A 13-Oxo-9,10-epoxytridecenoate phospholipid analogue of the genotoxic 4,5-epoxy-2E-decenal: detection in vivo, chemical synthesis, and adduction with DNA

Abstract

Often guided by analogy with nonphospholipid products from oxidative cleavage of polyunsaturated fatty acids, we previously identified a variety of biologically active oxidatively truncated phospholipids. Previously, 4,5-epoxy-2(E)-decenal (4,5-EDE) was found to be produced by oxidative cleavage of 13-(S)-hydroperoxy-9,11-(Z,E)-octadeca-dienoic acid (13-HPODE). 4,5-EDE reacts with deoxy-adenosine (dAdo) and deoxy-guanosine (dGuo) to form mutagenic etheno derivatives. We hypothesized that a functionally similar and potentially mutagenic compound, that is, 13-oxo-9,10-epoxytridecenoic acid (OETA), would be generated from 9-HPODE through an analogous fragmentation. We expected that an ester of 2-lysophosphatidylcoline (PC), OETA-PC, would be produced by oxidative cleavage of 9-HPODE-PC in biological membranes. An efficient, unambiguous total synthesis of trans-OETA-PC was first executed to provide a standard that could facilitate the identification of this phospholipid epoxyalkenal that was shown to be produced during oxidation of the linoleic acid ester of 2-lysoPC. Finally, trans-OETA-PC was detected in a lipid extract from rat retina. The identity of the naturally occurring oxidatively truncated phospholipid was further confirmed by derivatization with methoxylamine that produced characteristic mono and bis adducts. The average amount of trans-OETA-PC in rat retina, 0.33 pmol, is relatively low as compared to other oxidatively truncated PCs, for example, the 4-hydroxy-7-oxohept-5-enoic acid PC ester (2.5 pmol) or the 4-keto-7-oxohept-5-enoic acid PC ester (1.7 pmol), derived from the docosahexaenoic acid ester of 2-lysoPC. This, most likely, is because docosahexaenoate PCs are particularly abundant in the retina as compared to the linoleate PC ester precursor of OETA-PC. As predicted by analogy with 4,5-EDE, OETA-PC reacts with dAdo and dGuo, as well as with DNA, to form mutagenic etheno adducts.

Figure 1

Mass spectrometric fragmentation of OETA-PC.

Figure 2

LC/ESI-MS/MS analysis of OETA-PC from oxidized PL-PC liposomes. A. Synthetic OETA-PC standard (parent m/z 718.3), MRM chromatogram (daughter m/z 184.5); B. OETA-PC detected in oxidized PL-PC liposomes, MRM chromatogram (718.3 → 184.5).

Figure 3

LC/ESI-MS/MS analysis of OETA-PC derivatives. A. The methoxime of OETA-PC standard (parent m/z 747.9), MRM chromatogram (daughter m/z 184); B. The methoxime of OETA-PC detected in derivatized oxidized PL-PC liposomes, MRM chromatogram (747.9 → 184.5) C. The bis-methoxyl-amine adduct from OETA-PC standard (parent m/z 794.7), MRM chromatogram (daughter m/z 184); D. The bis-methoxylamine adduct of OETA-PC detected in derivatized oxidized PL-PC liposomes, MRM chromatogram (794.7 → 184.5)

Figure 4

Consumption of PL-PC under various oxidation conditions. Data are the average of two sets of independent experiments.

Figure 5

Evolution profile for production of OETA-PC from aerial oxidation of PL-PC liposomes promoted by Cu (II), UV or MPO at 37 °C. Quantification was achieved with LC/ESI-MS/MS. The yields were calculated by dividing the amount of each analyte by the amount of starting PL-PC. Data are the average of two sets of independent experiments.

Figure 6

LC/ESI-MS/MS detection of OETA-PC (718.3 → 184.5) from rat retina. (A) Synthetic standard OETA-PC. (B) Rat retina extract.

Figure 7

LC-APCI/MS analysis of reaction between OETA-PC and dGuo at 60 °C for 48 h.

Figure 8

LC-APCI/MS analysis of reaction between OETA-PC and dAdo at 60 °C for 48 h.

Figure 9

LC-APCI/MS analysis of reaction of OETA-PC with calf thymus DNA at 60 °C for 48 h.

Scheme 1

Formation of HNE and HOOA-PC from PA-PC.

Scheme 2

Formation of 4,5-EDE from 13-HPODE and postulated formation of OETA from 9-HPODE.

Scheme 3

Strategy for synthesis of trans-OETA-PC.

Scheme 4

Synthesis of OETA-PC.

Scheme 5

A plausible mechanism for formation of 1, N²-etheno-dGuo.