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KMID : 1177720190130010039
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Journal of Alternatives to Animal Experiments
2019 Volume.13 No. 1 p.39 ~ p.47
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A Simple Spectrophotometric Test to Identify Skin Sensitizers and Non-Sensitizers in Chemico
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Cha Dong-Ho
Nepal Mahesh Raj
Kim Geon-Ho
Kang Mi-Jeong
Jeong Tae-Cheon
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Abstract
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For the key event No. 1 in skin sensitization, a hapten should react with endogenous proteins for the initiation of immune reaction. Although Direct Peptide Reactivity Assay (DPRA) has been developed as an alternative method for key event 1, artificially synthesized peptides and HPLC should be employed in performance, which are somewhat inconvenient. In the present study, a simple spectrophotometrical method to identify skin sensitizers in chemico was developed by using cysteamine, an endogenous substance that contains amino and thiol groups. To quantitate remaining cysteamine following the reaction with skin sensitizers, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) was selected for the derivatization. The conditions optimized included: incubation time of cysteamine with test substances, molar ratios of cysteamine to test substances, stability of derivatized products of test substances with optimal concentration and incubation time of DTNB, and spectrophotometrical characteristics of the derivatized product. With the optimized conditions, 11 skin sensitizers and non-sensitizers were tested to know whether the method is feasible or not. Although further optimization is required, it would be a useful screening tool for determining skin sensitization potential of small molecules, because the present method employs simple endogenous cysteamine as an acceptor for sensitizers with a spectrophotometric detection system,.
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KEYWORD
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skin sensitization, cysteamine, DPRA, spectrophotometry, in chemico
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