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

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 biomarkerfor 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 GSTisoforms 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 proteomicspanel for human clinical toxicity studies, including comparison to traditional toxicity biomarkers in patients undergoing chemotherapy. PUBLIC HEALTH RELEVANCE 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 pharmaceuticals or to environmental or occupational toxins. This proposal expands on a successful Phase 1 feasibility study with the ongoing aim of developing a panel of highly specific and ultrasensitive tests to measure the levels of a family of proteins (the glutathione transferases) in blood and urine. Incorporating these tests into a comprehensive multiplexed panel will allow for high sensitivity organ-specific toxicology testing, for which there are many significant preclinical and clinical commercial applications.