Evaluating Photogenotoxicity using 3D Tissue Models

 描述（由申请人提供）： 本提案的目的是重点评价材料引起光遗传毒性的能力。光敏性是一种有害的反应，当暴露于紫外线或可见光时，皮肤或眼睛中的药物或化学物质会引起不希望的细胞损伤。光遗传毒性被用作筛选材料光致癌潜力的一种手段。遗传毒性是光毒素可能导致的后果之一。尽管非光遗传毒性具有标准化的检测方法，但目前还没有监管机构批准的光遗传毒性试验。将遗传毒性试验中使用的试验程序应用于光遗传毒性并不是一种简单的方法。在光致染色体断裂（紫外线诱导的染色体破坏）化合物的综述中，超过75%的在哺乳动物试验中被归类为光致染色体断裂的化合物在标准3 T3中性红更新（NRU）光毒性试验中呈阴性，表明假光致染色体断裂的发生率较高。许多这些测定的局限性是对细胞进行大量处理以分析DNA损伤，这可能需要低渗细胞处理或细胞裂解、DNA固定或解旋、染色或电泳，然后进行目视计数和评分。细胞系历来用于大多数实验室研究中的遗传毒性试验。尽管如此，3D组织模型的发展已经允许活组织的更大表示。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。将评价3D多细胞人肝微组织（InSphero）和3D分化的人表皮模型（EpiDerm”- MatTek Corp）预测光遗传毒素的能力。组蛋白磷酸化的评价 H2 AX将作为DNA损伤的指标进行定量测量。DNA损伤诱导组蛋白H2 AX羧基端的Ser 139磷酸化。β-H2 AX的检测可以通过流式细胞术和免疫荧光检测。监测DNA修复的机制终点将通过允许更多的定量终点来提高评价光遗传毒性的准确性。

 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.