The role that endogenously produced chemicals may play in the etiology of cancer has become increasingly important over the last decade. This is largely due to epidemiological data, which show that apart from tobacco smoke ad sunlight, exposure to genotoxic environmental carcinogens that are derived from lipid hydroperoxides. The formation of lipid hydroperoxides is a complex process, which involves a number of different racial intermediates. However lipid hydroperoxides are also readily formed as a consequence of LOX-mediated oxidation of endogenous polyunsaturated acids (PUFAs). Therefore, there are both enzymatic and non-enzymatic pathways by which lipid hydroperoxides can be formed. In view of their potential role as genotoxins, it is important to understand how structural modifications to DNA can occur from reactions with lipid hydroperoxides. Their genotoxic properties are thought to result from generation of bifunctional electrophiles such as malondialdehyde and 4- hydroxy-2-nonenal. These bifunctional electrophiles then react at electron rich sites of the DNA bases to form DNA-adducts. Evidence has been obtained for the presence of both malondialdehyde- and 4-hydroxy- nonenal-derived DNA-adducts in vivo. Transition metal ions are known to enhance the formation of bifunctional electrophiles through a homolytic process. The presence of transition metal cations bound to the polar sugar residues of DNA can potentially enhance the breakdown of lipid hydroperoxides to genotoxic bifunctional electrophiles. We have recently studied the decomposition of 13-hydroperoxylinoleic acid (a prototypic w- 6 PUFA hydroperoxide) in the presence of the DNA bases. From the structures of the resulting DNA-adducts, we proposed that the covalent modifications arose through the generation of 4-oxo-2-nonenal from 13- HPODE. We have also demonstrated that the same adducts were formed when the DNA-bases were treated with synthetic 4-oxo-2-nonenal. This lead us to speculate that 4-oxo-2-nonenal was the major breakdown product of lipid hydroperoxides rather than 4-hydroxy-2-nonenal. We have obtained definitive evidence that 4-oxo-2-nonenal is indeed a major breakdown product of lipid hydroperoxides. In addition, we have documented the unexpected formation of 4-hydroperoxy-2-nonenal as an additional lipid peroxidation breakdown product. We now propose to elucidate the mechanisms by which these bifunctional electophiles are formed and to define the relative importance of their formation as a consequence of lipid peroxidation. We plan to develop highly sensitive quantitative methodology for the analysis of these adducts in vivo using a novel mass spectrometry technique that we have discovered recently. We plan to examine the relative importance of DNA-adduct and RNA-adduct formation in a model of oxidative stress.
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