Genetic Toxicity And Carcinogenicity In 16 Human Bronchial Epithelial Cells Induced By Long Term And Low Dose Exposure Of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Objectives Lung cancer,also known as primary bronchogenic carcinoma,which is a kind of malignant tumors originated in the bronchial epithelial cells.It is one of the most common tumors,which is difficult to be treated.In China,lung cancer is the leading cause of cancer death for both men and women.More and more epidemiological evidences show that air pollution is positively correlated with the risk of lung cancer.However,few studies were reported on the ambient air pollution as causative factors for lung cancer,especially the levels of persistent organic pollutant dioxins in the air pollution.2,3,7,8-Tetrachlorodibenzo-p-dioxin?2,3,7,8-TCDD?,one of dioxin-like compounds,is known as the most toxic chemical in the world,and has the properties of persistence,long distance migration,carcinogenicity and mutagenicity.But until now,to the best of our knowledge,no study has been reported on the lung carcinogenic effects caused by long-term low-dose TCDD exposure.The aim of this thesis work was to investigate the effects of long-term TCDD exposure at air pollution levels on the genetic damage and malignant transformation of human immortalized bronchial epithelial cells?16HBE?,which may provide scientific evidence for the toxicity evaluation of dioxin-like substances,as well as provide a reference for the prevention and control of environmental pollution.Research contents and methods 1.The effects of TCDD exposure on proliferation of 16 HBE cells Proliferation of 16 HBE cells was determined by cell counting kit-8 kit?CCK-8?.Ambient air pollution levels of TCDD?0,1 aM,5 aM,5 fM and 5 pM?were tested in this study.Exposure experiments were conducted at the 5^th,10^th,15^th,20^th generations of 16 HBE cells.The cytotoxicity of TCDD exposure was evaluated by CCK-8 kit.2.Genetic damage in 16 HBE cells induced by long-term low-dose TCDD exposure DNA damage was detected by single cell gel electrophoresis?SCEG?in 16 HBE cells at the 5^th,10^th,15^th and 20^th generations after exposure to TCDD.Genetic damage at chromosome level was detected by micronucleus test?MNT?at the same generations in 16 HBE cells after exposure to TCDD.3.Epigenetic damage in 16 HBE cells induced by long-term low-dose TCDD exposure The changes of the global DNA methylation in 16 HBE cells at the 5^th,10^th,15^th and 20^th generations after long-term and low-dose TCDD exposure were detected by immunofluorescence assay.4.The effects of long-term and low-dose TCDD exposure on carcinogenesis in 16 HBE cells The carcinogenic effects of long-term and low-dose TCDD exposure were evaluated by soft agar colony forming assay.The 20^th generation of 16 HBE cells was tested after TCDD exposure.The migration ability of the 20^th generation of 16 HBE cells after exposure to TCDD was measured by cell migration assay.Results 1.No obvious growth inhibition effects were observed in 16 HBE cells within the tested doses of TCDD exposure,but increased proliferation trends were presented.No growth inhibition was found even under the condition of maximum concentration of 100 n M TCDD in 0.1% DMSO solution.2.No significant differences of DNA damage in 16 HBE cells were detected by single cell gel electrophoresis,and there were no significant differences between each generation of the treated 16 HBE cells.3.Comparing with the negative control group,significant differences of the micronucleus rates in the 5 p M,5 f M and 5 a M dose groups were obtained in 16 HBE cells after TCDD exposure for 15 generations or more.4.No significant differences of the global DNA methylation in 16 HBE cells between each TCDD treated groups were found by immunofluorescence,as well as no significant differences between each generation.5.Comparing with the negative control group,no statistical differences of the colony size and quantity of 16 HBE cells in each TCDD treated group.Also no significant differences of cell migration ability in each TCDD treated group.Conclusions 1.No obvious cytotoxicity effects were observed in 16 HBE cells treated by TCDD in the low-dose range under the tested conditions.2.Long-term and low-dose exposure of TCDD on 16 HBE cells induces genetic damage at the chromosome level,but not at DNA level.3.No detectable global DNA methylation differences were observed after long-term and low-dose exposure of 16 HBE cells to TCDD.4.Effects of TCDD on the malignant transformation and migration ability of 16 HBE cells were not detected after TCDD exposure for 20 generations,which suggests that 16 HBE cells may not be malignant transformed by TCDD expose alone or the progress of malignant transformation may require even more treatment time.