Early Biomarkers For Carcinogenesis And Mechanism Of Cell Malignant Transformation Induced By Coke Oven Emissions

There are sufficient epidemiological evidences suggesting an etiological link between lung cancer in coke-oven workers and exposure to polycyclic aromatic hydrocarbons(PAHs), which has been classified as a glass1carcinogens by the International Agency for Research on Cancer. Previous studies found numerous genetic changes, such as DNA and chromosome damages, in cases of lung cancer caused by COE. The role of epigenetic modifications, such as DNA methylation, in lung cancer progression is still not well understood.In this study, we investigated the relationships between methylation level of MGMT promoter region and genetic damage indexes and genetic polymorphisms of MGMT among83coke-oven workers and62non-COE exposed workers. We chose normal human bronchial epithelial cell(16HBE), which was exposed to the organic extracts from coke oven emission (OE-COE) repeatedly in order to induce malignant transformation and build the malignant cell transformation model in vitro. By analyzing the alteration of the genetic damages and apparent epigenetic modifications, such as the methylation of O6-methylguanine-DNA-methyltransferase (MGMT) and p16gene, in the different cell transformation phases and the malignant transformation cell colonies, we investigated the mechanism of these genetic damages and apparent epigenetic modifications in the transformation process. At the same times, The main results are as follows:1.The study on the methylation level of MGMT among83coke-oven workers and62non-COE exposed workers.Multivariate analysis of covariance with adjustment for urinary1-OHP, age, sex, smoking status revealed that the level of MGMT was low, which was related to the level of exposure and the CBMN frequency, detected by cytokinesis-block micronucleus (CBMN) assay. And we found that the sites of methylation in coke-oven workers was different from the coke-oven workers and non-COE exposed workers. But we could not find there were relationships between the level of MGMT promoter methylation and the Olive TM, detected by comet assay, and the polymorphisms of MGMT rs1625649. 2. The construction and identification of the cell malignant transformation2.1The collection of coke oven emissions, chemical analysis and risk assessment on lung cancerWe used fixed-point method to collect the coke oven emissions, and detected its concentration by the gas chromatography-mass spectrometry. According to the relative potency factor, we converted the Benzo[a]pyrene equivalent concentration(BaPeq) of COE was53.41μg/m3according to the relative potency factor and its risk of lung cancer was4.47.2.2The construction of the malignant cell transformation induced by OE-COE and its indificationWe plated the cells at a concentration of5×104cell/ml, and induced by a low dose of BaP for48h. Then replaced with the treatment fluid of Organic extracts of coke oven emission(OE-COE) with different dose of5,10,20,40μg/ml. After24h treatment, we harvested cells and conducted the second induction following procedures used in the first treatment. We chose20μmol/L BaP as positive control, and1%o DMSO as solvent control.2.3The assessment of the characteristics of malignant transformation cellWe selected the following methods to identify the characteristics of malignant cell transformation. First was to test whether the cells colonies could form subcutaneous tumors in nude mice. Tumors were generated in mice after20days, and the tumor was diagnosed as low differentiated Squamous cell carcinoma through histopathological examination and immunohistochemistry technique. The results of chromosome aberration showed that the number of chromosome was stable and that most mutations were deletions or shifts. There was a significantly mutation at the p53exon-9in the RF colony, and no mutation of other cell colonies was observed. The results of the cell cycle test showed that the G1phase was significantly shortened, S phase and the G2period were significantly longer among the cells colonies, and its growth curve revealed that the cells colonies had lost contact inhibition, appeared malignant hyperplasia and the cell average doubling time significantly shortened for28.72h. Taken together, the above results all affirm that the cell transformed into malignant cells and was therefore a suitable cells model in the correlation studies of malignant transformation.3. The genetic damages and DNA methylation changes in the process of malignant transformation induced by OE-COE.According to the results of the second part, we selected the following cell lines:three cell lines induced by20μg/ml OE-COE for50days,100days, and120days, with their corresponding control; ten cell colonies(RB, RC, RE, RF, RG, RJ, RK, RI, RN, RO) induced by20μg/ml OE-COE for120days; three cell lines induced by20μmol/L BaP for75days,150days, and255days, with their corresponding control; four cell colonies(RA, RE, RO, RQ) induced by20μmol/L BaP for255days. At the same times, we exposed continuously the cell to2.5,5,10,20μg/ml OE-COE for5days. And observing the genetic damage indexes and DNA methylation level of MGMT gene among the above cell lines, we elucidated the mechanism of genetic damage and DNA methylation in the procession of malignant cell transformation.The results of the genetic indexes showed that compared to the control groups, the frequency of Cytokinesis-block micronucleus cytome indexes, tail moment of comet Olive and the concentration of BPDE-DNA adducts increased significantly with the increase of the induction dose and duration, while the relative length of telomere declined (p<0.05), in the short-time and malignant transformation cell model. And we observed there were significant alteration of the concentration of BPDE-DNA adducts, the relative length of telomere and the frequency of nuclear bud. The result of methylation showed that the low level of MGMT methylation change among the short-time and the malignant transformation cell model, except RC(40.47%) and RF(14.76%) cell colonies induced by OE-COE, which indicated that there were heterogeneity among the cell colonies.In summary, the level of MGMT promoter methylation was very low in coke-oven workers. The low level of MGMT promoter methylation, the relative length of telomere and the frequency of nuclear bud might be the early molecular biological events occured in the process of cell malignant transformation. There was amount of substance of carcinogenicity and mutations in the COE, which could induce the normal human bronchial epithelial cell to undergo malignant transformation after120days at the concentration of20mg/ml. We found there was difference in the level of genetic damage and DNA methylation among the different cell colonies, which indicated that there was heterogeneity among the cell colonies with the characteristics of cell malignant transformation, and the method of selection of cell colonies was adopted.