Evidence for the catalysis of Dextromethorphan O-demethylation by a CYP2D6-like enzyme in pig liver

Abstract

The pig is increasingly being used in pharmacological and toxicological studies, and is the species of choice for future research into xenotransplantation, extracorporeal liver support and hepatocyte-based bioartificial liver. However, relatively little is known about xenobiotic-metabolizing enzymes in this species. In the present study, immunoblotting with polyclonal anti-rat and anti-human cytochrome P450 (CYP) antibodies revealed the presence of proteins in pig liver which cross-reacted with anti-human CYP1A2, CYP2D6 and CYP3A4, and with anti-rat CYP2E1 antibodies. Northern blot analysis demonstrated the presence of mRNA which hybridized to cDNA probes for human CYP2D6, CYP2E1 and CYP3A4, and to an oligonucleotide probe for pig CYP3A29. As there is a lack of a good animal model for CYP2D6, the presence of a CYP2D6-related protein in pig liver was of particular interest. Pig hepatocytes also demonstrated CYP2D6 immunoreactive protein, and mRNA hybridizable to a CYP2D6 cDNA probe. We investigated the ability of pig liver microsomes to catalyse dextromethorphan O-demethylation, a widely-used marker enzyme activity for CYP2D6. This enzyme activity demonstrated biphasic kinetics, with a high affinity apparent K m1 =6.9±3.6 μ m and V max1 =10.5±6.1 nmol/min/nmol CYP. The reaction was sensitive to inhibition by the CYP2D6-selective inhibitors quinidine, quinine, lobeline and norfluoxetine, whereas chemical inhibitors selective for other CYP isoforms failed to affect the reaction. We conclude that dextromethorphan O-demethylation is catalysed by a CYP2D enzyme which is remarkably similar to human CYP2D6, suggesting potential value of the pig as a model for predicting human metabolism of xenobiotics which undergo CYP2D6-dependent biotransformation.