Epigenetics mechanisms in toxicity and carcinogenicity of diesel exhaust particles - Particles induced variations in gene and microRNA expression in human bronchial epithelial cells
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Lung cancer is the leading cause of cancer related deaths worldwide. Several lines of evidence are confirming that air pollution significantly increase the risk of lung cancer. Exhaust from diesel engines is a component of air pollution and categorized as a human carcinogen by IARC. Diesel exhaust particles and the associated complex mixture of organic pollutants, such as PAHs and their derivatives are major components of diesel emissions, have been under surveillance to exert adverse effects on human health.The aim of the thesis was to identify DEP induced changes on the expression of genes and miRNAs over short period of time and with increasing concentrations of DEP (SRM 2975). An in vitro human bronchial epithelial cell (HBEC3-KT) model was set up and cells were exposed for a short span of time (6-72 h) with 100 μg/ml of DEP or with different concentrations of DEP (25-400 μg/ml) for a fixed time of forty eight hours. Additionally, variations in gene/miRNA expression when the HBEC3-KT cells were exposed for long term (27 weeks) to DEP (100 μg/ml) and for short term (6-72 h) exposure to DEP & B[a]P (100 μg/ml and 3μM respectively) were analyzed. Gene expression analysis was carried out on selected candidate genes and miRNAs, which are known to be involved in lung cancer and/or environmentally induced carcinogenesis. The analyses included CYP1A1, COX2, CDH1, IL1B, HO1, NRF2, miR17, miR21, miR27, miR31, miR146a and miR146b, respectively. In addition, secretion of IL-1-β to the cell culture medium was analyzed. Expression of genes and miRNA s was measured by RT-qPCR, while secretion of IL-1-β was analyzed by ELISA.CYP1A1, which is a phase 1 metabolizing enzyme involved in biotransformation of xenobiotic compounds, was found to be induced by the exposure of DEP both in the time course experiments (maximum at 48 h) and over a wide range of DEP concentrations. Heme oxygenase 1 (encoded by HO1) is involved in many biological processes such as regulation of angiogenesis, cell death, response to oxidative stress and many others. We found that HO1 expression might be up-regulated when HBEC3-KT was exposed to DEP (and benzo[a]pyrene; B[a]P), which gives4indications of oxidative stress on the cells. DEP has the ability to induce inflammatory response in cells. Interleukin-1beta (IL-1-β) is a key pro-inflammatory cytokine, which regulates the expression of several genes involved in inflammation. We found significant induction of IL1B by the exposure to DEP and B[a]P. Similarly, IL-1-β secretion from the cells was stimulated by DEP (and B[a]P) exposure, but no clear time or concentration patterns were observed. COX2 is a key enzyme involved in prostaglandin production and prostaglandin function as attractant of inflammatory cells. The COX2 gene was possibly induced by the DEP exposure to HBEC3-KT cells. In contrast, expression of CDH1, NRF2 and the tested miRNAs were found not to be significantly altered by the exposure to DEP.In conclusion, these data indicate that exposure of HBEC3-KT to DEP may affect genes involved in xenobiotic metabolism, induction of inflammatory response, and response to oxidative stress. However, further studies are needed for solid conclusions regarding several of the gene/miRNAs.