The multidrug transporters breast cancer resistance protein (BCRP), multidrug-resistance protein 1 (MDR1), and multidrugresistance-associated protein (MRP) 2 and 3 eliminate toxic compounds from tissues and the body and affect the pharmacokinetics of many drugs and other potentially toxic compounds. The food-derived carcinogen PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine) is transported by BCRP, MDR1, and MRP2. To investigate the overlapping functions of Bcrp1, Mdr1a/b, and Mrp2 in vivo, we generated Bcrp1;Mdr1a/b;Mrp2 2/2 mice, which are viable and fertile. These mice, together with Bcrp1;Mrp2;Mrp3 2/2 mice, were used to study the effects of the multidrug transporters on the pharmacokinetics of PhIP and its metabolites. Thirty minutes after oral or intravenous administration of PhIP (1 mg/kg), the PhIP levels in the small intestine were reduced 4-to 6-fold in Bcrp1;Mdr1a/b;Mrp2 2/ 2 and Bcrp1;Mrp2;Mrp3 2/2 mice compared with wild-type mice. Fecal excretion of PhIP was reduced 8-to 20-fold in knockouts. Biliary PhIP excretion was reduced 41-fold in Bcrp1;Mdr1a/b;Mrp2 2/2 mice. Biliary and small intestine levels of PhIP metabolites were reduced in Bcrp1;Mrp2-deficient mice. Furthermore, in both knockout strains, kidney levels and urinary excretion of genotoxic PhIPmetabolites were significantly increased, suggesting that reduced biliary excretion of PhIP and PhIP metabolites leads to increased urinary excretion of these metabolites and increased systemic exposure. Bcrp1 and Mdr1a limited PhIP brain accumulation. In Bcrp1;Mrp2;Mrp3 2/2 , but not Bcrp1; Mdr1a/b;Mrp 2/2 mice, the carcinogenic metabolites N2-OHPhIP (2-hydroxyamino-1-methyl-6-phenylimidazo [4,5-b]pyridine) and PhIP-5-sulfate (a genotoxicity marker) accumulated in liver tissue, indicating that Mrp3 is involved in the sinusoidal secretion of these compounds. We conclude that Bcrp1, Mdr1a/b, Mrp2, and Mrp3 significantly affect tissue disposition and biliary and fecal elimination of PhIP and its carcinogenic metabolites and may affect PhIP-induced carcinogenesis as a result.

BackgroundHepatic membrane-embedded proteins are involved in trafficking endogenous and exogenous compounds and may influence the pharmacokinetics of drugs. Transporter-specific age-related changes in pro-tein abundance were found in a pilot study (n=24), but now we aimed to elucidate the exact developmental pat-tern of clinically relevant hepatic transporters in a larger cohort of 63 fetuses, preterm and term neonates and in-fants and compare it with adults.MethodsProtein expression of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1-3, NTCP, OCT1, OATP1B1, OATP1B3, and OATP2B1 was quantified using UPLC-MS/MS, on snap-fro-zen post mortem fetal and infant liver samples and adult surgical liver samples. Protein expression was quantified in isolated crude membrane fractions. Pairwise compar-ison Kruskal-Wallis test was used to analyse a possible age-related difference.ResultsThirty-six fetal [median GA 23.4 weeks (range 15.3–41.3), no PNA], 12 premature neonatal [GA 30.2 weeks (24.9–36.7), PNA 1.0 weeks (0.14–11.4)], 11 term neonatal [GA 40.0 weeks (39.7–41.3), PNA 4.14 weeks (0.29-18.1)], 4 paediatric [PNA 4.13 years (1.08–7.44)] and 8 adult liver samples were studied. Expressions of BCRP, MCT1, OATP1B3, and OATP2B1 were similar in all age groups. MDR1, MRP1, MRP2, MRP3 and OCT1 expressions were low in fetus and high in adults (all p<0.05). Expression of BSEP increased from fetal to term newborn and to adult age (both p<0.01) and of NCTP increased over the whole age range (all p<0.05). GLUT1 and OATP1B1 expressions were high in fetuses and decreased towards newborns age (both p<0.01). GLUT1 expression decreased further in children’s and adult age (both p<0.05).ConclusionThese data further delineate transporter specific changes in protein abundance across the first months of age.