CHARACTERIZATION OF THE CYTOCHROME-P-450 MONOOXYGENASE SYSTEM IN NONCILIATED BRONCHIOLAR EPITHELIAL (CLARA) CELLS ISOLATED FROM MOUSE LUNG

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in futher defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immumocytochemistry of isolated mouse cells showed that the majority were positive with antibbodies against three mahor components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 usizymes 2 (HB), 5 (IVB), and NADPH cytochrome P-450 redutase, purified from rabbit lung. The isolated cells also showed a positive reacion with an antibody against the cytochrome P-450 isozyme that is active in the steroselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry usingk the antibody against cytochrome P-450 isozyme 6 (IAI), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gell electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demithylation of benzphjetamine and expoxidation of naphthalend occured at easily mesurble rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in exacts of Clara cells incubated with H-3-labeled substrate. We conclude: 1) cells isolated by this method resemble Clara cells untrastructurally and immunocytochemically, 2) the cells retain cytochrome P-450 monooxygenase activity, 3) the isolation procedure does not degrade proteins fo the cytochrome P-450 enzyme system 4) isolated mouse Clara cell preparations lack the cytochrome P-450 isozyme (IAI) primarily responsible for the metabolism of benzo(a)pyrene, and 5) the isolated cells are capable of metabolizing a cytoxicant for which they are the principal target.