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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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Amount of lipid peroxidation metabolites from CESS cells. A, 8-HETEs. B, 11-HETEs. C, 12-HETEs. D, 15-HETEs. NT, no treatment; CA, treated with 1.0 μm A23187; CA+VC, treated with 1.0 μm A23187 and 1.0 mm vitamin C; CA+MK, treated with 1.0 μm A23187 and 1.0 μm MK886; CA+ASP, treated with 1.0 μm A23187 and 200.0 μm aspirin. Analyses were performed by stable isotope dilution LC-ECAPCI/MRM/MS of PFB derivatives. Determinations were conducted in triplicate (means ± S.D.).
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Figure 5: Amount of lipid peroxidation metabolites from CESS cells. A, 8-HETEs. B, 11-HETEs. C, 12-HETEs. D, 15-HETEs. NT, no treatment; CA, treated with 1.0 μm A23187; CA+VC, treated with 1.0 μm A23187 and 1.0 mm vitamin C; CA+MK, treated with 1.0 μm A23187 and 1.0 μm MK886; CA+ASP, treated with 1.0 μm A23187 and 200.0 μm aspirin. Analyses were performed by stable isotope dilution LC-ECAPCI/MRM/MS of PFB derivatives. Determinations were conducted in triplicate (means ± S.D.).

Mentions: A variety of HETEs could be derived either from enzyme activities or nonenzymatically from ROS. The basal levels of 8(R)- and 8(S)-HETE in unstimulated CESS cells were 0.06 ± 0.03 and <0.01 pmol/107 cells, respectively (Figs.2 and 5 A). Calcium ionophore A23187 stimulated the cells to produce 0.33 ± 0.13 pmol/107 cells of 8(R)-HETE and 0.46 ± 0.12 pmol/107 cells of 8(S)-HETE (Figs. 4 and 5A). The basal levels of 11(R)- and 11(S)-HETE in unstimulated CESS cells were 0.17 ± 0.06 and 0.29 ± 0.05 pmol/107 cells, respectively (Figs.2 and 5B). Calcium ionophore A23187 treatment increased the production of 11(R)-HETE ∼5-fold to 0.82 ± 0.22 pmol/107 cells with no effect on the production of 11(S)-HETE (Figs.4 and5B). Basal levels of 12(R)- and 12(S)-HETE in unstimulated CESS cells were 0.09 ± 0.05 and 0.13 ± 0.04 pmol/107 cells, respectively (Figs. 2 and 5C). When the cells were stimulated with calcium ionophore, the amount of 12(R)-HETE was 0.70 ± 0. 17 pmol/107 cells, and the amount of 12(S)-HETE was similar at 0.75 ± 0.17 pmol/107 cells (Figs.4 and 5C). The basal levels of 15(R)- and 15(S)-HETE in unstimulated CESS cells were 0.05 ± 0.01 and 0.16 ± 0.01 pmol/107 cells, respectively (Figs.2 and 5D). With calcium ionophore A23187 treatment, the production of 15(R)- and 15(S)-HETE increased to 1.31 ± 0.17 and 1.59 ± 0.28 pmol/107 cells, respectively (Figs. 4 and 5D).


5-Lipoxygenase-mediated endogenous DNA damage.

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

Amount of lipid peroxidation metabolites from CESS cells. A, 8-HETEs. B, 11-HETEs. C, 12-HETEs. D, 15-HETEs. NT, no treatment; CA, treated with 1.0 μm A23187; CA+VC, treated with 1.0 μm A23187 and 1.0 mm vitamin C; CA+MK, treated with 1.0 μm A23187 and 1.0 μm MK886; CA+ASP, treated with 1.0 μm A23187 and 200.0 μm aspirin. Analyses were performed by stable isotope dilution LC-ECAPCI/MRM/MS of PFB derivatives. Determinations were conducted in triplicate (means ± S.D.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Amount of lipid peroxidation metabolites from CESS cells. A, 8-HETEs. B, 11-HETEs. C, 12-HETEs. D, 15-HETEs. NT, no treatment; CA, treated with 1.0 μm A23187; CA+VC, treated with 1.0 μm A23187 and 1.0 mm vitamin C; CA+MK, treated with 1.0 μm A23187 and 1.0 μm MK886; CA+ASP, treated with 1.0 μm A23187 and 200.0 μm aspirin. Analyses were performed by stable isotope dilution LC-ECAPCI/MRM/MS of PFB derivatives. Determinations were conducted in triplicate (means ± S.D.).
Mentions: A variety of HETEs could be derived either from enzyme activities or nonenzymatically from ROS. The basal levels of 8(R)- and 8(S)-HETE in unstimulated CESS cells were 0.06 ± 0.03 and <0.01 pmol/107 cells, respectively (Figs.2 and 5 A). Calcium ionophore A23187 stimulated the cells to produce 0.33 ± 0.13 pmol/107 cells of 8(R)-HETE and 0.46 ± 0.12 pmol/107 cells of 8(S)-HETE (Figs. 4 and 5A). The basal levels of 11(R)- and 11(S)-HETE in unstimulated CESS cells were 0.17 ± 0.06 and 0.29 ± 0.05 pmol/107 cells, respectively (Figs.2 and 5B). Calcium ionophore A23187 treatment increased the production of 11(R)-HETE ∼5-fold to 0.82 ± 0.22 pmol/107 cells with no effect on the production of 11(S)-HETE (Figs.4 and5B). Basal levels of 12(R)- and 12(S)-HETE in unstimulated CESS cells were 0.09 ± 0.05 and 0.13 ± 0.04 pmol/107 cells, respectively (Figs. 2 and 5C). When the cells were stimulated with calcium ionophore, the amount of 12(R)-HETE was 0.70 ± 0. 17 pmol/107 cells, and the amount of 12(S)-HETE was similar at 0.75 ± 0.17 pmol/107 cells (Figs.4 and 5C). The basal levels of 15(R)- and 15(S)-HETE in unstimulated CESS cells were 0.05 ± 0.01 and 0.16 ± 0.01 pmol/107 cells, respectively (Figs.2 and 5D). With calcium ionophore A23187 treatment, the production of 15(R)- and 15(S)-HETE increased to 1.31 ± 0.17 and 1.59 ± 0.28 pmol/107 cells, respectively (Figs. 4 and 5D).

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