A Comprehensive Investigation of Dog Cytochrome P450 3A (CYP3A) Reveals a Functional Role of Newly Identified CYP3A98 in Small Intestine.

Dogs are frequently used in drug metabolism studies, and their important drug-metabolizing enzymes, including cytochromes P450 (P450), have been analyzed. In humans, CYP3A4 is an especially important P450 due to its abundance and major roles in liver and intestine. In the present study, dog CYP3A98 and CYP3A99 were identified and characterized, along with previously identified CYP3A12 and CYP3A26. The dog CYP3A cDNAs contained open reading frames of 503 amino acids and shared high sequence identity (78%-80%) with human CYP3As. Among the dog CYP3A mRNAs, CYP3A98 mRNA was expressed most abundantly in small intestine. In contrast, dog CYP3A12 and CYP3A26 mRNAs were expressed in liver, where CYP3A12 mRNA was the most abundant. The four genes had similar gene structures and formed a gene cluster in the dog and human genomes. Metabolic assays of dog CYP3A proteins heterologously expressed in indicated that the dog CYP3As tested were functional enzymes with respect to typical human CYP3A4 substrates. Dog CYP3A98 efficiently catalyzed oxidations of nifedipine, alprazolam, and midazolam, indicating major roles of CYP3A98 in the small intestine. Dog CYP3A12 and CYP3A26 metabolizing nifedipine and/or midazolam would play roles in these reactions in the liver. In contrast, dog CYP3A99 showed minimal mRNA expression and minimal metabolic activity, and its contribution to overall drug metabolism is, therefore, negligible. These results indicated that newly identified dog CYP3A98, a testosterone 6 - and estradiol 16 -hydroxylase, was abundantly expressed in the small intestine and is likely the major CYP3A in the small intestine in combination with liver-specific CYP3A12. SIGNIFICANCE STATEMENT: Novel dog cytochromes P450 3A98 (CYP3A98) and CYP3A99 were identified and characterized to be functional and highly identical to human CYP3A4. Known CYP3A12 and new CYP3A98 efficiently catalyzed estradiol 16α-hydroxylation and midazolam 1'-hydroxylation. CYP3A98 mRNA was expressed in small intestine, whereas CYP3A12 mRNA was predominant in liver. Dog hepatic CYP3A12 and intestinal CYP3A98 are the enzymes likely responsible for the metabolic clearances of orally administered drugs, unlike human CYP3A4/5, which are in both the liver and intestine.