Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK; Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
Safety & Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, MK44 1LQ, UK.
Toxicology. 2020 Dec 1;445:152603. doi: 10.1016/j.tox.2020.152603. Epub 2020 Sep 28.
Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.
对代表皮肤致敏关键介质影响事件（MIE）的模型亲核试剂进行半抗原化，是常规的化学计量学测量方法，可为皮肤过敏风险评估提供数据。更好地了解亲核试剂在人体皮肤中的半抗原化动态，可以为提高化学物质致敏剂效力的确定提供所需的指标，从而有助于进行风险评估。我们之前已经证明了双重稳定同位素标记方法的适用性和敏感性，可用于检测蛋白质复杂混合物中低水平的半抗原化。在本研究中，我们在亚毒性浓度下，随着时间的推移，在相关的活细胞模型中研究了半抗原化。DNCB 是一种非常有效的敏化剂，在 HaCaT 细胞中，在亚毒性浓度（10μM）下作用 4 小时，仅引起总蛋白质差异表达的微小变化，并且使大约 0.25%的所有可用亲核试剂发生半抗原化。数据表明，最大半抗原化水平发生在 2 小时，DNCB 虽然是一种混杂的半抗原，但对 Cys 残基表现出偏好，尽管胺类亲核试剂的浓度要高得多。尽管一些高度丰富的蛋白质被半抗原化，但也在低丰度蛋白质上检测到许多半抗原化位点。与暴露于表面的亲核试剂相比，某些蛋白质在深埋于蛋白质结构内部的残基处被修饰，这些残基对半抗原化的可及性较低。埋藏残基的微环境可能是影响靶亲核试剂和半抗原反应性的几个因素的结果。
