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.

AIM:To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential ( Ψ Ψ Ψ Ψ Ψm). METHODS:Cell culture, cell counting, ELISA assay, TUNEL, flow cymetry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism.RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis.The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION:The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Ψ Ψ Ψ Ψ Ψm and JTE-522-induced apoptosis in AGS cells.