Nasal cytochrome P450 2A: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen.

Two members of the cytochrome P450 2A subfamily, CYP2A10 and 2A11, are abundant nasal enzymes previously characterized in rabbit olfactory microsomes. Rabbit CYP2A is active toward a number of nasal toxicants, including the rat nasal procarcinogen hexamethylphosphoramide (HMPA). While P450s immunochemically related to the rabbit CYP2As have been detected in rat and human nasal mucosa, confirmation of these enzymes as members of the CYP2A subfamily and efforts to characterize their ability to bioactivate toxicants have been limited. In the present study, the regional distribution and cell-specific expression of CYP2A in the rat nasal cavity were examined using an antibody to rabbit CYP2A10/11. In sections of the anterior nose, immunoreactive CYP2A was present in ciliated cells of the nasal respiratory epithelium and cuboidal epithelial cells of the nasal transitional epithelium, but was absent in squamous epithelial cells. The most intense immunostaining was observed in the posterior nose. Olfactory sustentacular cells and Bowman's gland cells in sections posterior to the nasal papilla stained most intensely. Western blot analysis revealed that anti-CYP2A10/11 recognized a sharp band of approximately 50 kDa in nasal respiratory and olfactory microsomes, supporting the premise that the antibody is reacting with a cytochrome P450 enzyme. The nasal expression of CYP2A6 mRNA--a member of the human CYP2A subfamily having a high degree of homology to rabbit 2A10 and 2A11--was examined in human surgical patients. Middle turbinectomy tissues--largely composed of nasal respiratory epithelia--from 11 patients were analyzed for the presence of CYP2A6 using reverse transcription-polymerase chain reaction (RT-PCR). Identification of CYP2A6 was confirmed by DNA sequencing of RT-PCR products. CYP2A6 mRNA was detected in all of the human samples analyzed. In additional experiments, human CYP2A6 metabolized HMPA to formaldehyde, suggesting that this compound might cause nasal toxicity in humans. The identification of CYP2A cytochromes in rat and human nasal tissues may have important implications for risk assessment of inhaled xenobiotics.

Baculovirus-mediated expression and characterization of rat CYP2A3 and human CYP2a6: role in metabolic activation of nasal toxicants.

Cytochrome P450 2A3 (CYP2A3) was previously identified in rat lung by cDNA cloning and recently found to be expressed at a high level in the olfactory mucosa. In the current study, CYP2A3 was expressed in insect cells lacking endogenous cytochrome P450 (P450) activity, and the substrate specificity of the recombinant cytochrome was characterized and compared with that of CYP2A6, a human ortholog of rat CYP2A3, which has been detected in human olfactory mucosa as well as in liver. The CYP2A3 and CYP2A6 cDNAs were cloned into baculovirus, and recombinant viruses were used to produce active enzymes in Spodoptera frugiperta (SF9) cells. The metabolic activities of S. frugiperta cell microsomal fractions containing CYP2A3 or CYP2A6 were studied in a reconstituted system with purified rabbit NADPH-P450 reductase. CYP2A3 was found to be active toward testosterone, producing 15 alpha-hydroxytestosterone and several other metabolites, but it had only low activity toward coumarin. On the other hand, CYP2A6 was active toward coumarin but not toward testosterone. However, both enzymes were active in the metabolic activation of hexamethylphosphoramide, a nasal procarcinogen, and 2,6-dichlorobenzonitrile (DCBN), a herbicide known to cause tissue-specific toxicity in the olfactory mucosa of rodents at very low doses. In addition, both enzymes were active toward 4-nitrophenol, a preferred substrate for CYP2E1. Consistent with CYP2A3 being a major catalyst in microsomal metabolism of DCBN, the activities of both CYP2A3 and rat olfactory microsomes in DCBN metabolism were inhibited strongly by metyrapone and methoxsalen (ID50 < 1 microM, with DCBN at 30 microM), but only marginally by 4-methylpyrazole, an inhibitor of CYP2E1. In contrast, the activity of CYP2A6 was only weakly inhibited by metyrapone or methoxsalen (ID50 > 50 microM). Thus, rat CYP2A3 and human CYP2A6 have differences in substrate specificity as well as tissue distributor. These findings should be taken into account when assessing the risk of exposure to potential nasal toxicants in humans.