COVALENT INTERACTIONS OF REACTIVE NAPHTHALENE METABOLITES WITH PROTEINS

Naphthalene produces selective necrosis of Clara cells in the mouse bu t not in the rat. The pulmonary toxicity depends on cytochrome P450-me diated metabolism; however, the selective pulmonary toxicity of naphth alene in the mouse does not correspond to tissue-selective covalent bi nding of reactive naphthalene metabolites in vivo. These studies compa re reactive metabolite binding in target and nontarget cells and in va rious subcompartments of mouse lung and characterize, by sodium dodecy l sulfate polyacrylamide gel electrophoresis, the proteins to which ar ylating metabolites are bound. Reactive metabolite binding was substan tially higher in incubations of [H-3]-naphthalene with distal bronchio les and isolated Clara cells than with explants of trachea or bronchus from the mouse. Likewise, binding was substantially higher in incubat ions of murine Clara cells than in identical incubations with mouse he patocytes (nontarget cells) or rat trachea cells (nonsusceptible speci es). These data show a good correlation between cellular Susceptibilit y to toxicity and the amount of reactive metabolite bound in vitro. Co ncentrations of adduct were highest in the medium and the nuclear/cell debris fraction (1000 x g pellet) of isolated Clara cells incubated w ith naphthalene; very small amounts of adduct were noted in pellets is olated at 20,000 or at 100,000 x g (mitochondrial and microsomal fract ions) or in cytosol. These observations were consistent with the findi ng that adduct concentrations in bronchoalveolar lavage were substanti ally higher than in the lung at low doses of naphthalene and suggest t hat monitoring adducts in lavage may serve as a useful biomarker of ex posure and effect. [H-3]-Naphthalene metabolites were bound primarily to three proteins: relative molecular weight, 14 to 15 (major), 30 and 45 kD (minor), in mouse Clara cell incubations; binding in hepatocyte incubations was solely to a protein of 14 to 15 kD. The chromatograph ic characteristics of the 14-kD adduct differed substantially from tho se of rat surfactant protein B or a secretory protein elaborated by Cl ara cells with a M(r) of 10 to 12 kD. Reactive metabolite binding to p roteins in both Clara cells and hepatocytes appears to be highly selec tive. These studies suggest that the amount of metabolite bound to the 14 to 15 kD protein may be an important determinant in cellular susce ptibility to naphthalene and that the nature of the major protein targ eted in murine Clara cells (susceptible) and hepatocytes (nonsusceptib le) is similar.