DNA adducts and altered protein expression profile in human lung cell line after environmentally relevant exposure to benzo(a)pyrene

2081

Several mutagens/carcinogens are inhaled by human lungs with particulate matter (PM) through the respiratory tract and contribute to lung cancer induction. PM is a complex mixture of compounds, of which the most toxicologically relevant is benzo(a)pyrene (BaP), a prototype of polycyclic aromatic hydrocarbons (PAH). It has been estimated that human inhaled dose of air-borne derived-BaP can range from 50 to 5000 ng BaP/24 h in urban settings. This preliminary study was undertaken to investigate the extent to which BaP exposure at environmentally relevant concentrations damages DNA and alters protein expression in an in vitro model and to determine which, if any, protein marker might be indicative of BaP carcinogenic mechanisms. To this end, detection of benzo(a)pyrene diolepoxide-DNA adducts (BPDE-DNA) by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry and comparative proteomic analysis of whole cellular proteins were performed in human lung adenocarcinoma A549 cell line after 24h exposure at 1 μM BaP, a dose comparable to an inhaled concentration of 5000 ng BaP/24 h. After 24h exposure, BPDE-DNA adducts in A549 cells were 82/10e8 nucleotides, without any significant effect on cell viability and showed a differential protein profile in comparison to their unexposed counterpart. Bidimensional gels image visualization and statistical analysis indicate that 13 protein species showed significant changes in abundance (p<0,05) following BaP exposure. These proteins were subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. We found that BaP appears to modulate the intracellular levels of proteins not previously connected to a cellular response to PAH. These proteins were shown to be involved in multiple cellular processes such as energy pathways and protein metabolism. For the first time, we observed a significantly increased expression in the elongation factor 2 (EF2) suggesting a deregulation of the translational system with possible implication on cell growth and protein synthesis. Interestingly, the significant decrease in the abundance of aldehyde dehydrogenase A1 (ALDH1) involved in the metabolism of retinoic acid, might account for the reported effect of BaP on retinoid signalling pathway. In summary, these preliminary data suggest that this approach will allow us to have a global perspective, at the protein expression level, of the many different effects of BaP. Some of these changes may lead to new insights into the mechanism of BaP carcinogenesis.