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Final Progress Reports: University of North Carolina-Chapel Hill: Biomarkers of Exposure versus Effect: Improving the Scientific Basis for Risk Assessment

Superfund Research Program

Biomarkers of Exposure versus Effect: Improving the Scientific Basis for Risk Assessment

Project Leader: James A. Swenberg
Grant Number: P42ES005948
Funding Period: 1995-2018
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Final Progress Reports

Year:   2017  2005  1999 

Many Superfund chemicals are thought to cause their toxicity through the production of reactive oxygen species (ROS) that can subsequently react with DNA, proteins and lipids. The production of increased ROS is often called oxidative stress, a condition that is thought to be causally involved in many chronic diseases including aging, cancer and neurodegeneration. A major focus of this research project has been the development of new, highly sensitive and accurate methods for measuring such damage, so that Dr. Swenberg and his research team can evaluate possible roles of Superfund chemicals in such diseases. The most common biomarker studied in oxidative stress is the direct oxidation of deoxyguanosine, one of the four nucleosides composing DNA. The oxidized product, 8-OH deoxyguanosine (8OHdG), is subject to artifactual oxidation during DNA isolation and analysis. In the past, project investigators have developed methods to minimize such artifact and to accurately measure this adduct. During the past year, efforts have focused on increasing the throughput of their analysis. In collaboration with the Chemistry and Analytical Core, the investigators have greatly simplified their clean-up and mass spectrometric analysis. Whereas it used to take a minimum of two days to process and analyze a set of samples, they have now developed a simple nucleoside digestion, followed by ultra high performance chromatography separation and mass spectrometry with a run time of under 15 minutes. A second biomarker of oxidative stress is M1G, an exocyclic DNA adduct that was thought to primarily arise from the reaction of malondialdehyde, a lipid peroxidation product. Using [13C]-DNA and mass spectrometry, the research team was able to demonstrate that most M1G actually arises from hydroxyl radical attack of the DNA backbone, yielding base propenal that reacts with guanine to form M1G. Furthermore, they were able to demonstrate that this secondary oxidative product is much less prone to artifactual formation during DNA isolation and analysis. Thus, M1G may be a more reliable biomarker for oxidative stress. The development of these and several additional biomarkers of oxidative stress will play a major role in the projects future studies on the role of oxidative stress in PAH and PCB toxicity and its application to risk assessment. They are continuing their basic research on DNA repair. An improved Glyoxal electrophoresis assay was developed to monitor SSB in genomic DNA extracted from cells. They have not been able to establish a Polβ-/-/Fen1-/- double gene knockout in DT40 cells, suggesting that this alteration may be lethal. In addition, preliminary data suggests that there may be a back-up pathway for polβ. Theinvestigators will continue studies to develop new cell lines and identify new pathways for their studies on DNA repair pathways of importance to oxidative stress. Finally, the real-time SSB assay they published in 2003 was accepted as a patent (US Patent#: 6,913,878, Issued on 7/5/2005).

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