Exploration of functional cytochrome P450 4F enzymes in liver, intestine, and kidney from dogs, cats, pigs, and tree shrews and comparison of their metabolic capacities with human P450 4F2 and 4F12

Abstract

Pigs are often used in drug metabolism studies because of their evolutionary proximity to humans, including similarities in their cytochromes P450 (P450s or CYPs). In the current study, the following cDNAs of novel CYP4Fs were isolated and characterized: dog CYP4F22 and CYP4F140; cat CYP4F22 and CYP4F140; pig CYP4F22, CYP4F52, CYP4F53, CYP4F54, CYP4F56, and CYP4F176; and tree shrew CYP4F22. Previously identified pig CYP4F55 cDNA was also isolated. These CYP4F cDNAs contained open reading frames of 522–531 amino acids and shared high sequence identities (60–92 %) with human CYP4Fs. Dog CYP4F3a and CYP4F3b cDNAs were also identified but lacked the 3′ end of the coding region. Phylogenetic analysis of amino acid sequences showed that these CYP4Fs were clustered in a species-dependent manner, except for CYP4F3, CYP4F22, and CYP4F140, which were clustered in an isoform-dependent manner. All CYP4F genes, containing 12 coding exons, formed a gene cluster at the corresponding location of the genome in each species. Among the tissue samples analyzed, dog and cat CYP4F140 mRNAs were more abundantly expressed in liver/testis and kidney, respectively. Preferential expression of pig CYP4F mRNAs were found in liver, small intestine, and/or kidney, where the most abundant were CYP4F56, CYP4F52, and CYP4F176 mRNAs, respectively. Enzyme assays using recombinant proteins revealed that all these CYP4Fs oxidized the human CYP4F substrate arachidonic acid at the ω-position, indicating that they are functional enzymes. These findings suggest that dog, cat, pig, and tree shrew CYP4Fs have similar functional characteristics to human CYP4Fs.