Identification of hydroxyalkenals formed from omega-3 fatty acids

The highly toxic lipid peroxidation product, 4-hydroxynonenal, is formed from the decomposition of hydroperoxides of omega-6 fatty acids. In this study the analogous hydroxyalkenals formed from the decomposition of hydroperoxides of omega-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) were isolated and identified using TLC densitometry, HPLC and GC/Mass Spectrometry. The major hydroxyalkenal formed from both fatty acids was a diene analog of 4-hydroxynonenal, 4-hydroxynona(2,6)dienal, while 4-hydroxyhexanal was a minor product. Measurement of specific omega-3 lipid peroxidation products may be important in studies using dietary fish oil.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.     

4-Hydroxyhexenal: a lipid peroxidation product derived from oxidized docosahexaenoic acid

4-Hydroxynonenal and 4-hydroxyhexenal are cytotoxic aldehydic products of lipid peroxidation with high biological activity. Peroxidation of n - 6 fatty acids produces 4-hydroxynonenal, but the origin of 4-hydroxyhexenal has been uncertain. We now present evidence that 4-hydroxyhexenal is generated by oxidation of docosahexaenoic acid, the most abundant n-3 fatty acid in tissues.     

Hydroxynonenal and uncoupling proteins: a model for protection against oxidative damage

In this mini review we summarize recent studies from our laboratory that show the involvement of superoxide and the lipid peroxidation product 4-hydroxynonenal in the regulation of mitochondrial uncoupling. Superoxide produced during mitochondrial respiration is a major cause of the cellular oxidative damage that may underlie degenerative diseases and ageing. Superoxide production is very sensitive to the magnitude of the mitochondrial protonmotive force, so can be strongly decreased by mild uncoupling. Superoxide is able to give rise to other reactive oxygen species, which elicit deleterious effects primarily by oxidizing intracellular components, including lipids, DNA and proteins. Superoxide-induced lipid peroxidation leads to the production of reactive aldehydes, including 4-hydroxynonenal. These aldehydic lipid peroxidation products are in turn able to modify proteins such as mitochondrial uncoupling proteins and the adenine nucleotide translocase, converting them into active proton transporters. This activation induces mild uncoupling and so diminishes mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of energy production.     

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determins, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.     

Aldehyde reductase gene expression by lipid peroxidation end products, MDA and HNE

Membrane lipid peroxidation results in the production of a variety of aldehydic compounds that play a significant role in aging, drug toxicity and the pathogenesis of a number of human diseases, such as atherosclerosis and cancer. Increased lipid peroxidation and reduced antioxidant status may also contribute to the development of diabetic complications. This study reports that lipid peroxidation end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) induce aldehyde reductase (ALR) gene expression. MDA and HNE induce an increase in intracellular peroxide levels; N-Acetyl-L-cysteine (NAC) suppressed MDA- and HNE-induced ALR gene expression. These results indicate that increased levels of intracellular peroxides by MDA and HNE might be involved in the upregulation of ALR.     

Formation of oxylipins by CYP74 enzymes

Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes. Products are hydroperoxy polyunsaturated fatty acids and metabolites derived there from collectively named oxylipins. They may either originate from chemical oxidation or are synthesized by the action of various enzymes, such as lipoxygenases. Cloning of many lipoxygenases and other key enzymes metabolizing oxylipins revealed new insights on oxylipin functions, new reactions and the first hints on enzyme mechanisms. These aspects are reviewed with respect to metabolism of fatty acid hydroperoxides by an atypical P450 subfamily: the CYP74. Up to now this protein family contains three different enzyme activities: (i) allene oxide synthase leading to the formation of unstable allene oxides which react to ketol and cyclopentenone fatty acids, (ii) hydroperoxide lyase producing hemiacetals decomposing to aldehydes and ω-oxo fatty acids and (iii) divinyl ether synthase which forms divinyl ethers. Signalling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among their numerous products.     

Aldehyde reductase gene expression by lipid peroxidation end products,MDA and HNE

Membrane lipid peroxidation results in the production of a variety of aldehydic compounds that play a significant role in aging, drug toxicity and the pathogenesis of a number of human diseases, such as atherosclerosis and cancer. Increased lipid peroxidation and reduced antioxidant status may also contribute to the development of diabetic complications. This study reports that lipid peroxidation end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) induce aldehyde reductase (ALR) gene expression. MDA and HNE induce an increase in intracellular peroxide levels; N-Acetyl-L-cysteine (NAC) suppressed MDA- and HNE-induced ALR gene expression. These results indicate that increased levels of intracellular peroxides by MDA and HNE might be involved in the upregulation of ALR.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Formation of 4-hydroxynonenal and further aldehydic mediators of inflammation during bromotrichlorornethane treatment of rat liver cells

Bromotrichloromethane (CBrCl(3)) treatment is a model for studies on molecular mechanisms of haloalkane toxicity with some advantages compared with CCl(4) treatment. The formation of 4-hydroxynonenal and similar aldehydic products of lipid peroxidation, which play a role as mediators of inflammatory processes, was clearly demonstrated in rat hepatocytes treated with CBrCl(3). It may be assumed that haloalkane toxicity is connected with the biological effects of those inflammation mediatory aldehydic compounds.     

Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes

The alteration of structural and biological properties of human plasma low density lipoprotein (LDL) exposed to oxidative conditions is in part ascribed to lipid peroxidation. The objective of this investigation was to measure quantitatively several parameters in oxidizing LDL indicative for lipid peroxidation. Exposure of freshly prepared EDTA-free LDL to an oxygen-saturated buffer led to a complete depletion of alpha- and gamma-tocopherol within 6 hr, thereafter lipid peroxidation commenced as indicated by the kinetics of the loss of linoleic (18:2) and arachidonic (20:4) acids, the formation of aldehydic lipid peroxidation products and fluorescent apoB. Within 24 hr of oxidation, on average 79 nmol of 18:2 (initial 345) and 12.8 nmol of 20.4 (initial 25.6) were oxidized per mg of LDL and the sample contained in total 7.1 nmol of aldehydes with the following molar distribution: 36.6% malonaldehyde, 25% hexanal, 8.9% propanal, 8.2% 4-hydroxynonenal, 7.6% butanal, 4.1% 2.4-heptadienal, 3.4% pentanal, 3.4% 4-hydroxyhexenal, and 2.5% 4-hydroxyoctenal. Malonaldehyde was predominantly (93%) in the aqueous phase, whereas the other aldehydes remained mostly (34-98%) within the LDL particle, where the total aldehyde concentration was in the range of 12 mM. Oxidized LDL exhibited a 1.6-fold enhanced electrophoretic mobility. Similarily, native LDL incubated for 5 hr with aldehydes showed increased electrophoretic mobility. At equal concentrations (5 mM) 4-hydroxynonenal was most effective, followed by 2,4-heptadienal, hexanal, and malonaldehyde. This study reports for the first time the rate and extent of the change of LDL constituents occurring during lipid peroxidation.     

Possible mutagens derived from lipids and lipid precursors

Free radicals can initiate the oxidative decomposition of cellular membranes by lipid peroxidation. In this process a great variety of reactive aldehydes are produced intracellularly. Some of them, such as 4-hydroxynonenal or malonaldehyde, are biologically very active and might be involved in free radical-mediated DNA damage. A short review of the effects of aldehydic lipid peroxidation products on isolated DNA, their genotoxic effect in prokaryotes and eukaryotes and their in vivo carcinogenicity is given. Additionally own experiments on cytotoxic and genotoxic effects of 4-hydroxynonenal, 2-nonenal and nonanal in primary cultures of rat hepatocytes are reported. 4-Hydroxynonenal was highly cytotoxic at 100 microM, at subcytotoxic concentrations of 0.1-10 microM 4-hydroxynonenal increased the frequency of micronuclei, chromosomal aberrations and sister-chromatid exchange. 2-Nonenal and nonanal were not cytotoxic at 100 microM, the maximum dose tested. At 100 microM 2-nonenal led to a slight increase in micronuclei; chromosomal aberrations were not significantly altered. Nonanal had no detectable genotoxic effects. The level of endogenous 4-hydroxynonenal in tissues is in the range of 0.1-3.0 microM and can increase to 10 microM in conditions of oxidative stress; such levels appear to be sufficiently high to produce DNA damages, whether such damages are transient or irreversible is not known.     

Chemistry and biochemistry of lipid peroxidation products

Abstract

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as ‘second messengers’ of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.     

Impairment of the cytotoxic and oxidative activities of 7 beta-hydroxycholesterol and 7-ketocholesterol by esterification with oleate

Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7 beta-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol- and 7 beta-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7 beta-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7 beta-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7 beta-hydroxycholesteryl-3-oleate. This finding supports the theory that 7 beta-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression.     

Adaptive response induced by lipid peroxidation products in cell cultures

The adaptive response induced by the lipid peroxidation products, such as phosphatidylcholine hydroperoxide, lysophosphatidylcholine (LysoPC), 15-deoxy-Delta(12,14)-prostaglandin J(2), 4-hydroxynonenal (4-HNE), hydroxyoctadecadienoic acid, 7-hydroxycholesterol, and cholesterol 5beta,6beta-epoxide, was investigated in this study. Although these products have been implicated in oxidative stress-related diseases, pretreatment with such compounds at sublethal concentrations significantly protected PC12 cells against subsequent oxidative stress induced by 6-hydroxydopamine. Moreover, 4-HNE and LysoPC also exhibited adaptive protection in human arterial endothelial cells. These findings suggest a general hormetic effect of such compounds in cell cultures and may lead to a reappraisal of the eventual role of reactive oxygen species and lipid peroxidation in organisms.     

Dietary polyunsaturated fatty acids and heme iron induce oxidative stress biomarkers and a cancer promoting environment in the colon of rats

The end products of polyunsaturated fatty acid (PUFA) peroxidation, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and isoprostanes (8-iso-PGF_2α), are widely used as systemic lipid oxidation/oxidative stress biomarkers. However, some of these compounds have also a dietary origin. Thus, replacing dietary saturated fat by PUFAs would improve health but could also increase the formation of such compounds, especially in the case of a pro-oxidant/antioxidant imbalanced diet. Hence, the possible impact of dietary fatty acids and pro-oxidant compounds was studied in rats given diets allowing comparison of the effects of heme iron vs. ferric citrate and of ω-6- vs. ω-3-rich oil on the level of lipid peroxidation/oxidative stress biomarkers. Rats given a heme iron-rich diet without PUFA were used as controls. The results obtained have shown that MDA and the major urinary metabolite of HNE (the mercapturic acid of dihydroxynonane, DHN-MA) were highly dependent on the dietary factors tested, while 8-iso-PGF_2α was modestly but significantly affected. Intestinal inflammation and tissue fatty acid composition were checked in parallel and could only explain the differences we observed to a limited extent. Thus, the differences in biomarkers were attributed to the formation of lipid oxidation compounds in food or during digestion, their intestinal absorption, and their excretion into urine. Moreover, fecal extracts from the rats fed the heme iron or fish oil diets were highly toxic for immortalized mouse colon cells. Such toxicity can eventually lead to promotion of colorectal carcinogenesis, supporting the epidemiological findings between red meat intake and colorectal cancer risk.Therefore, the analysis of these biomarkers of lipid peroxidation/oxidative stress in urine should be used with caution when dietary factors are not well controlled, while control of their possible dietary intake is needed also because of their pro-inflammatory, toxic, and even cocarcinogenic effects.     

Quantitative determination of hydroxy fatty acids as an indicator of in vivo lipid peroxidation: gas chromatography-mass spectrometry methods.

A new method has been developed for the quantitation of lipid peroxidation products by gas chromatography-mass spectrometry. An important advantage over existing gas chromatography-mass spectrometry methods is the elimination of autoxidation during sample preparation. The sensitivity is sufficient to permit measurement of lipid peroxidation products under normal physiological conditions on as little as 1 mg of tissue. Lipids from whole tissue samples or cell preparations are reduced by catalytic hydrogenation during extraction. The hydrogenation stabilizes the compounds by saturating the double bonds and reducing the hydroperoxides to hydroxy derivatives. The saturated lipids are then saponified and the resulting fatty acids are converted to pentafluorobenzyl esters. Hydroxy fatty acids are further converted to trimethylsilyl ether derivatives. Quantitation is accomplished by negative ion chemical ionization gas chromatography-mass spectrometry, using deuterated internal standards. Specific products from polyunsaturated fatty acids can be quantitated, and the method differentiates between products produced by free-radical and photooxidation mechanisms. Increased levels of lipid peroxidation products, above normal physiological levels, that result from prooxidant conditions, such as exposure of animals to carbon tetrachloride, can be measured.     

Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low-density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoprotein.

4-Hydroxynonenal (HNE) is a major aldehydic propagation product formed during peroxidation of unsaturated fatty acids. The aldehyde was used to modify freshly prepared human low-density lipoprotein (LDL). A polyclonal antiserum was raised in the rabbit and absorbed with freshly prepared LDL. The antiserum did not react with human LDL, but reacted with CuCl2-oxidized LDL and in a dose-dependent manner with LDL, modified with 1, 2 and 3 mM-HNE, in the double-diffusion analysis. LDL treated with 4 mM of hexanal or hepta-2,4-dienal or 4-hydroxyhexenal or malonaldehyde (4 or 20 mM) did not react with the antiserum. However, LDL modified with 4 mM-4-hydroxyoctenal showed a very weak reaction. Lipoprotein (a) and very-low-density lipoprotein were revealed for the first time to undergo oxidative modification initiated by CuCl2. This was evidenced by the generation of lipid hydroperoxides and thiobarbituric acid-reactive substances, as well as by a marked increase in the electrophoretic mobility. After oxidation these two lipoproteins also reacted positively with the antiserum against HNE-modified LDL.     

Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes

Incubation of rat striatal synaptosomes in ascorbic acid induced the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and 4-hydroxynonenal (4-HNE), a lipid peroxidation aldehydic product. Incubations with 4-HNE, used at a range of concentrations comparable to those obtained during peroxidation, induced a simultaneous, dose-dependent decrease of dopamine (DA) uptake and Na⁺/K⁺ ATPase activity and a loss of sulfhydryl (SH) groups. Similar results were observed in a previous study when lipid peroxidation was induced after incubation of synaptosomes in ascorbic acid. Taken together, these data suggest that 4-HNE is an important mediator of oxidative stress and may alter DA uptake after binding to SH groups of the DA transporter and to Na⁺/K⁺ ATPase. These toxic events may contribute to the onset and progression of Parkinsons disease.