Evaluating Photogenotoxicity using 3D Tissue Models

使用 3D 组织模型评估光遗传毒性

• 批准号: 9001682
• 负责人: Matthew J Troese
• 金额: $ 13.31万
• 依托单位: MB RESEARCH LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-25 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9001682
• 关键词: Adherent Culture; Agriculture; Amitrole; Biological Assay; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Chemical Exposure; Chemicals; Chlorpromazine; Chromosomal Stability; Chromosomes; Cosmetics; Cytolysis; DNA; DNA Damage; DNA Repair; Detection; Development; Drops; Drug or Chemical; Electrophoresis; Epidermis; Evaluation; Exposure to; Eye; Flow Cytometry; Health; Hepatocyte; Human; Immunofluorescence Immunologic; In Vitro; Laboratory Study; Life; Liver; Measures; Medical; Metabolic; Methods; Methoxsalen; Modeling; Monitor; Mutagenicity Tests; Mutagens; Neutral Red; Outcome; Phosphorylation; Photosensitivity; Phototoxicity; Physiological; Procedures; Reaction; Route; Skin; Staining method; Stains; Testing; Therapeutic; Tissue Model; Tissues; UV Radiation Exposure; UV induced; Ultraviolet Rays; Update; Visible Radiation; Visual; Zinc Oxide; dermal exposure; genotoxicity; human H2AX protein; in vitro Model; in vitro testing; keratinocyte; nanoparticle; sample fixation; three-dimensional modeling; titanium dioxide

 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.

 描述（由申请人提供）： 该提案的目的是专注于评估材料引起光生毒性的能力。光敏性是一种有害反应，当皮肤或眼睛中的药物或化学物质在暴露于紫外线或可见光时会导致不良的细胞损伤。光生毒性被用作筛选材料的光氧促潜能的一种手段。遗传毒性是光毒素引起的可能结果之一。尽管非遗传毒性具有标准化的测试方法，但目前尚无监管批准的光生毒性评估。将用于遗传毒性测试的测试程序应用于光源毒性并不是一种直接的方法。在综述的化合物（紫外线诱导的染色体破坏）化合物的综述中，在标准的3T3中性红色更新（NRU）光毒性测定中，被归类为哺乳动物分析的化合物中有75％以上为阴性，证明了pseudopopollasseplassogeniticity的出现。许多这些测定法的局限性是细胞的广泛处理来分析DNA损伤，这可能需要低音细胞处理或细胞裂解，DNA固定或放松，染色或电泳，然后进行视觉计数和评分。历史上，大多数实验室研究都将细胞系用于遗传毒性测定。但是，3D组织模型的发展允许对生物组织的更大表示。我们建议评估包括悬挂滴3D肝细胞培养和3D重建的人表皮（RHE）组织组成的体外模型。将评估3D多细胞人类肝微动物（Insphero）和3D分化的人表皮模型（Epiderm' -Mattek Corp）的预测能力。 H2AX将以定量测量作为DNA损伤的指标。 DNA损伤诱导组蛋白H2AX（-H2AX）的羧基末端的Ser139磷酸化。可以通过流式细胞仪和免疫荧光检测-H2AX的检测。监测DNA修复的机理终点将使通过更定量的终点来评估光源毒性的精确度。