Novel in vitro systems for prediction of veterinary drug residues in ovine milk and dairy products

Abstract

A new in vitro tool was developed for the identification of veterinary substrates of the main drug transporter in the mammary gland. These drugs have a much higher chance of being concentrated into ovine milk and thus should be detectable in dairy products. Complementarily, a cell model for the identification of compounds that can inhibit the secretion of drugs into ovine milk, and thus reduce milk residues, was also generated. The ATP-binding cassette transporter G2 (ABCG2) is responsible for the concentration of its substrates into milk. The need to predict potential drug residues in ruminant milk has prompted the development of in vitro cell models over-expressing ABCG2 for these species to detect veterinary drugs that interact with this transporter. Using these models, several substrates for bovine and caprine ABCG2 have been found, and differences in activity between species have been reported. However, despite being of great toxicological relevance, no suitable in vitro model to predict substrates of ovine ABCG2 was available. New MDCKII and MEF3.8 cell models over-expressing ovine ABCG2 were generated for the identification of substrates and inhibitors of ovine ABCG2. Five widely used veterinary antibiotics (marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin and difloxacin) were discovered as new substrates of ovine ABCG2. These results were confirmed for the bovine transporter and its Y581S variant using previously generated cell models. In addition, the avermectin doramectin was described as a new inhibitor of ruminant ABCG2. This new rapid assay to identify veterinary drugs that can be concentrated into ovine milk will potentially improve detection and monitoring of veterinary drug residues in ovine milk and dairy products.

Graphical Abstract

Keywords:

• ABCG2
• ABC transporters
• active transport
• transcellular transport
• sheep
• veterinary antibiotics

Acknowledgements

The authors thank Dr Alfred Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for kindly supplying the MDCKII and MEF3.8 cells. The authors are grateful to Professor James McCue for assistance in language editing.

Funding

This work was supported by the Spanish Ministry of Science and Innovation, the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund under [grant numbers AGL2009-11730, AGL2012-31116 and a predoctoral fellowship FPU – to L. González-Lobato].