Modification of the MTT method for the study of bilirubin cytotoxicity

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Abstract
We propose a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method to study the cytotoxicity of bilirubin. The original method involves reading the intensity of a purplish blue color resulting from the conversion of MTT to formazan crystals by the mitochondria of viable cells. We have found that when the method is applied to study the effect of bilirubin on growing cells, precipitation of the yellow bilirubin pigment interferes with the colorimetric reading. A human liver cell line was used. The interference of bilirubin deposition on the MTT assay was investigated by comparing the value of optical density of the MTT solution in the presence and absence of bilirubin. The effect of 0.04 mol/L HCL-isopropanol on the bilirubin precipitate was tested by recovering the amount of bilirubin from the wells after the isopropanol treatment. Bilirubin deposition increases MTT reading by 10-24%. Hydrochloride-isopropanol (0.04 mol/L) dissolves MTT formazan only without disturbing the bilirubin precipitates. The bilirubin extracted into the supernatant was less than 5% of the total bilirubin deposited. This indirect MTT assay, as developed in this study, could eliminate the interference of bilirubin deposits and serve as a good method for the study of bilirubin cytotoxicity.