Benzo(a)pyrene 於肺臟幹源性Clara 細胞造成之細胞反應

Abstract

Benzo(a)pyrene (BaP) belongs to polycyclic aromatic hydrocarbon (PAH) family of environmental carcinogens that are present in tobacco smoke , barbecued food and auto exhaust. BaP require cytochrome P450 and epoxide hydrolase mediated bio-activation for generation of their respective ultimate carcinogenic dio epoxide intermediates, (+)benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE). Covalent interation of BPDE with DNA is believed to be a critical step in BaP-induced tumorigenesis. BaP have been implicated in the etiology of lung cancer. In the lung, BaP was located in ciliated and non-ciliated Clara and alveolar type II cells, resulting in the experimental animals, such as rat, mice, or rabbit. Clara cells play important role in the metabolism of xenobiotic in the lung, because they have abundant cytochrome P450 monooxygenase. In previously studies, Clara related stem/progenitor cells were identified and generated in lung stem/progenitor cell. Although, the potential roles of Clara cells in metabolism of xenobiotic have been proposed, however, the molecular mechanism of BaP metabolically activation in the Clara related stem/progenitor cells still unclear. In this work, we set a lung stem/progenitor cell including Clara cell related stem/progenitor cells surrounding with mesenchymal stroma cells to examine the BaP-induced cell response. Different concentration of BaP treatments were applied to lung stem/progenitor cells. The MTT assay showed the high concentration of BaP cause cell proliferation in lung stem/progenitor cells. The cell cycle analysis revealed that S phase increased after BaP treatment for 24 hours. By BrdU incorporation experiment showed that BaP exposure was able to promote Clara related stem/progenitor cell entry to S phase. BaP is a ligand of aryl hydrocarbon receptor(AhR), binding leads to nuclear translocation and interaction with its partner protein AhR nuclear translocator (ARNT) will active xenobiotic metabolism and proliferation. By mRNA and immunocytochemistry analysis, BaP exposure caused AhR translocation to nuclei at 4 hours and activated CYP1A1, CYP1B1 expression at 12 and 24 hours. Further, BaP-induced c-myc, cyclin D1 expression up to 3.5 and 4.2 folds by real-time PCR and CDK4 protein level up to 3.5 folds by western blot analysis. Knockdown of c-myc in lung stem/progenitor cells by siRNA transfection, suppressed S phase distribution for BaP treatment. BaP can be metabolized into BPDE, a ultimate of carcinogen attach to DNA cause genotoxicity. From immunocytochemistry of BPDE DNA positive result reveal that BaP probably cause BPDE DNA adduct in Clara related stem/progenitor cells. And using comet assay, BaP has been shown to cause genotoxicity. The current study showed that BaP activates c-myc expression of lung stem/progenitor cell, and through cyclin D1/CDK4 mediated cell cycle entry to S phase. Meanwhile, BaP treatment also induced CYP1A1 expression and genotoxicity in Clara related stem/progenitor cells. Together, these results indicated that BaP presented the potent carcinogen for lung stem/progenitor cells to undergo tumorigenesis processes. And hinted that S phase increased caused from cell cycle progession by BaP-induced c-myc activation and might result in arrested by genotoxity induced repair mechanism.