Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity was measured in alveolar type II cells from control and beta-naphthoflavone (ip) treated-rats. Type II cells were isolated from collagenase/elastase-digested lung tissue and purified by centrifugal elutriation. The specificity of the cytochrome P450-dependent activity towards four alkoxyphenoxazones (methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone) was measured under conditions that minimized interference by cytosolic conjugating- and NADPH-dependent quinone reductase activities. Ethoxyphenoxazone dealkylase activity was induced 17-fold following beta-naphthoflavone treatment and was further characterized by its kinetic parameters and sensitivities toward in vitro inhibitors (Km(app) = 0.20 microM, Vmax = 1.74 pmoles resorufin min-1 (10(6) cells)-1 10(6) cells; I50 (alpha-naphthoflavone) = 0.025 microM, and I50 (metyrapone) = 72 microM). beta-Naphthoflavone pretreatment of the rats did not result in statistically significant changes in methoxy-, pentoxy-, or benzyloxyphenoxazone dealkylase activity of alveolar type II cells, although, a trend towards decrease activity was observed for benzyloxyphenoxazone. beta-Naphthoflavone pretreatment had no effect on oxygen consumption or trypan blue exclusion in alveolar type II cells and macrophage ethoxyphenoxazone dealkylase and benzyloxphenoxazone dealkylase activities were not affected by the beta-naththoflavone pretreatment. The results show that exposure to beta-naphthoflavone resulted in an increase in type II cell cytochrome P450-dependent ethoxyphenoxazone dealkylase activity but not in other alveolar type II cell or macrophage alkoxyphenoxazone dealkylase activities or in parameters that monitor viability and cell wall integrity.

Spectral properties of the cytochrome P-450 substrates, methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone (MeOPx, EtOPx, PeOPx, and BzOPx, respectively) were investigated from 350 to 600 nm in ethanol and aqueous buffer. In ethanol, each alkoxyphenoxazone displayed a lambda max at 460 nm and a shoulder around 390 nm. Extinction coefficients (EmM) in ethanol were calculated as MeOPx, 20.5; EtOPx, 20.4; PeOPx, 24.7; and BzOPx, 22.4. In aqueous buffer, only MeOPx obeyed the Lambert-Beer law (lambda max = 480 nm, EmM = 22.1). Three substrates, EtOPx, PeOPx, and BzOPx, displayed anomalous behavior in aqueous solution, wherein the lambda max shifted to lower wavelengths (480-430 nm) and EmM (apparent) decreased as the alkoxyphenoxazone concentration increased. This behavior was dependent on the side chain, and the concentrations at which the spectral changes took place were estimated as: BzOPx, 2 microM; PeOPx, 5 microM; EtOPx, 17 microM; and MeOPx, greater than 20 microM. The blue shift and decreased EmM (apparent) observed for PeOPx at high concentration in aqueous buffer was reversed at high temperature. Unlike EtOPx, PeOPx, and BzOPx, and like MeOPx, hydroxyphenoxazone (resorufin) and unsubstituted phenoxazone obeyed the Lambert-Beer law in aqueous buffer and ethanol. The data suggest that the pentoxy and benzyloxy substituents facilitated a self-association process among the phenoxazones in aqueous solution. The data further show that aqueous solutions should be avoided when spectral data are used to determine alkoxyphenoxazone concentrations.