Proteomic Analysis Of Dose-related And Time-related Cellular Response To Anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide In Human Amnion FL Cells

Benzo(a)pyrene is one of the polycyclic aromatic hydrocarbons(PAHs) environmental pollutants and can be generated from incomplete combustion of organic materials such as gasoline in motor vehicles, coal burning, cooking, and tobacco smoke. Benzo(a)pyrene is metabolically activated to form the ultimate carcinogen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) by microsome enzymes. Anti-BPDE is the most carcinogenetic form and the electrophilic species of it is able to interact with nucleophilic sites on cellular DNA, RNA and protein resulting in bulky-adduct damage. As a result, nucleotide excision repair, translesion DNA synthesis, activation of cell cycle checkpoint pathway and alterations of gene expression are induced.BPDE is a complete carcinogen, which plays a role in tumor initiation and promotion in two-stage carcinogenesis test. Extensive studies have been done in the mechanism of BPDE in mutagenesis. DNA damage occurs mainly by binding of C 10 position of anti-BPDE with N~2-dG in DNA to form the adduct (+)-trans-anti-BPDE-N~2-dG. The bulky adduct will block DNA replication. However, a DNA synthesis bypass way, translesion DNA synthesis can help cell to avoid the deadly fate of un-repaired DNA damage. As a result, mispairing base is often inserted opposite the lesion, which increases the risk of mutagenesis mainly G→T transversion mutation。However, the mechanism involved in the early responses induced by BPDE, which is thought to be mediated through initiating signal transduction pathways leading to activation of transcription factors and their target genes, is barelyunderstood.Studies have shown signaling pathway such as p53, PI-3K/Akt/JNKs, MAPKs/ AP-1, IKKbeta/NF-kappaB was activated after BPDE exposure. To investigate the direct effects of BPDE on gene expression, DNA immunoprecipitation technique was used to identify and clone BPDE-binding DNA fragments. A total of 67 fragments were sequenced and BLASTed in the GenBank database. The 67 fragments include DNA repair and apoptosis-related genes, zinc finger protein, cellular enzymes, expressed sequence tag clones, and CpG islands. These data further demonstrate that direct binding of BPDE with DNA of related genes is an important mechanism of BPDE-induced alterations in gene expression。 In addition, high-throughput gene microarray has been applied to detect the cellular global changes in gene and signaling pathways. After exposure to BPDE, Akennan et al used a human 350 gene array and found the genes with alterated expression were involved in cell cycle regulation, glutathione detoxification, cell apoptosis and etc.In study of responses to toxins, dose-related responses should be well considered. However, different doses belong to nontoxic, subtoxic or toxic ranges may actually induce dose dependent or completely different responses. In a proteomics study, Moller et al. found that treatment with daunorubicin of low, medium and high dose led to a significant up-regulation of 20 proteins, independent of the concentration used.In addition to exposure dose, the intervals after exposure is also should be taken into accounted. While the intervals elongates, the time related alteration in gene or protein expression could be discovered. It provides the possibility for the understanding of the mechanisms and finding of the biomarkers of the early and late response.In our laboratory, we have performed proteomic analysis in B[a]P-treatment in human amnion epithelial cells and a comprehensive alteration of protein expression profiles were found. The identified proteins included a number of zinc finger proteins and other transcription regulators. In order to eliminate the potential confounding effects of differential metabolic activation, we used the reactive metabolites of B[a]P for further study. Human amnion cells (FL cells) were exposed to 0.005μM BPDE in a secretome proteomic experiment, as a result, 3 and 16 protein spots were foundappearing and up-regulated in the cultured medium of BPDE treated cells as compared to the control group. Oligonucleotide microarray technique was also applied to detect the differential gene expression profiles after exposure to 0.005μM, 0.05 and 0.5μM BPDE, followed by quantitative real-time RT-PCR validation. There were few and robust gene expression changes, respectively, in response to the two lower doses and the higher dose of BPDE. The results in high dose exposure showed the alteration in expression level of genes related to cell cycle control, signaling molecules, transcription factors, and metabolic enzymes etc., signaling pathways such as p53-mediated, mitogen-activated protein kinases, MAPKs, Akt/PKB were activated. Additionally, Liu et al found 0.005μM and 0.05μM BPDE could trigger the endoplasmic reticulum stress in exposed cells, while 0.5μM BPDE could not induce such a response.All these findings raised our interests in revealing the cellular changes at different concentrations and time intervals after exposure. The rapid development of biological techniques has given us the possibility to try to understand the changes more comprehensively. Nowadays, many high-throughput methods including gene expression microarray assay, real-time RT-PCR analysis and bioinformatics are under way in this laboratory for the study of cell stress responses, and the preliminary results are inspiring. But these methods cannot provide direct information of how proteins are regulated at the translational or post-translational levels. Since proteins rather than mRNA are usually the functional executors in cells. Proteomic analysis, which combines 2-DE and mass Spectrometry, allows simultaneous monitoring of the expression of hundreds and even thousands of proteins in a sample following exposure to toxicant. Furthermore, it can study the post translation modification and protein interaction. Therefore, proteomic analysis is becoming a popular high throughput method of choice to detect differentially expressed proteins between profiles after exposure to toxicants.In our present studies, 2-DE combining MS was undertaken to identify the differentially expressed proteins following exposure to different doses of BPDE as well as at different time intervals after BPDE exposure. The differentially expressed proteins were classified according to their function through bioinformatics analysisand the functional implications of these proteins were discussed.In order to identify the proteins of differential expression in the FL cells after BPDE treatment, the IPG strips (24 cm, pH 4-7) and Ettan DALT II vertical electrophoresis system which allow a higher loading capacity and provide a longer separation distance for micropreparative runs were used to separate the whole cellular proteins of the FL cells treated with BPDE and DMSO, respectively. Then the 2D gels were visualized by silver staining; the digitized images were analyzed with 2D analysis software. In dose response study, 65 protein spots showed significant changes in BPDE-treated cells compared to control cells (DMSO treatment). There was 1 protein spot detected after 0.05μM and 0.5μM BPDE treatment, 1 protein spot detected only after 0.5μM BPDE treatment. Moreover, another 24 protein spots were found to be affected by 0.005μM BPDE(11 were up-regulated, 13 were down-regulated), 24 protein spots were found to be affected by 0.05μM BPDE(16 were up-regulated, 8 were down-regulated), 25 protein spots were found to be affected by 0.5μM BPDE(14 were up-regulated, 11 were down-regulated); however,10 protein spots were found to be affected by BPDE in two doses, no protein spot was affected by BPDE in three doses. These protein spots were cut from the gels and subjected to in-gel digestion with trypsin. The peptides were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass Spectrometry to obtain the peptide mass fingerprinting (PMF). And based on the information of PMF, some of the proteins were identified by searching non-redundant NCBI protein database using MASCOT software. Forty six protein spots were identified successfully. These identified proteins are involved in a variety of cellular process including transcription regulation, cell-cycle control, cell proliferation, signal transduction, cell skeleton, development, metabolism and some proteins with unknown functions. Based on the result, BPDE exposure induced comprehensive cellular responses.One hundred and twenty eight protein spots showed significant differential expression in the time response study. 36 protein spots were found to be affected by 0.05μM BPDE(11 were up-regulated, 25 were down-regulated) at 3 hour after exposure, 29 protein spots were found to be affected by 0.05μM BPDE(20 wereup-regulated, 9 were down-regulated) at 12 hour, 69 protein spots were found to be affected by 0.05μM BPDE(35 were up-regulated, 34 were down-regulated)at 24 hour; however,6 protein spots were found to be affected by BPDE in two time intervals, no protein spot was affected by BPDE in three time intervals. Eighty four protein spots were identified successfully. Similar to the dose response study, these identified proteins are involved in a variety of cellular process.Conclusion: There are comprehensive responses in the FL cells after exposure to BPDE. Many proteins are involved in BPDE-induced cellular responses in mammalian cells. These proteins take part in a variety of cellular processes. This work provides the new insight into the mechanisms of BPDE and the possibility of new biomarkers for evaluating the exposure to environmental carcinogen. Little dose-dependent or time-dependent manner could be observed in the proteins in the dose response study or time response study. Therefore, the biological responses of high dose exposure can't be considered as only an amplification of low dose response. BPDE may activate different pathway to regulate the cell response machinery followed exposure to different doses as well as at different time intervals after exposure.