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/107 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 (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/107 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 HϵdGuo-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 HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells.
Highlights
Western blot analysis was employed to profile the expression of lipid peroxidation enzymes in CESS cells
The cells were stimulated with calcium ionophore A23187 to activate the enzymes to generate lipid hydroperoxides, which were measured as reduced and secreted forms in the cell culture medium
MK886 as an inhibitor of FLAP, aspirin as an inhibitor of COX, or vitamin C as a mediator of lipid hydroperoxide decomposition was employed to elucidate the different pathways of lipid peroxidation
Summary
Material and Reagents—CESS cells was obtained from ATCC (Manassas, VA). RPMI 1640, fetal bovine serum, and penicillin-streptomycin were supplied by Invitrogen. Cells were incubated for 1 h, and the medium was collected for lipid analysis. The remaining sample solution was added with 20.0 ng of 15N5-H⑀dGuo as internal standards and applied to LC-18 solid phase extraction tube (1 g, 6 ml), which was preconditioned with acetonitrile (18 ml) followed with water (18 ml). A certain amount of authentic H⑀dGuo (0, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 5.00 ng of each) was mixed with internal standards and subjected to the same procedure as that of samples from this step. The eluate was evaporated to dryness under nitrogen and dissolved in 300 l of acetonitrile containing PFB-Br (20:300, v/v) and diisopropylethylamine (4:300, v/v) and incubated at room temperature for 1.5 h. DNA adduct levels were calculated by interpolation from the calibration curve and normalized with the total base content in each sample. Converted to H⑀dGuo-adducts/107 normal bases from the DNA base analysis data
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