CYP3As catalyze nifedipine oxidation in pig liver microsomes: Enzyme kinetics, inhibition and functional expression

doi:10.1016/j.catcom.2010.12.031

Catalysis Communications

Volume 12, Issue 8, 31 March 2011, Pages 694-697

https://doi.org/10.1016/j.catcom.2010.12.031 Get rights and content

Abstract

Cytochrome P450 (CYP) 3As have received much attention because of their importance in drug metabolism. However, information on CYP3As in pigs is still largely incomplete. Herein, we focused on the involvement of CYP3As in nifedipine oxidation in pig liver microsomes. Enzyme kinetics, inhibition and immunochemical studies indicated that CYP3As might catalyze nifedipine oxidation and that ketoconazole could strongly inhibit this reaction in a noncompetitive and time-dependent manner. Furthermore, pig CYP3A46, showing the highest amino acid sequence identity to human CYP3A4, exhibited nifedipine oxidation activity. Collectively, these results provide strong evidence that CYP3As catalyze nifedipine oxidation in pig liver microsomes.

Graphical Abstract

Research Highlights

►Ketoconazole noncompetitively and time-dependently inhibited nifedipine oxidation in pig liver microsomes. ►Recombinant CYP3A46 exhibited nifedipine oxidation activity. ►CYP3As catalyze nifedipine oxidation in pig liver microsomes.

Introduction

Cytochrome P450 (CYP) enzymes represent a large superfamily of membrane bound monooxygenases, which are widely distributed and highly conserved from bacteria to mammals. These enzymes catalyze the synthesis and metabolism of numerous compounds from the biotransformation of natural endogenous compounds, such as steroids and cholesterol, to the detoxification of many exogenous compounds, including drugs and environmental pollutants [1], [2]. In humans, CYP3As are the most abundant CYP enzymes in the liver and can metabolize a wide range of pharmacologically, physiologically and toxicologically important agents [3].

In contrast to the extensive data available for human and rodent CYP3A enzymes, information on CYP3As in pigs is still incomplete despite the presence of enzyme activities known to be CYP3As-associated in other species [4], [5], [6], [7], [8]. The few investigations conducted by research groups are difficult to compare, as the breed, methods and model substrates used differed substantially. Additionally, the substrates and inhibitors were selected on the basis of the available knowledge from rodent or human studies, although there is no direct evidence indicating that pig CYP3As show the same catalytic specificities as rodent and human CYP3As [5]. Moreover, enzyme kinetic or enzyme inhibition kinetic parameters of pig CYP3As, such as K_m and V_max values, have been determined only in a very few studies [5], [7]. Defining the contribution of a CYP isoform in the metabolism of a specific drug is important not only for veterinary pharmacology and toxicology, but also for human health because of the possible presence of residues in animal products.

Nifedipine is a vasodilator and calcium agonist that has been widely used in patients with arterial hypertension, angina and cardiac arrhythmias [9]. In humans, CYP3A4 is involved in the oxidation of nifedipine [10]. Nifedipine oxidation activity is also detectable in pig liver microsomes, which had a good correlation with the apoprotein level measured using anti-human CYP3A in an immunoblotting assay [8]. Among the four isoforms of CYP3As found in pigs (CYP3A22, CYP3A29, CYP3A39 and CYP3A46), CYP3A46 shows the highest amino acid sequence identity to human CYP3A4 [11]. However, whether this CYP isoenzyme is involved in the oxidation of nifedipine in pig liver microsomes is still unclear.

In this study, nifedipine oxidase activity and kinetic parameters were first determined in pig liver microsomes. Inhibition profile was then determined with the chemical inhibitor ketoconazole. Finally, pig CYP3A46 was confirmed to display nifedipine-metabolizing activity through functional expression in vitro. Together, our results demonstrate that CYP3As are involved in nifedipine oxidation in pig liver microsomes. The present study deepens our understanding of CYP3As in pigs, which may provide valuable knowledge for veterinary medicine.

Section snippets

Chemicals, reagents and plasmids

Nifedipine, oxidized nifedipine, ketoconazole and NADPH were purchased from Sigma. All other chemicals and reagents used were of analytical grade.

The open reading frame (ORF) region of pig CYP3A46 (NM_001134824) was cloned and inserted into Not I/Hind III sites of pcDNA™3.1/myc-His(−)A (Invitrogen) vector for eukaryotic expression.

Animals and preparation of pig liver microsomes

Danish Landrace × Yorkshire × Duroc cross-breed pigs (4–5 months old) were purchased from the College of Veterinary Medicine at South China Agricultural University (SCAU).

Results and discussion

Using pig liver microsomes, enzyme kinetic parameters of nifedipine oxidase and inhibition kinetic parameters of ketoconazole on nifedipine oxidation were systematically studied. The formation of oxidized nifedipine was observed to follow simple Michaelis-Menten kinetics (Fig. 1A). Moreover, the nifedipine oxidase activity measured was similar to that previously described in human microsomes [17], accounting to 97.5 ± 5.5 μM (mean ± standard error) for K_m and 1.173 ± 0.0187 nmol min^− 1 mg^− 1 protein (mean ±

Conclusion

In summary, our results provide strong evidence that pig CYP3As catalyze nifedipine oxidation, which have similar characteristics to humans with regard to enzyme activity, inhibition profile and immunochemical characteristics. The present study hopes to deepen our understanding of CYP3As in pigs, which may provide valuable knowledge for veterinary medicine.

The following is the supplementary material related to this article.

. Western blot detection of CYP3A in pig liver microsomes. Thirty

Acknowledgements

This work was supported by the National Basic Research Program of China (973 Program), No. 2009CB118802; the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2009); the Program for New Century Excellent Talents in University, No. NCET-08-0643; the Guangdong Province Universities and Colleges Special Funds for Talents Introduction (2008); and the China Postdoctoral Science Foundation, No. 20100470930.

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Short communicationCYP3As catalyze nifedipine oxidation in pig liver microsomes: Enzyme kinetics, inhibition and functional expression

Abstract

Graphical Abstract

Research Highlights

Introduction

Section snippets

Chemicals, reagents and plasmids

Animals and preparation of pig liver microsomes

Results and discussion

Conclusion

Acknowledgements

Biochem. Biophys. Res. Commun.

Eur. J. Pharmacol.

J. Biol. Chem.

Anal. Biochem.

Mol. Immunol.

Catal. Commun.

Toxicol. Lett.

Am. J. Med.

Drug Metab. Pharmacokinet.