Supplementary Material for: Differential Distribution of CYP2A6 and CYP2A13 in the Human Respiratory Tract

Background: Human CYP2A6 and CYP2A13 play important roles in metabolic activation of many pulmonary carcinogens and thus their expression and distribution may determine the pulmonary susceptibility to metabolically activated carcinogens and the following lung cancer development. Because of the 93.5% of amino acid identity between CYP2A6 and CYP2A13, generation of antibodies specific to CYP2A6 or CYP2A13 has limited immunohistochemical (IHC) analysis of CYP2A6 and CYP2A13 levels in the respiratory tract. Objectives: This study aimed to determine the differential distribution of CYP2A6 and CYP2A13 in human respiratory tissue with IHC analysis. Methods: With computer-aided protein sequence analyses, candidate epitopes of 15 amino acids in the C-terminal domains of CYP2A6 and CYP2A13 were selected for antibody generation. Specificity of these two antibodies was confirmed with immunoblot and immunofluorescence analyses. With these two selective antibodies, the differential distribution of CYP2A6 and CYP2A13 in human respiratory tissues, including tracheae, bronchi, bronchioles and alveoli, was determined. Results: IHC results showed that both CYP2A6 and CYP2A13 were markedly expressed in epithelial cells of tracheae and bronchi and that only CYP2A6 was detected in bronchiolar epithelial cells of peripheral lungs. A limitation of the present study is the cross-reactivity of our CYP2A6 antibody to the functional inactive CYP2A7. Conclusions: The differential distribution patterns of CYP2A6 and CYP2A13 in the respiratory tract are of importance in considering the pulmonary susceptibility to carcinogens and the following lung cancer development.