Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549).

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 µM), 1-NP (1 and 10 µM) and 3-NBA (0.5 and 5 µM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

Impact of air pollution on oxidative DNA damage and lipid peroxidation in mothers and their newborns.

Ambient air particulate matter (PM) represents a class of heterogeneous substances that form one component of air pollution. Oxidative stress has been implicated as an important action mechanism for PM on the human organism. Oxidative damage induced by reactive oxygen species (ROS) may affect any cellular macromolecule. The aim of our study was to investigate the impact of air pollution on oxidative DNA damage [8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)] and lipid peroxidation [15-F2t-isoprostane (15-F2t-IsoP)] in the urine and blood from mothers and newborns from two localities with different levels of air pollution: Ceske Budejovice (CB), a locality with a clean air, and Karvina, a locality with high air pollution. The samples from normal deliveries (38-41 week+) of nonsmoking mothers and their newborns were collected in the summer and winter seasons. Higher PM2.5 concentrations were found in Karvina than in CB in the summer 2013 (mean±SD: 20.41±6.28 vs. 9.45±3.62µg/m(3), P<0.001), and in the winter 2014 (mean±SD: 53.67±19.76 vs. 27.96±12.34µg/m(3), P<0.001). We observed significant differences in 15-F2t-IsoP levels between the summer and winter seasons in Karvina for newborns (mean±SD: 64.24±26.75 vs. 104.26±38.18pg/ml plasma, respectively) (P<0.001). Levels of 8-oxodG differed only in the winter season between localities, they were significantly higher (P<0.001) in newborns from Karvina in comparison with CB (mean±SD: 5.70±2.94 vs. 4.23±1.51 nmol/mmol creatinine, respectively). The results of multivariate regression analysis in newborns from Karvina showed PM2.5 concentrations to be a significant predictor for 8-oxodG excretion, PM2.5 and B[a]P (benzo[a]pyrene) concentrations to be a significant predictor for 15-F2t-IsoP levels. The results of multivariate regression analysis in mothers showed PM2.5 concentrations to be a significant predictor of 8-oxodG levels.

DNA demethylating agent 5-azacytidine inhibits myeloid-derived suppressor cells induced by tumor growth and cyclophosphamide treatment.

MDSCs represent one of the key players mediating immunosuppression. These cells accumulate in the TME, lymphoid organs, and blood during tumor growth. Their mobilization was also reported after CY therapy. DNMTi 5AC has been intensively studied as an antitumor agent. In this study, we examined, using two different murine tumor models, the modulatory effects of 5AC on TU-MDSCs and CY-MDSCs tumor growth and CY therapy. Indeed, the percentage of MDSCs in the TME and spleens of 5AC-treated mice bearing TRAMP-C2 or TC-1/A9 tumors was found decreased. The changes in the MDSC percentage were accompanied by a decrease in the Arg-1 gene expression, both in the TME and spleens. CY treatment of the tumors resulted in additional MDSC accumulation in the TME and spleens. This accumulation was subsequently inhibited by 5AC treatment. A combination of CY with 5AC led to the highest tumor growth inhibition. Furthermore, in vitro cultivation of spleen MDSCs in the presence of 5AC reduced the percentage of MDSCs. This reduction was associated with an increased percentage of CD11c+ and CD86+/MHCII+ cells. The observed modulatory effect on MDSCs correlated with a reduction of the Arg-1 gene expression, VEGF production, and loss of suppressive capacity. Similar, albeit weaker effects were observed when MDSCs from the spleens of tumor-bearing animals were cultivated with 5AC. Our findings indicate that beside the direct antitumor effect, 5AC can reduce the percentage of MDSCs accumulating in the TME and spleens during tumor growth and CY chemotherapy, which can be beneficial for the outcome of cancer therapy.

Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.