| As an environmental and social issue, atmospheric particulate matter(PM) is becoming a growing concern of more governments and healthagencies all over the world. The harm of PM2.5 fine particles is especially serious, for its potential risk of causing a series of health problem in respiratory and cardiovascular system, reproductive and developmental toxicity, and even the teratogenic, mutagenic, carcinogenic risks that leading immune, not only affects the health of parents but also their offspring.In recent years, studies on toxicity of PM mostly use A549, Beas-2B or other non normal orimmortalized cell lines, while the CHL cell line is a more conventional model for evaluation of genetic toxicity. The other cell lines had disadvantages such as genetic material change, species differences, thus the establishment of toxicity evaluation system in normal cells of human source is the trend of further research. In this study, EBf-H9, which derived from human embryonic stem cell lines, with its unique advantages such as the standard culturing and differentiation conditions, the more similar genetic characteristics to the human normal fibroblasts, and a smaller batch differences, a more subcultured capacity, a stable karyotype and strong repeatability, the EBf-H9 is able to reflect a more actual reaction of human cells to the toxicants than the other cells.Objective: This study used EBf-H9 cells as a toxicity assessment model of DNA damage study caused by Hanyang’s PM2.5, intending to clarify the patern and mechanism of Hanyang PM2.5 caused DNA damage, and also to provide a new alternative cell model for PM2.5 risk assessment, so as to provide some theoretical basis for further study.Methods: The particle morphology of PM2.5 is valued with transmission electron microscopy. Hanyang PM2.5 exposed liquid was formulated into concentration of 6.25 g/ml、12.5 g/ml、25 g/ml、50 g/ml、100 g/ml、200 g/ml、400 g/ml. 24 h of cell viability and LDH leakage rate caused by Hanyang PM2.5 in EBf-H9 were valued with CCK-8 kit and LDH kit. The morphology of 24 h contaminated EBf-H9 cells by Hanyang PM2.5 were achieved by HE staining; the total ROS content of EBf-H9 cells contaminated 1h by Hanyang PM2.5 is valued with DCFH-DA probe; the total SOD, total GPx, total MDA content of EBf-H9 cells caused by 6h of Hanyang PM2.5 contamination were detected using SOD kit, GPx kit, MDA kit respectively; Selecting the cell non-toxic dose in which cell viability is more than 90%, then evaluate the DNA and chromosomal damage degree of EBf-H9 cells caused by 24 h of Hanyang PM2.5 contamination with single cell gel electrophoresis assay and micronucleus test; DNA damage associated gene p53 of EBf-H9 cells contaminated by Hanyang PM2.5 for 6h was detected with RT-PCR; DNA damage associated H2 AX and PARP protein gamma of cells contaminated by Hanyang PM2.5 for 6h was detected with Western blot。Results: By PM2.5 characterization that can be seen, Hanyang PM2.5 contains the ball shaped coal fly ash, the dense shapedsoot aggregates, and irregular shaped mineral salts.The results of EBf-H9 cytotoxicity test showed that, with the increasing doses of PM2.5, cell survival rate decreasing in a negative relation; the cell model is more sensitive comparing to human lung fibroblast a more obvious cytotoxic effect. LDH leakage rate increased first and decreased later in a dose-response relationship. The the leakage rate of LDH decline at high doses cells may be caused by cell death which leading to the release of LDH decrease. Through the observation of cell morphology after contamination, the number of cells decreased gradually as dose increases; cell vacuolar degeneration appeared at lower doses, and nuclear fragmentation and nuclear pyknosis showed at higher doses.Reactive oxygen species(ROS) is a general term for a class of oxygenated compounds in theorganism, including oxygen ion, peroxide and oxygen radical etc. ROS plays a great role in cell signal transduction and maintain body balance. In the ROS detecting result from flow cytometer, an increased total cell ROS is observed with the contamination dose increasing in a positive correlation. Superoxide dismutase(SOD) can scavenge redundant oxygen free radicals in the body and protect cells from oxidative injury. Glutathione peroxidase(GPx) protects the structure and function of cell membrane by reducing the generation of toxic peroxide into harmless hydroxyl compounds. When the oxygen free radicals generated by the enzyme system and non enzymatic system overload the capability of the cleaning, the lipid peroxidation will happen to form lipid peroxide such as malondialdehyde(MDA). From the results of indexes of oxidative damage we observed the decreasing SOD and GPx with the dose increased in a negative correlation, while MDA increased with the contamination dose increasing in a positive correlation, both in a dose-response pattern. It shows that with the increase of the dose cells will produce a large number of ROS in short time. When ROS cannot be eliminated, SOD and GPx of EBf-H9 cells begin to activate, resisting excessive oxygen free radical intracellular. Ultimately the formation of product MDA induced apoptosis and even lead to cell death.Single cell gel electrophoresis assay and binucleate cells micronucleus test are regular methods to evaluate the genetic toxicity in DNA and chromosomes level. The results showed that with the increasing PM2.5 dose, comet tails rate and binuclear cellmicronucleus rate gradually increased, and there is an obvious dose-response relationship, indicating DNA and chromosome damage of EBf-H9 cell caused by Hanyang PM2.5, showing that PM2.5 can lead to cell DNA integrity, chromosome integrity and chromosome separation changes and having certain genetic toxicity. Comparing with our previous research results, we find that EBf-H9 cells were more sensitive than CHL cell line, which is the present evaluation model of the common genetic toxicity.RT-PCR and Western blot are widely used in detection of the gene and protein content. With RT-PCR we detected EBf-H9 cell DNA damage gene p53. The results showed that its expression was increased firstly and then decreased; Western blot detecting of γH2AX protein showed an first increased and then decreased tendency, while PARP protein 89 kd is in the upward trend throughout. This shows that the DNA damage of EBf-H9 cell through activation of γH2AX by p53 causes DNA double strand breaks, and activate the repair protein PARP. The repair capacity of cells decreased with increasing dose, yet the cell DNA damage is a complex pathway system involving multiple pathways and a variety of proteins, it needs further study to fully reveal its function mode and channe. Conclusion: This study shows EBf-H9 is a new candidate cell model of PM2.5 risk assessment. Hanyang PM2.5 has some genotoxic; the Hanyang PM2.5 caused DNA damage pattern and mechanism on EBf-H9 cell, confirm that the EBf-H9 cells produce a large number of ROS through external PM2.5 stimulation, and then trigger the cells generate a series of oxidative injury that finally cause cells suffer DNA damage and chromosome damage. In DNA damage repairing pathway it can be seen that DNA damage is the activation of γH2AX by p53, and start the repair protein PARP, against oxidative damage caused by ROS. |
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