(Ϫ/Ϫ)/Abcb1b(Ϫ/Ϫ) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also ϳ35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (Ն37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.Phase II metabolism, including sulfation and glucuronidation, occurs primarily in the liver. Conjugation of a substrate with a sulfate or glucuronide moiety increases its hydrophilicity to promote excretion from the body. These conjugates are typically too polar to undergo passive diffusion from hepatocytes after their intracellular formation and therefore require carrier-mediated transport for excretion across the hepatic canalicular (apical) membrane into bile and across the basolateral membrane into sinusoidal blood. Most sulfate and glucuronide metabolites are inactive; notable exceptions include morphine-6-glucuronide, minoxidil sulfate, SN-38-glucuronide, and troglitazone sulfate (Zamek-Gliszczynski et al., 2006b). Altered hepatic export of pharmacologically and toxicologically active sulfate and glucuronide metabolites formed in the liver can have profound pharmacodynamic and toxic implications, underscoring the importance of understanding the mechanisms of metabolite excretion (Meisheri et al., 1993;Funk et al., 2001;Horikawa et al., 2002;. Despite the importance of active transport in hepatic excretion of phase II metabolites, mechanisms responsible for excretion of sulfate and glucuronide conjugates have not been elucidated fully.

Cisplatin resistant (CP-r) cells often show decreased uptake of cisplatin in association with reduced cell surface proteins and decreased endocytosis. In this report, two major [14C]carboplatin-binding proteins were identified as filamin and actin by photoaffinity labeling and mass spectrometry. Decreased expression of these two proteins was found in two different human CP-r cell lines (KB-CP20 and 7404-CP20), in comparison with their parental cell lines (KB-3-1 and BEL-7404), respectively. Disorganization of beta-actin and filamin 250 and 90 was also detected in these CP-r cells by confocal microscopy. Transfection of a wild-type actin-enhanced green fluorescent protein (EGFP) expression vector into 7404-CP20 cells resulted in a nonfilamentous actin-EGFP distribution compared with a normal distribution in the cisplatin-sensitive BEL-7404 cells, suggesting that cytoskeletal organization is disturbed in the CP-r cells. The identification of actin and filamin as [14C]carboplatin-binding proteins and decreased expression and disorganization of several cytoskeletal proteins in CP-r cells provide a molecular and cellular basis for the known defect in endocytosis in these cells.

The multidrug resistance-associated protein (MRP) family plays a major role in the hepatic excretion of organic anions. The expression, localization, and function of Mrp2 (Abcc2), a canalicular multispecific organic anion transport protein, were studied in sandwich-cultured rat hepatocytes. The amount of Mrp2 protein remained constant in sandwich-cultured rat hepatocytes over 4 days in culture, but the molecular mass increased ϳ10 kDa from 190 to 200 kDa. Mrp2 was internalized initially after hepatocyte isolation and was gradually sorted to the canalicular membrane. Disposition of 5-(6)-carboxy-2Ј,7Ј-dichlorofluorescein (CDF), an Mrp2 substrate, confirmed the changes in Mrp2 localization. CDF was localized predominantly inside hepatocytes at day 0 and gradually localized to the canalicular domain over time in culture. By day 4 in culture, CDF was localized exclusively in the canalicular networks. Tunicamycin, an inhibitor of glycosylation, decreased the molecular mass and simultaneously impaired the trafficking of Mrp2 to the canalicular membrane. Treatment of lysates from both day 0 (Mrp2, 190 kDa) and day 4 (Mrp2, 200 kDa) sandwichcultured rat hepatocytes with peptide N-glycosidase F, a deglycosylation agent, resulted in a band of 180 kDa, suggesting that Mrp2 from both day 0 and day 4 was glycosylated, but Mrp2 on day 4 was more glycosylated than on day 0. In conclusion, these data support the hypothesis that glycosylation of Mrp2 is responsible for the increase in molecular mass and may be involved in directing the canalicular localization of Mrp2 in sandwich-cultured rat hepatocytes over days in culture.