Advanced GST Proteomics for Early Stage Organ-Specific Toxicity Screening

描述(申请人提供)：目前用于检测器官毒性的生物标志物往往不够灵敏，不足以检测急性器官损伤的早期阶段，即在减少或消除暴露于毒素可以防止器官损伤或疾病过程进展的阶段。当在生物体液中检测到当前的毒性生物标记物时，长期接触有毒化合物已经对器官造成了严重的、往往是不可逆转的损害。例如，血液中的转氨酶(ALT和AST)水平是检测肝脏损伤的一种被广泛接受的做法。然而，这些目前的毒性生物标记物通常在低水平肝损伤的血液中检测不到，需要相对较长的毒素暴露时间才能检测到。胞液谷胱甘肽转移酶(GST)家族具有许多必需的特性，如组织特异性定位、低毒性(高敏感性)和高细胞内浓度，为器官损伤的生物标志物提供了一个更可靠的替代选择。GST蛋白家族由几个类别(如A、M、P)组成，有些类别包含多种异构体。许多GST亚型在组织分布上表现出显著的差异。例如，GSTA1-1和A2-2是在肝脏中发现的主要GST酶，而GSTA3-3主要在类固醇合成组织中表达，而GSTA4-4在所有被检测的组织中都表达。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害的一个有价值的指标。然而，目前可用的GSTs抗体不能区分不同的GSTA亚型。对特定GST亚型的检测将使对外源暴露造成的器官损伤的分辨率和敏感性更高。最近发现了几个额外的细胞质GST类别(S、O和Z)，这为进一步扩大GST检测的毒理学应用提供了额外的机会，开发了针对特定组织相关特定异构体的免疫检测方法。第一阶段可行性研究的目标是开发(A)高度特异的抗体，能够区分三种GSTA亚型，即GSTA1-1/2-2、GSTA3-3和GSTA4-4，以及(B)对这些生物标志物进行超灵敏的免疫分析。此外，在第一阶段，我们将(C)使用动物模型来验证这些检测方法是检测器官特异性毒性的可靠方法。长期目标(第二阶段)包括开发一个全面的GST蛋白质组学小组，用于高灵敏度的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：非常需要更灵敏、微创的方法来检测因接触药物或由于环境或职业暴露而对特定器官造成的早期毒性。这项拟议研究的目标是开发一套高度特异和超敏感的测试来监测血液中一系列蛋白质(谷胱甘肽转移酶)的水平，并将这些测试纳入一个具有重要临床前和临床商业应用的高灵敏度器官特异性毒理学测试的综合小组。

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.