Advanced GST Proteomics for Early Stage Organ-Specific Toxicity Screening. Phase

描述：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进程进展的阶段，也不能充分区分对一个器官系统和另一个器官系统的损害。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，在肝脏损伤程度较低的情况下，这些酶通常在血液中检测不到，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)家族为器官损伤提供了一种更可靠的生物标志物，因为它们表现出许多这一用途所需的特征，即组织特异性定位，低毒性水平(高敏感性)释放到血液中，以及高细胞内浓度。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。GST类及其亚型在组织分布上表现出明显的差异。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。此外，GSTP1-1是主要在血小板中发现的GST。因此，测量血液中特定的GST类别和异构体的水平将是特定器官或组织受损的一个有价值的指标。然而，目前可用的GST抗体往往不能区分GST类别的不同亚型，在某些情况下，不足以区分不同的GST类别。检测方法的可用性 特定的GST类别及其异构体，以及许多GST异构体的多重面板，将使对外源暴露造成的器官损害的分辨率和敏感度大大提高。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检测在毒理学测试中的应用提供了机会，开发了针对特定组织的特定类别和异构体的免疫检测方法。在第一阶段，我们成功地开发了针对人类GST A亚型的抗体和免疫分析方法，并证明了它们在啮齿动物毒性测试和非侵入性监测人类尿中GST亚型中的作用。第二阶段计划包括：(A)开发针对其他人类GST类别及其异构体的高度特异性抗体；(B)开发针对这些生物标记物的超灵敏免疫分析；(C)为一组GST免疫分析开发一个复杂、快速、易于使用的多重平台；(D)确定该GST蛋白质组用于动物模型的临床前器官特异性毒性测试；以及(E)确定GST蛋白质组学小组用于人类临床毒性研究的实用性，包括与化疗患者的传统毒性生物标志物进行比较。

DESCRIPTION: Biomarkers currently employed for 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, nor do they adequately discriminate damage to one organ system vs. another. For example, the level of aminotransferases (ALT and AST) in blood is a widely accepted practice for detecting liver damage. However, these enzymes 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 for that use, i.e. tissue specific localization, release into the blood at low leves 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 GST classes and their 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. Additionally, GSTP1-1 is the GST predominantly found in platelets. Therefore, measuring the level of specific GST classes and isoforms in blood would be a valuable indicator of damage to a particular organ or tissue. However, currently available antibodies for GSTs are often not capable of distinguishing among the different isoforms of a GST class and, in some cases, are insufficiently specific to distinguish among GST classes. The availability of assays for specific GST classes and their isoforms, and multiplex panels for many GST isoforms, would enable a much greater 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 the opportunity to further expand the applications of GST assays for toxicology testing with the development of immunoassays that are specific for given classes and isoforms associated with specific tissues. In Phase 1 we succeeded in developing antibodies and immunoassays specific for human GST A subforms, and demonstrated their utility for rodent toxicity testing and for non-invasive monitoring of GST isoforms in human urine. Phase 2 plans include (a) the development of highly specific antibodies for additional human GST Classes and their isoforms (b) development of ultrasensitive immunoassays for these biomarkers, (c) development of a sophisticated, rapid, easy to use multiplex platform for a panel of GST immunoassays, (d) determination of the utility of this GST proteomics panel for pre-clinical organ-specific toxicity testing in animal models, and (e) determination of the utility of a GST proteomics panel for human clinical toxicity studies, including comparison to traditional toxicity biomarkers in patients undergoing chemotherapy.