Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene

The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L-3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day-old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in cotyledons.     

Molecular Characterization of an Arabidopsis Gene Encoding Hydroperoxide Lyase, a Cytochrome P-450 That Is Wound Inducible

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.     

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (S_Ni) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.     

A Lipoxygenase Pathway Is Activated in Rice after Infection with the Rice Blast Fungus Magnaporthe grisea

Lipoxygenase (LOX) and lipid hydroperoxide-decomposing activity (LHDA) markedly increased in the fifth leaves of rice (Oryza sativa cv Aichiasahi) after infection with the rice blast fungus, Magnaporthe grisea. The increases in the enzyme activities were significantly higher in response to infection with an incompatible strain (race 131) compared with infection with a compatible strain (race 007) of the fungus. Using ion-exchange chromatography, we isolated three LOX activities (leaf LOX-1, -2, -3) from both uninoculated and infected leaves. The activity of leaf LOX-3, in particular, increased in the incompatible race-infected leaves. The leaf LOX-3 had a pH optimum of 5.0 and produced preferentially 13-l-hydroperoxy-9,11 (Z,E)-octadecadienoic acid (13-HPODD) from linoleic acid. 13-HPODD and 13-l-hydroxy-9,11 (Z,E)-octadecadienoic acid, one of the reaction products from 13-HPODD by LHDA, were highly inhibitory to the germination of conidia of the fungus. The present study provides correlative evidence for important roles of LOX and LHDA in the resistance response of rice against the blast fungus.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Characterization of factors involved in the production of 2(E)-nonenal during mashing

To characterize the factors involved in the production of volatile aldehydes during mashing, a model mashing experiment was done. After we inactivated the endogenous lipoxygenase (LOX) activity in the mash by mashing at 70 degrees C for 30 min, further incubation with recombinant barley LOX-1 stimulated the accumulation of 2(E)-nonenal; however, this effect was significantly reduced by boiling the mash sample. The result suggests that both LOX-1 and a heat-stable enzymatic factor are involved in the production of 2(E)-nonenal during mashing. Malt contained fatty acid hydroperoxide lyase-like activity (HPL-like activity) that transformed 9-hydroperoxy-10(E), 12(Z)-octadecadienoic and 13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid into 2(E)-nonenal and hexanal, respectively. Proteinase K sensitivity tests showed that they are distinct factors. 9-HPL-like activity survived through the mashing at 70 degrees C for 30 min but was inactivated by boiling, suggesting it will be the heat-stable enzymatic factor found in the model mashing experiment.     

Cationic substrates of soybean lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.     

On the reaction specificity of the lipoxygenase from tomato fruits

A lipoxygenase was purified 300-fold from a homogenate supernatant of ripe tomato fruits by fractionated ammonium sulfate precipitation and anion exchange fast protein liquid chromatography. The specific linoleate oxygenase activity of the final enzyme preparation was 1300 nkat per mg protein at pH 6.8 and 25°C in the absence of any detergent. The enzyme oxygenated linoleic acid and α-linolenic acid at comparable rates, whereas γ-linolenic acid, arachidonic acid, 11,14-eicosadienoic acid and 11,14,17-eicosatrienoic acid were poor substrates. Linoleic acid was converted to 9(S)-hydroperoxy-10E,12Z-octadecadienoic acid, whereas 5(S)-HpETE, 11(S)-HpETE and 8(S)-HpETE were identified as major oxygenation products from arachidonic acid. The tomato lipoxygenase did not react with either dilinoleyl phosphatidylcholine or the lipid extract from beef heart mitochondria. The possible biological importance of the reaction of tomato lipoxygenase with arachidonic acid is discussed.     

Effect of endocannabinoids on soybean lipoxygenase-1 activity

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a K_i value of 3.7 μM and 6.2 μM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with K_in value of 27 μM and 24 μM, respectively, and k₃ value of 0.12 min⁻¹ and 0.35 min⁻¹, respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

Oxygenation of the 5-lipoxygenase product 5S-hydroxyeicosatetraenoic acid by cyclooxygenase-2 yields a bicyclic di-endoperoxide. The di-endoperoxide contains two peroxides spanning from carbons 9 to 11 and 8 to 12, and two hydroxyls at carbons 5 and 15 of arachidonic acid (Schneider C., et al. 2006. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2. J. Am. Chem. Soc. 128: 720). Here, we report that treatment of the di-endoperoxide with hematin or ferrous chloride results in cleavage of both peroxide O-O bonds and of the bonds between the carbons that carry the peroxide groups, producing the aldehydes 4-hydroxy-2E-nonenal (4-HNE), 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde (MDA). The hematin- and ferrous iron-catalyzed transformation of the di-endoperoxide proceeded with a similar yield of products as the cleavage of the prostaglandin endoperoxide PGH₂ to 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid and MDA. Chiral phase HPLC analysis of the 4-HNE cleavage product showed greater than 98% 4S and thus established the S configuration of the 15-carbon of the di-endoperoxide that had not previously been assigned. This transformation of the 5-lipoxygenase/cyclooxygenase-2 derived di-endoperoxide invokes the possibility of a novel pathway to formation of the classic lipid peroxidation products 4-HNE and MDA.     

Vanadium-catalyzed transformation of 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid: Structural studies on epoxy alcohols and trihydroxy acids

Treatment of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate with vanadium oxyacetylacetonate led to the formation of two diastereometric α,β-epoxy alcohols, i.e. methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate and methyl 11(S), 12(S)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate. The epoxy alcohols underwent spontaneous hydrolysis into isomeric trihydroxyesters. The first mentioned epoxy alcohol afforded methyl 9(R), 12(S), 13(S)- and methyl 9(S), 12(S), 13(S)-trihydroxy-10(E)-octadecenoates as major hydrolysis products whereas the latter epoxy alcohol afforded methyl 9(R), 12(R), 13(S)- and methyl 9(S), 12(R)-13(S)-trihydroxy-10(E)-octadecenoates as major compounds. Smaller amounts of diastereomeric methyl 11,12,13-trihydroxy-9-octadecenoates were also formed from both epoxy alcohols. The vanadium-catalyzed conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13(S)HPOD) (methyl ester) into α,β-epoxy alcohols and their further conversion into trihydroxy derivatives offers a model system for similar transformations of certain poly-unsaturated fatty acids recently described in the fungus, Saprolegnia parasitica.     

Sialic acid attenuates the cytotoxicity of the lipid hydroperoxides HpODE and HpETE

Reduction of peroxide molecular species is an essential function in living organisms. In previous studies, we proposed a new function for the sialic acid N-acetylneuraminic acid (Neu5Ac)—that of antioxidant/hydrogen peroxide scavenging agent. On the basis of the reaction scheme, Neu5Ac is thought to act as a general antioxidant of all hydroperoxide-type species (R-OOHs). The concentration of tert-butyl hydroperoxide (t-BuOOH) decreased after co-incubation with N-acetylneuraminic acid. Neu5Ac also decreased the R-OOH concentration in solutions of peroxylinolenic acid (13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid, HpODE) and peroxyarachidonic acid (15(S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, HpETE)—two lipid hydroperoxides that participate in many physiological events. Moreover, the cytotoxicity of both these lipid hydroperoxides was attenuated by reaction with Neu5Ac acid. Our results suggest that N-acetylneuraminic acid is a potential antioxidant of most hydroperoxides that accumulate in organisms.     

Payne rearrangement during analysis of epoxyalcohols of linoleic and alpha-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry

Hydroperoxides of polyunsaturated fatty acids can be transformed to epoxyalcohols and keto fatty acids by metal enzymes, hematin, and various catalysts. In the current study, we used hematin to transform 9-hydroperoxy-10E,12Z-octadecadienoic acid and 13-hydroperoxy-9Z,11E-octadecadienoic acid to epoxyalcohols (with trans epoxide configuration) and to keto fatty acids. The products were separated by normal phase high-performance liquid chromatography (NP-HPLC) and analyzed using postcolumn addition of isopropanol/water and online negative ion electrospray ionization mass spectrometry (MS). The tandem MS (MS/MS) spectra were studied using analogs prepared from [9,10,12,13-²H₄]linoleic acid (18:2n−6) and from α-linolenic acid (18:3n−3). We also studied the MS/MS spectra of epoxyalcohols formed from 11-hydroperoxy- and 8-hydroperoxy-9Z,12Z-octadecadienoic acids. Results were confirmed by MS/MS analysis of a series of authentic standards. MS/MS ions of 9-keto-10E,12Z-octadecadienoic acid and 13-keto-9Z,11E-octadecadienoic acid could be explained by keto-enol tautomerism. MS/MS spectra of regioisomeric allylic epoxyalcohols differed in relative intensities of characteristic ions. The MS/MS spectra of the epoxyalcohols with 1-hydroxy-2,3-epoxy-4Z-pentene or 3-hydroxy-1,2-epoxy-4Z-pentene elements were virtually identical and showed two characteristic ions that differed by 30 in m/z values (CH(OH)). The results suggested that epoxide migration (Payne rearrangement) occurred during collision-induced dissociation. We conclude that regioisomeric allylic epoxyalcohols can be identified by their MS/MS spectra, whereas regioisomeric epoxyalcohols can be identified by MS/MS in combination with their retention times on NP-HPLC.     

Biosynthesis,isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.     

Secretion of two novel enzymes,manganese 9S-lipoxygenase and epoxy alcohol synthase,by the rice pathogen Magnaporthe salvinii

The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion.     

5-Lipoxygenase-mediated Endogenous DNA Damage

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.PUFAs² can be converted into lipid hydroperoxides enzymatically by the action of LOs (1) and COXs (2) or nonenzymatically by reactive oxygen species (ROS) (3). Arachidonic acid, one of the essential PUFAs present in cell membranes, is released from phospholipids by different phospholipase A₂ isoforms upon diverse physical, chemical, inflammatory, and mitogenic stimuli (4). The free arachidonic acid then serves as a substrate for LOs, COXs, or ROS to produce a variety of lipid hydroperoxides (5, 6). ROS, 12-LO, and 15-LO can also act directly upon arachidonic acid esterified in phospholipids to produce lipid hydroperoxides (7), which are reduced (8), hydrolyzed by phospholipase A₂ (4), and then secreted as the corresponding free HETEs (9). COX-2-mediated (10) and 15-LO-1-mediated (11) metabolism of linoleic acid results in the formation of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, which is rapidly reduced to 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid (HODE) and secreted from cells. Arachidonic acid is specifically metabolized by 5-LO into 5(S)-HpETE, which is either reduced to 5(S)-HETE or serves as precursor to the formation of leukotrienes (LTs) (Scheme 1) (12). In contrast, ROS-mediated reactions produce racemic mixtures of all possible regioisomers of HpETEs and 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acids (3) that are subsequently secreted from cells as complex mixtures of racemic HETEs and HODEs. Therefore, the ability to analyze different HETE and HODE enantiomers and regioisomers is important for elucidating specific cellular lipid peroxidation pathways (13).Open in a separate windowSCHEME 1.5-LO-mediated formation of arachidonic acid metabolites and dGuo-adducts. HPNE, 4-hydroperoxy-2(E)-nonenal; DOOE, dioxo-6-octenoic acid.The conversion of arachidonic acid to 5(S)-HpETE by 5-LO is critically dependent upon the presence of FLAP (14). 5-LO and FLAP are expressed primarily in inflammatory cells such as polymorphonuclear leukocytes, monocytes, macrophages, and mast cells (12, 15–17). Therefore, 5-LO-mediated LT formation is thought to play a critical role in inflammation and allergic disorders (18–21). In addition, a number of studies have implicated 5-LO-derived arachidonic acid metabolites as mediators of atherogenesis and heart disease (12, 22, 23). The 5-LO pathway of arachidonic acid metabolism has also been proposed to play a role in prostate and pancreatic cancer (24–26).Lipid hydroperoxides undergo homolytic decomposition into bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal, ONE, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy-2(E)-nonenal (27). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH) (28, 29), DNA, (5, 6), and proteins (30, 31). Our previous in vitro studies characterized the bifunctional electrophiles ONE and 4-hydroperoxy-2(E)-nonenal as major products arising from the homolytic decomposition of 5-LO-derived 5(S)-HpETE (32). Reactions with DNA resulted in the formation of etheno-2′-deoxyguanosine (ϵdGuo) from 4-hydroperoxy-2(E)-nonenal and heptanone-ϵdGuo (HϵdGuo) from ONE (Scheme 1). 5,8-Dioxo-6-octenoic acid, a bifunctional electrophile from the carboxyl terminus of 5(S)-HpETE, gave rise to the novel DNA-adduct carboxypentanone-ϵdGuo (CPϵdGuo)-adduct as shown in Scheme 1.Previous studies have demonstrated that lipid hydroperoxides generated by COX-2 could lead to the formation of endogenous DNA adducts in epithelial cells (6). Cellular 5-LO, like COX-2, synthesizes lipid hydroperoxides on the nuclear membrane. Therefore, it is highly possible that 5-LO could also mediate the formation of lipid hydroperoxide-derived endogenous DNA adducts in cells. CESS is a human lymphoblastic cell line that expresses both 5-LO and FLAP, and they have been used as a model for inflammatory cells to examine the role of 5-LO metabolites in signal transduction (33, 34). In the present study, CESS cells provided an ideal model to elucidate the relationship of 5-LO mediated-lipid peroxidation and DNA-adduct formation in a cellular setting. Stable isotope dilution chiral LC-electron capture (EC) APCI/MRM/MS (13) was used to monitor the concomitant formation of lipid hydroperoxides in the presence of different enzyme stimulator or inhibitors. DNA-adduct formation in the same cells was measured by a stable isotope dilution APCI/MRM/MS method. The powerful tool of chiral lipid analysis enabled us to dissect the complicated lipid peroxidation pathways and to correlate them with endogenous DNA-adduct formation. The results demonstrated that 5-LO-mediated lipid peroxidation could cause HϵdGuo formation in cells. This novel finding provided additional explanation for the previous observation that increased 5-LO activity was associated with cancers and cardiovascular diseases (24–26).     

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of ∼50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-²H]18:1n-9, [7R-²H]18:2n-6, and [8R-²H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.     

Long chain lipid hydroperoxides increase the glutathione redox potential through glutathione peroxidase 4

BackgroundPeroxidation of PUFAs by a variety of endogenous and xenobiotic electrophiles is a recognized pathophysiological process that can lead to adverse health effects. Although secondary products generated from peroxidized PUFAs have been relatively well studied, the role of primary lipid hydroperoxides in mediating early intracellular oxidative events is not well understood.MethodsLive cell imaging was used to monitor changes in glutathione (GSH) oxidation in HAEC expressing the fluorogenic sensor roGFP during exposure to 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HpODE), a biologically important long chain lipid hydroperoxide, and its secondary product 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). The role of hydrogen peroxide (H₂O₂) was examined by direct measurement and through catalase interventions. shRNA-mediated knockdown of glutathione peroxidase 4 (GPx4) was utilized to determine its involvement in the relay through which 9-HpODE initiates the oxidation of GSH.ResultsExposure to 9-HpODE caused a dose-dependent increase in GSH oxidation in HAEC that was independent of intracellular or extracellular H₂O₂ production and was exacerbated by NADPH depletion. GPx4 was involved in the initiation of GSH oxidation in HAEC by 9-HpODE, but not that induced by exposure to H₂O₂ or the low molecular weight alkyl tert-butyl hydroperoxide (TBH).ConclusionsLong chain lipid hydroperoxides can directly alter cytosolic E_GSH independent of secondary lipid oxidation products or H₂O₂ production. NADPH has a protective role against 9-HpODE induced E_GSH changes. GPx4 is involved specifically in the reduction of long-chain lipid hydroperoxides, leading to GSH oxidation.SignificanceThese results reveal a previously unrecognized consequence of lipid peroxidation, which may provide insight into disease states involving lipid peroxidation in their pathogenesis.