HEPATOBILIARY EXCRETION OF ACETAMINOPHEN GLUTATHIONE CONJUGATE AND ITS DERIVATIVES IN TRANSPORT-DEFICIENT (TR^-) HYPERBILIRUBINEMIC RATS

Abstract

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., ...

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

“…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).…”

“…In rats, excretion of APAP-GLUC and APAP-GSH metabolites from the liver occurs predominantly via the biliary route, whereas the APAP-SULF metabolite predominantly undergoes sinusoidal efflux and subsequent elimination in the urine (Gregus et al, 1988). Importantly, all APAP metabolites require efflux transport to be excreted from the liver, and each can be detected in both bile and urine (Gregus et al, 1988;Xiong et al, 2002;Chen et al, 2003;Manautou et al, 2005;Zamek-Gliszczynski et al, 2006a).…”

Systems Biology Investigation to Discover Metabolic Biomarkers of Acetaminophen-Induced Hepatic Injury Using Integrated Transcriptomics and Metabolomics

“…Taken together, the results indicated that bile acids may play important roles in energy expenditure through regulating the mobility/storage of free fatty acids. The up-regulation of the expression of ATP-binding cassette genes Mrp2 and Mrp3 (also named as Abcc2 and Abcc3) may also be an attempt by the hepatocytes to transport residual APAP metabolites into the bile and portal blood [33,34]. APAP-GSH, APAP-NAC and APAP-Glu conjugates are substrates of MRP2 and MRP3 [33,34].…”

“…The up-regulation of the expression of ATP-binding cassette genes Mrp2 and Mrp3 (also named as Abcc2 and Abcc3) may also be an attempt by the hepatocytes to transport residual APAP metabolites into the bile and portal blood [33,34]. APAP-GSH, APAP-NAC and APAP-Glu conjugates are substrates of MRP2 and MRP3 [33,34]. These results are consistent with a xenobiotic detoxification metabolism wherein the expressions of Mrp and Mdr transporters are increased to facilitate the detoxification process [35][36][37].…”