The effects of benzene exposure on apoptosis in epithelial lung cells: localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene products.

Although benzene, a well-known human carcinogen, has been shown to induce apoptosis in vitro, no studies have been carried out to confirm and characterize its role in activating apoptosis in vivo. The present study investigated the effects of benzene inhalation on the epithelial cells lining the respiratory tract including bronchioles, terminal bronchioles, respiratory bronchioles and alveoli of male Sprague-Dawley rats. Inhalation of benzene 300 ppm for 7 days induced apoptotic changes in the parenchymal components in the lung that significantly exceeded the events of programmed cell death in normal control tissues. Apoptosis was confirmed by the electrophoretic analysis of internucleosomal DNA fragmentation of benzene-exposed lung tissues, which exhibited 180-200 bp laddering subunits indicative of genomic DNA degradation. Furthermore, semi-quantitative analysis of intracellular localization of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling TUNEL) showed a significant (p < 0.001) increase in the apoptotic index calculated for bronchiolar 73.5%, terminal bronchiolar (65%), and respiratory bronchiolar 60.8% segmental epithelial components as well as alveolar (55%) epithelia. Analysis of immunohistochemical expression of apoptosis-related gene products also supported the hypothesis that benzene can induce apoptosis in chemosensitive target cells in the lung parenchyma. Quantitative immunhistochemistry showed a statistically significant increase p < 0.001 in the immunoreactive staining index for cytochrome c, Apaf-1 (apoptosis activating factor-1), DNA fragmentation factor, and representative cysteine proteases including caspase-1, caspase-2L, caspase-8 and caspase-9. Thus this is the first study of the respiratory system that demonstrates that benzene inhalation induces lung cell apoptosis as confirmed by DNA electrophoresis, in situ nick end labeling, and the upregulation of apoptosis-related gene products that facilitate caspase-cleaved enzymes which lead to cell degradation via programmed cell death. These responses may represent an important defense mechanism within the parenchymal cells of the respiratory system that reduce mutational hazard and the potential carcinogenic effects of benzene-initiated pathogenesis.

Immunocytochemical localization of insulin-immunoreactive cells in the pancreatic ducts of rats treated with trypsin inhibitor.

It is well known that soybean trypsin inhibitor exerts trophic effects on the exocrine pancreas, resulting in the hypertrophy of acinar cells. Some evidence also exists for hyperplasia in acinar tissue, the ductal epithelium and islet tissue. Rats maintained for 3 weeks on an oral administration of soybean trypsin inhibitor (200 mg/50 ml drinking water) were compared with untreated animals. Significant changes were noted in treated animals (p less than 0.01). Trypsin inhibitor-treated rats showed an increase in pancreatic weight (2.33 +/- 0.46 g). The volume ratio of acinar, islet and connective tissue as measured by the stereology point-count technique remained the same in both groups. Ductal tissue, however, exhibited an increase in volume ratio, 3.77 +/- 4.38% per 2714 micron2 area of tissue, in trypsin inhibitor-treated animals. All tissue components showed an increase in the experimental animals: acinar (125%), islet (144%), ductal (325%) and connective tissue (94%). Increased size of acinar cell nuclei, as measured by average cord length, 6.20 +/- 0.13 micron, and a decreased nuclear density of acinar cells, 28 +/- 4.74 per 150 micron2 area of tissue, indicated hypertrophic changes in these cells of the experimental animals. Using immunohistochemical localization and the point-count technique, a significant fraction of the total pancreatic volume in experimental animals was represented by ducts containing immunoreactive cells. The percent of volume ratio, 0.42 +/- 0.15% per 2714 micron2 area of tissue, was calculated for ducts containing insulin-immunoreactive cells within their epithelium.