Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins

Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and malonaldehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5-dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Schiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanals, alk-2-enals and alka-2,4-dienals can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.     

Secondary products of lipid oxidation

In the last decade, a multitude of secondary products have been identified from the radical and photosensitized oxidations of polyunsaturated lipids. These secondary products consist of oxygenated monomeric materials including epoxy-hydroperoxides, oxo-hydroperoxides, hydroperoxy epidioxides, dihydroperoxides, hydroperoxy bis-epidioxides, and hydroperoxy bicycloendoperoxides. More recently, higher molecular weight dimeric compounds have been identified from autoxidized methyl linoleate and linolenate. Decomposition of these oxidation products form a wide range of carbonyl compounds, hydrocarbons, furans, and other materials that contribute to the flavor deterioration of foods and that are implicated in biological oxidation. The interaction of some of these degradation products with DNA may be involved in cell-damaging reactions.     

Fluorescence formation from the interaction of DNA with lipid oxidation degradation products

To clarify the mechanism of fluorescence formation between DNA and lipid degradation products in the presence of ferric chloride and ascorbic acid, a number of carbonyl compounds and decomposition products of pure methyl linolenate hydroperoxides were examined. Keto derivatives of methyl ricinoleate, linoleate, and oleate, alkanals and 2-alkenals produced little or no fluorescence with DNA in the presence of ferric chloride-ascorbic acid. 2,4-Alkadienals were more active and 2,4,7-decatrienal was the most active. Mixtures of volatile aldehydes prepared from linolenate hydroperoxide decomposed either thermally or with iron and ascorbate had the same activity as 2,4,7-decatrienal. Higher molecular-weight products from the decomposition of methyl linolenate hydroperoxides showed relatively low activity. beta-Carotene, alpha-tocopherol and other antioxidants effectively reduced the amount of fluorescence formed by linolenate hydroperoxides. The results suggest that, in addition to hydroperoxide decomposition products, singlet oxygen and/or free radical species contribute significantly to the fluorescence formed from the interaction of methyl linolenate hydroperoxides with DNA in the presence of ferric chloride and ascorbic acid.     

Peroxynitrite-mediated lipid oxidation and nitration: Mechanisms and consequences

Lipid oxidation and nitration represents a novel area of research of relevance in the understanding of inflammatory processes. Peroxynitrite, the product of the diffusion-limited reaction between nitric oxide and superoxide anion, mediates oxidative modifications in lipid systems including cell membranes and lipoproteins. In this review, we discuss the mechanisms of lipid oxidation and nitration by peroxynitrite as well as the influence of physiological molecules and cell targets to redirect peroxynitrite reactivity. We also provide evidence to support that oxidation/nitration of lipids results in the formation of novel signaling modulators of key lipid-metabolizing enzymes.     

Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species

Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-Delta12,14 -prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.     

Systematic isolation and identification of membrane lipid oxidation products

This report discusses the analytical procedure by which it is possible to isolate and identify the oxidation products of cellular and subcellular membrane lipids. The key point of this procedure is the method used for the transmethylation of the lipid material isolated from the tissues. In effect, both the conversion of the glycerides into methyl esters and the reduction of the hydroperoxyl groups into the corresponding hydroxyl groups is performed in one step, without breaking any oxirane rings that may be present. The methyl esters containing functional groups introduced by oxidative processes are separated from the non-modified ones by preparative TLC and are identified by GLC and GC-MS.     

Small reactive carbonyl compounds as tissue lipid oxidation products; and the mechanisms of their formation thereby

Small reactive carbonyl compounds (RCCs) such as formaldehyde, acetaldehyde, acrolein, crotonaldehyde, glyoxal, methylglyoxal, glycolaldehyde, glycidaldehyde, and 2-butene-1,4-dial are involved in carbonyl and oxidative stress-related physiological disorders. While some evidence indicates that lipid oxidation could be an important source of these compounds in vivo, this has sometimes been doubted because the mechanisms of their formation thereby are poorly understood. Here, representative literature supporting the significant formation of these compounds during lipid oxidation under physiologically relevant conditions are highlighted, and the strengths and weaknesses of previously proposed mechanisms of their formation thereby are considered. In addition, based on the current understanding of lipid oxidation chemistry, some new pathways of their formation are suggested. The suggested pathways also generate 4-hydroxy-2-butenal, a precursor of the carcinogen furan, whose endogenous formation in tissues has hitherto not been seriously considered.     

DNA polymerase V allows bypass of toxic guanine oxidation products in vivo

Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.     

MutS recognition of exocyclic DNA adducts that are endogenous products of lipid oxidation.

The ability of the methyl-directed mismatch repair system to recognize and repair the exocyclic adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G), the major adduct derived from the endogenous mutagen malondialdehyde, has been assessed both in vivo and in vitro. Both adducts were site-specifically incorporated into M13MB102 DNA, and the adducted genomes were electroporated into wild-type or mutS-deficient Escherichia coli strains. A decrease in mutations caused by both adducts was observed in mutS-deficient strains, suggesting that MutS was binding to the adducts and blocking repair by nucleotide excision repair. This hypothesis was supported by the differences in mutation frequency observed when hemimethylated genomes containing PdG on the (-)-strand were electroporated into a uvrA(-) strain. The ability of purified MutS to bind to PdG- or M(1)G-containing 31-mer duplexes in vitro was assessed using both surface plasmon resonance and gel shift assays. MutS bound to M(1)G:T-containing duplexes with similar affinity to a G:T mismatch but less strongly to M(1)G:C- and PdG-containing duplexes. Dissociation from each of the adduct-containing duplexes occurred at a faster rate than from a G:T mismatch. The present results indicate that MutS can bind to exocyclic adducts resulting from endogenous DNA damage and trigger their removal by mismatch repair or protect them from removal by nucleotide excision repair.     

The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bishydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.     

Stability of lipid oxidation products in the rat liver and pathways of their utilization

The ability of liver homogenates to utilize various lipid peroxidation products was studied. Conjugated dienes and TBA-reactive products of unsaturated fatty acid phospholipids and triglycerides were found to be more stable that the corresponding lipid hydroperoxides. It was shown that decomposition of lipid hydroperoxides in liver homogenates is due to their reduction to corresponding oxycompounds without activation of free radical reactions. The ability of lipid hydroperoxides to be reduced in liver homogenates is determined by their chemical structure and decreases in the following order: polyunsaturated fatty acids--phospholipids--triglycerides--cholesterol esters.     

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.     

AβP1-42 incorporation and channel formation in planar lipid membranes: the role of cholesterol and its oxidation products

Amyloid beta peptide (AβP) is a natural peptide, normally released into the cerebrospinal fluid (CSF), that plays a key role in Alzheimer’s disease. The conversion of the peptide from a native soluble form to a non-native and often insoluble form, such as small and large aggregates, protofibrils and fibrils of AβP appears to be implicated in the pathogenesis of AD. Although the molecular mechanisms of AβP neurotoxicity are not fully understood, a large body of data suggests that the primary target of amyloid peptides is the cell membrane of neurons, that may modulate the structural and functional conversion of AβP into assemblies involved in pathological processes. In our study, we provide a systematic investigation of AβP1-42’s ability to incorporate and form channel-like events in membranes of different lipid composition and focus on cholesterol and its oxidation products. We propose that cholesterol and its oxidation products can be considered neuroprotective factors because a) by favouring AβP1-42 insertion into membranes, the fibrillation/clearance balance shifts toward clearance; b) by shifting channel selectivity toward anions, the membrane potential is moved far from the threshold of membrane excitability, thus decreasing the influx of calcium into the cell.     

Inhibitory effects of some natural products on metal-induced lipid oxidation in cooked fish

Divalent metal ions (Fe²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Mn²⁺) induced lipid oxidation in cooked, but not in raw fish. The extent of lipid oxidation, measured by the production of thiobarbituric acid reactive substances (TBRS), was increased with higher concentrations of iron, zinc, and nickel, but was decreased with increasing concentrations of copper and manganese. The natural products: ellagic acid, tannic acid, myricetin, and quercetin, inhibited lipid oxidation in cooked fish. The enhanced lipid oxidation caused by cupric ions (10³ pmol/100 g fish) was also inhibited by the natural products. The degree of inhibition in copper-treated fish, however, was less than that in fish that had no added copper. The inhibition was concentration dependent. The antioxidative potency of the various natural products was independent of the type of metal ion-induced lipid oxidation. Ellagic acid was the most potent antioxidant (75.7–83.9%), followed by tannic acid (60.4–77.3%), myricetin (52.9–70.4%), and quercetin (32.6–44.2%).