Characterization of 4-Oxo-2-nonenal as a Novel Product of Lipid Peroxidation Seon Hwa Lee and Ian A. Blair* Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160 Received May 5, 2000 Fe II -mediated decomposition of 13-[S-(Z,E)]-9,11-hydroperoxyoctadecadienoic (hydroperoxy- linoleic) acid resulted in the formation of three R,-unsaturated aldehydes. At low Fe II concentrations or at early time points after the addition of Fe II , two major products were observed. The least polar product had chromatographic properties that were identical with those of 4-oxo-2-nonenal. Conversion of this product to its bis-oxime derivative with hydroxyl- amine hydrochloride resulted in two syn- and two anti-oxime isomers that had chromatographic and mass spectral properties identical with the properties of products derived from an authentic standard of 4-oxo-2-nonenal. This confirmed for the first time that 4-oxo-2-nonenal is a major product of the Fe II -mediated breakdown of lipid hydroperoxides. The more polar product had chromatographic properties that were similar to those of 4-hydroperoxy-2-nonenal. LC/MS analysis of its syn- and anti-oxime isomers confirmed this structural assignment. Thus, 4-hydroperoxy-2-nonenal is a previously unrecognized major product of lipid hydroperoxide decomposition. At high Fe II concentrations and at longer incubation times, a third more polar product was observed with chromatographic properties that were identical to those of 4-hydroxy- 2-nonenal. The syn- and anti-oxime isomers had chromatographic and mass spectral properties identical with the properties of products derived from an authentic standard of 4-hydroxy-2- nonenal. It appears that 4-hydroperoxy-2-nonenal is formed initially and that it is then converted to 4-hydroxy-2-nonenal in the presence of high Fe II concentrations or by extended incubations in the presence of low Fe II concentrations. It is conceivable that some of the 4-hydroperoxy-2-nonenal is also converted to 4-oxo-2-nonenal. However, we cannot rule out the possibility that it is also formed by a concerted mechanism from a rearrangement product of 13-[S-(Z,E)]-9,11-hydroperoxyoctadecadienoic acid. Introduction The role that endogenously produced chemicals may play in the etiology of cancer has become increasingly important over the past decade (1). This is largely due to epidemiological data, which show that apart from tobacco smoke and sunlight, exposure to genotoxic envi- ronmental carcinogens accounts for only a minority of human cancers (2). Lipid peroxidation has long been thought to produce endogenous genotoxins that are derived from lipid hydroperoxides (3). The formation of lipid hydroperoxides is a complex process, which involves a number of different radical intermediates (4, 5). How- ever, lipid hydroperoxides are also readily formed as a consequence of LOX-mediated oxidation of endogenous polyunsaturated fatty acids (PUFAs) 1 (6). Therefore, there are both enzymatic and nonenzymatic 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 the generation of bifunctional electrophiles such as malon- dialdehyde (7) and 4-hydroxy-2-nonenal (8). These bi- functional electrophiles then react at electron rich sites of the DNA bases to form DNA adducts (1). Evidence has been obtained for the presence of both malondialdehyde- (9, 10) and 4-hydroxynonenal-derived DNA adducts (11) in vivo. Transition metal ions are known to enhance the formation of bifunctional electrophiles through a ho- molytic process (12). The presence of transition metal cations bound to the polar sugar residues of DNA can potentially enhance the breakdown of lipid hydroperox- ides to genotoxic bifunctional electrophiles (13). We have recently studied the decomposition of 13-HPODE (a prototypic ω - 6 PUFA) in the presence of the DNA bases dGuo and dAdo. From the structures of the resulting DNA adducts, we proposed that the covalent modifica- tions arose through the generation of 4-oxo-2-nonenal from 13-HPODE (14, 15). We have also demonstrated that the same adducts were formed when the DNA bases were treated with synthetic 4-oxo-2-nonenal (14, 16). Surprisingly, we were unable to detect covalent modifica- tions resulting from 4-hydroxy-2-nonenal. This led us to speculate that 4-oxo- * To whom correspondence should be addressed: Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 1254 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160. Fax: (215) 573-9889. E-mail: ian@spirit.gcrc.upenn.edu. 1 Abbreviations: ESI, electrospray ionization; 4-HNE, 4-hydroxy-2- nonenal; 4-HPNE, 4-hydroperoxy-2-nonenal; 13-HPODE, 13-hydro- peroxy-[S-(Z,E)]-9,11-octadecadienoic acid; 13-(E,E)-HPODE, 13-hy- droperoxy-(E,E)-9,11-octadecadienoic acid; 9-(E,Z)-HPODE, 9-hydro- peroxy-(E,Z)-10,12-octadecadienoic acid; 9-(E,E)-HPODE, 9-hydroperoxy- (E,E)-10,12-octadecadienoic acid; LC/MS, liquid chromatography/mass spectrometry; LOX, lipoxygenase; MS n , multiple tandem mass spec- trometry; 4-ONE, 4-oxo-2-nonenal; PUFA, polyunsaturated fatty acid; TIC, total ion current. 698 Chem. Res. Toxicol. 2000, 13, 698-702 10.1021/tx000101a CCC: $19.00 © 2000 American Chemical Society Published on Web 07/18/2000