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분광광도법을 이용한 간단한 in Chemico 피부감작성 시험법의 개발
차동호 ( Dong-ho Cha ),네팔마헤시 ( Mahesh Raj Nepal ),김건호 ( Geon-ho Kim ),강미정 ( Mi-jeong Kang ),정태천 ( Tae-cheon Jeong ) 한국동물실험대체법학회 2019 동물실험대체법학회지 Vol.13 No.1
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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치올기 함유 저분자 화합물과의 반응성을 이용하는 간단한 in chemico 피부감작성 시험법의 개발 가능성 평가
김건호 ( Geon Ho Kim ),차동호 ( Dong Ho Cha ),구형모 ( Hyung Mo Gu ),박재성 ( Jae Sung Park ),정재훈 ( Jae Hoon Jeong ),황진선 ( Jin Sum Hwang ),네팔마헤시 ( Mahesh Raj Nepal ),정태천 ( Tae Cheon Jeong ) 한국동물실험대체법학회 2020 동물실험대체법학회지 Vol.14 No.1
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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