mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates.
1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S-warfarin (CYP2C9 in humans) by dog liver microsomes (mean +/- SD from 12 (six male and six female) dogs = 10.8 +/- 1.9 fmol mg(-1) protein min(-1)) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4'-hydroxylation of S-mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 +/-1.5 pmol mg(-1) protein min(-1)) compared with human liver. In contrast, the rate of 4'-hydroxylation of the R-enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K(m) or K(0.5) 15-22 micro M, V(max) 35-59 pmol mg(-1) protein min(-1), n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S-mephenytoin, dog liver microsomes 5'-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K(m) 42-64 micro M, V(max) 22-46 pmol mg(-1) protein min(-1), n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmol mg(-1) protein min(-1) (p < 0.02); S-warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmol mg(-1) protein min(-1) (p < 0.02); R-mephenytoin 4'-hydroxylation: female = 16-35, male = 11.5-19 pmol mg(-1) protein min(-1) (p < 0.01); omeprazole 5'-hydroxylation: female = 15-20, male 13-22 pmol mg(-1) protein min(-1) (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4'-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S-warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.
Graham
M J
MJ
Safety Assessment UK, AstraZeneca R&D Charnwood, Loughborough LE11 5RH, UK.
Bell
A R
AR
Crewe
H K
HK
Moorcraft
C L
CL
Walker
L
L
Whittaker
E F
EF
Lennard
M S
MS
D000465:Algorithms; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000378:metabolism*; D003915:Dextromethorphan / Q000031:analogs & derivatives* / Q000378:metabolism; D004285:Dogs; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008617:Mephenytoin / Q000031:analogs & derivatives* / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009853:Omeprazole / Q000031:analogs & derivatives / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis*; D014859:Warfarin / Q000031:analogs & derivatives* / Q000378:metabolism
D007527:Isoenzymes; D012333:RNA, Messenger; C400322:cytochrome P-450 CYP2C subfamily; C028657:3-methoxymorphinan; D014859:Warfarin; C025687:4-hydroxymephenytoin; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; C087837:7-hydroxywarfarin; C475779:cytochrome P-450 CYP3A12 (canine); D009853:Omeprazole; D008617:Mephenytoin