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치올기 함유 저분자 화합물과의 반응성을 이용하는 간단한 in chemico 피부감작성 시험법의 개발 가능성 평가

A Possible Simple in Chemico Test to Identify Skin Sensitizers and Non-Sensitizers by Using Low Molecular Weight Compounds Containing Thiol Group

동물실험대체법학회지 2020년 14권 1호 p.41 ~ 48
김건호, 차동호, 구형모, 박재성, 정재훈, 황진선, Nepal Mahesh Raj, 정태천,
소속 상세정보
김건호 ( Kim Geon-Ho ) - College of Pharmacy Yeungnam University
차동호 ( Cha Dong-Ho ) - College of Pharmacy Yeungnam University
구형모 ( Gu Hyung-Mo ) - College of Pharmacy Yeungnam University
박재성 ( Park Jae-Sung ) - College of Pharmacy Yeungnam University
정재훈 ( Jeong Jae-Hoon ) - College of Pharmacy Yeungnam University
황진선 ( Hwang Jin-Sun ) - College of Pharmacy Yeungnam University
 ( Nepal Mahesh Raj ) - College of Pharmacy Yeungnam University
정태천 ( Jeong Tae-Cheon ) - College of Pharmacy Yeungnam University

Abstract


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.

키워드

skin sensitization; glutathione; monobromobimane; spectrofluorometry; in chemico

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