Carcinogen and Anticancer Drug Transport by Mrp2 in Vivo: Studies UsingMrp2(Abcc2) Knockout Mice

Abstract

The ATP-binding-cassette (ABC) transporter multidrug resistance protein (MRP) 2 (ABCC2) forms a natural barrier and efflux system for various (conjugates of) drugs, other xenotoxins, and endogenous compounds. To obtain insight in the pharmacological and physiological functions of Mrp2, we generated Mrp2 knockout mice, which were viable and fertile but suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin monoglucuronides into bile. The mice also had an 80-fold decreased biliary glutathione excretion and a 63% reduced bile flow. Levels of Mrp3 (Abcc3) in liver and Mrp4 The multidrug transporter MRP2 (ABCC2, cMOAT), a member of the ATP-binding-cassette (ABC) superfamily, confers resistance to a range of anticancer drugs (Borst and Oude Elferink, 2002;Chan et al., 2004). The protein is mainly present in the apical membranes of polarized cells in liver, small intestine, and kidney and mediates active transport of both endogenous and xenobiotic compounds to bile, urine, or feces (Chan et al., 2004). MRP2 is functionally deficient in patients with the Dubin-Johnson syndrome (Zimniak, 1993), in TR Ϫ rats (Jansen et al., 1985), and Eisai hyperbilirubinemic rats (EHBRs) (Hosokawa et al., 1992), which all show impaired secretion of bilirubin glucuronides into the bile and as a consequence suffer from conjugated hyperbilirubinemia (Jansen et al., 1985; Hosokawa et al., 1992;Zimniak, 1993). The mutant rat strains also show substantially reduced biliary excretion of glutathione and glutathione conjugates (Paulusma et al., 1999).Besides its role in transport of endogenous compounds, MRP2 plays an important role in the transport of various 1 These authors contributed equally to this work. Article, publication date, and citation information can be found at

The apical conjugate efflux pump ABCC2 (MRP2)

“…Two distinct naturally occurring sequence variants in the rat Abcc2 gene, either at codon 401 (GY/ TR − ) [133] or at codon 855 (EHBR) [60], lead to premature stop codons and to a lack of the Abcc2 protein from the hepatocyte canalicular membrane [13,114,133]. Recently, Abcc2-knock-out mice have been generated and characterized by several groups [18,121,177]. These Abcc2-deficient mice are apparently healthy and fertile, as are As recommended by the Human Genome Variation Society (http://www.hgvs.org/mutnomen) and by ref [26], nucleotide position +1 is the A of the ATG of the translation initiation codon in the ABCC2 cDNA sequence, "c." describes a nucleotide change in relation to the ABCC2 cDNA sequence, "p." describes a change in relation to the deduced ABCC2 protein sequence, and "X" denotes a premature stop codon b If not indicated otherwise, the diagnosis of Dubin-Johnson syndrome was based on the histopathology of the liver and on elevated serum levels of conjugated bilirubin c PolyPhen online tool for assessing potential effects of amino acid substitutions, http://genetics.bwh.harvard.edu/pph d Single nucleotide polymorphism database, http://www.ncbi.nlm.nih.gov/SNP; data based on NCBI dbSNP build 125, regular updates available e Identified in cell lines derived from Japanese individuals f Probably reduced ABCC2 mRNA levels due to nonsense-mediated mRNA decay [167] the Abcc2-deficient rats.…”

“…Today, many sequence variants in the ABCC2 gene have been identified, as described below, but only some of them cause DubinJohnson syndrome. The hereditary deficiency of ABCC2 in humans, or of Abcc2 in the mutant rats mentioned above, and the recently generated Abcc2 knock-out mouse strains [18,121,177] illustrate the key function of this apical efflux pump in the elimination of anionic conjugates from the body. It should be noted, however, that this loss of ABCC2 function is usually well tolerated and compensated by the upregulation of other membrane transporters, particularly of ABCC3 in the basolateral membrane of hepatocytes [30,82,91].…”

Bcrp1;Mdr1a/b;Mrp2 Combination Knockout Mice: Altered Disposition of the Dietary Carcinogen PhIP (2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine) and Its Genotoxic Metabolites

“…Besides endogenous compounds and a wide range of drugs, the dietary heterocyclic amine carcinogens PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline) are also substrates for ABC transporters in vitro and in vivo (Dietrich et al, 2001a,b;van Herwaarden et al, 2003van Herwaarden et al, , 2006Leslie et al, 2005;Vlaming et al, 2006;Enokizono et al, 2008). PhIP is the most abundant heterocyclic amine in fried or cooked meat, chicken, and fish.…”

“…Using Bcrp1-or Mrp2-deficient mice and rats, it was previously shown that Bcrp1 and Mrp2 significantly influence the pharmacokinetics of [ 14 C]PhIP in vivo (Dietrich et al, 2001b;van Herwaarden et al, 2003van Herwaarden et al, , 2006Vlaming et al, 2006). However, since in several studies only radioactivity was measured, no differentiation between parent PhIP and its mutagenic metabolites could be made.…”

“…To investigate the overlapping or complementary roles of Bcrp1, Mdr1a/b, Mrp2, and Mrp3 in vivo, we generated a set of compound knockout mice, deficient in up to three of these ABC transporters (Schinkel et al, 1997;Jonker et al, 2002;Vlaming et al, 2006Vlaming et al, , 2008Vlaming et al, , 2009a. These strains proved to be useful tools for pharmacokinetic studies.…”