Effects Of Lipid Peroxidation Products Research Articles

Lipid Peroxidation Products Influence Calpain-1 Functionality In Vitro by Covalent Binding

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 μM MDA but not with 500 and 1000 μM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 μM. No difference in calpain-1 autolysis was observed between the control and 1000 μM MDA. However, 1000 μM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

Effects of 4-Oxo-2-nonenal on biochemical properties of bovine heart mitochondria.

The effects of lipid peroxidation products 4‐Hydroxy‐2‐nonenal (4‐HNE) and 4‐oxo‐2‐nonenal (4‐ONE) were evaluated using bovine heart mitochondria. Oxygen consumption rate (OCR), ultrastructure, antioxidant activity, and membrane permeability were examined to compare their effects on isolated mitochondria from beef cardiac muscle. For the mitochondrial morphology, the final concentration of mitochondria and 4‐ONE or 4‐HNE in the reaction tube were 10 mg/ml and 1 mM, respectively. For the OCR experiment, mitochondria (2.5 mg/ml) were incubated with 0.20 mM ONE or in a Clark electrode chamber at 25°C. Mitochondrial membrane permeability was determined by incubating 0.5 mg/ml of mitochondrial protein with either 0.05 mM ONE or HNE or ethanol control at pH 5.6 and 7.4 at 25°C. Transmission electron microscopy (TEM) revealed that the size of 4‐ONE treated mitochondria at pH 7.4 increased (p < .05), as did permeability (p < .05), unlike ethanol controls. However, mitochondria incubated with 4‐ONE at pH 5.6 showed a decrease in volume (p < .05). Incubating mitochondria with 4‐ONE at pH 5.6 and pH increased oxygen consumption rate 7.4 caused less oxygen consumption than either 4‐HNE treatment or ethanol control. The hydrogen peroxide assay (H2O2), ferric reducing antioxidant properties (FRAP), and 2,2’‐azinobis (3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS.+) assays revealed that 4‐ONE is a more potent inhibitor of the endogenous antioxidant system of mitochondria than 4‐HNE (p < .05).

Lipid Peroxidation Induces Reactive Astrogliosis by Activating WNT/β-Catenin Pathway in Hydrocephalus.

BackgroundHydrocephalus induces mechanical and biochemical changes in neural cells of the brain. Astrogliosis, as the hallmark of cellular changes in white matter, is involved in demyelination process, re-myelination inhibitory effect, and inhibition of axonal elongation and regeneration. The pathophysiology of this process is not well understood. The purpose of the present study is to elucidate the effect of lipid peroxidation product on astrogliosis through WNT/ β-catenin in kaolin-induced hydrocephalic rats.MethodsThe study used kaolin-induced hydrocephalic rats. Obstructive hydrocephalus was expected to develop within seven days after induction. The hydrocephalus animals were killed at day 7, 14 and 21 after induction. One group of the saline-injected animals was used for sham-treatment.ResultsWe demonstrated that the hydrocephalic rats exhibited a high expression of 4-hydroxynonenal (4-HNE) in the periventricular area. The expression of β-catenin also increased, following the pattern of 4-HNE. Reactive astrocyte, expressed by positive glial fibrillary acidic protein (GFAP), was upregulated in an incremental fashion as well as the microglia.ConclusionThis work suggests that lipid peroxidation product, 4-HNE, activated the WNT/β-catenin pathway, leading to the development of reactive astrocyte and microglia activation in hydrocephalus.

4-Hydroxyhexenal and 4-hydroxynonenal are mediators of the anti-cachectic effect of n-3 and n-6 polyunsaturated fatty acids on human lung cancer cells

Cachexia, the most severe paraneoplastic syndrome, occurs in about 80% of patients with advanced cancer; it cannot be reverted by conventional, enteral, or parenteral nutrition. For this reason, nutritional interventions must be based on the use of substances possessing, alongside nutritional and energetic properties, the ability to modulate production of the pro-inflammatory factors responsible for the metabolic changes characterising cancer cachexia. In light of their nutritional and anti-inflammatory properties, polyunsaturated fatty acids (PUFAs), and in particular n-3, have been investigated for treating cachexia; however, the results have been contradictory.Since both n-3 and n-6 PUFAs can affect cell functions in several ways, this research investigated the possibility that the effects of both n-3 and n-6 PUFAs could be mediated by their major aldehydic products of lipid peroxidation, 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE), and by their anti-inflammatory properties. An “in vitro” cancer cachexia model, consisting of human lung cancer cells (A427) and murine myoblasts (C2C12), was used.The results showed that: 1) both n-3 and n-6 PUFAs reduced the growth of lung cancer cells without causing cell death, increased lipid peroxidation and Peroxisome Proliferator-Activated Receptor (PPAR)α, and decreased TNFα; 2) culture medium conditioned by A427 cells grown in the absence of PUFAs blocked myosin production and the differentiation of C2C12 muscle cells; conversely, muscle cells grown in culture medium conditioned by the same cells in the presence of PUFAs showed myosin expression and formed myotubes; 3) adding HHE or HNE directly to C2C12 cells maintained in culture medium conditioned by A427 cells in the absence of PUFAs stimulated myosin production and myotube formation; 4) putative consensus sequences for (PPARs) have been found in genes encoding fast isoforms of myosin heavy chain, by a bioinformatics approach.The overall results show, first, the ability of both n-3 and n-6 PUFAs and their lipid peroxidation products to prevent the blocking of myosin expression and myotube formation caused in C2C12 cells by medium conditioned by human lung tumour cells. The C2C12 cell differentiation can be due to direct effect of lipid peroxidation products, as evidenced by treating C2C12 cells with HHE and HNE, and to the decrease of pro-inflammatory TNFα in A427 cell culture medium. The presence of consensus sequences for PPARs in genes encoding the fast isoforms of myosin heavy chain suggests that the effects of PUFAs, HHE, and HNE are PPAR-mediated.

Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress.

We performed molecular dynamics simulations to investigate the effect of lipid peroxidation products on the structural and dynamic properties of the cell membrane. Our simulations predict that the lipid order in a phospholipid bilayer, as a model system for the cell membrane, decreases upon addition of lipid peroxidation products. Eventually, when all phospholipids are oxidized, pore formation can occur. This will allow reactive species, such as reactive oxygen and nitrogen species (RONS), to enter the cell and cause oxidative damage to intracellular macromolecules, such as DNA or proteins. On the other hand, upon increasing the cholesterol fraction of lipid bilayers, the cell membrane order increases, eventually reaching a certain threshold, from which cholesterol is able to protect the membrane against pore formation. This finding is crucial for cancer treatment by plasma technology, producing a large number of RONS, as well as for other cancer treatment methods that cause an increase in the concentration of extracellular RONS. Indeed, cancer cells contain less cholesterol than their healthy counterparts. Thus, they will be more vulnerable to the consequences of lipid peroxidation, eventually enabling the penetration of RONS into the interior of the cell, giving rise to oxidative stress, inducing pro-apoptotic factors. This provides, for the first time, molecular level insight why plasma can selectively treat cancer cells, while leaving their healthy counterparts undamaged, as is indeed experimentally demonstrated.

Oxidative Stress Regulation of Regulator of G‐Protein Signaling 4 (RGS4)

Regulators of G‐protein signaling (RGS) proteins are a family of proteins that regulate G‐protein coupled receptors (GPCRs) by acting as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of hydrolysis of GTP to GDP in select Gα subunits, including Gαo, Gαi, and Gαq but not Gαs. One of these proteins, RGS4, has been implicated in several neurological diseases. In this study, we analyze the effect of oxidative stress on RGS4 regulation in neuronal cells and the effect of lipid peroxidation products, such as 4‐hydroxy‐nonenal (4HNE), on RGS4 activity. Select RGS4 isoforms were induced during oxidative stress with H2O2. 4HNE forms covalent adducts on reactive amino acids such as cysteine. Previous research revealed that modification only occurs at select cysteine residues. Several mutants were made containing only one of the modified cysteines, either C71, C148, and, C183, and were treated with increasing amounts of 4HNE and then assayed for coupling to the native binding partner Gαo by ALPHA‐Screen. The wild type construct showed sensitivity to 4HNE but the cysteine null mutant did not. Of the single cysteine containing mutants, only C148 conferred sensitivity to RGS4. Research support provided by the Pharmacological Sciences Training Grant (NIH/NIGMST32GM067795).

Two faces of lipid peroxidation products: the -Yin and Yang- principles of oxidative stress

Oxidants and antioxidants, like many other things, have two faces. Lipids, important biological substances of aerobic organisms, are oxidized by reactive oxygen and nitrogen species formed inevitably in vivo to give diverse products. The emerging evidence suggests that lipid peroxidation products have also two faces. Lipid peroxidation products alter biological membrane organization and modify proteins and nucleic acids, which result in a number of dysfunctional states in vivo. Many studies show that the levels of lipid peroxidation products increase with the development of acute and chronic diseases. Lipid peroxidation products may modulate signal transduction pathways and mediate biological processes through receptors or receptor-independent pathways. At the same time, it has been shown that the lipid peroxidation products induce adaptive response and increase tolerance against forthcoming oxidative stress by upregulating defense capacity. In this brief review, the examples of such dual opposite effects of lipid peroxidation products are shown and the underlying mechanisms are suggested.

The effect of lipid peroxidation products on reactive oxygen species formation and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages

Lipid peroxidation induced by oxidants leads to the formation of highly reactive metabolites. These can affect various immune functions, including reactive oxygen species (ROS) and nitric oxide (NO) production. The aim of the present study was to investigate the effects of lipid peroxidation products (LPPs) – acrolein, 4-hydroxynonenal, and malondialdehyde – on ROS and NO production in RAW 264.7 macrophages and to compare these effects with the cytotoxic properties of LPPs. Macrophages were stimulated with lipopolysaccharide (0.1 μg/ml) and treated with selected LPPs (concentration range: 0.1–100 μM). ATP test, luminol-enhanced chemiluminescence, Griess reaction, Western blotting analysis, amperometric and total peroxyl radical-trapping antioxidant parameter assay were used for determining the LPPs cytotoxicity, ROS and NO production, inducible nitric oxide synthase expression, NO scavenging, and antioxidant properties of LPPs, respectively. Our study shows that the cytotoxic action of acrolein and 4-hydroxynonenal works in a dose- and time-dependent manner. Further, our results imply that acrolein, 4-hydroxynonenal, and malondialdehyde can inhibit, to a different degree, ROS and NO production in stimulated macrophages, partially independently of their toxic effect. Also, changes in enzymatic pathways (especially NADPH-oxidase and nitric oxide synthase inhibition) and NO scavenging properties are included in the downregulation of reactive species formation.

Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action

In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 muM resulting in a reduction of enzyme activities by 88-94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.

Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells

Several lines of evidence suggest that progressive dysfunction of the retinal pigment epithelium (RPE) is central to the pathogenesis of age-related macular degeneration (AMD). We previously demonstrated that protein modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), induce lysosomal dysfunction in RPE cells in vitro. Here, we investigated whether phagocytosis of modified photoreceptor outer segments (POS) affects lipofuscinogenesis and autophagy, two interrelated processes directly connected to lysosomal function. Incubation of human RPE cells with HNE- and MDA-modified POS resulted in pronounced intracellular accumulation of granular material with lipofuscin-like autofluorescence. After daily treatment with modified POS for 7 days, cellular autofluorescence increased 8.2-fold as quantified by flow cytometry. In the presence of the lysosomal inhibitor ammonium chloride, unmodified POS likewise induced an 8.0-fold increase in autofluorescence. Spectral profiles of cellular autofluorescence after incubation with modified POS were unchanged compared to incubation with native POS. Autophagy activity, measured as turnover of metabolically radiolabeled endogenous proteins, was reduced by both HNE- and MDA-modified POS by 40%. Autophagy inhibition by 3-methyladenine and lysosomal inhibition by ammonium chloride induced lipofuscinogenesis even in the absence of POS. In summary, our results demonstrate that induction of lysosomal dysfunction by lipid peroxidation-derived protein modifications results in increased lipofuscinogenesis and reduced autophagy activity in RPE cells in vitro. These mechanisms may contribute to RPE cell dysfunction and degeneration in AMD.

Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action

In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 μM resulting in a reduction of enzyme activities by 88–94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.

TRPA1: irritant detector of the airways

The respiratory tract is innervated by primary afferent sensory neurons that respond to mechanical and chemical stimuli (Taylor-Clark & Undem, 2006). The majority of these neurons are unmyelinated C-fibres that respond to noxious chemicals, such as reactive oxygen species (ROS), the main irritants of tear gas, air pollution and burning vegetation (Kollarik & Undem, 2004). Activation of airway sensory neurons leads to centrally mediated afferent reflexes including shortness of breath, cough and changes in breathing patterns. Sensory neuron activation also triggers the local release of inflammatory mediators that sensitize the airways to future chemical and mechanical stimuli. While normally protective, airway inflammation can exacerbate respiratory disorders such as asthma, emphysema or bronchitis. In this issue of The Journal of Physiology, Taylor-Clark et al. (2008) investigate the role of the transient receptor potential (TRP) channels, TRPV1 and TRPA1, as sensors for oxidative stress in the airways. The heat and capsaicin receptor, TRPV1, has long been thought to play a role in the detection of noxious chemicals in the airway. However, mice lacking the TRPV1 ion channel display only partial defects in nocifensive responses to inhaled irritants (Kollarik & Undem, 2004). Thus, other receptors are required for the detection of irritants by vagal neurons. The ion channel TRPA1 has now emerged as a leading candidate for an airway irritant detector. TRPA1 is highly expressed in a subset of TRPV1-expressing primary sensory afferents and has been shown to play a key role in pain and neurogenic inflammation. TRPA1 is activated by reactive environmental irritants such as mustard oil, cinnemaldehyde and the α,β-unsaturated aldehyde, acrolein, an airway irritant present in tear gas, vehicle exhaust and smoke (Jordt & Ehrlich, 2007). Recent studies have identified a number of reactive endogenous molecules that activate TRPA1 including 4-HNE, 4-HHE and 4-ONE, products of oxidative stress-induced lipid peroxidation (Macpherson et al. 2007; Trevisani et al. 2007; Andersson et al. 2008). Taylor-Clark et al. (2008) use a variety of techniques to examine the role of TRPA1 in the airways. Using calcium imaging they show that cultured vagal neurons innervating the respiratory tract are potently activated by the lipid peroxidation product 4-ONE. At lower concentrations of 4-ONE, calcium responses are absent in neuronal cultures generated from TRPA1-deficient mice. However, at higher concentrations, 4-ONE elicits robust calcium influx in TRPA1-deficient neurons; these responses are attenuated in TRPV1-deficient neurons. These results suggest that both TRPA1 and TRPV1 contribute to cellular sensitivity of lipid peroxidation products. Further analysis of the relative contributions of these channels to 4-ONE sensitivity must await the characterization of neurons from animals lacking both TRPV1 and TRPA1. To address the role of TRPA1 in the airways in situ, Taylor-Clark et al. measure the effects of lipid peroxidation products on contractions of isolated bronchi. They find that 4-ONE induces robust bronchi contraction via sensory neuron-mediated release of neuropeptides. Strikingly, 4-HNE had little effect on bronchi contraction. The authors also use an ex vivo vagally innervated lung preparation to record action potential discharge in response to irritant application. While 4-ONE robustly evoked action potential discharge, 4-HNE had no effect on fibre firing. Responses to 4-ONE were not observed in lung preparations isolated from TRPA1-deficient mice, demonstrating that TRPA1 is required for 4-ONE airway sensitivity. It is surprising that 4-HNE has little effect on bronchi contraction or action potential firing in lung afferents, as this chemical robustly stimulates calcium influx through TRPA1 in cultured sensory neurons and TRPA1-transfected cell lines (MacPherson et al. 2007; Trevisani et al. 2007; Andersson et al. 2008; Taylor-Clark et al. 2008). Also, 4-HNE was shown to elicit robust TRPA1-dependent nocifensive behaviour when injected subcutaneously into the hindpaw of mice (Trevisani et al. 2007). This suggests that TRPA1 sensitivity to irritants may differ depending on the tissue environment. In addition, tissue-specific signalling pathways may differentially modulate sensory neuron excitability in response to ROS. Taken together these findings suggest that TRPA1 activation by 4-ONE is a key mechanism that links oxidative stress to nocifensive responses in the airway. A recent study by Bessac et al. (2008) also probes the role of TRPA1 in airway irritancy. Their study demonstrates that TRPA1 is required for nocifensive airway responses to the reactive oxygen species, hydrogen peroxide. Individuals with altered respiratory function display hypersensitivity to environmental irritants that robustly activate TRPA1. Thus, TRPA1 may represent a new target for the development of drugs that suppress neuronal hypersensitivity in individuals with airway disease.

Effect of lipid peroxidation products on the activity of human retinol dehydrogenase 12 (RDH12) and retinoid metabolism

Mutations in human Retinol Dehydrogenase 12 (RDH12) are known to cause photoreceptor cell death but the physiological function of RDH12 in photoreceptors remains poorly understood. In vitro, RDH12 recognizes both retinoids and medium-chain aldehydes as substrates. Our previous study suggested that RDH12 protects cells against toxic levels of retinaldehyde and retinoic acid [S.A. Lee, O.V. Belyaeva, I.K. Popov, N.Y. Kedishvili, Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12, J. Biol. Chem. 282 (2007) 35621–35628]. Here, we investigated whether RDH12 can also protect cells against highly reactive medium-chain aldehydes. Analysis of cell survival demonstrated that RDH12 was protective against nonanal but not against 4-hydroxynonenal. At high concentrations, nonanal inhibited the activity of RDH12 towards retinaldehyde, suggesting that nonanal was metabolized by RDH12. 4-Hydroxynonenal did not inhibit the RDH12 retinaldehyde reductase activity, but it strongly inhibited the activities of lecithin:retinol acyl transferase and aldehyde dehydrogenase, resulting in decreased levels of retinyl esters and retinoic acid and accumulation of unesterified retinol. Thus, the results of this study showed that RDH12 is more effective in protection against retinaldehyde than against medium-chain aldehydes, and that medium-chain aldehydes, especially 4-hydroxynonenal, severely disrupt cellular retinoid homeostasis. Together, these findings provide a new insight into the effects of lipid peroxidation products and the impact of oxidative stress on retinoid metabolism.

Anticancer properties of oxidation products of docosahexaenoic acid

Docosahexaenoic acid (DHA) is the longest, most unsaturated, and hence, most oxidizable fatty acid commonly found in nature. The mechanisms behind DHA's many biological functions remain a subject of much debate. Here we review one important, but often unstudied, aspect of DHA function, namely, the potential role of its many oxidation products. We divide this review into camps, enzymatic and non-enzymatic oxidations, and report their effects primarily on induction of apoptosis in cancer cells. We conclude that the study of the effects of lipid peroxidation products on biochemical function will be a difficult but highly rewarding area for future studies.

Abstract 1313: Pro-atherogenic Effects Of Lipid Peroxidation Products: Role Of The Unfolded Protein Response

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are associated with atherosclerosis. Expression of UPR target genes such as activating transcription factor 3 (ATF3) and ATF4 is markedly increased in human atherosclerotic lesions. Staining for these proteins co-localizes with the staining with antibodies that recognize the aldehydic epitopes of oxidized LDL, suggesting that lipid-derived aldehydes could be involved in mediating ER stress and UPR. We examined the role of phospholipid aldehyde, 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine (POVPC), unsaturated lipid-derived aldehydes- 4-hydroxy, trans -2-nonenal (HNE) and acrolein in the induction of ER-stress and UPR in human aortic endothelial cells (HAEC) and human umbical vein endothelial cells (HUVEC). POVPC, HNE and acrolein (10 –25 μM) increased the phosphorylation of eIF2α (eukaryotic initiation factor-2α) by 1.5–5 fold (P&lt;0.001) and induced its downstream effector proteins - ATF4 (1.5–3.5 fold; P&lt;0.001) and ATF3 (4–10 fold; P&lt;0.0001). Incubation of HAEC with these aldehydes also increased the adhesion of THP-1 cells (monocyte) to HAEC by 1.4–1.6 fold (P&lt;0.01). Moreover, incubation of endothelial cells with POVPC increased the mRNA level of the pro-inflammatory cytokine IL-8 by &gt;25 fold (P&lt;0.0001). Chemical chaperone, phenyl butyric acid (PBA), diminished aldehydes-induced expression of ATF3 and ATF4 proteins, endothelial cell-monocyte adhesion and IL-8 formation by 80–95% (P&lt;0.001). POVPC (10–25 μM) also activated JNK by (3–6 fold) in HAEC. Reduction of POVPC to its corresponding alcohol, 1-palmitoyl-2-(5-hydroxyvaleroyl)- sn -glycero-3-phosphocholine (PHVPC) inhibited JNK activation by 74 ± 14 % (P&lt;0.001). Pharmacological inhibition of JNK, inhibited the aldehyde-induced induction of ATF3 and ATF4 proteins by 70–90 % (P&lt;0.001) but not the phosphorylation of eIF2α, and PBA inhibited the POVPC-induced JNK activation by 85 ± 11 % (P&lt;0.001). These data suggest that lipoprotein oxidation products activate endothelial cells in part by inducing ER-stress and their inflammatory signaling could be attenuated by chemical chaperones of protein folding.

Clastogenic and cytotoxic effects of lipid peroxidation products generated in culinary oils submitted to thermal stress

In rats, the administration of oil submitted to high temperatures is teratogenic. To understand the mechanisms involved, the clastogenic and cytotoxic effects were studied respectively in lymphocytes, hepatocytes (HepG2) and in human umbilical vein endothelial cells (HUVEC) cultured with a water extract obtained from heated oil (HO). In lymphocytes incubated with HO containing 0.075 or 0.15 μM of thiobarbituric reacting substances, the rate of chromosomal breakage was 18.4% and 23.1%, compared to 8.7% and 6.6%, or 8.1% and 9.2%, respectively in lymphocytes incubated with the same volume of a water extract from non-heated oil (NHO) or distilled water (DW). In HepG2 or HUVEC cells, the cytotoxic properties of the HO were dose dependent, the cytotoxicity beginning at concentrations as low as 0.25 μM. In contrast, the same volume of NHO or DW was non-toxic for these cells. The results obtained show that a water extract obtained from heated oil is clastogenic and, in higher doses, cytotoxic.

Comet Fluorescence in situ Hybridization Analysis for Oxidative Stress-Induced DNA Damage in Colon Cancer Relevant Genes

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products

cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni–NTA silica. GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 μmol min−1 mg−1). GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 μmol min−1 mg−1 . Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates. Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure.

The effects of a dietary oxidized oil on lipid metabolism in rats.

This study was carried out to investigate the effects of a dietary oxidized oil on lipid metabolism in rats, particularly the desaturation of fatty acids. Two groups of rats were fed initially for a period of 35 d diets containing 10% of either fresh oil or thermally treated oil (150 degrees C, 6 d). The dietary fats used were markedly different for lipid peroxidation products (peroxide value: 94.5 vs. 3.1 meq O2/kg; thiobarbituric acid-reactive substances: 230 vs. 7 micromol/kg) but were equalized for their fatty acid composition by using different mixtures of lard and safflower oil and for tocopherol concentrations by individual supplementation with DL-alpha-tocopherol acetate. In the second period which lasted 16 d, the same diets were supplemented with 10% linseed oil to study the effect of the oxidized oil on the desaturation of alpha-linolenic acid. During the whole period, all the rats were fed identical quantities of diet by a restrictive feeding system in order to avoid a reduced food intake in the rats fed the oxidized oil. Body weight gains and food conversion rates were only slightly lower in the rats fed the oxidized oil compared to the rats fed the fresh oil. Hence, the effects of lipid peroxidation products could be studied without a distortion by a marked reduced food intake and growth. To assess the rate of fatty acid desaturation, the fatty acid composition of liver and heart total lipids and phospholipids was determined and ratios between product and precursor of individual desaturation reactions were calculated. Rats fed the oxidized oil had reduced ratios of 20:4n-6/18:2n-6, 20:5n-3/18:3n-3, 20:4n-6/20:3n-6, and 22:6n-3/22:5n-3 in liver phospholipids and reduced ratios of 20:4n-6/18:2n-6, 22:5n-3/18:3n-3, and 22:6n-3/18:3n-3 in heart phospholipids. Those results suggest a reduced rate of desaturation of linoleic acid and alpha-linolenic acid by microsomal delta4-, delta5-, and delta6-desaturases. Furthermore, liver total lipids of rats fed the oxidized oil exhibited a reduced ratio between total monounsaturated fatty acids and total saturated fatty acids, suggesting a reduced delta9-desaturation. Besides those effects, the study observed a slightly increased liver weight, markedly reduced tocopherol concentrations in liver and plasma, reduced lipid concentrations in plasma, and an increased ratio between phospholipids and cholesterol in the liver. Thus, the study demonstrates that feeding an oxidized oil causes several alterations of lipid and fatty acid metabolism which might be of great physiologic relevance.

Cellular Response to the Redox Active Lipid Peroxidation Products: Induction of Glutathione S-Transferase P by 4-Hydroxy-2-nonenal

Membrane lipid peroxidation is known to produce various aldehydic compounds which cause a wide range of biological effects including heart disease, aging, and cancer. To investigate the effect of lipid peroxidation products on the expression of glutathione S-transferases (GSTs), which catalyze the conjugation of reactive chemicals with glutathione and play an important role in protecting cells, normal rat liver epitherial cells (RL34) were exposed to a variety of aldehydic compounds. We found that the GST activity in RL34 cells was induced by α,β-unsaturated aldehydes, such as acrolein (1.3-fold), crotonaldehyde (1.3-fold), 4-hydroxy-2-hexenal (HHE) (1.4-fold), and 4-hydroxy-2-nonenal (HNE) (1.7-fold). The induction of GST activity by HNE was time-dependent, reaching a plateau after 16 h. The immunoblot analysis using the polyclonal antibodies against GST isozymes demonstrated that GST-P (π-class), a well-known tumor marker, was significantly induced 16 h after the HNE treatment. Also, immunostaining for the presence of GST-P confirmed the enhanced expression of GST-P in the cytoplasm of the cells. Northern blot analysis revealed that the HNE treatment of RL34 cells for 1 h enhanced the expression of GST-P mRNA, which returned to the control level after 16 h. These data suggest that the induction of GST-P by HNE may represent an important cellular defense mechanism against oxidative injury.