DNA Adducts with Lipid Peroxidation Products (2008)

Homolytic decomposition of PUFA 2The abbreviations used are: PUFA, polyunsaturated fatty acid; DDE, trans, trans-2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; HNE, 4-hydroxy-(2E)-nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; MDA, malondialdehyde; ONE, 4-oxo-(2E)-nonenal; DODE, 9,12-dioxo-(10E)-dodecenoic acid; MS, mass spectrometry; ROS, reactive oxygen species; COX, cyclooxygenase; LOX, lipoxygenase; GST, glutathione S-transferase; LC-MRM-MS, liquid chromatography-multiple reaction monitoring-mass spectrometry; LA, linoleic acid; HPODE, hydroperoxy-(9Z),(11E)-octadecadienoic acid; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; APCI, atmospheric pressure chemical ionization; MS/MS, tandem MS; ESI, electrospray ionization; dGuo, 2′-deoxyguanosine; ∈DNA, etheno-DNA; H∈, heptanone-etheno; dAdo, 2′-deoxyadenosine; dCyd, 2′-deoxycytidine; C∈, carboxynonanone-etheno; GC, gas chromatography. 2The abbreviations used are: PUFA, polyunsaturated fatty acid; DDE, trans, trans-2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; HNE, 4-hydroxy-(2E)-nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; MDA, malondialdehyde; ONE, 4-oxo-(2E)-nonenal; DODE, 9,12-dioxo-(10E)-dodecenoic acid; MS, mass spectrometry; ROS, reactive oxygen species; COX, cyclooxygenase; LOX, lipoxygenase; GST, glutathione S-transferase; LC-MRM-MS, liquid chromatography-multiple reaction monitoring-mass spectrometry; LA, linoleic acid; HPODE, hydroperoxy-(9Z),(11E)-octadecadienoic acid; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; APCI, atmospheric pressure chemical ionization; MS/MS, tandem MS; ESI, electrospray ionization; dGuo, 2′-deoxyguanosine; ∈DNA, etheno-DNA; H∈, heptanone-etheno; dAdo, 2′-deoxyadenosine; dCyd, 2′-deoxycytidine; C∈, carboxynonanone-etheno; GC, gas chromatography.-derived lipid hydroperoxides results in formation of the α,β-unsaturated aldehydic bifunctional electrophiles DDE, EDE, HNE, HPNE, MDA (shown as β-hydroxyacrolein), ONE, DODE, and 5,8-dioxo-(10E)-octenoic acid (see Fig. 1) (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). Intracellular formation of the bifunctional electrophiles can then result in the formation of GSH, protein, and DNA adducts (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar, 5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar). The analysis of lipid hydroperoxide-derived DNA adducts can facilitate molecular epidemiology studies by providing insight into the amount of a genotoxin that has reached the DNA of the tissue under study (6Kensler T.W. Qian G.S. Chen J.G. Groopman J.D. Nat. Rev. Cancer. 2003; 3: 321-329Crossref PubMed Scopus (179) Google Scholar, 7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). DNA repair enzymes, such as those involved in base excision repair, are able to excise the DNA adducts so that they can potentially be excreted in the urine (7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). This suggests that non-invasive MS-based techniques could be used to monitor urinary DNA adducts arising from lipid hydroperoxide-mediated DNA damage. Unfortunately, to date, the analysis of urinary DNA adducts of lipid hydroperoxide-derived bifunctional electrophiles has not been particularly successful. ONE-derived DNA adducts, which can arise only from lipid peroxidation, have now been characterized in the tissues of mouse models (8Williams M.V. Lee S.H. Pollack M. Blair I.A. J. Biol. Chem. 2006; 281: 10127-10133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Therefore, it might eventually be possible to detect these specific lipid hydroperoxide-derived DNA adducts after they have been excised from the DNA and excreted in the urine (see Fig. 2).FIGURE 2Formation of ∈DNA and H∈DNA adducts through homolytic decomposition of lipid hydroperoxides. BER, base excision repair.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oxidative stress represents one of the major cellular responses to toxic insults, carcinogenic chemicals, and environmental agents such as viruses and mycotoxins (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar). During oxidative stress, there is a decrease in the amount of intracellular GSH and an increase in GSSG (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar) with a concomitant increase in ROS and reactive nitrogen species (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar) and an increase in the activity of COX-2 (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and LOXs (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar), involved in lipid peroxidation (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar). ROS are generated constantly in vivo by a variety of endogenous processes, including normal mitochondrial aerobic respiration, phagocytosis of bacteria- or virus-containing cells, and peroxisome-mediated degradation of fatty acids (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar). Increased ROS production occurs in inflammation; during radiation; or during metabolism of hormones, drugs, and environmental toxins. This can overwhelm endogenous protective mechanisms and increase ROS-mediated damage to GSH, proteins, DNA, and lipids. The resulting ROS-derived lipid hydroperoxides can then undergo homolytic decomposition to various bifunctional electrophiles that form DNA adducts with different lipid modifications (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar;5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar). Alternatively, the bifunctional electrophiles can arise from homolytic decomposition of COX- and LOX-derived lipid hydroperoxides (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 13Jian W. Lee S.H. Arora J.S. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2005; 18: 599-610Crossref PubMed Scopus (24) Google Scholar). Intracellular lipid hydroperoxides are detoxified by GSH peroxidase-mediated reduction to lipid alcohols, with GSH providing the reducing equivalents (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar). Peroxidized phospholipids are detoxified by peroxiredoxin 6 to the corresponding phospholipid alcohols in a GSH-dependent manner (14Manevich Y. Fisher A.B. Free Radic. Biol. Med. 2005; 38: 1422-1432Crossref PubMed Scopus (322) Google Scholar). GSH also readily forms adducts with lipid hydroperoxide-derived bifunctional electrophiles, providing an additional protective pathway (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 15Jian W. Lee S.H. Mesaros C. Oe T. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1008-1018Crossref PubMed Scopus (44) Google Scholar). Formation of the GSH adducts is generally facilitated by GSTs, and the resulting adducts are exported from cells by ATP-binding and non-ATP-binding cassette transporters (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). Total GSH and GSSG concentrations are modulated in cells by low extracellular concentrations of lipid hydroperoxide-derived bifunctional electrophiles such as HNE. This can increase GSH concentrations by up-regulation of γ-glutamylcysteine ligase, a key enzyme involved in GSH biosynthesis. In contrast, high concentrations of bifunctional electrophiles deplete GSH (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar) through a direct GST-mediated reaction as well as by inhibition of enzymes involved in GSH biosynthesis. GSH/GSSG homeostasis plays an important role in maintaining cellular redox status, and changes of the half-cell reduction potential of the 2GSH/GSSG couple correlate with the biological status of the cell (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). It is difficult to accurately quantify intracellular GSH and GSSG concentrations because of the ease with which free sulfhydryl groups are oxidized during the lysis of cells. To overcome this problem, a stable isotope dilution LC-MRM-MS method was developed using 4-fluoro-7-sulfamoylbenzofurazan derivatization of GSH (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). Increased lipid peroxidation resulting from transfection of the human 15-LOX-1 gene into a mouse macrophage cell line was found to increase intracellular GSH biosynthesis. This resulted in a lower resting cellular redox potential and provided protection against exogenous lipid hydroperoxide-derived bifunctional electrophiles. Therefore, increased intracellular lipid peroxidation can induce a protective adaptive response (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). ROS-mediated oxidation of esterified LA-containing lipids and free LA results in the formation of four HPODE isomers as enantiomeric pairs. They are subsequently reduced to the corresponding hydroxyoctadecadienoic acids. Similarly, ROS-mediated oxidation of AA-containing lipids and free AA results in the formation of a complex mixture of HPETEs that are reduced to racemic hydroxyeicosatetraenoic acids (HETEs), including (15S)- and (15R)-HETE (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 16Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar). Lipid hydroperoxides can also be formed by the action of LOXs (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar) and COXs (17Porter N.A. Caldwell S.E. Mills K.A. Lipids. 1995; 30: 277-290Crossref PubMed Scopus (996) Google Scholar) on PUFAs. High sensitivity, stable isotope dilution, normal phase, chiral LC-electron capture APCI-MS/MS methodology (18Singh G. Gutierrez A. Xu K. Blair I.A. Anal. Chem. 2000; 72: 3007-3013Crossref PubMed Scopus (180) Google Scholar) makes it possible to separate and quantify all of the enantiomers and regioisomers of the PUFA alcohols derived from LA and AA (19Lee S.H. Williams M.V. Dubois R.N. Blair I.A. Rapid Commun. Mass Spectrom. 2003; 17: 2168-2176Crossref PubMed Scopus (156) Google Scholar, 20Lee S.H. Blair I.A. Methods Enzymol. 2007; 433: 159-174Crossref PubMed Scopus (34) Google Scholar). Eicosanoids and isoprostanes can be quantified at the same time. This method revealed that endogenous (15S)-HPETE (as measured by (15S)-HETE release) was formed in amounts that were similar to prostaglandin E2 in unstimulated rat intestinal epithelial cells stably expressing COX-2 (RIES cells) (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar). When exogenous AA was added to the RIES cells, the profile of eicosanoids was found to be dependent upon the time of incubation as well as the AA concentration (12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar). Interestingly, COX-2-mediated biosynthesis of prostaglandin E2 and (15S)-HETE was found to increase linearly with the addition of increasing amounts of AA to the cells. From the intercepts on the y axes of the regression lines, it was possible to calculate the amount of each eicosanoid formed from endogenous AA in resting cells. The calculated values were very close to the experimentally determined values. It was also possible to estimate how much endogenous AA (0.56 μm) had been mobilized by phospholipases in the unstimulated RIES cells. The ability to determine how much of an eicosanoid metabolite is formed in the absence of exogenous AA stimulation is particularly useful when endogenous production of the metabolite is below the detection limit of the assay that is being utilized. Studies on DNA adducts resulting from lipid hydroperoxide-derived bifunctional electrophiles have relied heavily on the use of reversed-phase LC-ESI-MS methodology (3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). However, many of the bifunctional electrophiles themselves are poorly ionized under ESI conditions. Aldehydic bifunctional electrophiles can be converted to oxime derivatives to improve ESI efficiency. However, this results in syn- and anti-oxime isomers together with extremely complex LC chromatograms (22Lee S.H. Blair I.A. Chem. Res. Toxicol. 2000; 13: 698-702Crossref PubMed Scopus (230) Google Scholar). Therefore, normal phase LC-APCI-MS was employed to quantify the lipid hydroperoxide-derived bifunctional electrophiles (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar, 23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar, 24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). HNE was more readily ionized than the other bifunctional electrophiles, so its APCI response was almost an order of magnitude more intense compared with the other bifunctional electrophiles. HPNE, trans-EDE, and ONE were the major products identified from the Fe(II)-mediated homolytic decomposition of (13S)-HPODE (24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). The possible formation of ONE and HNE from HPNE was confirmed by treating HPNE with increasing concentrations of Fe(II). Vitamin C was more than twice as efficient at initiating the decomposition of 13-HPODE to bifunctional electrophiles compared with transition metal ions. It also induced the conversion of HPNE to ONE and HNE. Normal phase LC-APCI-MS methodology also showed that ONE was a major product from both Fe(II)-and vitamin C-mediated homolytic decomposition of (15S)-HPETE (23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar). A number of DNA adducts arising from lipid hydroperoxide-derived bifunctional electrophiles (Fig. 1) have been identified by LC-MS (3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). The reaction between (13S)-HPODE and dGuo was shown to result in the formation of a single ∈DNA adduct, H∈dGuo (Fig. 2) (25Rindgen D. Nakajima M. Wehrli S. Xu K. Blair I.A. Chem. Res. Toxicol. 1999; 12: 1195-1204Crossref PubMed Scopus (136) Google Scholar). The initially formed ethano adducts arose from highly regioselective nucleophilic addition of N-2 of the dGuo to the C-1 aldehyde of ONE, followed by reaction of N-1 at C-2 of the resulting α,β-unsaturated ketone. H∈dAdo DNA adducts were also detected in the reaction between dAdo and 13-HPODE-derived ONE (Fig. 2) (26Rindgen D. Lee S.H. Nakajima M. Blair I.A. Chem. Res. Toxicol. 2000; 13: 846-852Crossref PubMed Scopus (70) Google Scholar, 27Lee S.H. Rindgen D. Bible Jr., R.H. Hajdu E. Blair I.A. Chem. Res. Toxicol. 2000; 13: 565-574Crossref PubMed Scopus (103) Google Scholar). Furthermore, the reaction between ONE and dCyd resulted in formation of H∈dCyd (Fig. 2) as a major product (28Pollack M. Oe T. Lee S.H. Silva Elipe M.V. Arison B.H. Blair I.A. Chem. Res. Toxicol. 2003; 16: 893-900Crossref PubMed Scopus (79) Google Scholar). The endogenous formation of unsubstituted ∈DNA adducts was suggested originally to arise from lipid hydroperoxide-mediated epoxidation of HNE to 2,3-epoxy-4-hydroxynonanal and subsequent reaction of this bifunctional electrophile with DNA bases (29Sodum R.S. Chung F.L. Cancer Res. 1991; 51: 137-143PubMed Google Scholar). Subsequently, it was suggested that this reaction was unlikely to occur in cells because of the relatively high pKa of the lipid hydroperoxides (30Douki T. Odin F. Caillat S. Favier A. Cadet J. Free Radic. Biol. Med. 2004; 37: 62-70Crossref PubMed Scopus (78) Google Scholar). Unsubstituted etheno adducts were also formed from the reaction of lipid hydroperoxide-derived DDE with dAdo or dGuo in the presence of peroxides (31Loureiro A.P. Di M.P. Gomes O.F. Medeiros M.H. Chem. Res. Toxicol. 2000; 13: 601-609Crossref PubMed Scopus (74) Google Scholar). This pathway of endogenous ∈DNA adduct formation would require intracellular epoxidation of DDE to occur more rapidly than its detoxification by GSTs and aldoketoreductases (32Jian W. Arora J.S. Oe T. Shuvaev V.V. Blair I.A. Free Radic. Biol. Med. 2005; 39: 1162-1176Crossref PubMed Scopus (46) Google Scholar). Unsubstituted etheno adducts were formed when DNA bases were treated with EDE and HPNE (Fig. 2) (33Lee S.H. Oe T. Blair I.A. Chem. Res. Toxicol. 2002; 15: 300-304Crossref PubMed Scopus (74) Google Scholar, 34Lee S.H. Arora J.A. Oe T. Blair I.A. Chem. Res. Toxicol. 2005; 18: 780-786Crossref PubMed Scopus (61) Google Scholar). However, HPNE was much more reactive to DNA bases compared with EDE. Based on this increased reactivity, it appears that HPNE is most likely the major lipid hydroperoxide-derived bifunctional electrophile responsible for the formation of unsubstituted ∈DNA adducts (Fig. 2) (34Lee S.H. Arora J.A. Oe T. Blair I.A. Chem. Res. Toxicol. 2005; 18: 780-786Crossref PubMed Scopus (61) Google Scholar). Previous studies have shown that environmental chemicals such as vinyl chloride, vinyl fluoride, and chloroethylene oxide form the same unsubstituted ∈DNA adducts (Fig. 2) (35Barbin A. Friesen M. O'Neill I.K. Croisy A. Bartsch H. Chem. Biol. Interact. 1986; 59: 43-54Crossref PubMed Scopus (13) Google Scholar, 36Swenberg, J. A., Bogdanffy, M. S., Ham, A., Holt, S., Kim, A., Morinello, E. J., Ranasinghe, A., Scheller, N., and Upton, P. B. (1999) in Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis (Singer, B., and Bartsch, H., eds) Volume 150, pp. 29–43, IARC Science Publications, Scholar). Therefore, unsubstituted ∈DNA adducts not arise from lipid It has been that a of 13-HPODE would to the formation of a of K. Lipid Res. 2003; PubMed Scopus Google Scholar). is to rapidly form HPNE, which in is converted to ONE and HNE (23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. 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Toxicol. 2000; 13: PubMed Scopus (74) Google Scholar). this and specific it was shown that was in at much lower in human DNA than found by the The between the capture chemical and studies was suggested to result from formation of during the and derivatization This to the with high of lipid hydroperoxide-derived DNA adducts in human tissue COX-2 can AA into which intracellular reduction to (15S)-HETE (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar, Y. Fisher A.B. Free Radic. Biol. Med. 2005; 38: 1422-1432Crossref PubMed Scopus (322) Google Scholar). In of oxidative stress, reducing are that (15S)-HPETE might to undergo homolytic decomposition to bifunctional electrophiles. COX-2 is to the which the potential of bifunctional electrophiles into the RIES cells, which stably it was possible to that endogenous production of (15S)-HPETE resulted in the formation of H∈dGuo adducts (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). from in studies (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar, 23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar), there was a increase in endogenous H∈dGuo adduct formation when vitamin C was added to the RIES cells (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). H∈dGuo adduct formation and (15S)-HPETE biosynthesis were both by a specific COX-2 and by The role of COX-2 induced DNA damage in vivo was using the mouse of (8Williams M.V. Lee S.H. Pollack M. Blair I.A. J. Biol. Chem. 2006; 281: 10127-10133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The in is to of This results in increased together with of Therefore, a useful to the of increased in vivo COX-2 DNA was from the of and and then in the presence of isotope dilution was to and In separate it was shown that the H∈DNA adducts were not generated as during and by (Fig. The use of DNA a method to an endogenous DNA adduct can arise during and of DNA or during LC-MS H∈dGuo was increased from normal bases in to normal bases in H∈dCyd was much This suggested that base excision repair H∈dAdo and H∈dCyd H∈dGuo in the DNA and that the excised adducts might be excreted in the Interestingly, adducts were which suggests that peroxidation of LA not to DNA damage that was to the In with this could the only when esterified (32Jian W. Arora J.S. Oe T. Shuvaev V.V. Blair I.A. Free Radic. Biol. Med. 2005; 39: 1162-1176Crossref PubMed Scopus (46) Google Scholar). The of DNA repair that have to adducts arising from endogenous has been in of biological to L.J. 2000; PubMed Scopus Google Scholar). DNA is very of the DNA damage results in (7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). This that DNA adducts, which are and in the can be used as of DNA damage. and which are formed by lipid hydroperoxide-mediated DNA might eventually be useful urinary (Fig. It is that in with high stable isotope dilution LC-MRM-MS be for the specific of urinary H∈DNA adducts Anal. Chem. 2001; PubMed Scopus Google Scholar, M. L.J. J. Mass Spectrom. 2004; 39: PubMed Scopus Google Scholar). the that H∈dCyd is highly in human cells M. Blair I.A. M. Chem. Res. Toxicol. 2006; 19: PubMed Scopus Google Scholar) suggests that the of urinary H∈DNA adducts might the of human that are at for through lipid DNA damage.