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• W1584137966 abstract "The major objective of this study was to evaluate whether the environmental contaminant benzo[a]pyrene B[a]P, one of the most important polycyclic aromatic hydrocarbons (PAH), is capable of mediating DNA damage in urinary bladder epithelial cells and hence potentially bladder carcinogenesis. To pursue this goal, effects of B[a]P on urinary bladder epithelial cells were investigated by applying a proteomic approach with the purpose of identifying proteins and pathways involved in B[a]P toxicity. First, the ability of bladder epithelial cells for B[a]P uptake and metabolism was determined. Secondly, a proteome map of primary porcine urinary bladder epithelial cells (PUBEC), the cell model used in the majority of the studies, was established as basis for comparative investigations. In the same model, investigations on time- and concentration-dependent expression changes of proteins after B[a]P exposure followed. The proteins were separated by using 2D gel electrophoresis and identified by MALDI-TOF-MS analysis in these studies. Finally, to elucidate mechanisms by which B[a]P mediates its toxicity, signaling pathways were studied in RT4 cells by using Blue Native PAGE analysis. Besides offering some insights into B[a]P-mediated toxic effects, the studies also point towards the possibility of bladder cancer development induced by B[a]P exposure.B[a]P is a ubiquitous environmental pollutant formed during the combustion of fossil fuels, grilling, barbecuing, and smoking of food. Although much information is available on the carcinogenic properties of B[a]P, the mechanism by which this chemical is taken up by cells is still not known. In Chapter 3 of this thesis, attempts were made to investigate the dynamics of B[a]P uptake, subcellular distribution, and metabolism in PUBEC. It was found that exposure to 0.5 μM B[a]P led to an increase in intracellular concentration of B[a]P in bladder epithelial cells in a time-dependent manner but without approaching saturation. Also, a marked difference in B[a]P uptake was observed among various PUBEC pools used for the studies. Subcellular partitioning studies of B[a]P by using confocal microscopy revealed that a significant amount of B[a]P accumulated in the cell membrane of PUBEC, while only a slight but significant increase in B[a]P fluorescence intensity was observed in the cytosol and nucleus. Quantification of B[a]P uptake by bladder epithelial cells by spectrofluorometric and gas chromatographic-mass spectrometric analysis yielded intracellular concentrations ranging from 7.28 μM to 35.07 μM in cells exposed to 0.5 μM B[a]P and from 29.9 μM to 406.64 μM in cells exposed to 10 μM B[a]P. The formation of 3-OH-B[a]P in all of the B[a]P-exposed PUBEC determined by GC-MS analysis demonstrated for the first time that oxygenated B[a]P metabolites are actually formed in this cell model. These results indicate that bladder epithelial cells are capable of a strong accumulation and metabolic activation of B[a]P and suggest that B[a]P may act as a bladder cancer-inducing chemical. Also, the differences in B[a]P uptake by the various PUBEC pools is an explanation for the inter-individual variation in PAH toxicity as observed in humans. Urinary bladder epithelial cells (also known as transitional epithelial cells) are the innermost cells of the bladder which are involved in accommodating the fluctuation of liquid volume in this organ and also help to protect it against caustic/toxic effects of urine. These cells are also the first ones to come in contact with urinary toxicants and thus account for 90 % of bladder cancers known as transitional cell carcinomas. As a prerequisite for proteomic studies, the first reference proteome and phosphoproteome maps of porcine bladder epithelia cells were generated by applying 2DE gel electrophoresis. This is discussed in Chapter 4. A total of 120 selected protein spots were identified by MALDI-TOF-MS analysis, among which 31 phosphoproteins were enriched by using a method based on the precipitation with lanthanum ions (La3+). All identified proteins were bioinformatically annotated according to their physiochemical characteristic, subcellular location, and function. Most of the proteins were distributed in an area of pI 4-10 and a molecular mass range between 20 kDa and 100 kDa. The 2DE map with the complete range of expressed proteins, especially with information about phosphoproteins, provides a valuable resource for comparative proteomic analysis of normal and pathological conditions affecting the bladder function. The studies described in Chapter 5 of the thesis deal with a series of events leading from DNA damage to apoptosis that were investigated by using a proteomic approach. 2DE gel electrophoresis mapped the differences between cells exposed to 0.5 μM B[a]P and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by MALDI-TOF-MS analysis. A concentration-dependent increase in DNA damage was observed after an exposure period of 24 h. The expression of VDAC2, CTSD, HSP27, and HSP70 indicated towards the intrinsic apoptotic mitochondrial pathway, although the analysis of mitochondrial dysfunction pointed towards an alternate pathway of cell death: The mitochondrial membrane potential (MMP), although disturbed during the initial exposure period, was nearly retained after 24 h of B[a]P treatment. In conclusion, the studies indicated DNA damage caused by B[a]P at low concentrations during an exposure period of 24 h and also shed light on a possible apoptotic mechanism induced by DNA damage. Studies on protein-protein interactions involved in B[a]P toxicity are described in Chapter 6. A comparative analysis of proteomic complexes involving the two AhR ligands B[a]P and TCDD was carried out by using 2D BN/SDS-PAGE. For the enrichment of the protein complexes, a subcellular fractionation of unexposed cells and cells exposed to B[a]P and TCDD was carried out. BN/SDS-PAGE of these fractions revealed an effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major differences in the protein maps obtained from samples of control cells and cells exposed to B[a]P and TCDD, respectively, concerned the expression of many calcium- and iron-containing proteins. On the basis of these findings, the intracellular calcium content of cells exposed to TCDD and B[a]P was evaluated, revealing an increase only after 24 h of exposure but with no transient elevation. The cells exposed to TCDD also showed an alteration in the labile iron pool (LIP) of the cells, but no such changes were observed in B[a]P-exposed cells. The increase in the LIP was strongly inhibited by the calmodulin (CaM) antagonist W-7 (10 μM). These results point towards a possible interaction between the iron and calcium signaling of the cells. The analysis of nitric oxide generation by using the Griess assay revealed a substantial increase in NO content of both B[a]P- and TCDD-exposed cells. Also in these cells, the basal NO generation was inhibited when the cells were blocked with the CaM antagonist W-7. The results led to the conclusion that alterations in calcium and iron homeostasis upon exposure to TCDD and B[a]P is linked by NO that is produced by CaM-activated nitric oxide synthase (NOS). The NO thus produced by interacting with the iron centers of IRPAs modulated the activity of TfR1 and FTH1 which in turn changed the LIP of the cells and hence the toxicity. Although some new mechanistic insights into the mechanisms of B[a]P- and TCDD-induced toxicity were provided by these studies, further investigations are still required for the validation of these initial results." @default.
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• W1584137966 date "2013-01-28" @default.
• W1584137966 modified "2023-09-27" @default.
• W1584137966 title "Proteomic Analysis of Benzo[a]pyrene-Mediated Bladder Toxicity" @default.
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