Metabolism of bromobenzene to glutathione adducts in lung slices from mice treated with pneumotoxicants

Recent studies showing that the bronchiolar Clara cell and alveolar Type II cell are major loci of cytochrome P-450 monooxygenases in the lung suggested that measurement of xenobiotic metabolizing enzyme activity might provide a useful and sensitive index of injury to these cell types. Accordingly, an assay has been developed for quantitating the rate of formation of reactive bromobenzene metabolites in lung slices which is based upon measuring the rate of formation of bromobenzene glutathione adducts. To demonstrate that monitoring adduct formation would yield quantitatively similar data to the traditional covalent binding assay for measuring the formation of reactive bromobenzene intermediates, covalent binding and conjugate formation were assayed in incubations of phenobarbital-induced hepatic microsomes conducted in the presence of various cytochrome P-450 monooxygenase inhibitors. Incubation conditions which decreased the rate of covalent binding (incubations done in the absence of glutathione) resulted in similar decreases in conjugate formation (incubations done in the presence of glutathione). In lung slices, the metabolism of bromobenzene to glutathione conjugates was linear for 20 min and continued to increase with time over the entire 160 min of the study. The formation of bromobenzene glutathione adducts in lung slices from piperonyl butoxide-treated animals occurred at a significantly lower rate than control. Likewise, lung slices from animals treated with butylated hydroxytoluene or carbon tetrachloride, agents known to injure alveolar epithelial cells, metabolized bromobenzene to glutathione conjugates at significantly slower rates than control. In contrast, treatment with naphthalene or dichloroethylene, agents which damage the bronchiolar epithelial cells, had little or no effect on conjugate formation. Similarly, there were no significant differences in the rate of bromobenzene glutathione conjugate formation between lungs of air- and ozone-exposed (1.0 ppm × 4 hr) mice killed 2,24,48,72, or 120 hr after exposure. These studies suggest that monitoring the rate of bromobenzene glutathione conjugate formation in lung slices may be a useful and sensitive biochemical index of injury to certain cells of the lung but that severe damage to the nonciliated bronchiolar epithelial cells has little effect on the rate of metabolic activation of this aromatic hydrocarbon.