Application of tryptophan fluorescence to assess sensitizing potentials of chemicals

There are too many chemical substances around our living space. However, the toxicity of most of them has not been reported, especially regarding their sensitizing potentials. We aimed to develop a simple in vitro method to quantitatively predict the sensitizing potentials of chemicals by measuring the fluorescence of chemical-human serum albumin (HSA) complexes. HSA was treated with test chemicals and then analyzed by tryptophan fluorescence and protein concentration measurement. Four commonly designated sensitizers, two possible sensitizers, and two nonsensitizers were examined using the tryptophan fluorescence assay. HSA fluorescence at 280 nm excitation and 340 nm emission was reduced by toluene 2,4-diisocyanate (TDI), dose dependently. The addition of TDI immediately reduced the fluorescence, and it was stable for 6 h to 21 days after treatment, with a slight decrease. The reduction of HSA fluorescence by chemicals was in the order: commonly designated sensitizers > possible sensitizers > nonsensitizers. Chemical treatment at 0.05 and 0.5 mM led to optimal separation among the three groups. o-Phthalaldehyde (OPA), which has not been evaluated regarding its sensitization potential by any of the authorized organizations, reduced HSA fluorescence as much as the commonly designated sensitizer at final concentrations of the chemical of 0.05 and 0.5 mM. According to our method, OPA is evaluated as a commonly designated sensitizer. The treatment of all test chemicals did not lead to marked differences in the total protein concentrations by either the Lowry or the Bradford method. The assay utilizing tryptophan fluorescence loss of HSA after chemical treatment is a promising method to evaluate the sensitizing potentials of chemicals.