Abstract 1789: Effects of benzo(a)pyrene-7,8-dione and cadmium on early response to DNA damage in primary human bronchoepithelial cells.

Abstract Polycyclic aromatic hydrocarbons (PAHs) and heavy metals often co-exist in the environment, but despite their significant toxicities little is known about their combined effects. Cadmium (Cd), a known human carcinogen, interferes with cellular responses to DNA damage, through inhibition of DNA repair systems, disturbance of tumor suppressor functions, or increased resistance to apoptosis. Cd has been linked to lung cancer in humans and the most common exposure scenarios in humans involve co-exposure with PAHs. However, studies on combined effects of Cd and PAHs are mostly limited to marine invertebrates or soil bacteria. Both PAHs and Cd induce formation of reactive oxygen species (ROS) in the cell. Mitochondria are the main target for oxidative DNA damage and also play an important role in programmed cell death. Cytochrome c is an important component of apoptosis and its translocation from mitochondria to the cytosol is an indicator of apoptotic activity in intact cells. Another indicator of the early response to apoptotic signals is the phosphorylation of H2AX, which also reflects DNA damage. Here, we propose that the exposure of primary human bronchoepithelial cells (hBEC) to the mixture of benzo(a)pyrene-7,8-dione (BaPD) and cadmium chloride (CdCl2) will significantly augment DNA damage and disrupt mitochondrial functions involving apoptosis. Methods: Cytotoxicity was analyzed by the WST-8 assay. hBEC were exposed to CdCl2 (5 μg/ml) and BaPD (10 μg/ml) for 24h. Levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and cytochrome c were quantified using ELISA. Quantification of H2AX phosphorylation (Ser139, γ-H2AX) was performed by western blotting. Statistical tests were conducted using GraphPad Prism software at a significance level of 0.05. Mean levels across treatment groups were compared using analysis of variance (ANOVA). Results: IC50 values at 24h for CdCl2 and BaPD were 8.7 and 12.5 μg/ml, respectively. Levels of 8-OHdG in nuclear DNA were significantly higher in hBEC exposed to CdCl2 or BaPD when compared to controls. We observed an increased release of cytochrome c during the first few hours after exposure to CdCl2 and BaPD, followed by significant reductions at 24h. Furthermore, cytochrome c levels in cells exposed to the combination of BaPD and CdCl2 were substantially lower than those observed with either treatment alone. Phosphorylation of H2AX increased in hBEC exposed to CdCl2 or BaPD, with higher levels observed in cells treated with BaPD. Conclusion: Our results suggest increased DNA damage in hBEC after 24h of exposure to Cd and BaPD. We also observed decreased cytochrome c levels after exposure to BaPD and/or Cd, suggesting disruption of mitochondrial functions. This effect was more pronounced in cells exposed to the mixture. Citation Format: Jibanananda Mishra, Berrin Serdar. Effects of benzo(a)pyrene-7,8-dione and cadmium on early response to DNA damage in primary human bronchoepithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1789. doi:10.1158/1538-7445.AM2013-1789