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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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Representative of LC-APCI/MRM/MS chromatograms of HϵdGuo-PFB (m/z 584 → m/z 468) and 15N5-HϵdGuo-PFB (m/z 589 → m/z 473). A, scheme of derivatization of HϵdGuo-PFB. B, chromatograms from unstimulated CESS cells. C, chromatograms from CESS cells treated with 1.0 μm A23187. D, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 mm vitamin C. E, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 μm MK886. F, chromatograms from CESS cells treated with 1.0 μm A23187 and 200.0 μm aspirin.
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Figure 6: Representative of LC-APCI/MRM/MS chromatograms of HϵdGuo-PFB (m/z 584 → m/z 468) and 15N5-HϵdGuo-PFB (m/z 589 → m/z 473). A, scheme of derivatization of HϵdGuo-PFB. B, chromatograms from unstimulated CESS cells. C, chromatograms from CESS cells treated with 1.0 μm A23187. D, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 mm vitamin C. E, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 μm MK886. F, chromatograms from CESS cells treated with 1.0 μm A23187 and 200.0 μm aspirin.

Mentions: HϵdGuo and its 15N5 internal standard were converted to PFB-ester derivatives before analysis by stable isotope dilution LC-APCI/MRM/MS (Fig. 6A). The basal level of HϵdGuo in CESS cells was 2.41 ± 0.35 adducts/107 normal bases (Figs. 6B and 7). When treated with calcium ionophore for 24 h, the DNA-adduct level increased to 6.31 ± 0.73 adducts/107 normal bases (Figs. 6C and 7). Vitamin C, in the presence of calcium ionophore, further increased the HϵdGuo-adduct formation by 16% to 7.33 ± 0.60 adducts/107 normal bases (Figs. 6D and 7).


5-Lipoxygenase-mediated endogenous DNA damage.

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

Representative of LC-APCI/MRM/MS chromatograms of HϵdGuo-PFB (m/z 584 → m/z 468) and 15N5-HϵdGuo-PFB (m/z 589 → m/z 473). A, scheme of derivatization of HϵdGuo-PFB. B, chromatograms from unstimulated CESS cells. C, chromatograms from CESS cells treated with 1.0 μm A23187. D, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 mm vitamin C. E, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 μm MK886. F, chromatograms from CESS cells treated with 1.0 μm A23187 and 200.0 μm aspirin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Representative of LC-APCI/MRM/MS chromatograms of HϵdGuo-PFB (m/z 584 → m/z 468) and 15N5-HϵdGuo-PFB (m/z 589 → m/z 473). A, scheme of derivatization of HϵdGuo-PFB. B, chromatograms from unstimulated CESS cells. C, chromatograms from CESS cells treated with 1.0 μm A23187. D, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 mm vitamin C. E, chromatograms from CESS cells treated with 1.0 μm A23187 and 1.0 μm MK886. F, chromatograms from CESS cells treated with 1.0 μm A23187 and 200.0 μm aspirin.
Mentions: HϵdGuo and its 15N5 internal standard were converted to PFB-ester derivatives before analysis by stable isotope dilution LC-APCI/MRM/MS (Fig. 6A). The basal level of HϵdGuo in CESS cells was 2.41 ± 0.35 adducts/107 normal bases (Figs. 6B and 7). When treated with calcium ionophore for 24 h, the DNA-adduct level increased to 6.31 ± 0.73 adducts/107 normal bases (Figs. 6C and 7). Vitamin C, in the presence of calcium ionophore, further increased the HϵdGuo-adduct formation by 16% to 7.33 ± 0.60 adducts/107 normal bases (Figs. 6D and 7).

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