Toxicity of diesel exhaust: Biological effects of occupational exposure and chemical in vitro transformation of human bronchial epithelial cells

The issue of urban air pollution and lung cancer risk became a concern already in the 1950s. Of particular health concern has long been occupational settings where heavy-duty diesel powered equipment is used in enclosed areas such as underground mining and tunnel construction. Exposure to diesel exhaust (DE), including DE particles (DEP), is associated with lung and cardiovascular diseases. In 2012, exposure to DE/DEP was classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer. This classification was predominantly based on evidence of increased risk of lung cancer from studies in non-metal miners and truck drivers. Lung cancer is the leading cause of cancer worldwide and the five-year survival rate remains low. The main risk factor for lung cancer is tobacco smoking, although contributions from environmental and occupational exposures including DE/DEP is of importance. More knowledge is needed on the toxic effects of DE/DEP and molecular alterations associated with chemically induced lung carcinogenesis to aid in establishing occupational exposure limits and the continued development of lung cancer treatments. In this thesis, the main objective was to investigate biological effects associated with exposures to DE/DEP, both in vivo in Norwegian tunnel finishing workers (TFW) and in vitro using human bronchial epithelial cells (HBEC). In addition, a comparative in vitro study was performed to assess toxic effects of DEP, cigarette smoke condensate (CSC) and benzo[a]pyrene (B[a]P). Despite reduction efforts, the level of DE/DEP emitted from diesel powered equipment remains relatively high during tunnel finishing work in Norway. TFW are fitters performing work related to e.g. electrical installations, rock support, and water- and frost protection. In this thesis, increased levels of bulky DNA adducts, deregulated expression of microRNA (miRNA) in peripheral blood mononuclear cells, in addition to altered profiles of plasma arachidonic acid and eicosanoids are reported in TFW compared with a reference group. Long-term in vitro exposure of a HBEC line to DEP induced the ability of these cells to form colonies in soft agar assay. A stable DEP transformed cell line, T2-HBEC3, was established. The further analyses indicated the occurrence of epithelial-to-mesenchymal transition (EMT) based on morphological alterations and expression of EMT marker genes. Parental HBEC3 and T2-HBEC3 showed basal differences in gene expression profiles, and altered sensitivity to DEP in short-term exposure experiments particularly regarding genes involved in xenobiotic and lipid metabolism, and inflammation. Gene- and miRNA expression profiles of T2-HBEC3 were compared with those of transformed cell lines previously established from long-term exposures to CSC and B[a]P. Both general and specific changes in gene- and miRNA expression profiles were identified in the transformed cell lines, which were mainly associated with carcinogenesis, EMT, and the extracellular matrix. Moreover, alterations in the aryl hydrocarbon receptor pathway and inflammatory responses were identified. In conclusion, the studies conducted for this thesis add information of toxic effects of diesel exhaust both in vivo and in vitro, in addition to contributing to an increased understanding of molecular events during chemically induced transformation of human bronchial epithelial cell lines