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

  • Toxicokinetics and Metabolism
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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.

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Abbreviations

ABCG2:

ATP-binding cassette subfamily G member 2

DAPI:

4′-6-Diamidino-2-phenylindole

ER:

Efflux ratio

FTC:

Fumitremorgin C

Ko143:

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

NSAID:

Nonsteroidal anti-inflammatory drug

MEM:

Minimum essential medium

MDCKII:

Madin-Darby canine kidney epithelial cells

P app :

Apparent permeability coefficient

PhIP:

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine

SDS:

Sodiumdodecylsulfate

TEER:

Transepithelial electrical resistance

ZO-1:

Tight-junction-associated zona occludens 1 protein

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Acknowledgments

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.

Author information

Authors and Affiliations

  1. Faculty of Veterinary Medicine, Institute of Pharmacology, Pharmacy and Toxicology, Universität Leipzig, Leipzig, Germany

    Louise Wassermann, Sandra Halwachs, Daniela Baumann & Walther Honscha

  2. Molecular Cell Therapy, Faculty of Medicine, Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany

    Ingo Schaefer & Peter Seibel

Authors
  1. Louise Wassermann
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  2. Sandra Halwachs
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  3. Daniela Baumann
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  4. Ingo Schaefer
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  5. Peter Seibel
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  6. Walther Honscha
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Correspondence to Louise Wassermann.

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Wassermann, L., Halwachs, S., Baumann, D. et al. Assessment of ABCG2-mediated transport of xenobiotics across the blood–milk barrier of dairy animals using a new MDCKII in vitro model. Arch Toxicol 87, 1671–1682 (2013). https://doi.org/10.1007/s00204-013-1066-9

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