Heterogeneity of clara cell glutathione. A possible basis for differences in cellular responses to pulmonary cytotoxicants.

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.

Hypoxia-inducible mammalian gene expression analyzed in vivo at a TATA-driven promoter and at an initiator-driven promoter.

We have analyzed protein-DNA interactions in vivo at transcriptional control elements for two hypoxia-inducible genes in mouse hepatoma cells. The promoter for the phosphoglycerate kinase 1 (PGK1) gene contains an initiator element, but no TATA sequence, whereas the promoter for the glucose transporter 1 (Glut1) gene contains a TATA element but no initiator sequence. Our findings reveal hypoxia-inducible, Arnt-dependent occupancy of DNA recognition sites for hypoxia-inducible factor 1 (HIF-1) upstream of both target genes. The conserved recognition motif among the five recognition sites is 5'-CGTG-3'. The PGK1 promoter exhibits constitutive occupancy of a binding site for an unknown protein(s); however, we detect no protein-DNA interaction at the initiator element, in either uninduced or induced cells. The Glut1 promoter also exhibits constitutive protein binding; in addition, the TATA element exhibits partial occupancy in uninduced cells and increased occupancy under hypoxic conditions. We find no evidence for hypoxia-induced changes in chromatin structure of either gene. Time-course analyses of the Glut1 gene reveal a temporal relationship between occupancy of HIF-1 sites and TATA element occupancy. Our findings suggest that the promoters for both hypoxia-responsive genes constitutively maintain an accessible chromatin configuration and that HIF-1 facilitates transcription by recruiting and/or stabilizing a transcription factor(s), such as TFIID, at both promoters.

Metabolism and cytotoxicity of naphthalene and its metabolites in isolated murine Clara cells.

Nonciliated bronchiolar epithelial (Clara) cells of mice are highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type expresses high levels of cytochrome P450 monooxygenases. To establish the capability of these cells to metabolize an agent that causes Clara cell-selective toxicity in vivo, we evaluated the metabolism of naphthalene in isolated cells under two distinct conditions, i.e., in homogenized cell preparations supplemented with glutathione and glutathione S-transferases and in intact cells. In homogenized cell preparations naphthalene was metabolized to dihydrodiol (minor) and a single glutathione adduct (major) derived from the 1R,2S-epoxide. In intact cells the rate of formation of glutathione adduct was much lower and dihydrodiol predominated. Approximately 3-10% of racemic naphthalene oxide added to isolated homogenized cells was converted to glutathione adducts and dihydrodiol in 3-min incubations. At high concentrations of naphthalene oxide (0.25 and 0.5 mM), formation of the adduct derived from the 1R,2s-epoxide was favored. The intracellular glutathione concentration, measured by high performance liquid chromatography as the fluorescence of the monobromobimane-glutathione derivative, was 1.14 +/- 0.13 nmol/10(6) cells. To determine whether Clara cell injury results from cytotoxic metabolites of naphthalene, we assessed viability of intact cells in response to different concentrations of naphthalene and naphthalene metabolites. At high naphthalene concentrations (0.5 and 1.0 mM) cell viability decreased to 63% or less of control, whereas lower concentrations (0.1 or 0.05 mM) did not alter viability significantly. Naphthalene-induced decreases in cell viability were blocked by preincubation of Clara cells with the cytochrome P450 monooxygenase inhibitor piperonyl butoxide. The cytotoxicity of naphthalene metabolites varied. Incubation of cells with 0.5 mM dihydrodiol, 1-naphthol, or 1,2-naphthoquinone decreased cell viability to an extent similar to that produced by 0.5 mM naphthalene. In contrast, 0.5 mM naphthalene oxide and 1,4-naphthoquinone significantly decreased viability more than the parent compound. Preincubation of Clara cells with piperonyl butoxide did not affect the loss in cell viability associated with naphthalene oxide. We conclude that isolated Clara cells 1) are capable of metabolizing naphthalene, a Clara cell-specific cytotoxicant, to two major metabolites, 2) have a detectable intracellular glutathione pool, and 3) are more susceptible to specific naphthalene metabolites than to the parent compound naphthalene.

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 further 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. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), 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 gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable 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 extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)