Advanced GST Proteomics for Early Stage Organ-Specific Toxicity Screening

描述（由申请人提供）：目前用于检测器官毒性的生物标志物通常不够灵敏，无法检测急性器官损伤的早期阶段，即在减少或消除毒素暴露可以预防器官损伤或疾病过程进展的阶段。当在生物体液中检测到目前的毒性生物标志物时，由于长期暴露于有毒化合物，已经对器官造成了显著的、往往是不可逆的损害。例如，血液中的转氨酶（ALT和AST）水平是检测肝损伤的广泛接受的实践。然而，这些目前的毒性生物标志物在低水平的肝损伤时通常在血液中检测不到，并且在检测到它们之前需要相对长的毒素暴露。细胞溶质谷胱甘肽转移酶（GST）家族的酶提供了作为器官损伤的生物标志物的更可靠的替代物，因为它们表现出许多所需的特征，即组织特异性定位、以低毒性水平（高灵敏度）释放到血液中以及高细胞内浓度。GST蛋白家族由几个类别（例如A、M、P）组成，其中一些类别含有多种同种型。许多GST同工型在组织分布中表现出显著差异。例如，GSTA 1 -1和A2-2是在肝脏中发现的主要GST酶，而GSTA 3 -3主要在类固醇生成组织中表达，而GSTA 4 -4在已检查的所有组织中表达。因此，测量血液中特定GST亚型的水平将是对特定器官或组织损伤的有价值的指标。然而，目前可用的GST抗体不能区分不同的GSTA同种型。特异性GST亚型的测定将能够实现对由于外源性暴露引起的器官损伤的更高程度的分辨率和灵敏度。最近鉴定的几个额外的胞质GST类（S，O和Z）提供了一个额外的机会，进一步扩大GST检测的毒理学应用与特定组织相关的特定亚型特异性的免疫测定的发展。I期可行性研究的目标是开发（a）能够区分三种GSTA同种型，即GSTA 1 -1/2-2、GSTA 3 -3和GSTA 4 -4的高度特异性抗体和（B）用于这些生物标志物的超灵敏免疫测定。此外，在第一阶段，我们将（c）采用动物模型来验证这些检测方法是否是检测器官特异性毒性的可靠方法。长期目标（第二阶段）涉及开发一个全面的GST蛋白质组学小组，用于高灵敏度器官特异性毒理学测试，具有重要的临床前和临床商业应用。 公共卫生相关性：非常需要更敏感、最小侵入性的方法来检测由于暴露于药物或由于环境或职业暴露而对特定器官产生的早期毒性。拟议研究的目标是开发一套高度特异性和超灵敏度的测试，以监测血液中蛋白质家族（谷胱甘肽转移酶）的水平，并将这些测试纳入高灵敏度器官特异性毒理学测试的综合小组，具有重要的临床前和临床商业应用。

DESCRIPTION (provided by applicant): Current biomarkers employed in the detection of organ toxicity are often not sensitive enough to detect the early stages of acute organ damage, i.e. at a stage when reducing or eliminating exposure to a toxin could prevent progression of organ damage or a disease process. By the time current toxicity biomarkers are detected in biological fluids there is already significant, often irreversible, damage to organs resulting from prolonged exposure to toxic compounds. For example, the level of aminotransferases (ALT and AST) in blood is a widely accepted practice for detecting liver damage. However, these current toxicity biomarkers are typically not detectable in blood at low levels of liver damage and require a relatively long exposure to a toxin before they are detected. The cytosolic glutathione transferase (GST) family of enzymes offer a more reliable alternative as a biomarker for organ damage as they exhibit many of the required characteristics, i.e. tissue specific localization, release into the blood at low levels of toxicity (high sensitivity), and a high intracellular concentration. The GST protein family is comprised of several classes (e.g. A, M, P) with some classes containing multiple isoforms. The many GST isoforms exhibit marked differences in tissue distribution. For example, GSTA1-1 and A2-2 are the predominant GST enzymes found in the liver, whereas GSTA3-3 is mainly expressed in steroidogenic tissues and GSTA4-4 is expressed in all tissues that have been examined. Therefore, measuring the level of specific GST isoforms in blood would be a valuable indicator of damage to a particular organ or tissue. However, current available antibodies for GSTs are not capable of distinguishing among the different GSTA isoforms. Assays for specific GST isoforms would enable a higher degree of resolution and sensitivity to organ damage due to xenobiotic exposure. Recent identification of several additional cytosolic GST classes (S, O and Z) provides an additional opportunity to further expand the toxicology applications of GST assays with the development of immunoassays that are specific for specific isoforms associated with specific tissues. The goal of Phase I feasibility studies is to develop (a) highly specific antibodies capable of distinguishing between three GSTA isoforms, namely GSTA1-1/2-2, GSTA3-3 and GSTA4-4 and (b) ultrasensitive immunoassays for these biomarkers. In addition, in Phase I we will (c) employ animal models to validate these assays as a reliable way to detect organ-specific toxicity. The long-term goal (Phase II) involves development of a comprehensive GST Proteomics panel for high sensitivity organ-specific toxicology testing that has significant preclinical and clinical commercial applications. PUBLIC HEALTH RELEVANCE: There is a great need for more sensitive, minimally-invasive methods to detect the early stages of toxicity to specific organs from exposure to drugs, or as the result of environmental or occupational exposure. The goal of the proposed research is to develop a set of highly specific and ultrasensitive tests to monitor the levels of a family of proteins (the glutathione transferases) in blood, and to incorporate these tests into a comprehensive panel for high sensitivity organ-specific toxicology testing that has significant preclinical and clinical commercial applications.