Assessment of ABCG2-mediated transport of xenobiotics across the blood–milk barrier of dairy animals using a new MDCKII in vitro model

Abstract

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.

Novelin vitrosystems for prediction of veterinary drug residues in ovine milk and dairy products

“…Despite being a highly conserved transporter (Wu et al 2008), there is an increasing number of studies that describe differences in function and specificity of ABCG2 in different species (Schnepf & Zolk 2013;Wassermann, Halwachs, Baumann, et al 2013). In vitro cell models have been generated for the identification of ABCG2 substrates using transcellular transport mediated by the caprine and bovine ABCG2 and its Y581S variant Wassermann, Halwachs, Baumann, et al 2013).…”

“…Our previous in vitro models stably expressing the wild-type bovine ABCG2 and its Y581S variant showed an increased transcellular transport of drugs by this polymorphism . Assessment of caprine ABCG2-mediated transport of drugs has been recently reported, including species differences in drug transport (Wassermann, Halwachs, Baumann, et al 2013). In the case of ovine ABCG2, several drug interactions with this transporter using the Hoescht accumulation assay have been shown (Wassermann, Halwachs, Lindner, et al 2013).…”

A model of secreting murine mammary epithelial HC11 cells comprising endogenous Bcrp/Abcg2 expression and function

“…Madine-Darby Canine Kidney II (MDCK II), human intestinal epithelial cells (Caco-2), human breast adenocarcinoma cells (MCF7), human choriocarcinoma cells (BeWo), and human bone osteosarcoma cells (SAOS-2) constitute examples of models used to characterize the function and substrate specificities of efflux proteins such as BCRP (Doyle et al 1998;Jonker et al 2000;Ceckova et al 2006;Matsson et al 2007;Li et al 2011;Wassermann et al 2013). However, cancer cell lines and non-tumorigenic cell lines deriving from tissues other than the alveolar lining of the mammary glands harbor different sets of transporter protein panels at both the apical and basolateral membranes which may affect secretion, and net accumulation of substrates may therefore differ (Krajci 2013).…”

“…Transfected Madin-Darby Canine Kidney II (MDCKII) epithelial cells stably expressing human, bovine, and caprine BCRP have been used to identify new BCRP substrates as well as the impact of the common Y581S point mutation in BCRP function (Jonker et al 2000;Real et al 2011;Wassermann et al 2013;Halwachs et al 2014). However, expression and function of endogenous BCRP in non-tumorigenic mammary epithelial cell lines have not yet been examined.…”

In vitro systems: current limitations and future perspectives

“…A particularly high number of in vitro studies focus on hepatotoxicity (e.g., Hadi et al 2013;Schaap et al 2012;Burkhardt et al 2012). Goals of these studies are to improve differentiated functions by 3D cultivation conditions (Messner et al 2013;Schyschka et al 2013) to study metabolites (Sierra-Santoyo et al 2012;Watzek et al 2013;Abdelhamid et al 2013;Tolosa et al 2013) and drug transporters hepatocytes (Wassermann et al 2013). Moreover, zebrafish-based test systems have been studied as an alternative method to identify hepatotoxic compounds (Driessen et al 2013;Scholz 2013).…”