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5-Lipoxygenase-mediated endogenous DNA damage.

Jian W, Lee SH, Williams MV, Blair IA - J. Biol. Chem. (2009)

Bottom Line: The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)).FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels.In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.

ABSTRACT
Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 +/- 0.01 to 45.50 +/- 4.05 pmol/10(7) cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2'-deoxyguanosine (HepsilondGuo) from 2.41 +/- 0.35 to 6.31 +/- 0.73 adducts/10(7) normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HepsilondGuo DNA-adduct in CESS cells.

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Chromatograms from targeted lipidomics analysis using LC-ECAPCI/MRM/MS for analysis of lipid metabolites from CESS cells. Chromatograms are shown for 5(R,S)-HETE (m/z 319 → m/z 115), 5(S)-[2H8]HETE (m/z 327 → m/z 116), 12(R,S)-HETE (m/z 319 → m/z 179), 12(S)-[2H8]HETE (m/z 327 → m/z 184), 15(R,S)-HETE (m/z 319 → m/z 219), 15(S)-[2H8]HETE (m/z 327 → m/z 226), 11(R,S)-HETE (m/z 319 → m/z 167), 8(R,S)-HETE (m/z 319 → m/z 155), 13(R,S)-HODE (m/z 295 → m/z 195), 13(S)-[2H4]HODE (m/z 299 → m/z 198), LTB4 (m/z 335 → m/z 195), [2H4]LTB4 (m/z 339 → m/z 197), PGE2 (m/z 351 → m/z 271), PGD2 (m/z 351 → m/z 271), [2H4]PGE2 (m/z 355 → m/z 275), [2H4]PGD2 (m/z 355 → m/z 275), PGF2α (m/z 353 → m/z 309), [2H4]PGF2α (m/z 357 → m/z 313).
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Figure 2: Chromatograms from targeted lipidomics analysis using LC-ECAPCI/MRM/MS for analysis of lipid metabolites from CESS cells. Chromatograms are shown for 5(R,S)-HETE (m/z 319 → m/z 115), 5(S)-[2H8]HETE (m/z 327 → m/z 116), 12(R,S)-HETE (m/z 319 → m/z 179), 12(S)-[2H8]HETE (m/z 327 → m/z 184), 15(R,S)-HETE (m/z 319 → m/z 219), 15(S)-[2H8]HETE (m/z 327 → m/z 226), 11(R,S)-HETE (m/z 319 → m/z 167), 8(R,S)-HETE (m/z 319 → m/z 155), 13(R,S)-HODE (m/z 295 → m/z 195), 13(S)-[2H4]HODE (m/z 299 → m/z 198), LTB4 (m/z 335 → m/z 195), [2H4]LTB4 (m/z 339 → m/z 197), PGE2 (m/z 351 → m/z 271), PGD2 (m/z 351 → m/z 271), [2H4]PGE2 (m/z 355 → m/z 275), [2H4]PGD2 (m/z 355 → m/z 275), PGF2α (m/z 353 → m/z 309), [2H4]PGF2α (m/z 357 → m/z 313).

Mentions: 5-HETE and LTB4 are major lipid peroxidation products from 5-LO activity. Formation of 5-HETE and LTB4 were measured to monitor the 5-LO activity in the cells treated with calcium ionophore A23187. Unstimulated CESS cells produced similar amounts of 5(R)-HETE (0.06 ± 0.01 pmol/107 cells) and 5(S)-HETE (0.07 ± 0.01 pmol/107 cells) (Figs. 2 and 3A). After stimulation with 1 μm calcium ionophore A23187, 5(S)-HETE increased more than 3 orders of magnitude to 45.50 ± 4.05 pmol/107 cells, whereas the production of 5(R)-HETE only increased ∼20-fold to 1.42 ± 0.07 pmol/107 cells (Figs. 3A and 4). LTB4 secreted by unstimulated CESS cells was below the detection limit of the assay (B). The level of LTB4 increased to 48.10 ± 4.60 pmol/107 cells (Figs. 3B and 4) with the treatment of calcium ionophore.


5-Lipoxygenase-mediated endogenous DNA damage.

Jian W, Lee SH, Williams MV, Blair IA - J. Biol. Chem. (2009)

Chromatograms from targeted lipidomics analysis using LC-ECAPCI/MRM/MS for analysis of lipid metabolites from CESS cells. Chromatograms are shown for 5(R,S)-HETE (m/z 319 → m/z 115), 5(S)-[2H8]HETE (m/z 327 → m/z 116), 12(R,S)-HETE (m/z 319 → m/z 179), 12(S)-[2H8]HETE (m/z 327 → m/z 184), 15(R,S)-HETE (m/z 319 → m/z 219), 15(S)-[2H8]HETE (m/z 327 → m/z 226), 11(R,S)-HETE (m/z 319 → m/z 167), 8(R,S)-HETE (m/z 319 → m/z 155), 13(R,S)-HODE (m/z 295 → m/z 195), 13(S)-[2H4]HODE (m/z 299 → m/z 198), LTB4 (m/z 335 → m/z 195), [2H4]LTB4 (m/z 339 → m/z 197), PGE2 (m/z 351 → m/z 271), PGD2 (m/z 351 → m/z 271), [2H4]PGE2 (m/z 355 → m/z 275), [2H4]PGD2 (m/z 355 → m/z 275), PGF2α (m/z 353 → m/z 309), [2H4]PGF2α (m/z 357 → m/z 313).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719316&req=5

Figure 2: Chromatograms from targeted lipidomics analysis using LC-ECAPCI/MRM/MS for analysis of lipid metabolites from CESS cells. Chromatograms are shown for 5(R,S)-HETE (m/z 319 → m/z 115), 5(S)-[2H8]HETE (m/z 327 → m/z 116), 12(R,S)-HETE (m/z 319 → m/z 179), 12(S)-[2H8]HETE (m/z 327 → m/z 184), 15(R,S)-HETE (m/z 319 → m/z 219), 15(S)-[2H8]HETE (m/z 327 → m/z 226), 11(R,S)-HETE (m/z 319 → m/z 167), 8(R,S)-HETE (m/z 319 → m/z 155), 13(R,S)-HODE (m/z 295 → m/z 195), 13(S)-[2H4]HODE (m/z 299 → m/z 198), LTB4 (m/z 335 → m/z 195), [2H4]LTB4 (m/z 339 → m/z 197), PGE2 (m/z 351 → m/z 271), PGD2 (m/z 351 → m/z 271), [2H4]PGE2 (m/z 355 → m/z 275), [2H4]PGD2 (m/z 355 → m/z 275), PGF2α (m/z 353 → m/z 309), [2H4]PGF2α (m/z 357 → m/z 313).
Mentions: 5-HETE and LTB4 are major lipid peroxidation products from 5-LO activity. Formation of 5-HETE and LTB4 were measured to monitor the 5-LO activity in the cells treated with calcium ionophore A23187. Unstimulated CESS cells produced similar amounts of 5(R)-HETE (0.06 ± 0.01 pmol/107 cells) and 5(S)-HETE (0.07 ± 0.01 pmol/107 cells) (Figs. 2 and 3A). After stimulation with 1 μm calcium ionophore A23187, 5(S)-HETE increased more than 3 orders of magnitude to 45.50 ± 4.05 pmol/107 cells, whereas the production of 5(R)-HETE only increased ∼20-fold to 1.42 ± 0.07 pmol/107 cells (Figs. 3A and 4). LTB4 secreted by unstimulated CESS cells was below the detection limit of the assay (B). The level of LTB4 increased to 48.10 ± 4.60 pmol/107 cells (Figs. 3B and 4) with the treatment of calcium ionophore.

Bottom Line: The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)).FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels.In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.

ABSTRACT
Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 +/- 0.01 to 45.50 +/- 4.05 pmol/10(7) cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2'-deoxyguanosine (HepsilondGuo) from 2.41 +/- 0.35 to 6.31 +/- 0.73 adducts/10(7) normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HepsilondGuo DNA-adduct in CESS cells.

Show MeSH
Related in: MedlinePlus