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Evaluation of the hepatocyte-derived cell line BFH12 as an in vitro model for bovine biotransformation

  • Alexander Gleich
  • Bastian Kaiser
  • Walther Honscha
  • Herbert Fuhrmann
  • Axel SchoenigerEmail author
Original Article
  • 29 Downloads

Abstract

The knowledge of drug metabolising enzymes (DMEs) in cattle is rather limited. The capability of the bovine foetal hepatocyte-derived cell line BFH12 to serve as model for biotransformation was evaluated. Gene expression analysis of DMEs was performed by reverse transcription PCR (RT-PCR). The presence of efflux transporters was visualised by immunocytochemistry, and functional induction of cytochrome P450 (CYP) 1A was assessed by the ethoxyresorufin-O-deethylase (EROD) assay. The production of bile acids was measured by liquid chromatography-tandem mass spectrometry (LC–MS/MS). RT-PCR revealed the expression of cytochromes 1A1, 1A2, 3A4 and phase II enzymes UGT1A1, UGT1A6 and GSTM1. Immunofluorescence demonstrated efflux transporters ABCG2 and ABCC1. The EROD assay revealed a dose-dependent CYP1A induction after treatment with benzo[a]pyrene (BP). LC–MS/MS analysis of cell culture supernatants showed the production of bile acids including taurocholic acid, tauro-chenodeoxycholic acid, taurodeoxycholic acid and taurolithocholic acid. The results strongly suggest the applicability of the cell line BFH12 for subsequent experiments in the emerging field of bovine biotransformation.

Keywords

Biotransformation Cytochrome P450 Bovine hepatocyte Cattle In vitro model Bile acid 

Abbreviations

ABCC1

ATP-binding cassette sub-family C member 1

ABCG2

ATP-binding cassette sub-family G member 2

ACTB

β-actin

AhR

aryl hydrocarbon receptor

ANOVA

Analysis of variance

ARNT

AhR nuclear translocator

BP

benzo[a]pyrene

cDNA

complementary DNA

Cy2

Cyanine

Cy3

Indocarbocyanine

CYP

Cytochrome P450

CYP1A1

Cytochrome P450 1A1

CYP1A2

Cytochrome P450 1A2

CYP2B6

Cytochrome P450 2B6

CYP2C19

Cytochrome P450 2C19

CYP2C9

Cytochrome P450 2C9

CYP3A4

DAPI, 4′,6-diamidino-2-phenylindole

DMEs

drug metabolising enzymes

EDTA

Ethylenediaminetetraacetic acid

EROD

7-Ethoxy-resorufin-O-deethylase

FBS

Foetal bovine serum

GLUT2

Glucose transporter 2

GSTM1

Glutathione S-Transferase M1

LC/MS–MS

Liquid chromatography tandem mass spectroscopy

MRL

Maximum residue level

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NTCP

Sodium-taurocholate cotransporting polypeptide

OAT

Organic anion transporter

OATP

Organic anion-transporting polypeptide

OATP1B3

Organic anion-transporting polypeptide 1B3

OCT1

Organic cation transporter 1

P-gp

P-glycoprotein

PAH

Polycyclic aromatic hydrocarbons

PCR

Polymerase chain reaction

RT-PCR

Reverse transcription polymerase chain reaction

RT-qPCR

Quantitative reverse transcription PCR

SD

Standard deviation

SDS

Sodium dodecyl sulfate

SLC22A1

Solute Carrier Family 22 Member 1

SLCO1B3

Solute carrier organic anion transporter family member 1B3

SV40LTAg

SV40 large-T antigen

TCA

Taurocholic acid

TCDCA

Taurochenodeoxycholic acid

TDCA

Taurodeoxycholic acid

TLCA

Taurolithocholic acid

UGT1A1

UDP glucuronosyltransferase 1 family polypeptide A1

UGT1A6

UDP glucuronosyltransferase 1 family polypeptide A6

Notes

Acknowledgements

We gratefully thank the group of Professor Michaela Schulz-Siegmund of the Institute of Pharmaceutical Technology (University of Leipzig) for discussions and feedback. Adult liver tissue samples were obtained from the group of Professor Alexander Starke of the Medizinische Tierklinik (University of Leipzig). We also thank the University Hospital Regensburg for conducting LC/MS–MS measurements of bile acids. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10616_2018_279_MOESM1_ESM.docx (814 kb)
Supplementary material 1 (DOCX 814 kb)

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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Institute of BiochemistryUniversity of LeipzigLeipzigGermany
  2. 2.Institute of Veterinary PhysiologyUniversity of LeipzigLeipzigGermany
  3. 3.Institute of Veterinary Pharmacology and ToxicologyUniversity of LeipzigLeipzigGermany

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