Secretomic Analysis Of Human Amnion Epithelial Cells Exposed To Low Concentration Of Anti-7, 8-dihydroxy-9, 10-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]Pyrene

B[a]P is a ubiquitous environmental contaminant which is mainly found in tobacco smoke, broiled foods, diesel exhausts, and polluted environments, and induces various pathogenic effects in vivo, especially carcinogenesis. It is metabolized by cytochrome P4501A1 (CYP1A1) into various metabolites, among which anti-BPDE exhibits the predominant mutagenic and carcinogenic activities resulted from the adduct formation or oxidative damage on DNA. The formation of BPDE-DNA adducts triggers a series of cellular responses, including activation of signaling pathways, apoptosis, cell cycle arrest, DNA repair, translesion DNA synthesis, etc. Some apoptosis-associated MAPK, such as JNK and p38, as well as p53 proteins, which regulate the transcription of genes involved in apoptosis, are activated after BPDE exposure. Meanwhile, the elevated intracellular Ca(2+) may result in the activation of Ras/Erk/AP-1 and PI-3K/Akt signaling pathways, both of which exhibit anti-apoptotic activity. Chk1, which mediates S-phase checkpoint response to bulky adducts is phosphorylated and activated in BPDE treated cells. The consequently activation of p53 and p21 genes results in the inhibition of CDKs which drive cell cycle progression. Thus the blockage of DNA synthesis and even inhibition of initiation and elongation steps ofDNA replication occur according to different BPDE concentrations. The un-repaired bulky DNA adducts may result in translesion DNA synthesis which facilitates mutagenesis because of the low fidelity of alternative DNA polymerases. p53 tumor suppressor and K-ras proto-oncogene are two most frequently mutated genes in smoking-induced lung tumors, both of which are linked to the formation of N2-BPDE-dG, since a strong correlation between the sites of increased BPDE-induced DNA damage and "hot spots" in those two genes is observed. In addition, BPDE also induces chromatin aberrations.In this laboratory, hundreds genes and proteins, which are involved in transcription, translation, cell cycle regulation, cell signaling, metabolism and detoxification, etc. have been found either up or down regulated in global genomic and proteomic analysis of human amnion FL cells exposed to BPDE.Since BPDE is a confirmed carcinogen, the population-based monitoring of it is an important strategy to evaluate health risk especially among people under occupational exposure or with health risk behaviors, which urges a batch of sensitive and specific biomarkers. Biomarkers are measurable indicators of specific biological states, particularly ones relevant to the risk of contractions, the presence or the stages of diseases. They are widely applied in early diagnose, subtype classification of disease, progression monitoring, prognosis of disease, therapeutic effect assessment of drugs, and epopulation health survailance, etc.Proteins, as the real functional executors and also the most ubiquitously affected targets in disease, response and recovery, are the most focused and studied biomarkers. Proteomic analysis has become the preferential strategy for biomarker discovery, characterization, and evaluation because of its capacity of globally examining the protein expression profiles. The preferable sample types for population monitoring should be obtained through noninvasive procedures, thus the best biomarkers should be peptides/proteins shed or secreted from the exposed cells to the biofluids. However, many biomarkers in blood are massively diluted from the tissue derived and are always masked by the abundant constitutive proteins. Thus the supernatant fluid ofcultured cells, which mimics body fluid to some degree, is an acceptable surrogate of the latter.In the present study, we employed 2-DE combining with MALDI-TOF to analyze the differential secretome between BPDE treated and control cells as well as probe candidate biomarkers. Human amnion epithelial cells were either exposed to 5nmol/L anti-BPDE or DMSO as solvent control. Culture medium of each repetition from both groups was harvested. After ultrafilteration and precipitation by acetone at -20°C, proteins were re-suspended in lysis buffer followed by quantification. All protein samples were subjected to IEF using IPG strips (pH 4-7 linear, 24cm). The second separation was carried out on 12.5% SDS slab gels which were silver-stained later. The digitized images were then analyzed with PDQuest version 4.7.0 in order to construct the differential secretomic profile. As a result, there were 3 protein spots only detected in the BPDE treatment group, and 16 protein spots up-regulated in the same one according to their relative volumes (p<0.05). Differentially expressed protein spots were picked from the gels and were subjected to in-gel digestion with trypsin. Peptide mixtures were analyzed with voyager-DE STR MALDI-TOF mass spectrometer using a delayed ion extraction and ion mirror reflector mass spectrometer to obtain PMFs. Peptide fingerprinting mapping was performed using on-line soft ware MSCOT and nrNCBI protein sequence database. Totally, 12 proteins were successfully identified with high confidence, including 14-3-3ζ, the appearing protein, as well as annexin III, annexin V and hydroxypyruvate isomerase homolog which were up-regulated in response to BPDE. Correspondingly, 14-3-3ζ and annexin III were found down-regulated in the cell lysates of BPDE treatment groups. They participates in multiple biological processes (e.g. carcinogenesis), though the exact mechanisms remain far from known.In conclusion, proteomic analysis involving 2-DE and MS is proved to be an effective strategy in biomarker discovery. The differential secretome revealed the complexity of cellular response network triggered by BPDE exposure from the perspective of exocytosis, and referred some proteins as candidate biomarkers formore detailed study.