Toxicoproteomics is the use of global protein expression technologies in toxicology to gain a better understanding of environmental and genetic factors in acute episodes of toxicant exposure and in long term development of disease. The formulation of a strategy to integrate transcript, protein and toxicology data is a major objective for the young field of Toxicogenomics. The National Center for Toxicogenomics is pursuing a strategy of conducting parallel DNA microarray and proteomic analyses on the same tissues from each Toxicogenomics study. The advantage of this approach is to bring more information to bear on toxicological problems in identifying affected, biochemical and regulatory pathways that can lead to biomarker discovery. Both DNA microarray and proteomic technologies are driven by measuring differential expression of transcripts and proteins after toxicant exposure. The high level of information density and gene discovery potential in microarray analysis can be enhanced by proteomics technologies, which allow identification of toxicant-altered proteins based on subcellular localization as well as characterization of post-translational modifications that occur in signaling pathway proteins. Further, proteomic platforms appear particularly well suited for biomarker development in blood in that the appearance of new polypeptides in the serum proteome may reveal early stage disease and organ toxicity. Intramural toxicoproteomic research is a coordinated effort between two research groups, the Proteomics Group within the NCT and the Mass Spectrometry Group in the Laboratory of Structural Biology. Proteomics technologies used in this integrated program involve two-dimensional (2D) gel electrophoresis and mass spectrometry for global protein separation and protein identification, respectively. Surface retentate mass spectrometry or surface enhanced laser desorption ionization, SELDI, is being used by the Proteomics Group for serum biomarker development and classification of control and toxicant-exposed experimental animals and also in clinical studies with the epidemiology branch to classify control and diseased populations. Progress in toxicoproteomics occurred in several areas during the year in 2003. A primary goal has been biomarker development in liver and serum using proteomic approaches in response to hepatotoxic chemicals. While these studies have been primarily conducted in experimental animals, new studies using SELDI will use human serum from clinically documented patients with disease. Proteomics Resource Contract: The contract to assist the NCT in proteomics in toxiciology began last year in July, 2003. The contract in 2003 has focused upon two major sample studies submitted by my group involving i) acetaminophen hepatotoxicity and ii) lipopolysacharide (LPS) -induced endotoxemia and hepatotoxicity. The first four months were spent finalizing SOPs with test samples and the last 8 months have involved sample preparation of serum and liver tissue for 2D gel separation and MS identification. Data is currently being received by NCT that will be publishable. Alex Merrick and Ken Tomer serve as technical project officers on this contract. Intramural Research: In FY03, group research was published in Archives of Biochemistry and Biophysics demonstrating the presence of NFkB pathway components in the cellular membrane component, specifically in endoplasmic reticulum and golgi. A collaborative project with Teddy Devereux?s group was published in Carcinogenesis describing the genomic and proteomic effects in liver of non-genotoxic carcinogens. Progress was made on proteomic mapping and phosphorylation staining of liver and cell culture nuclei with the MS/microchemistry core facility, led by Ken Tomer. Collaborative work with Rick Fannin and Rick Paules of the NIEHS Microarray Group, was performed with the inflammagen, lipopolysaccharide (LPS), to establish blood-born biomarkers of LPS treated rats. SELDI: A proof of principle study in serum biomarker development was performed using serum from acutely treated rats with LPS as an in vivo model for endotoxemia. Serum biomarkers that differentiated control from treated serum were successfully detected. Development of data mining algorithms and immunohistochemical validation of LPS toxicity were conducted in collaboration with Leping Li and Julie Foley, respectively.
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