Culturing of primary bovine mammary epithelial cells and validation of biotransformation capacity in experiments with enrofloxacin

Abstract

Many drugs and toxic compounds are subjected to disposition and metabolism in bovine mammary epithelial cells (bMECs). For rapid investigation of different compounds and their possible interactions, validated in vitro models are needed. Therefore, the first objective of described experiments was to develop the techniques for cell isolation, purification and culturing of bMECs. The second objective was the application of these cell cultures in a well-known substrate for one of the major biotransformation enzymes in epithelial cells. To this end, the metabolism of enrofloxacin (ENR) into its active metabolite ciprofloxacin (CPR), was studied. This conversion is known to be catalysed by enzymes of the cytochrome P4501A and P4503A family. The expression profile of these enzymes shows a close correlation with cellular ABC-efflux transporters. Primary bMECs were isolated from healthy udders of lactating cows (n=5 animals). mRNA levels of α-casein, b-lactoferrin and cyclophilin B were determined as markers of cell identity of purity of the cultures. Subsequently, bMECs cultures were incubated with ENR (10 µM). Concentrations of ENR and its main metabolite CPR in the medium and in the cells were determined by HPLC-FL analysis. Gene expression of CYP1A1, CYP1A2 and CYP3A4, bovine ABCG2 was detected by qRT-PCR. Results showed that ENR penetrated into bMECs and was converted to CPR. CPR was excreted in the medium suggesting participation of ABCG2 in fluoroquinolone efflux. In conclusion, the data showed that the established bMEC cultures expressed major CYP450 enzymes as well as the most relevant efflux transport ABGG2. This model should be further validated and can serve as an interesting model for further studies on site-specific drug/toxin metabolism and transport in the bovine mammary gland.