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

Abstract

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.

Deregulation of apoptosis mediators' p53 and bcl2 in lung tissue of COPD patients

“…A recent study by Weaver and Liu [24] in rats after exposure to benzene, a ubiquitous environmental pollutant and a cigarette smoking by-product, showed significant up-regulation of pro-apoptotic p53 in lung epithelia of benzene-exposed rats compared to controls, whereas no statistical difference was found in the expression of bcl2 in airway epithelial cells in both study groups [24]. Other groups describe similar findings with an increase in apoptosis of alveolar epithelial cells in patients with emphysema compared to smokers without COPD [25,26] while the anti-apoptotic protein bcl2 was not detected in either normal or emphysematous lung tissue [25].…”

Effect of Benzene on liver functions in rats (Rattus norvegicus)

“…In a study, it was shown that properties of membrane phospholipid were changed (Engelke et al 1993), some structural and functional changes occur under benzene treatment in rat microsomes (Sukhodub and Padalko 1999). In another study, Weaver et al (2007) investigated the effects of benzene inhalation on the epithelial cells lining the respiratory tract including bronchioles, terminal bronchioles, respiratory bronchioles and alveoli of male rats. They were shown that 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.…”

The role of catechols and free radicals in benzene toxicity: An oxidative DNA damage pathway

“…Related to this, Chen et al [2007] described that mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by ROS. According to this, a relation between mitochondria as a source of ROS and apoptosis has been reported [Martínez-Velázquez et al, 2006;Kim et al, 2007;Weaver et al, 2007;Chen and Gibson, 2008;Mosca et al, 2008;Scherz-Shouval and Elazar, 2007]. Several lines of evidence suggest that endogenous catechol and also exogenous molecules bearing a catechol moiety are inhibitors of mitochondrial respiration: (i) endogenous cysteinyl catechols are potent inhibitors of mitochondrial complex I activity in vitro [Montine et al, 1997]; (ii) dopamine and its metabolite 3,4-dihydroxyphenylacetic acid can also inhibit brain mitochondrial state 3 NADH-linked respiration [Gluck and Zeevalk, 2004]; and (iii) flavonoids with a catechol moiety in their structure are inhibitors of state 2 FADH 2 -and Environmental and Molecular Mutagenesis.…”