Assessment of ABCG2-mediated transport of xenobiotics across the blood–milk barrier of dairy animals using a new MDCKII in vitro model
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The ATP-binding cassette (ABC) efflux transporter ABCG2 represents the main route for active secretion of drugs and toxins across the blood–milk barrier, thereby producing a potential health risk for dairy consumers through formation of relevant residues in milk. However, no suitable in vitro model is as yet available to systematically investigate ABCG2-mediated transport of xenobiotics into milk of dairy animals. We recently cloned ABCG2 from the lactating mammary gland of dairy cows (bABCG2) and goats (cABCG2). Thus, the objective of this study was to generate a suitable blood–milk barrier in vitro model using polarized MDCKII monolayers stably expressing mammary bABCG2 or cABCG2. ABCG2 protein was localized by confocal microscopy to the apical and lateral plasma membrane of polarized MDCKII cells. Intact barrier function of MDCKII-bABCG2 and MDCKII-cABCG2 monolayers was confirmed by determination of cell permeability of transcellular marker propranolol and paracellular marker atenolol which was ≤1 %. In flux assays, ABCG2 substrate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) showed preferential basolateral to apical (B > A) transport in ABCG2-MDCKII cells. This apically directed PhIP transport was significantly inhibited by ABCG2 inhibitor fumitremorgin C (FTC) or the flavonoid equol. PhIP B > A transport in MDCKII-bABCG2 monolayers was additionally decreased by ABCG2 inhibitor Ko143. The fluoroquinolone antibiotic enrofloxacin was identified as a substrate of ruminant mammary ABCG2. The analgesic drug sodium salicylate was shown to be substrate of bABCG2 but not of cABCG2. Thus, the generated mammary ABCG2-expressing MDCKII cells represent a valuable tool to study active secretion of drugs and toxins into milk.
KeywordsABCG2/BCRP Transport Blood–milk barrier Drugs Dairy animals MDCKII cells
ATP-binding cassette subfamily G member 2
3-(3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12 a-octahydro-pyrazino [1,2:1,6] pyrido[3,4-b] indol-3-l)-propionic acid tert-butyl ester
Nonsteroidal anti-inflammatory drug
Minimum essential medium
Madin-Darby canine kidney epithelial cells
Apparent permeability coefficient
Transepithelial electrical resistance
Tight-junction-associated zona occludens 1 protein
We kindly thank Pablo Steinberg (Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany) for donating PhIP.
Conflict of interest
The authors declare that they have no conflict of interest.
- Allen JD, van Loevezijn A, Lakhai JM, van der Valk M, van Tellingen O, Reid G, Schellens JH, Koomen GJ, Schinkel AH (2002) Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 1:417–425PubMedCrossRefGoogle Scholar
- Lindner S, Halwachs S, Wassermann L, Honscha W (2013) Expression and subcellular localization of efflux transporter ABCG2/BCRP in important tissue barriers of lactating dairy cows, sheep and goats. J Vet Pharmacol Ther (in press)Google Scholar
- Maubach J, Bracke ME, Heyerick A, Depypere HT, Serreyn RF, Mareel MM, De Keukeleire D (2003) Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 784:137–144PubMedCrossRefGoogle Scholar
- Pavek P, Merino G, Wagenaar E, Bolscher E, Novotna M, Jonker JW, Schinkel AH (2005) Human breast cancer resistance protein: interactions with steroid drugs, hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine. J Pharmacol Exp Ther 312:144–152PubMedCrossRefGoogle Scholar