Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

Orsolya Palócz, Orsolya Farkas, Paul Clayton, György Csikó

Abstract

As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h) cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital) and 2 inhibitors (alpha-naphthoflavone and ketoconazole). In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in vivo. CYP1A1 activity was decreased by ketoconazole in vitro and increased in vivo. This is the first report of the inducer effect of ketoconazole on rabbit cytochrome isoenzymes in vivo. Our data support the use of a luciferin-based assay in short-term primary hepatocytes as an appropriate tool for xenobiotic metabolism assays and short-term toxicity testing in rabbits.

 


Keywords

CYP450; hepatocyte; ketoconazole; luminescence; rabbit

Full Text:

PDF

References

Beigel J., Fella K., Kramer P.J., Kroeger M., Hewitt P. 2008. Genomics and proteomics analysis of cultured primary rat hepatocytes. Toxicol. in Vitro, 22: 171-181. https://doi.org/10.1016/j.tiv.2007.06.019

Boek-Dohalská L., Hodek P., Šulc M., Stiborová M. 2001. α-Naphthoflavone acts as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6. Chem-Biol. Interact., 138: 85-106. https://doi.org/10.1016/S0009-2797(01)00263-0

Cali J.J., Ma D., Sobol M., Simpson D.J., Frackman S., Good T.D., Daily W.J., Liu D. 2006. Luminogenic cytochrome P450 assays. Expert Opin. Drug Met., 2: 629-645. https://doi.org/10.1517/17425255.2.4.629

Cho U.S., Park E.Y., Dong M.S., Park B.S., Kim K. Kim K.H. 2003. Tight-binding inhibition by α-naphthoflavone of human cytochrome P450 1A2. BBA-Proteins Proteom., 1648: 195-202. https://doi.org/10.1016/S1570-9639(03)00148-1

Eeckhoutte C., Albo A.G., Carletti M., Giaccherino A.R., Galtier P., Nebbia C., Dacasto M. 2002. Time-dependent variations of drug-metabolising enzyme activities (DMEs) in primary cultures of rabbit hepatocytes. Toxicol. in Vitro, 16: 375-382. https://doi.org/10.1016/S0887-2333(02)00018-8

Ekroos M., Sjögren T. 2006. Structural basis for ligand promiscuity in cytochrome P450 3A4. In Proc.: National Academy of Sciences of the United States of America, 103: 13682-13687. https://doi.org/10.1073/pnas.0603236103

Elsherbiny M.E., El-Kadi A.O., Brocks D.R. 2008. The metabolism of amiodarone by various CYP isoenzymes of human and rat, and the inhibitory influence of ketoconazole. J. Pharm. Pharm. Sci., 11: 147-159. https://doi.org/10.18433/J3SG66

Fink-Gremmels J. 2008. Implications of hepatic cytochrome P450-related biotransformation processes in veterinary sciences. Eur. J. Pharmacol., 585: 502-509. https://doi.org/10.1016/j.ejphar.2008.03.013

Franklin M.R. 1995. Enhanced rates of cytochrome P450 metabolic-intermediate complex formation from nonmacrolide amines in rifampicin-treated rabbit liver microsomes. Drug Metab. Dispos., 23: 1379-1382.

Guillouzo A. 1998. Liver cell models in in vitro toxicology. Environ. Health Perspect., 106: 511-532. https://doi.org/10.2307/3433803

Korashy H.M., Shayeganpour A., Brocks D.R., El-Kadi A.O.S. 2007. Induction of cytochrome P450 1A1 by ketoconazole and itraconazole but not fluconazole in murine and human hepatoma cell lines. Toxicol. Sci., 97: 32-43. https://doi.org/10.1093/toxsci/kfm012

Lahoz A., Donato M.T., Castell J.V., Gomez-Lechon M.J. 2008. Strategies to in vitro assessment of major human CYP enzyme activities by using liquid chromatography tandem mass spectrometry. Curr. Drug Metab., 9: 12-19. https://doi.org/10.2174/138920008783331112

Lan S.F., Safiejko-Mroczka B., Starly B. 2010. Long-term cultivation of HepG2 liver cells encapsulated in alginate hydrogels: A study of cell viability, morphology and drug metabolism. Toxicol. in Vitro, 24: 1314-1323. https://doi.org/10.1016/j.tiv.2010.02.015

LeCluyse E.L. 2001. Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. European J. Pharma. Sci., 13: 343-368. https://doi.org/10.1016/S0928-0987(01)00135-X

Lee J.T., Pao L.H., Hsiong C.H., Huang P.W., Shih T.Y., Yoa-Pu Hu O. 2013 Validated liquid chromatography–tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDPglucuronosyltransferases. Talanta, 106: 220-228. https://doi.org/10.1016/j.talanta.2012.12.023

Masters B.S.S., Williams Jr. C.H., Kamin H. 1967. [92] The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. Method. Enzimol., 10: 565-573. https://doi.org/10.1016/0076-6879(67)10098-0

Moeller T.A., Ma D., Dennel S., Cali J.J. 2011. Comparison of novel luciferin-1A2 substrate and phenacetin for measuring CYP1A2 induction in human hepatocytes. Drug Metabol. Rev., 43: 158-158.

Moore L.B., Maglich J.M., McKee D.D., Wisely B., Willson T.M., Kliewer S.A., Lambert M.H., Moore J.T. 2002. Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Mol. Endocrinol., 16: 977-986. https://doi.org/10.1210/mend.16.5.0828

Mueller D., Krämer L., Hoffmann E., Klein S., Noor F. 2014. 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicol. in Vitro, 28: 104-112. https://doi.org/10.1016/j.tiv.2013.06.024

Nash T. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J., 55: 416-421. https://doi.org/10.1042/bj0550416

Nebbia C., Ceppa L., Dacasto M., Nachtmann C., Carletti M. 2001. Oxidative monensin metabolism and cytochrome P450 3A content and functions in liver microsomes from horses, pigs, broiler chicks, cattle and rats. J. Vet. Pharmacol. Ther., 24: 399-403. https://doi.org/10.1046/j.1365-2885.2001.00362.x

Nebbia C., Dacasto M., Rossetto Giaccherino A., Giuliano Albo A., Carletti M. 2003. Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species. Vet. J., 165: 53-64. https://doi.org/10.1016/S1090-0233(02)00174-0

Neunzig I., Drăgan C.A., Widjaja M., Schwaninger A.E., Peters F.T., Maurer H.H., Bureik M. 2011. Whole-cell biotransformation assay for investigation of the human drug metabolizing enzyme CYP3A7. BBA-Proteins Proteom., 1814: 161-167. https://doi.org/10.1016/j.bbapap.2010.07.011

Noel G., Le Vee M., Moreau A., Stieger B., Parmentier Y., Fardel O. 2013. Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes. Eur. J. Pharm. Sci., 49: 39-50. https://doi.org/10.1016/j.ejps.2013.01.013

Novotna A., Krasulova K., Bartonkova I., Korhonova M., Bachleda P., Anzenbacher P., Dvorak Z. 2014. Dual effects of ketoconazole cis-enantiomers on CYP3A4 in human hepatocytes and HepG2 Cells. PLoS One, 9: e111286. https://doi.org/10.1371/journal.pone.0111286

Ohno M., Motojima K., Okano T., Taniguchi A. 2009. Induction of drug-metabolizing enzymes by phenobarbital in layered co-culture of a human liver cell line and endothelial cells. Biol. Pharm. Bull., 32: 813-817. https://doi.org/10.1248/bpb.32.813

Paine A.J. 1996. Validity and reliability of in vitro systems in safety evaluation. Environ. Toxicol. Pharmacol., 2: 207-212. https://doi.org/10.1016/S1382-6689(96)00057-9

Paine M.F., Schmiedlin-Ren P., Watkins P.B. 1999. Cytochrome P-450 1A1 expression in human small bowel: interindividual variation and inhibition by ketoconazole. Drug Metab. Dispos., 27: 360-364.

Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T.L. 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13: 134-144. https://doi.org/10.1186/1471-2105-13-134

Yueh M.F., Kawahara M., Raucy J. 2005. Cell-based highthroughput bioassays to assess induction and inhibition of CYP1A enzymes. Toxicology in Vitro, 19: 275-287. https://doi.org/10.1016/j.tiv.2004.10.003

Zlokarnik G., Grootenhuis P.D.J., Watson J.B. 2005. High throughput P450 inhibition screens in early drug discovery. Drug Discov. Today, 10: 1443-1450. https://doi.org/10.1016/S1359-6446(05)03580-4

Abstract Views

1727
Metrics Loading ...

Metrics powered by PLOS ALM


 

Cited-By (articles included in Crossref)

This journal is a Crossref Cited-by Linking member. This list shows the references that citing the article automatically, if there are. For more information about the system please visit Crossref site

1. Alteration of avian hepatic cytochrome P450 gene expression and activity by certain feed additives
Orsolya Palócz, Géza Szita, György Csikó
Acta Veterinaria Hungarica  vol: 67  issue: 3  first page: 418  year: 2019  
doi: 10.1556/004.2019.042



 

 Universitat Politècnica de València

 

Official journal of the World Rabbit Science Association (WRSA)

 

e-ISSN: 1989-8886     ISSN: 1257-5011   https://doi.org/10.4995/wrs