Azo dyes and carcinogenic aromatic amines in cell cultures

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Azo dyes are widely used in the food, pharmaceutical, cosmetic, textile and leather industries. They can be reduced by azoreductases in intestinal bacteria, liver cells and skin surface microflora so that aromatic amines are released. In this study an analytical system for the determination of carcinogenic aromatic amines at the picogram to femtogram level and a cell culture assay to evaluate the toxicological effects of azo dyes and aromatic amines is presented. For the assays, the commercial azo dye Resacor Blue 2F (Color Index: Direct Blue 15), a 3,3′-dimethoxybenzidine-based dye, was used. The released carcinogenic aromatic amine, 3,3′-dimethoxybenzidine, was extracted with diethylether derivatized with pentafluoropropionic anhydride and analyzed by capillary gas chromatography/mass spectrometry. In addition, the cytotoxicity of Resacor Blue 2F on cultured kidney epithelial cells and on two hepatocyte cell lines (Hep G2 and Chang liver) was evaluated. For this purpose, the cells were exposed for 72 h to varying concentrations of Resacor Blue 2F. The results show that the azo dye inhibits the proliferation of the kidney epithelial cells much more than the proliferation of the two hepatocyte cell lines. As calculated from the dose-response curves, the EC50 (effective concentration reducing cell proliferation by 50%) for kidney epithelial cells was 40 μg/ml, whereas the EC50 for both hepatocyte cell lines was more than 250 μg/ml. Higher concentrations of 3,3′-dimethoxybenzidine were found in kidney epithelial and in Hep G2 culture supernatants; only small amounts were found in the Chang liver culture supernatant. In summary, it was demonstrated that released 3,3′-dimethoxybenzidine was found in the cell culture supernatants, but it did not accumulate within the cells. For an interpretation of the toxicological results, cell-specific transport systems and osmotic effects of the commercial azo dye which contains several inorganic and organic additives has to be considered.