The analysis of DNA-adducts is important for molecular dosimetry studies, and for monitoring treatment options because they can provide insight into the amount of genotoxin that that has reached the DNA in the tissue under study. No specific biomarkers of endogenous lipid hydroperoxide-mediated DNA damage in vivo are available in spite of an intensive research effort spanning several decades. Our studies over the last six years have identified three heptanone-etheno-DNA-adducts that can only arise from endogenously formed lipid hydroperoxides. We have now shown that heptanone-etheno-2'-deoxyguanosine heptanone-etheno-21- deoxy-cytidine adducts are formed in the DNA of rat intestinal epithelial cells that stably express COX-2 and that the adducts are present in mammalian tissue DNA. We have also discovered a new class of cyclic glutathione (GSH)-adducts derived from endogenous lipid hydroperoxides. Lipid hydroperoxides undergo homolytic decomposition to the highly reactive bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). Lipid hydroperoxide-derived ONE is responsible for the formation of both heptanone-etheno- adducts and the novel cyclic GSH-adducts. Our recent discovery that electron capture atmospheric chemical ionization mass spectrometry can be used to analyze chiral lipids with high sensitivity and specificity makes it possible to analyze the lipid hydroperoxide precursors of DNA- and glutathione- adducts. Therefore, we are poised to make some significant advances in understanding the role of oxidative stress in the etiology of cancer. We propose to address the following hypotheses: 1. Endogenous GSH-adducts and their metabolites represent a new class of biomarkers that will complement isoprostanes as biomarkers of oxidative stress. 2. Endogenous DNA-, RNA-, and cyclic GSH-adducts adducts can arise from cyclooxygenase-mediated pathways. 3. Endogenous DNA-, RNA-, and cyclic GSH-adducts can arise from 5-lipoxygenase but not 15-lipoxygenase. 4. Heptanone- etheno-DNA- and RNA-adducts, together with metabolites of endogenous cyclic GSH-adducts are dosimeters of colon cancer. The proposed research will be conducted under four specific aims.
Aim 1. To examine the enzymatic formation and metabolism of endogenous lipid hydroperoxide-derived GSH- adducts.
Aim 2. To compare ROS- and COX-2-mediated formation of endogenous DNA-, RNA-, and GSH-adducts with lipid hydroperoxide formation in cellular models.
Aim 3. To compare ROS- and LOX- mediated formation of endogenous DNA-, RNA-, and GSH-adducts with lipid hydroperoxide formation in cellular models.
Aim 4. To quantify endogenous lipid hydroperoxide-derived DNA-adducts, RNA- adducts, GSH-adducts as potential cancer biomarkers in animal models of colon cancer and in human colon tissue. ? ? ?
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