Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Abstract

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) (ϳ100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (ϳ0.2 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 (ϳ0.2 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.

Characterization of CYP2B6 in a CYP2B6-Humanized Mouse Model: Inducibility in the Liver by Phenobarbital and Dexamethasone and Role in Nicotine Metabolism In Vivo

“…Furthermore, in the lung, where the other two transgenes, CYP2A13 and CYP2F1, are expressed (Wei et al, 2012), the expression of CYP2A13 and CYP2F1 was not increased by PB or DEX.…”

“…The final melting curve analysis was carried out at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The following primers were used: GAPDH, forward 59-tgtgaacggatttggccgta-39 and reverse 59-tcgctcctggaagatggtga-39 (Wei et al, 2012); CYP2B6, forward 59-cattcttccggggattatggtg-39 and reverse 59-cctcatagtggtcacagagaatcg-39 (Rencurel et al, 2005); CYP3A11, forward 59-ggatgagatcgatgaggctctg-39 and reverse 59-caggtattccatctccatcacagt-39; CYP2B10, forward 59-caggtgatcggctcacacc-39 and reverse 59-tgactgcatctgagtatggcatt-39 (Pan et al, 2000).…”

“…Genotypic analysis for the TG mouse and the Cyp2abfgs-null mouse was performed as described (Wei et al, 2012;Li et al, 2014). Microsomes were prepared as described previously (Ding and Coon, 1990).…”

“…We recently reported the generation and initial characterization of a CYP2A13/2B6/2F1-transgenic (TG) mouse model (Wei et al, 2012). In this mouse model, whereas the CYP2A13 and CYP2F1 genes are expressed mainly in the respiratory tract, the CYP2B6 transgene was mainly expressed in the liver.…”

Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice

“…The final melting curve analysis was carried out at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The following primers were used: glyceraldehyde 3-phosphate dehydrogenase, forward 59-tgtgaacggatttggccgta-39 and reverse 59-tcgctcctggaagatggtga-39 (Wei et al, 2012); CYP3A11, forward 59-ggatgagatcgatgaggctctg-39 and reverse: 59-caggtattccatctccatcacagt-39; CYP2B10, forward 59-caggtgatcggctcacacc-39 and reverse: 59-tgactgcatctgagtatggcatt-39 (Pan et al, 2000); CYP2A5, forward 59-TCTGTTGCTCATGAAGTACC-39 and reverse 59-TTGTCATCTAGGAAGT-GCTT-39; and CYP2C29, forward 59-GGGCTCAAAGCCTACTGTCA-39 and reverse 59-AACGCCAAAACCTTTAA-39 (Zhang et al, 2003).…”