Porcine CYP2A polymorphisms and activity.
ABSTRACT 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.
SourceAvailable from: Galia Zamaratskaia[Show abstract] [Hide abstract]
ABSTRACT: Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint.09/2014; 11(19). DOI:10.1016/j.csbj.2014.09.003
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ABSTRACT: Abstract 1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown. 2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans. 3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences. 4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine. 5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.
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ABSTRACT: Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough-29 intact male pigs at the age of 1 day and 2 weeks and the castrated male pigs at the age of 5, 10, and 20 weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5 weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1’-hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20 weeks were about five times higher than those at the age of 5 weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20 weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development.Journal of Veterinary Pharmacology and Therapeutics 09/2014; 38(2). DOI:10.1111/jvp.12163 · 1.32 Impact Factor