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Published on: September 25, 2017
Characterization of mouse small intestinal cytochrome P450 expression
Qing-Yu Zhang1, Debbie Dunbar, Laurence S Kaminsky
1New York State Department of Health, Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509, USA.
Abstract:
The expression of biotransformation enzymes in mouse small intestine is poorly characterized, which limits the utility of transgenic or knockout mouse models for first-pass drug metabolism studies. In response, we have systematically examined the composition and inducibility of cytochrome P450 (P450) protein and mRNA in mouse small intestinal epithelial cells (enterocytes). RNA-PCR was conducted to confirm the expression and identity of CYP1A1, 1B1, 2B10, 2B19, 2B20, 2C29, 2C38, 2C40, 2E1, 3A11, 3A13, 3A16, 3A25, and 3A44 in the enterocytes of untreated mice, but CYP1A2, 2A4/5, 2A12, 2C37, 2C39, and 2F2 were not detected. The inducibility of CYP2B, 2C, and 3A subfamily forms was determined by real-time quantitative RNA-PCR. All five CYP3A forms were induced, in a range from 1.7- to 4.5-fold, by dexamethasone (DEX). Phenobarbital (PB) induced CYP2B9, CYP2B10, and CYP2B20 mRNAs and suppressed CYP2B19 mRNA levels. PB also induced CYP2C29 and CYP2C40, but not CYP2C38 mRNA. At the protein level, CYP1A1, CYP1B1, CYP2B, CYP2C, CYP2E1, and CYP3A were detected in enterocytes from untreated mice by immunoblot analysis. CYP1A1 was inducible by beta-naphthoflavone (BNF), CYP2B and CYP2C by PB, and CYP3A by DEX. CYP2B, 2C, and 3A proteins were all expressed at high levels proximally, and decreased distally. The inducibility of CYP1A1 followed a similar pattern. Intestinal P450 expression was compared between C57BL/6 (B6) and 129/sv (129) mice, strains commonly used in the preparation of transgenic and knockout mouse models. There was no significant strain difference in constitutive levels or induction patterns for CYP2B, 2C, and 3A protein. However, CYP1A1 was induced to a high level by BNF in B6 mice, but was not induced in the 129 mice.
Insights
This study characterizes cytochrome P450 (P450) enzyme expression and induction in mouse small intestines, crucial for drug metabolism research. Key P450s are identified, and their responses to inducers like dexamethasone and phenobarbital are detailed.
Area of Science:
- Pharmacology
- Biochemistry
- Genetics
Background:
- Biotransformation enzyme expression in the mouse small intestine is not well understood.
- This knowledge gap hinders the use of mouse models for studying drug metabolism.
Purpose of the Study:
- To systematically analyze the composition and inducibility of cytochrome P450 (P450) protein and mRNA in mouse enterocytes.
- To compare P450 expression between commonly used mouse strains (C57BL/6 and 129/sv).
Main Methods:
- RNA-PCR and real-time quantitative RNA-PCR were used to identify and quantify P450 mRNA.
- Immunoblot analysis was performed to detect P450 protein levels.
- Enzyme induction was studied using dexamethasone (DEX), phenobarbital (PB), and beta-naphthoflavone (BNF).
Main Results:
- Multiple CYP isoforms (CYP1A1, 1B1, 2B, 2C, 2E1, 3A) were detected in mouse enterocytes.
- Dexamethasone induced all five CYP3A forms, phenobarbital induced specific CYP2B and CYP2C forms, and beta-naphthoflavone induced CYP1A1.
- CYP2B, 2C, and 3A proteins, along with CYP1A1 inducibility, showed proximal high expression decreasing distally.
- No significant strain differences were observed in CYP2B, 2C, and 3A expression or induction between C57BL/6 and 129/sv mice, but CYP1A1 induction by BNF differed.
Conclusions:
- This study provides a comprehensive characterization of P450 expression and induction in mouse small intestinal enterocytes.
- The findings are valuable for selecting appropriate mouse models in drug metabolism research.
- Differences in CYP1A1 inducibility between mouse strains warrant consideration in experimental design.
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