Multiple Mechanisms Are Involved in the Biliary Excretion of Acetaminophen Sulfate in the Rat: Role of Mrp2 and Bcrp1

Abstract

ABSTRACT:Previous reports have demonstrated that sulfate metabolites may be excreted into bile by the multidrug resistance-associated protein 2 (Mrp2, Abcc2). Although recombinant human breast cancer resistance protein (BCRP, ABCG2) has affinity for sulfated xenobiotics and endobiotics, its relative importance in biliary excretion of sulfate metabolites in the intact liver is unknown. In the present studies, the potential contribution of Bcrp1 to the biliary excretion of acetaminophen sulfate (AS) was examined following acetaminophen administration (66 mol, bolus) to isolated perfused livers ; TR؊ and wild-type, respectively). In vitro assays indicated that impaired AS biliary excretion in the presence of GF120918 was due to inhibition of Bcrp1, and not P-glycoprotein. In conclusion, Mrp2 and, to a lesser extent, Bcrp1 mediate biliary excretion of AS in the intact liver.Hepatic sulfation of xenobiotics is a common phase II metabolic reaction that increases hydrophilicity of the molecule prior to biliary excretion or efflux across the hepatic basolateral membrane for subsequent renal clearance. Xenobiotic inactivation and/or detoxification may occur by direct sulfation of the parent compound (e.g., acetaminophen) or may follow phase I oxidation (e.g., phenobarbital). However, hepatic xenobiotic sulfation also can lead to activation of prodrugs [e.g., minoxidil (Buhl et al., 1990)] and hepatotoxins [e.g., covalent microsomal protein binding of phenacetin sulfate (Mulder et al., 1977); formation of DNA adducts with tamoxifen sulfate (Glatt et al., 1998); potent bile salt export pump inhibition by troglitazone sulfate leading to cholestasis (Funk et al., 2001)]. Considering that sulfated xenobiotics may exhibit pharmacologic or toxicologic activity, perturbations in transport mechanisms responsible for excretion of sulfate conjugates may have important therapeutic and toxic implications.Previous studies have demonstrated that the canalicular multispecific organic anion transporter, multidrug resistance-associated protein 2 (Mrp2, Abcc2), is responsible for the biliary excretion of glucuronide and glutathione conjugates of xenobiotics, but that it only partially mediates biliary excretion of sulfate metabolites. Transport of the sulfate metabolite of the dual inhibitor of 5-lipooxygenase and thromboxane A 2 synthase, E3040, was largely maintained in canalicular liver plasma membrane vesicles (ϳ75% of wild-type) and isolated perfused livers [(IPLs) comparable to wild-type] from Mrp2-deficient rats, whereas translocation of E3040 glucuronide across the canalicular membrane was negligible in the absence of Mrp2 (Takenaka et al., 1995). Cumulative biliary excretion of phenobarbital sulfate in IPLs from Mrp2-deficient rats was ϳ30% of that observed in livers from wild-type rats, whereas biliary excretion of the glucuronide conjugate was not observed in the absence of Mrp2 . Similarly, biliary excretion of phenolphthalein disulfate, but not glucuronide, was partially (ϳ15% of wild-type) maintained in rats lacking Mrp2 (Og...

The Role of Canalicular ABC Transporters in Cholestasis

“…Its endogenous substrates include tetrahydroxylated bile acids (Megaraj et al, 2010), divalent bile acids dmd.aspetjournals.org (Kuipers et al, 1988), glutathione (Oude Elferink et al, 1990), bilirubin glucuronosides (Paulusma et al, 1997), eicosanoids (prostaglandin E2, leukotriene C 4 ) , and conjugated steroids [estrone 3-sulfate (Kopplow et al, 2005), estradiol-17b-glucuronate ]. Exogenous ABCC2 substrates are mostly conjugated, either with glucuronic acid [e.g., phytoestrogens (Krumpochova et al, 2012), acetaminophen (Xiong et al, 2000), indomethacin (Kouzuki et al, 2000), morphine (van de Wetering et al, 2007)], sulfuric acid [e.g., acetaminophen (Zamek-Gliszczynski et al, 2005), resveratrol (Kaldas et al, 2003)], or with glutathione [e.g., acetaminophen (Chen et al, 2003a), bromosulfophthalein (Jansen et al, 1987), dinitrophenyl (Elferink et al, 1989)]. However, ABCC2 also transports unconjugated anionic drugs, such as pravastatin (Yamazaki et al, 1997), ampicillin (Verkade et al, 1990), and methotrexate (Hooijberg et al, 1999).…”

Efflux Transporter Expression and Acetaminophen Metabolite Excretion Are Altered in Rodent Models of Nonalcoholic Fatty Liver Disease

“…In vivo disposition studies and in vitro functional transport experiments indicate that Mrp2, Mrp3, Mrp4, and Bcrp each have the ability to transport a variety of unconjugated and conjugated drugs, including APAP metabolites (Büchler et al, 1996;Xiong et al, 2000;Nakanishi et al, 2003;Zamek-Gliszczynski et al, 2005. It is important to emphasize the distinctive location of these four transporters in hepatocytes, as well as their respective APAP metabolite substrates.…”

“…Mrp2 and Bcrp are localized to the canalicular (apical) membrane of hepatocytes from which they excrete their substrates into the bile canaliculi. Accordingly, in a healthy liver, biliary excretion of the SULF, GLUC, and GSH conjugates of APAP is predominantly mediated by Mrp2, whereas Bcrp appears also to contribute to excretion of APAP-SULF conjugates (Büchler et al, 1996;König, 1997, 2000;Borst et al, 2000;Chen et al, 2003;Zamek-Gliszczynski et al, 2005). Mrp3 and Mrp4 are expressed at the sinusoidal (basolateral) membrane of hepatocytes and cholangiocytes from which they expel their substrates into the blood (König et al, 1999;Donner and Keppler, 2001;Soroka et al, 2001).…”

Efflux Transport Is an Important Determinant of Ethinylestradiol Glucuronide and Ethinylestradiol Sulfate Pharmacokinetics

“…However, TR Ϫ rats and the Mrp2-and Bcrp-knockout mice used in the studies presented here were thoroughly characterized with respect to their glucuronidation, sulfation, sulfatase, and uptake and efflux transport properties at the protein expression and functional levels. Similar or modestly increased hepatic glucuronidation and sulfation activities have been demonstrated with various substrates in TR Ϫ rats, although increased UDP-glucuronosyltransferase (no change in intestine, no change to Ͻ4-fold in liver, and 5-fold in kidney) and sulfotransferase (Ͻ2-fold) protein expression has been reported in TR Ϫ rats (Xiong et al, 2000;Zamek-Gliszczynski et al, 2005, 2006aJohnson et al, 2006). Glucuronidation and sulfation activities are notably not decreased in TR Ϫ rats.…”