Breast Cancer Resistance Protein mediates the efflux of pyrene conjugates in human intestinal Caco-2 cells

The gastrointestinal tract is a main route of exposure to polycyclic aromatic hydrocarbons (PAH). Carcinogenic PAH are predominantly metabolised to biologically active dihydrodiol epoxides capable to form stable DNA adducts. Detoxification of dihydrodiol epoxides through glutathione (GSH) conjugate formation and their subsequent removal from the cell by transport proteins of the ATP-binding cassette (ABC) superfamily is a major process to control DNA adduct levels.Specific LC-ESI-MS/MS methods were established in order to determine GSH conjugates of the carcinogenic PAH benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP). Caco-2 cells served as a model system to investigate the intestinal metabolism of both PAH and its metabolite transport (Hessel et al. 2010). Medium_ and cell extract samples from Caco-2 cell cultures incubated with the particular PAH dihydrodiol or dihydrodiol epoxide were cleaned up by a solid phase extraction (SPE) step. LC-MS/MS analysis operated in the selected reaction monitoring mode (SRM) appeared to have the best specificity and sensitivity for the analysis of these GSH conjugates whose molecular ions are fragmented during this process to their associated daughter ions. Additional mass spectrometric scan modes like the daughter scan mode were used for structure determinations.Detoxification by GSH conjugation could be successfully analysed for the class of PAH by means of its representatives BP and DBP in the Caco-2 cell system with the described method. The focus was based on the detoxification of the ultimate carcinogenic dihydrodiol epoxides (+)-anti-BPDE and (-)-anti-DBPDE. This process included the formation of the GSH conjugates as well as their excretion from the Caco-2 cells into the surrounding medium. In the Transwell¶TM -system the conjugates reached both, the basolateral compartment signifying an excretion into the blood stream and the apical side representing the intestinal lumen.In conclusion, the specified method allows the procedure of transport experiments of detoxification products in cell culture with non-labeled compounds. Interestingly, Transwell¶TM -system experiments point to an equal excretion into the blood stream and the intestinal lumen.


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