Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes Using Label-Free Standard-Free Analysis Reveals Interbreed Variability

• Published in: Drug metabolism and disposition 2019-11, Vol.47 (11), p.1314-1324
• Main Authors: Martinez, Stephanie E, Shi, Jian, Zhu, Hao-Jie, Perez Jimenez, Tania E, Zhu, Zhaohui, Court, Michael H
• Format: Article
• Language: English
• Subjects: Animals; Breeding; CYP1A2 protein; Cytochrome; Cytochrome b5; Cytochrome P-450 Enzyme System - analysis; Cytochrome P-450 Enzyme System - genetics; Cytochrome P450; Cytochromes P450; Dogs; Dogs - metabolism; Enzymes; Estimates; Female; Genotype; Genotypes; Greyhounds; Liver; Male; Microsomes; Microsomes, Liver - enzymology; Models, Biological; Mutation; Oxidoreductase; Proteins; Proteomics; Quantitation; Shotguns; Species Specificity; Stop codon; Variance analysis
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.119.088070

Dogs are commonly used in human and veterinary pharmaceutical development. Physiologically based pharmacokinetic modeling using recombinant cytochrome P450 (CYP) enzymes requires accurate estimates of CYP abundance, particularly in liver. However, such estimates are currently available for only seven CYPs, which were determined in a limited number of livers from one dog breed (beagle). In this study, we used a label-free shotgun proteomics method to quantitate 11 CYPs (including four CYPs not previously measured), cytochrome P450 oxidoreductase, and cytochrome b5 in liver microsomes from 59 dogs representing four different breeds and mixed-breed dogs. Validation included showing correlation with CYP marker activities, immunoquantified protein, as well as CYP1A2 and CYP2C41 null allele genotypes. Abundance values largely agreed with those previously published. Average CYP abundance was highest (>120 pmol/mg protein) for CYP2D15 and CYP3A12; intermediate (40-89 pmol/mg) for CYP1A2, CYP2B11, CYP2E1, and CYP2C21; and lowest (