Student Research: Stephen Krival

, Environmental Toxicology (Tox), 2009
Faculty Advisor: Terrance J. Kavanagh

Principle Components Characterization of Liver Tissue Metabolites in Glutamate Cysteine Ligase Transgenic Mice Treated with Acetaminophen


Abstract

Metabolomics is a new field of research that is playing an important role in the identification of metabolites and regulatory pathways involved in pathogenesis. The term “metabolomics” describes the development of methods “intended to define all the metabolites within the cell, tissue or organism during a genetic alternation or physiological stimulus.” 1 In mammals, the liver is the principle organ of toxicant metabolism, and glutathione (GSH) is a simple tri-peptide that plays a central role in detoxifying many compounds. Dr. Kavanagh’s laboratory has developed genetically modified mice for the purpose investigating the role of glutathione in preventing toxicity. These mice differ in how much glutathione is available in the liver (and systemically) for detoxifying environmental chemicals and drugs, (such as acetaminophen; APAP), due to their differing abilities to make the rate-limiting enzyme responsible for the manufacture of glutathione, glutamate cysteine ligase (GCL). GCL is a heterodimer of a catalytic subunit (GCLC) and a modifier subunit (GCLM). A principle objective of my work is to determine if the metabolites in mouse liver samples may be classified based on their GCL and APAP treatment status. Mouse liver samples are analyzed using High Performance Liquid Chromatography (HPLC) coupled with CoulArray Electrochemical Detection analysis. CoulArray software was used to conduct peak alignment, concatenation, and data compression of the HPLC-EC prior to analyses using pattern recognition techniques including Principle Component Analysis (PCA), and Hierarchical Cluster Analysis (HCA). Initial results in one experiment show that mouse liver samples separate on GCL status alone. PCA results showed that male WT mice cluster separately from male GCLM-null mice when both groups are treated with APAP and a compound used to rescue subjects from acute APAP over-dose, N-acetyl cysteine (NAC). In a second experiment, mouse liver samples separated on both GCL status and treatment status. The sample groups in this experiment were GCLM-null mice, and GCL transgenic mice having extra copies of both GCLC and GCLM. Each group was further divided into those receiving APAP treatment or the vehicle (saline). These results suggest that the mouse acetaminophen model is amenable to metabolomic investigation using pattern recognition techniques.