Evaluating Photogenotoxicity using 3D Tissue Models

 描述(由申请人提供)： 这项提议的目标是重点评估材料引起光遗传毒性的能力。光敏是一种有害反应，当皮肤或眼睛暴露在紫外线或可见光下时，皮肤或眼睛中的药物或化学物质会导致不良的细胞损害。光遗传毒性被用作筛选材料的光致癌潜力的一种手段。遗传毒性是光毒素引起的可能后果之一。虽然非光遗传毒性已经有了标准化的检测方法，但目前还没有监管部门批准的光遗传毒性检测方法。将遗传毒性测试中使用的测试程序应用于光遗传毒性并不是一种简单的方法。在对光致裂解(UV诱导的染色体破坏)化合物的综述中，在哺乳动物实验中被归类为光致裂解的化合物中，超过75%的化合物在标准的3T3中性红更新(NRU)光毒实验中为阴性，表明假光致裂解的发生率很高。其中许多检测方法的局限性是需要对细胞进行广泛的处理来分析DNA损伤，这可能需要低渗细胞处理或细胞裂解、DNA固定或解开、染色或电泳法，然后进行目视计数和评分。细胞系历来被用于大多数实验室的遗传毒性检测。尽管如此，3D组织模型的发展已经允许更多地代表活组织。我们建议评估由悬滴、3D肝细胞培养和3D重建人表皮(RHE)组织组成的体外模型。3D多细胞人肝微组织(InSphero)和人表皮3D分化模型(Epiderm-Mattek Corp)将评估它们预测光遗传毒素的能力。组蛋白磷酸化的评价 H_2AX将作为DNA损伤的一个指标进行定量测量。组蛋白H_2AX(-H_2AX)的羧基末端Ser139发生磷酸化。流式细胞术和免疫荧光法均可检测-H_2AX。监测DNA修复的机械性终点将允许通过允许更定量的终点来更准确地评估光遗传毒性。

 DESCRIPTION (provided by applicant): The objective of this proposal is to focus on evaluating materials for their ability to cause photogenotoxicity. Photosensitivity is a harmful reaction that occurs when drugs or chemicals in the skin or eyes cause undesirable cellular damage when exposed to UV or visible light. Photogenotoxicity is used as a means to screen a material for its photocarcinogenic potential. Genotoxicity is one of the possible outcomes caused by phototoxins. Although non-photo genotoxicity has standardized testing methods, currently there are no regulatory approved photogenotoxicity assays. Applying the testing procedures used in genotoxicity testing towards photogenotoxicity is not a straightforward approach. In a review of photoclastogenic (UV induced chromosome disruption) compounds, more than 75% of compounds that were classified as photoclastogenic in mammalian assays were negative in the standard 3T3 Neutral Red Update (NRU) phototoxicity assay, demonstrating the high occurrence of pseudophotoclastogenicity. A limitation of many of these assays is the extensive processing of cells to analyze DNA damage, which can require hypotonic cell treatment or cell lysis, DNA fixation or unwinding, staining or electrophoresis, then visual counting and scoring. Cell lines have historically been used in most laboratory studies for genotoxicity assays. Nonetheless, the development of 3D tissue models have allowed for a much greater representation of living tissues. We propose to evaluate in vitro models consisting of hanging drop 3D liver cell culture and 3D reconstructed human epidermal (RHE) tissues. 3D Multi-cell human liver microtissues (InSphero) and 3D differentiated model of the human epidermis (EpiDerm" - MatTek Corp) will be evaluated for their ability to predict photogenotoxins. Evaluation of phosphorylation of histone H2AX will be quantitatively measured as an indicator of DNA damage. DNA damage induces phosphorylation of Ser139 of the carboxy terminus of histone H2AX (-H2AX). Detection of -H2AX can be detected by both flow cytometry and immunofluorescence. Monitoring a mechanistic endpoint of DNA repair will allow for a greater accuracy of evaluating Photogenotoxicity by allowing for a more quantitative endpoint.