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Generation and characterization of a CYP2A13/2B6/2F1-transgenic mouse model
1Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509, USA.
Abstract:
CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (∼100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (∼0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10- and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (∼0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.
Insights
A new transgenic mouse model expressing human cytochrome P450 genes CYP2A13, CYP2B6, and CYP2F1 was created. This model accurately reflects human gene expression patterns and shows functional activity, aiding research into drug metabolism and toxicity.
Area of Science:
- Pharmacogenomics
- Toxicology
- Biotechnology
Background:
- Cytochrome P450 enzymes CYP2A13, CYP2B6, and CYP2F1 are crucial for metabolizing drugs, toxicants, and carcinogens.
- These genes are located adjacently on human chromosome 19, posing challenges for studying their individual functions and regulation.
- A need exists for a reliable animal model to investigate the roles of these specific human P450 enzymes.
Purpose of the Study:
- To develop a transgenic mouse model expressing human CYP2A13, CYP2B6, and CYP2F1 genes.
- To characterize the tissue distribution and protein expression of these human transgenes in mice.
- To assess the functional activity of the expressed human P450 enzymes in the transgenic model.
Main Methods:
- Generation of CYP2A13/2B6/2F1-transgenic (TG) mice carrying all *1 alleles.
- Analysis of mRNA expression and protein detection via immunoblotting in various tissues.
- Assessment of enzyme activity using in vitro and in vivo bioactivation assays with a known CYP2A13 substrate.
Main Results:
- Transgenic mice exhibited normal development and fertility.
- Tissue distribution of transgene mRNA mirrored human expression patterns: respiratory tract for CYP2A13/CYP2F1 and liver for CYP2B6.
- Functional CYP2A13 activity was confirmed by enhanced bioactivation of its substrate in the nasal mucosa of TG mice compared to wild-type.
Conclusions:
- The developed CYP2A13/2B6/2F1 transgenic mouse model successfully recapitulates human gene expression and function.
- This model provides a valuable tool for studying the roles of these specific human P450 enzymes in drug metabolism, toxicity, and carcinogen activation.
- The findings support the utility of this model for future research in respiratory toxicology and pharmacogenomics.
