Gene expression and immunochemical localization of major cytochrome P450 drug-metabolizing enzymes in bovine nasal olfactory and respiratory mucosa

Abstract

Despite tremendous advancement in the characterization of nasal enzyme expression, knowledge of the role of the nasal mucosa in the metabolism of xenobiotics is still inadequate, primarily due to the limited availability of in vitro models for nasal metabolism screening studies. An extensive knowledge of the oxidative and conjugative metabolizing capacity of the cattle (Bos taurus) olfactory and respiratory mucosa can aid in efficient use of these tissues for pre-clinical investigations of the biotransformation and toxicity of therapeutic agents following nasal administration or inhalation. Cows are also exposed to a variety of airborne pollutants and pesticides during their lifetime, the metabolism of which can have profound toxicological and ecological consequences. The aim of the present study was to characterize cytochrome P450 (CYP) enzyme expression in the bovine nasal mucosa. Amplification of the specific genes through RT-PCR confirmed expression of several CYP enzymes in bovine hepatic and nasal tissues. The results demonstrate that bovine nasal olfactory and respiratory mucosal and liver tissues express similar populations, families, and distributions of CYP enzymes, as has been previously reported with other species, including humans. Bovine ex vivo tissues can serve as a readily available reference tissue to elucidate preclinical toxico-kinetic effects resulting from exposure to substances in the environment or following drug administration.

Keywords: Cytochrome P450; Real Time-PCR; gene expression; immunohistochemistry; metabolism; nasal mucosa.

Figure 1

Gel electrophoretic analysis of RT-PCR products obtained during amplification of the selected CYP genes from bovine hepatic, olfactory, and respiratory tissues extracted from six different cows.

Figure 2

Gene expression levels of the selected CYP enzymes normalized with respect to GAPDH expression in bovine hepatic, olfactory, and respiratory tissues analyzed using real time RT-PCR. Results are presented as mean ± standard deviation for 6 animals. Error bars represent standard deviations. * represents statistically significant difference in the enzyme expression levels for liver vs. the nasal (olfactory or respiratory) mucosa at p<0.05. No statistically significant differences between the mRNA expression levels were observed between the olfactory and the respiratory mucosa for any of the enzymes studied.

Figure 3

Gel electrophoretic analysis of RT-PCR products from amplification of CYP1A2 gene in bovine hepatic, olfactory, and respiratory tissues. Amplification of the CYP1A2 gene from mRNA extracted from several bovine respiratory and olfactory mucosal tissues resulted in multiple PCR products. Lanes showing multiple bands following amplification are highlighted with ovals.

Figure 4

Gene expression levels of the CYP1A2-like gene normalized with respect to GAPDH expression in bovine hepatic, olfactory, and respiratory tissues analyzed using real time RT-PCR. Real time PCR monitoring of one of six olfactory tissues and four of six respiratory tissues resulted in an indeterminate C_T value for the desired transcript of CYP1A2.

Figure 5

Brightfield image of a formalin-fixed, paraffin-embedded, and hematoxylin and eosin stained section (5 μm) of bovine olfactory mucosa. Scale bar: 200 μm.

Figure 6

Brightfield image of a formalin-fixed, paraffin-embedded, and hematoxylin and eosin stained section (5 μm) of bovine respiratory mucosa. Scale bar: 200 μm.

Figure 7

Immunohistochemical staining of CYP1A2 (A), CYP2A6 (B–C), CYP2C (D), and CYP3A4 (E) in bovine olfactory mucosa. A representative negative control image is shown in F. Green fluorescence indicates positive immunoreactivity; red coloration indicates nuclei (To-Pro3). Scale bar: 20 μm.

Figure 8

Immunohistochemical staining of CYP1A2 (A), CYP2A6 (B–C), CYP2C (D), and CYP3A4 (E) in bovine respiratory mucosa. A representative negative control image is shown in F. Green fluorescence indicates positive immunoreactivity; red coloration indicates nuclei (To-Pro3). Scale bar: 20 μm.