Porcine CYP2A polymorphisms and activity.
CYP2A6 in man catalyzes the oxidation of nicotine-forming cotinine and 7-hydroxylation of coumarin, which is used as test substrate for CYP2A6 in man. Large interindividual differences are found in man and some are due to genetic polymorphism. The 7-hydroxylation of coumarin is present in pigs, and an inter-individual variation has been found that might be due to polymorphisms. To enable the finding of polymorphism in pigs, the minipig cDNA was sequenced. Two cDNAs were found and translated to a 494 and a 487 amino acid long protein, both cDNAs were found in all but one pig. The 494 a.a. protein showed high homology to the human and 100% homology to the conventional pig CYP2A19 protein. In the wild type protein, all 6 substrate recognition sites were found, whereas the short protein only contained the first 5 substrate recognition sites. SSCP analysis revealed 3 polymorphisms. In order to study the effect of these polymorphisms on enzyme activity, microsomes were incubated with nicotine and coumarin. The polymorphisms appeared to have no effect on either enzyme activity as the specific enzyme activity towards nicotine and coumarin were approximately the same for all pigs. The specificity of pig CYP2A was investigated and it was found that the formation of cotinine correlated with the immunochemical level of CYP2A as did the coumarin hydroxylation. Anti-human CYP2A inhibitory antibody inhibited coumarin 7-hydroxylation by about 90% and formation of cotinine by 44--60% and 85--100% at substrate concentrations of 500 microM and 50 microM respectively, showing that coumarin and nicotine (50 microM) are very specific substrates for CYP2A in pigs, whereas the CYP2A only is responsible for about 50% of the cotinine formation at a 500 microM nicotine incubation concentration. These results show that the large interindividual differences in porcine CYP2A activity are not caused by polymorphisms but transcriptional regulation and the coumarin 7-hydroxylation is as specific a reaction for porcine CYP2A as for human CYP2A6.
Department of Veterinary Pathobiology, Laboratory of Toxicology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark. mts@kvl.dk
Mette T
MT
Skaanild
Christian
C
Friis
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D065637:Cytochrome P-450 CYP2A6; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009538:Nicotine / Q000378:metabolism; D010084:Oxidation-Reduction; D011110:Polymorphism, Genetic / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D013552:Swine; D013556:Swine, Miniature
D018076:DNA, Complementary; D009538:Nicotine; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6