Impact of Abcc2 (Mrp2) and Abcc3 (Mrp3) on the In vivo Elimination of Methotrexate and its Main Toxic Metabolite 7-hydroxymethotrexate

Abstract

Purpose: ATP-binding cassette sub-family C member 2 [ABCC2; multidrug resistanceâ ssociated protein 2 (MRP2)] and ABCC3 (MRP3) mediate the elimination of toxic compounds, such as drugs and carcinogens, and have a large overlap in substrate specificity.We investigated the roles of Abcc2 and Abcc3 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) in vivo. Experimental Design: Abcc2;Abcc3 -/-mice were generated, characterized, and used to investigate possibly overlapping or complementary roles of Abcc2 and Abcc3 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Results: Abcc2;Abcc3 -/-mice were viable and fertile. In Abcc2 -/-mice, the plasma area under the curve (AUC i.v. ) for MTX was 2.0-fold increased compared with wild type, leading to 1.6-fold increased urinary excretion, which was not seen in Abcc2;Abcc3 -/-mice. Biliary excretion of MTX was 3.7-fold reduced in Abcc2 -/-but unchanged in Abcc2;Abcc3 -/-mice. The plasma AUC i.v. s of 7OH-MTX were 6.0-fold and 4.3-fold increased in Abcc2 -/-and Abcc2;Abcc3 -/-mice, respectively, leading to increased urinary excretion. The biliary excretion of 7OH-MTX was 5.8-fold reduced in Abcc2 -/-but unchanged in Abcc2;Abcc3 -/-mice. 7OH-MTX accumulated substantially in the liver of Abcc2 -/-and especiallyAbcc2;Abcc3 -/-mice. Conclusions: Abcc2 is important for (biliary) excretion of MTX and its toxic metabolite 7OH-MTX.When Abcc2 is absent, Abcc3 transports MTX and 7OH-MTX back from the liver into the circulation, leading to increased plasma levels and urinary excretion. Variation in ABCC2 and/or ABCC3 activity may therefore have profound effects on the elimination and severity of toxicity of MTX and 7OH-MTX after MTX treatment of patients.The multidrug resistance proteins (MRP) ATP-binding cassette sub-family C member 2 (ABCC2; MRP2) and ABCC3 (MRP3) are members of the ATP-binding cassette (ABC) transporter super-family. ABCC2 is present in the apical membranes of hepatocytes and the epithelial cells of the small intestine and kidney, and is involved in the elimination of both endogenous and exogenous compounds from the body (1). ABCC3 is also found in the liver, kidney, and small intestine as well as in the adrenal glands and pancreas. In contrast to ABCC2, ABCC3 localizes to the basolateral membrane of polarized cells (2). Both transporters are expressed in various tumors and can transport a range of (anticancer) drugs (1, 2). There is a large overlap in the substrate specificity of ABCC2 and ABCC3. They can, for example, both transport bilirubin glucuronides (3 -5) and, in patients with the Dubin-Johnson syndrome, who have functionally deficient ABCC2 (6, 7), both plasma bilirubin glucuronide levels and ABCC3 protein levels are increased. It has been speculated that these two findings are related: ABCC3 up-regulation in the absence of ABCC2 would allow increased basolateral efflux of bilirubin glucuronides from the liver (7). Because Abcc2 and Abcc3 are both invol...

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

“…Cyp3a11 was also virtually undetectable in wild-type and Bcrp1;Mrp2;Mrp3 2/2 kidneys and increased 64-fold in Bcrp1;Mdr1a/b;Mrp2 2/2 kidneys, but it was still very low compared with expression levels in liver tissue (Supplemental Table 1). None of the other tested genes was significantly changed, with the exception of a 12-fold increase in Mrp4 RNA (P , 0.05) in Bcrp1;Mdr1a/b; Mrp2 2/2 kidneys, in line with what was found before for Mrp4 protein levels in kidney of other Mrp2-deficient strains (Vlaming et al, , 2008(Vlaming et al, , 2009a. Overall, changes in gene expression in these strains were remarkably limited given their genetic deficiencies in major detoxifying transporters.…”

“…Despite the increased liver size, detailed microscopic analysis of liver sections did not reveal obvious pathologic changes in either strain. An increase (somewhat smaller) in liver weight was previously seen in other Mrp2-deficient mouse strains (Vlaming et al, , 2008(Vlaming et al, , 2009aTian et al, 2008).…”

“…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.…”

Organic anion transporting polypeptide 1a/1b–knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs

“…Hepatic glucuronidation by Ugt1a1 is likely the most important detoxification pathway for bilirubin in mice, although it has to be noted that in humans gastrointestinal glucuronidation of bilirubin might also play a (minor) role (17). Bilirubin glucuronides formed within the liver can be transported into bile by Abcc2, Abcg2, and some other, as yet unidentified, canalicular transporter(s) (18,19) or, under pathological conditions (cholestasis), back into the circulation by Abcc3 (15,20). Our results lead us to hypothesize the unexpected existence of a substantial and, at first sight, seemingly futile cycling of bilirubin glucuronides in normal, healthy liver, in which bilirubin conjugated in the liver is substantially secreted across the sinusoidal membrane by Abcc3.…”

“…In humans and mice, urinary excretion is the prominent route of ultimate MTX elimination, although in mice still approximately 20% of the MTX is excreted unchanged in the feces (15). We collected urine and feces of WT and Slco1a/1b -/-mice for 24 hours after i.v.…”