Showing papers by "Per E. Schwarze published in 1996"

Expression of Cyp2B1 in Freshly Isolated and Proliferating Cultures of Epithelial Rat Lung Cells

Abstract: Bronchiolar Clara cells and alveolar type 2 cells of the lung are known to express relatively high levels of P450 enzymes compared to other pulmonary cells. Populations of enriched type 2 cells and Clara cells were isolated from rat lung by a procedure including lung perfusion, protease digestion, centrifugal elutriation, and differential attachment. Alveolar macrophages were removed by lavage. The purity of the type 2 cell-enriched population was approximately 90%, and the purity of the Clara cell-enriched population was 40-50%. Both type 2 cells and the cells of the Clara cell-enriched population proliferated in culture. CYP2B1 mRNA was expressed approximately to the same level in type 2 cells and the Clara cell-enriched population. The mRNA levels remained roughly constant for both cell types throughout the culture period, except for an early transient reduction. The apoenzyme level of CYP2B1 was 2-3 times higher in freshly isolated cells of the Clara cell-enriched population than in the type 2 cells. Both epithelial cell types showed decreased level of CYP2B1 apoenzyme in culture. The differences in the CYP2B1 mRNA and apoenzyme expression levels in freshly isolated cells and cultured cells suggest the existence of a post-transcriptional regulatory mechanism for CYP2B1 expression in lung cells. The characterization of specific functions of lung cells in culture, such as P450 gene expression, provides necessary information for the use of the cells in in vitro pulmonary toxicology.

Differential distribution of Met and epidermal growth factor receptor in normal and carcinogen-treated rat liver.

Abstract: Transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) are strong hepatocyte mitogens and important regulators of liver regeneration. The TGF-alpha receptor EGFr appears primarily to mediate a proliferative signal, whereas mitogenic, motogenic, and morphogenic effects have been attributed to activation of the HGF receptor Met. We have studied the localization of Met and EGFr in normal and carcinogen-treated rat livers. Oval cells and preneoplastic lesions were induced by diethylnitrosamine initiation, followed by promotion with 2-acetylaminofluorene combined with a partial hepatectomy. Different liver cell populations and their receptor expression were characterized by two-color immunofluorescence and confocal laser scanning microscopy. Hepatocytes were detected by keratin K8 staining, and oval cells and bile ducts were recognized by keratin K19 expression. Enzyme-altered preneoplastic lesions ere identified by expression of placental glutathione S-transferase (GST-pi). Staining for these cellular markers was combined with immunodetection of EGFr and Met. Normal liver exhibited strong staining for EGFr in hepatocytes, whereas blood vessels, bile ducts, and some sinusoidal cells were Met-positive. In carcinogen-treated livers, oval cells showed Met but not EGFr immunostaining. GST-pi-positive foci displayed EGFr immunostaining at a similar intensity as surrounding hepatocytes, whereas Met was not detected. Our data indicate that putative liver cells (oval cells) have a growth receptor phenotype similar to that of bile ducts, whereas preneoplastic live lesions appear hepatocyte-like. These results indicate that the preferential proliferation of preneoplastic liver lesions compared to surrounding hepatocytes is not associated with an altered EGFr or Met phenotype.

The use of isolated lung cells in in vitro pulmonary toxicology: studies of DNA damage, apoptosis and alteration of gene expression.

Abstract: Isolated lung cells constitute a valuable system for studying mechanisms involved in chemically induced toxicity in the lung. Different lung cells isolated from various species may be studied. Bronchiolar Clara and alveolar type 2 cells produce important lung-specific proteins, hold a major role in the metabolism of xenobiotics and serve as progenitor cells for other lung cell types. They are possible target cells in lung carcinogenesis. Alveolar macrophages play an important role in lung defence and in inflammatory responses. In the present study we have characterised chemically induced DNA damage, apoptosis, changes in cell cycle progression, transformation and alterations in gene expression in these specific lung cells isolated from rat, rabbit and human. Major differences between the cell types and the various species in the induction of DNA damage by chemicals were found, as measured by the 32P-postlabelling and alkaline filter elution techniques. Benzo(a)pyrene and hydrogen fluoride were found to induce apoptosis in the isolated cells as measured by microscopical analysis and flow cytometry. The function of various important tissue- or cell type specific proteins (CYP 2B1, Clara cell protein) and/or cellular signal transduction pathways constitute important targets that may be affected by exposure to toxic compounds. Using immunological and molecular techniques the differential expression of specific proteins/RNAs and their activity can be studied. Among other proteins, c/ebp is involved in the regulation of transcription at the end of signal pathways. The protein is differentially expressed in rat lung cells and thus could be suitable for studying differential toxic effects in various lung cells. In humans, bronchoalveolar lavage (BAL) fluid from human volunteers can be readily obtained and examined after exposure to different chemical compounds. An increase in the percentage of CD3-positive cells (T-lymphocytes) was found after exposure to hydrogen fluoride. The number of certain cell types and cytokines may be used to estimate the degree of inflammatory reaction. In conclusion, the use of in vitro data including the use of specific, primary human lung cell types may contribute considerably to the quality of risk assessment, together with in vivo data from animals and man.