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KMID : 1177720200140010041
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Journal of Alternatives to Animal Experiments
2020 Volume.14 No. 1 p.41 ~ p.48
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A Possible Simple in Chemico Test to Identify Skin Sensitizers and Non-Sensitizers by Using Low Molecular Weight Compounds Containing Thiol Group
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Kim Geon-Ho
Cha Dong-Ho
Gu Hyung-Mo
Park Jae-Sung
Jeong Jae-Hoon
Hwang Jin-Sun
Nepal Mahesh Raj
Jeong Tae-Cheon
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Abstract
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For the initiation of skin sensitization reaction, a sensitizer should react with endogenous proteins. During the haptenization of a sensitizer with proteins, it is also possible for a sensitizer to react with small molecules that contain either amino or thiol group, indicating a possibility to develop a simple skin sensitization test using the reactivity of test compounds with thiol-containing low molecular weight compounds. In the present study, 10 compounds that contain a thiol group were pre-tested to determine whether the reactivity with a thiol-detecting monobromobimane would be in concentration-dependent manners. Subsequently, among thiol compounds, a simple spectrofluorometrical method to identify skin sensitizers in chemico was tested with glutathione, an endogenous substance that contains amino and thiol groups. To flurorometrically quantitate the remaining thiol-containing compounds following a reaction with skin sensitizers, monobromobimane was employed. The conditions optimized included: incubation conditions of thiol compounds including glutathione with monobromobimane, molar ratios of thiol compounds to monobromobimane, optimal concentration and incubation time of monobromobimane, and pH and incubation time with test compounds. With a tentatively optimized conditions, 4 skin sensitizers and 2 non-sensitizers were tested to know whether the method could correctly identify skin sensitizers and non-sensitizers. The results indicated a good possibility. Although further optimization is required, it would be a useful screening tool for determining skin sensitization potential of test compounds, because the present method employs a simple endogenous thiol compound as an acceptor for sensitizers with a spectrofluorometric detection system requiring neither experimental animals nor cell cultures.
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KEYWORD
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skin sensitization, glutathione, monobromobimane, spectrofluorometry, in chemico
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