Molecular cloning, expression, and initial characterization of members of the CYP3A family in horses.
The use of performance-enhancing drugs in the horse racing industry combined with the need for more rational approaches in the use of therapeutic agents in equids necessitates additional studies on the spectrum, content, and catalytic activities of hepatic cytochrome P450 monooxygenases in this species. In this study, three cytochrome P450 (P450) monooxygenases in the 3A family were cloned from, sequenced, and expressed in a baculovirus expression system. The proteins were designated CYP3A89, CYP3A96, and CYP3A97. Expression studies produced various results among the three proteins. CYP3A89 appears to undergo post-translational modification, producing a truncated protein, and although metabolically active, CYP3A97 did not have a detectable P450 spectrum. Expression of CYP3A96 produced a full-length, catalytically active protein. CYP3A96 catalyzed testosterone, and nifedipine metabolism was 20- and 10-fold slower, respectively, compared with the human counterpart, CYP3A4. Relative hepatic expression levels of each member of the CYP3A family, determined using quantitative reverse transcription-polymerase chain reaction, varied more than 1000-fold in individual horses. The results demonstrate substantial interspecies variability in metabolism of substrates by members of the CYP3A family in the horse and human and support the need to fully characterize 450-mediated metabolism in equids. These studies provide a framework for screening therapeutically useful drugs and provide a method for determination of metabolites of illegal performance-enhancing drugs without the time and expense of either in vivo studies or obtaining liver samples for in vitro analysis.
Knych
Heather K DiMaio
HK
California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California-Davis, West Health Science Drive, Davis, CA 95616, USA. hkknych@ucdavis.edu
McKemie
Daniel S
DS
Stanley
Scott D
SD
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006736:Horses; D006801:Humans; D066298:In Vitro Techniques; D007313:Insecta; D007527:Isoenzymes; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009543:Nifedipine / Q000378:metabolism / Q000493:pharmacokinetics; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D013045:Species Specificity; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism / Q000493:pharmacokinetics; D019155:Veterinary Drugs / Q000378:metabolism / Q000493:pharmacokinetics*
D007527:Isoenzymes; D019155:Veterinary Drugs; D013739:Testosterone; D051544:Cytochrome P-450 CYP3A; D009543:Nifedipine