Extraction tool and matrix effects on arsenic speciation analysis in cell lines

Arsenic glutathione (As–GSH) complexes have been suggested as possible metabolites in arsenic (As) metabolism. Extensive research has been performed on the toxicological and apoptotic effects of As, while few reports exist on its metabolism at the cellular level due to the analytical challenges. In this study, an efficient extraction method for arsenicals from cell lines was developed. Evaluation of extraction tools; vortex, ultrasonic bath and ultrasonic probe and solvents; water, chemicals (methanol and trifluoroacetic acid), and enzymes (pepsin, trypsin and protease) was performed. GSH effect on the stability of As–GSH complexes was studied. Arsenic metabolites in dimethylarsino glutathione (DMA(GS)) incubated multiple myeloma cell lines were identified following extraction. Intracellular GSH concentrations of myeloma cell lines were imitated in the extraction media and its corresponding effect on the stability and distribution of As metabolites was studied. An enhancement in both extraction recoveries and time efficiency with the use of the ultrasonic probe was observed. Higher stabilities for the As species in water, pepsin and trypsin were obtained. The presence of 0.5 mM GSH in the extraction media (PBS, pH 7.4) could not stabilize the As–GSH complexes compared to the 5 mM GSH, where high stabilization of the complexes was observed over a 5 day storage study. Finally, the speciation analysis of the DMA(GS) culture incubated cell lines in the presence or absence of GSH revealed the important role GSH plays in the preservation of DMA(GS) identity. Hence, caution is required during the extraction of arsenicals especially the As–GSH complexes, since their identification is highly dependent on GSH concentration.

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► Sonication probe can be used efficiently to extract As species from cell lines with high recoveries (>92%).
► Water or PBS is the optimum solvents that prevent the decomposition of arsenicals upon storage.
► The stability of As–GSH complexes in cell extracts is highly dependent upon glutathione (GSH) concentration.
► Upon extraction, GSH concentration in cell lines decreases (diluted by PBS). Therefore, biological conditions (GSH concentration) should be mimicked to maintain the stability of DMA(GS) in the extracts.