Abstract
Benzo[a]pyrene (B[a]P), a well-known polyaromatic hydrocarbon, is known for its lung carcinogenicity, however, its role in bladder cancer development is still discussed. Comparative two-dimensional blue native SDS-PAGE analysis of protein complexes isolated from subcellular fractions of 0.5 µM B[a]P-exposed cells indicated a differential regulation of proteins involved in carbohydrate, fatty acid, and nucleotide metabolism, suggesting a possible metabolic flux redistribution. It appeared that B[a]P exposure led to a repression of enzymes (fructose-bisphosphate aldolase A, glucose-6-phosphate isomerase, lactate dehydrogenase) involved in glycolysis, and an up-regulation of proteins (glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconolactonase) catalyzing the pentose phosphate pathway and one carbon metabolism (10-formyltetrahydrofolate dehydrogenase, bifunctional purine biosynthesis protein). Untargeted metabolomics further supported the proteomic data, a lower concentration of glycolytic metabolite was observed as compared to glutamine, xylulose and fatty acids. The analysis of the glutathione and NADPH/NADP+ content of the cells revealed a significant increase of these cofactors. Concomitantly, we did not observe any detectable increase in the production of ROS. With the present work, we shed light on an early phase of the metabolic stress response in which the urothelial cells are capable of counteracting oxidative stress by redirecting the metabolic flux from glycolysis to pentose phosphate pathway.
Highlights
Benzo[a]pyrene (B[a]P) is a widely distributed environmental contaminant
By combining 2D BN/SDS-PAGE technique and metabolomics, we observed alterations of proteins and metabolites involved in cellular metabolism, of those involved in maintaining the redox homeostasis of the cells
The analysis revealed a significant number of proteins (27%) assigned to metabolic pathways, along with pathways involved in carbon metabolism, pentose phosphate pathway, one carbon metabolism and pathways related to amino sugar and nucleotide sugar metabolism
Summary
B[a]P, which like other polycyclic aromatic hydrocarbons, is produced as a result of incomplete combustion of organic matter, fossil fuel, tar deposits and charbroiled food It is one of the most studied components of cigarette smoke and is known for its mutagenic and carcinogenic properties[1]. We extended this precedent work by analyzing the shift in cellular metabolism that the cells undergo to sustain the hostile environment generated by B[a]P-induced toxicity For this purpose we applied the two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE) technique to elucidate the network of protein-protein interactions that regulate cellular metabolism and the toxicity of B[a]P. The untargeted acquisition was used to gain a holistic view of the changes in metabolites occurring upon B[a]P exposure in bladder epithelial cells This technique was preferred because of its comprehensive approach of taking into account the information of both anticipated and non-anticipated compounds during identification. It appeared that by alteration of proteins involved in detoxification, and by redirecting energetic substrates and metabolic intermediates into the biochemical pathways that generate key antioxidant molecules, mainly pentose phosphate pathway (PPP) derived NADPH or glutaminolysis-derived GSH, B[a]P exposed cells can directly promote ROS detoxification mechanism
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