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In Situ Alkylation of Reconstructed Human Epidermis by Methyl Methanesulfonate: A Quantitative HRMAS NMR Chemical Reactivity Mapping
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-11-16 , DOI: 10.1021/acs.chemrestox.0c00362
Hassan Srour 1 , François-Marie Moussallieh 1 , Karim Elbayed 2 , Elena Giménez-Arnau 1 , Jean-Pierre Lepoittevin 1
Affiliation  

Allergic contact dermatitis (ACD) is a reaction of the immune system resulting from skin sensitization to an exogenous hazardous chemical and leading to the activation of antigen-specific T-lymphocytes. The adverse outcome pathway (AOP) for skin sensitization identified four key events (KEs) associated with the mechanisms of this pathology, the first one being the ability of skin chemical sensitizers to modify epidermal proteins to form antigenic structures that will further trigger the immune system. So far, these interactions have been studied in solution using model nucleophiles such as amino acids or peptides. As a part of our efforts to better understand chemistry taking place during the sensitization process, we have developed a method based on the use of high-resolution magic angle spinning (HRMAS) NMR to monitor in situ the reactions of 13C substituted chemical sensitizers with nucleophilic amino acids of epidermal proteins in reconstructed human epidermis. A quantitative approach, developed so far for liquid NMR applications, has not been developed to our knowledge in a context of a semisolid nonanisotropic environment like the epidermis. We now report a quantitative chemical reactivity mapping of methyl methanesulfonate (MMS), a sensitizing methylating agent, in reconstructed human epidermis by quantitative HRMAS (qHRMAS) NMR. First, the haptenation process appeared to be much faster in RHE than in solution with a maximum concentration of adducts reached between 4 and 8 h. Second, it was observed that the concentration of cysteine adducts did not significantly increase with the dose (2.07 nmol/mg at 0.4 M and 2.14 nmol/mg at 1 M) nor with the incubation time (maximum of 2.27 nmol/mg at 4 h) compared to other nucleophiles, indicating a fast reaction and a potential saturation of targets. Third, when increasing the exposure dose, we observed an increase of adducts up to 12.5 nmol/mg of RHE, excluding cysteine adducts, for 3112 μg/cm2 (1 M solution) of (13C)MMS. This methodology applied to other skin sensitizers could allow for better understanding of the potential links between the amount of chemical modifications formed in the epidermis in relation to exposure and the sensitization potency.

中文翻译:

甲磺酸甲酯对重建人表皮的原位烷基化:定量 HRMAS NMR 化学反应性图谱

过敏性接触性皮炎 (ACD) 是一种免疫系统反应,由皮肤对外源性有害化学物质致敏引起,并导致抗原特异性 T 淋巴细胞活化。皮肤致敏的不良结果通路 (AOP) 确定了与这种病理机制相关的四个关键事件 (KE),第一个是皮肤化学致敏剂修饰表皮蛋白以形成抗原结构的能力,这将进一步触发免疫系统. 到目前为止,已经使用模型亲核试剂(例如氨基酸或肽)在溶液中研究了这些相互作用。作为我们努力更好地了解敏化过程中发生的化学反应的一部分,我们开发了一种基于使用高分辨率魔角旋转 (HRMAS) NMR 的方法来监测原位反应13C 在重建的人表皮中用表皮蛋白的亲核氨基酸取代化学敏化剂。迄今为止,针对液体 NMR 应用开发的定量方法,据我们所知尚未在半固体非各向异性环境(如表皮)的背景下开发。我们现在报告了通过定量 HRMAS (qHRMAS) NMR 在重建的人类表皮中甲基磺酸甲酯 (MMS)(一种敏化甲基化剂)的定量化学反应性映射。首先,RHE 中的半抗原化过程似乎比溶液中快得多,加合物的最大浓度在 4 到 8 小时之间达到。其次,观察到半胱氨酸加合物的浓度没有随着剂量(0.4 M 时为 2.07 nmol/mg 和 1 M 时为 2.14 nmol/mg)和孵育时间(最大 2. 27 nmol/mg at 4 h) 与其他亲核试剂相比,表明快速反应和潜在的目标饱和。第三,当增加暴露剂量时,我们观察到加合物增加到 12.5 nmol/mg 的 RHE,不包括半胱氨酸加合物,为 3112 μg/cm2 (1 M 溶液) ( 13 C) MMS。这种应用于其他皮肤致敏物的方法可以更好地了解与暴露有关的表皮中形成的化学修饰量与致敏效力之间的潜在联系。
更新日期:2020-12-21
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