Advanced GST Proteomics for Early Stage Organ-Specific Toxicity Screening

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 tofurther 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-specifictoxicology testing that has significant preclinical and clinical commercial applications.