linoleic-acid and 13-hydroxy-9-11-octadecadienoic-acid

Development of a diet that provides adequate nutrition and effective cancer prevention is an important goal in nutrition and cancer research. A confounding aspect of dietary control of tumor growth is the fact that some nutrients may up-regulate tumor growth, whereas other nutrients and nonnutrients down-regulate growth. Both up- and down-regulators may be present in the same foodstuff. Identification of these substances, determination of their mechanisms of action and potencies, as well as the interactions among the different mechanisms are topics of ongoing research. In this review, we describe results obtained in vivo or during perfusion in situ using solid tissue-isolated rodent tumors and human cancer xenografts in nude rats. Linoleic acid (LA), an essential n-6 polyunsaturated fatty acid (PUFA), was identified as an agent in dietary fat that is responsible for an up-regulation of tumor growth in vivo. Tumor LA uptake, mediated by high intratumor cAMP, stimulated formation of the mitogen, 13-hydroxyoctadecadienoic acid (13-HODE) and also increased ERK1/2 phosphorylation, [(3)H]thymidine incorporation and growth. A mechanism for control of this growth-promoting pathway was revealed during studies of the effects of dietary nutrients and nonnutrients known to inhibit tumor growth. These included four groups of lipophilic agents: n-3 fatty acids, melatonin, conjugated LA isomers and trans fatty acids. Each of these agents activated an inhibitory G protein-coupled receptor-mediated pathway that specifically suppressed tumor uptake of saturated, monounsaturated and n-6 PUFAs, thereby inhibiting an early step in the LA-dependent growth-promoting pathway.

Topics: Amino Acids; Animals; Blood Glucose; Dietary Fats, Unsaturated; Fish Oils; Humans; Ketone Bodies; Lactic Acid; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Male; Melatonin; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Signal Transduction; Trans Fatty Acids; Transplantation, Heterologous

In the skin epidermis, the metabolism of polyunsaturated fatty acids (PUFAs) is highly active. Dietary deficiency of linoleic acid (LA), the major 18-carbon n-6 PUFA in normal epidermis, results in a characteristic scaly skin disorder and excessive epidermal water loss. Because of the inability of normal skin epidermis to desaturate LA to gamma-linolenic acid, it is transformed by epidermal 15-lipoxygenase to mainly 13-hydroxyoctadecadienoic acid, which functionally exerts antiproliferative properties in the tissue. In contrast, compared with LA, arachidonic acid (AA) is a relatively minor 20-carbon n-6 PUFA in the skin and is metabolized via the cyclooxygenase pathway, predominantly to the prostaglandins E(2), F(2)(alpha), and D(2). AA is also metabolized via the 15-lipoxygenase pathway, predominantly to 15-hydroxyeicosatetraenoic acid. At low concentrations, the prostaglandins function to modulate normal skin physiologic processes, whereas at high concentrations they induce inflammatory processes. PUFAs derived from other dietary oils are also transformed mainly into monohydroxy fatty acids. For instance, epidermal 15-lipoxygenase transforms dihomo-gamma-linolenic acid (20:3n-6) to 15-hydroxyeicosatrienoic acid, eicosapentaenoic acid (20:5n-3) to 15-hydroxyeicosapentaenoic acid, and docosahexaenoic acid (22:6n-3) to 17-hydroxydocosahexaenoic acid, respectively. These monohydroxy acids exhibit antiinflammatory properties in vitro. Thus, supplementation of diets with appropriate purified vegetable oils, fish oil, or both may generate local cutaneous antiinflammatory and antiproliferative metabolites which could serve as less toxic in vivo monotherapies or as adjuncts to standard therapeutic regimens for the management of inflammatory skin disorders.

Topics: Anti-Inflammatory Agents; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Docosahexaenoic Acids; Epidermis; Fatty Acids, Unsaturated; Fish Oils; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Plant Oils; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Skin Diseases

Large scale human epidemiological studies indicate that high intakes of linoleic acid protect against the development of cancer. One mechanism may be the generation of 13-HODE from linoleic acid. 13-HODE prevents cell adhesion to endothelial cells and can inhibit cancer metastasis. 13-HODE synthesis is enhanced by cyclic AMP. Gamma-linolenic acid, a desaturated metabolite of linoleic acid, causes substantial stimulation of 13-HODE synthesis. A fall in gamma-linolenic acid synthesis with age may be related to the age-related fall in 13-HODE formation.

Topics: Animals; Anticarcinogenic Agents; Cell Adhesion; Cyclic AMP; Endothelium, Vascular; gamma-Linolenic Acid; Humans; Linoleic Acid; Linoleic Acids; Neoplasm Metastasis

The skin epidermis displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA) and 18-carbon (n-6) PUFA results in characteristic scaly skin disorder and excessive epidermal water loss. Arachidonic acid, a 20-carbon (n-6) PUFA is metabolized via the cyclooxygenase pathway into predominantly prostaglandin E2 (PGE2) PGF2 alpha, and PGD2 and via the lipoxygenase pathway into predominantly 15-hydroxyeicosatetraenoic acid (15-HETE). The prostaglandins modulate normal skin physiological processes at low concentrations and inflammatory reactions at high concentrations. Similarly, the very active epidermal 15-lipoxygenase transforms dihomogammalinolenic acid (DGLA) into 15-hydroxy eicosatrienoic acid (15-HETrE), eicosapentaenoic acid (EPA) into 15-hydroxyeicosapentaenoic acid (15-HEPE) and docosahexaenoic acid (DHA) into 17-hydroxydocosahexaenoic acid (17-HDoHE), respectively. These monohydroxy acids exhibit anti-inflammatory properties. In contrast, the 18-carbon (n-6) PUFA is transformed into 13-hydroxy-9,11-octadecadienoic acid (13-HODE), which exerts antiproliferative properties in the tissue. Thus, the supplementation of diets with appropriate purified vegetable oils and/or fish oil may generate local cutaneous anti-inflammatory metabolites which could serve as a less toxic in vivo monotherapy or as adjuncts to standard therapeutic regimens for the management of skin inflammatory disorders.

Topics: Animals; Cell Division; Dietary Fats, Unsaturated; Epidermis; Fatty Acids, Unsaturated; Humans; Linoleic Acid; Linoleic Acids; Lipoxygenase; Oxygen

It has been shown recently that troglitazone exerts an anti-inflammatory effect, in vitro, and in experimental animals. To test these properties in humans, we investigated the effect of troglitazone on the proinflammatory transcription factor nuclear factor-kappaB and its inhibitory protein IkappaB in mononuclear cells (MNC) and plasma soluble intracellular adhesion molecule-1, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and C-reactive protein. We also examined the effect of troglitazone on reactive oxygen species generation, p47(phox) subunit expression, 9-hydroxyoctadecadienoic acid (9-HODE), 13-HODE, o-tyrosine, and m-tyrosine in obese patients with type 2 diabetes. Seven obese patients with type 2 diabetes were treated with troglitazone (400 mg/day) for 4 weeks. Blood samples were obtained at weekly intervals. Nuclear factor-kappaB binding activity in MNC nuclear extracts was significantly inhibited after troglitazone treatment at week 1 and continued to be inhibited up to week 4. On the other hand, IkappaB protein levels increased significantly after troglitazone treatment at week 1, and this increase persisted throughout the study. Plasma monocyte chemoattractant protein-1 and soluble intracellular adhesion molecule-1 concentrations did not decrease significantly after troglitazone treatment, although there was a trend toward inhibition. Reactive oxygen species generation by polymorphonuclear cells and MNC, p47(phox) subunit protein quantities, plasminogen activator inhibitor-1, and C-reactive protein levels decreased significantly after troglitazone intake. 13-HODE/linoleic acid and 9-HODE/linoleic acid ratios also decreased after troglitazone intake. However, o-tyrosine/phenylalanine and m-tyrosine/phenylalanine ratios did not change significantly. These data show that troglitazone has profound antiinflammatory effects in addition to antioxidant effects in obese type 2 diabetics; these effects may be relevant to the recently described beneficial antiatherosclerotic effects of troglitazone at the vascular level.

Topics: Adult; Anti-Inflammatory Agents; Blood Glucose; C-Reactive Protein; Chemokine CCL2; Cholesterol; Chromans; Diabetes Mellitus; Diabetes Mellitus, Type 2; Female; Humans; I-kappa B Proteins; Insulin; Intercellular Adhesion Molecule-1; Leukocytes, Mononuclear; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Male; Middle Aged; NADPH Oxidases; Neutrophils; NF-kappa B; Obesity; Phenylalanine; Phosphoproteins; Plasminogen Activator Inhibitor 1; Reactive Oxygen Species; Thiazoles; Thiazolidinediones; Triglycerides; Troglitazone; Tyrosine

Topics: Adult; Dermatitis, Atopic; Dietary Supplements; Double-Blind Method; Eczema; Epidermis; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Female; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Male; Pilot Projects; Placebos; Skin

Linoleic acid (LNA)-derived 13-hydroxyoctadecadienoic acid (13-HODE) is a bioactive lipid mediator that regulates multiple signaling processes in vivo. 13-HODE is also produced when LNA is oxidized during food processing. However, the absorption and incorporation kinetics of dietary 13-HODE into tissues is not known. The present study measured unesterified d4-13-HODE plasma bioavailability and incorporation into rat liver, adipose, heart and brain following gavage or intravenous (IV) injection (n = 3 per group). Mass spectrometry analysis revealed that d4-13-HODE was absorbed within 20 min of gavage, and continued to incorporate into plasma esterified lipid fractions throughout the 90 min monitoring period (incorporation half-life of 71 min). Following IV injection, unesterified d4-13-HODE was rapidly eliminated from plasma with a half-life of 1 min. Analysis of tracer incorporation kinetics into rat tissues following IV injection or gavage revealed that the esterified tracer preferentially incorporated into liver, adipose and heart compared to unesterified d4-13-HODE. No tracer was detected in the brain. This study demonstrates that dietary 13-HODE is absorbed, and incorporated into peripheral tissues from esterified plasma lipid pools. Understanding the chronic effects of dietary 13-HODE exposure on peripheral tissue physiology and metabolism merits future investigation.

Topics: Adipose Tissue; Animals; Brain; Esterification; Linoleic Acid; Linoleic Acids; Liver; Male; Myocardium; Rats; Rats, Inbred F344

Different fatty acids have distinct effects on the survival of breast cancer cells, which could be mediated by fatty acid binding proteins (FABPs), a family of lipid chaperones. Due to the diverse structures of the members of FABP family, each FABP demonstrates distinct binding affinities to different fatty acids. Of note, FABP7 is predominantly expressed in triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. Yet, the role of FABP7 in modulating the effects of fatty acids on TNBC survival was unclear. In contrast to the high expression of FABP7 in human TNBC tumours, FABP7 protein was undetectable in TNBC cell lines. Hence, a FABP7 overexpression model was used for this study, in which the transduced TNBC cell lines (MDA-MB-231 and Hs578T) were treated with various mono- and polyunsaturated fatty acids. Oleic acid (OA), docosahexaenoic acid (DHA) and arachidonic acid (AA) inhibited TNBC cell growth at high concentrations, with no differences resulted from FABP7 overexpression. Interestingly, overexpression of FABP7 augmented linoleic acid-induced cell death in MDA-MB-231 cells. The increased cell death may be explained by a decrease in 13-HODE, a pro-tumorigenic oxidation product of linoleic acid. The phenotype was, however, attenuated with a rescue treatment using 25 nM 13-HODE. The decrease in 13-HODE was potentially due to fatty acid partitioning modulated by FABP7, as demonstrated by a 3-fold increase in fatty acid oxidation. Our findings suggest that linoleic acid could be a potential therapeutic strategy for FABP7-overexpressing TNBC patients.

Topics: Arachidonate 15-Lipoxygenase; Cell Death; Cell Line, Tumor; Cell Survival; Down-Regulation; Fatty Acid-Binding Protein 7; Fatty Acids; Humans; Linoleic Acid; Linoleic Acids; Lipid Droplets; Triple Negative Breast Neoplasms; Tumor Suppressor Proteins

The relationship between glioblastoma (GBM) and fatty acid metabolism could be the key to elucidate more effective therapeutic targets. 15-lipoxygenase-1 (15-LOX), a linolenic acid and arachidonic acid metabolizing enzyme, induces both pro- and antitumorigenic effects in different cancer types. Its role in glioma activity has not yet been clearly described. The objective of this study was to identify the influence of 15-LOX and its metabolites on glioblastoma cell activity.. GBM cell lines were examined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to identify 15-LOX metabolites. GBM cells treated with 15-LOX metabolites, 13-hydroxyoctadecadeinoic acid (HODE) and 9-HODE, and two 15-LOX inhibitors (luteolin and nordihydroguaiaretic acid) were also examined. Dose response/viability curves, RT-PCRs, flow cytometry, migration assays, and zymograms were performed to analyze GBM growth, migration, and invasion.. Higher quantities of 13-HODE were observed in five GBM cell lines compared to other lipids analyzed. Both 13-HODE and 9-HODE increased cell count in U87MG. 15-LOX inhibition decreased migration and increased cell cycle arrest in the G2/M phase.. 15-LOX and its linoleic acid (LA)-derived metabolites exercise a protumorigenic influence on GBM cells in vitro. Elevated endogenous levels of 13-HODE called attention to the relationship between linoleic acid metabolism and GBM cell activity.

Topics: Arachidonate 15-Lipoxygenase; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; G2 Phase Cell Cycle Checkpoints; Glioblastoma; Glioma; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase Inhibitors

Linoleic acid (LA; 18:2 n-6), the most abundant polyunsaturated fatty acid in the US diet, is a precursor to oxidized metabolites that have unknown roles in the brain. Here, we show that oxidized LA-derived metabolites accumulate in several rat brain regions during CO

Topics: Animals; Brain Ischemia; Brain Stem; Cerebellum; Cerebral Cortex; Chromatography, Liquid; Dinoprostone; Hippocampus; Linoleic Acid; Linoleic Acids; Male; Oxidation-Reduction; Oxylipins; Rats; Synaptic Transmission; Tandem Mass Spectrometry

Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis.

Topics: Animals; Arachidonate 15-Lipoxygenase; Binge Drinking; Body Composition; Cytokines; Dietary Fats; Disease Models, Animal; Ethanol; Inflammation; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Liver; Macrophages; Metabolome; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Oxidative Stress; RAW 264.7 Cells

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Arachidonic Acid; Carcinogenesis; Carcinogens; Dinoprostone; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Neoplasms; Rats

Lipid peroxidation is involved in many disorders and diseases such as cardiovascular disease, cancers, neurodegenerative diseases, and even aging. Lipid peroxidation products existing in blood or bodily fluids are very important biomarkers for the diagnosis of such diseases. In particular, 13(R,S)-hydroxy-9(E),11(E)-octadecadienoic acid (13-(E,E)-HODE) is an oxidiation product of linoleic acid, which is an important biomarker for many diseases such as diabetes and Alzheimer's disease. In this study, we successfully displayed the antigen-binding fragment of an antibody produced by hybridoma 1213-1 on the M13 phage and performed analysis of the antibody variable region genes. The blast results suggested that it is a novel antibody. We also developed a phage-antibody-based competitive ELISA and a novel Open Sandwich ELISA (OS ELISA) for the detection of 13-(E,E)-HODE. The OS ELISA showed a limit of detection (LOD) of 15.6 nM of 13-(E,E)-HODE and low cross-reactivity with other HODE such as 9-(E,E)-HODE. Another format of the open sandwich ELISA with purified maltose binding protein-fused V

Topics: Enzyme-Linked Immunosorbent Assay; Fatty Acids, Unsaturated; Humans; Linoleic Acid; Linoleic Acids; Lipid Peroxidation

A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg(-1) was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency (k cat/K m) were the highest for linoleic acid (k cat of 93.7 s(-1) and K m of 41.5 μM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l(-1) linoleic acid, 2.5 g l(-1) enzyme, 0.1 mM Cu(2+), and 6% (v/v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l(-1) 13-HODE (70.2 mM) from 20 g l(-1) linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5% and productivity of 10.4 g l(-1) h(-1). The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158% higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme.

Topics: Amino Acid Sequence; Arachidonic Acid; Burkholderia; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cloning, Molecular; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Linoleic Acid; Linoleic Acids; Lipoxygenase; Molecular Sequence Data; Molecular Weight; Recombinant Proteins; Sequence Homology; Substrate Specificity; Temperature

Linoleic acid-derived oxidation products are found in experimental pain models. However, little is known about the levels of such oxylipins in human pain. In consequence, in the present study, we have undertaken a lipidomic profiling of oxylipins in blood serum from patients with Achilles tendinopathy and controls.. A total of 34 oxylipins were analysed in the serum samples. At a significance level of P<0.00147 (<0.05/34), two linoleic acid-derived oxylipins, 13-hydroxy-10E,12Z-octadecadienoic (13-HODE) and 12(13)-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) were present at significantly higher levels in the Achilles tendinopathy samples. This difference remained significant when the dataset was controlled for age, gender and body-mass index. In contrast, 0/21 of the arachidonic acid- and 0/4 of the dihomo-γ-linolenic acid, eicosapentaenoic acid or docosahenaenoic acid-derived oxylipins were higher in the patient samples at this level of significance. The area under the Receiver-Operator Characteristic (ROC) curve for 12,13-DiHOME was 0.91 (P<0.0001). Levels of four N-acylethanolamines were also analysed and found not to be significantly different between the controls and the patients at the level of P<0.0125 (<0.05/4).. It is concluded from this exploratory study that abnormal levels of linoleic acid-derived oxylipins are seen in blood serum from patients with Achilles tendinopathy. Given the ability of two of these, 9- and 13-HODE to activate transient receptor potential vanilloid 1, it is possible that these changes may contribute to the symptoms seen in Achilles tendinopathy.

Topics: 8,11,14-Eicosatrienoic Acid; Achilles Tendon; Adult; Aged; Biomarkers; Case-Control Studies; Ethanolamines; Female; Humans; Linoleic Acid; Linoleic Acids; Male; Middle Aged; Oxidation-Reduction; Oxylipins; Risk Factors; ROC Curve; Tendinopathy; Young Adult

Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p < 0.01, 30 μM) and 13 HODE (p < 0.01, 30 μM), and the equivalent cell viability was also decreased (p < 0.001). Both 9-HODE and 13-HODE (but not LA or ALA) markedly increased caspase-3/7 activity (p < 0.001) in both monocytes and adherent THP-1 cells, with 9-HODE the more potent. In addition, 9-HODE and 13-HODE both increased Annexin-V labelling of cells (p < 0.001). There was no effect of LA, ALA, or the PPARγ agonist rosiglitazone (1 μM), but the effect of HODEs was replicated with apoptosis-inducer camptothecin (10 μM). Only 9-HODE increased DNA fragmentation. The pro-apoptotic effect of HODEs was blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent--and specific--regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

Topics: alpha-Linolenic Acid; Apoptosis; Caspase 3; Caspase 7; Cell Cycle Proteins; Cell Line; Cell Survival; DNA Fragmentation; Fatty Acid-Binding Proteins; Fatty Acids, Unsaturated; Gene Expression Regulation; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Monocytes; PPAR gamma; Receptors, G-Protein-Coupled; RNA, Small Interfering; Rosiglitazone; Signal Transduction; Thiazolidinediones

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Eicosanoids; Ganglia, Spinal; Hydroxyeicosatetraenoic Acids; Hyperalgesia; Linoleic Acid; Linoleic Acids; Lysophospholipids; Mice; Rats; TRPV Cation Channels; Ultraviolet Rays

Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous agonists to the transient receptor potential V1 (TRPV1) receptor. Although TRPV1 mediates inflammatory heat hyperalgesia, it is not known if the OLAMs contribute to the peripheral activation of this receptor during tissue inflammation. In the present study, we evaluated whether the OLAM system is activated during inflammation and whether cytochrome P450 enzymes mediate OLAM contributions to heat hyperalgesia using the complete Freund's adjuvant (CFA) model of inflammation.. Our results demonstrate that the intraplantar (ipl) injection of anti-OLAM antibodies significantly reversed CFA-induced heat hyperalgesia. Moreover, application of lipid extracts from inflamed rat skin to cultured sensory neurons triggered a significant release of iCGRP that is blocked by co-treatment with I-RTX, a TRPV1 antagonist. To determine the role of CYP enzymes in mediating OLAM effects, we used a broad spectrum CYP inhibitor, ketoconazole. Pretreatment with ketoconazole inhibited the release of TRPV1 agonists in lipid extracts from inflamed skin and significantly reversed CFA-induced heat hyperalgesia by a peripheral mechanism of action. Moreover, the ipl injection of linoleic acid to rats 24 hr after CFA evoked spontaneous nocifensive behaviors that were significantly reduced by capsazepine, by knockout of the TRPV1 gene, or by pretreatment with either anti-OLAM antibodies or ketoconazole.. Taken together, our data suggests that OLAMs contribute to inflammatory nociception in the periphery and that cytochrome P450 enzymes play a crucial role in mediating OLAM contributions to inflammatory heat hyperalgesia.

Topics: Animals; Antibodies; Behavior, Animal; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Freund's Adjuvant; Hot Temperature; Hyperalgesia; Inflammation; Ketoconazole; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Mice; Mice, Inbred C57BL; Nociception; Oxidation-Reduction; Pain; Rats; Rats, Sprague-Dawley; TRPV Cation Channels

Fatty acid-binding proteins (FABPs) are postulated to serve as lipid shuttles that solubilize hydrophobic fatty acids and deliver them to appropriate intracellular sites. Epidermal FABP (E-FABP/FABP5) is predominantly expressed in keratinocytes and is overexpressed in the actively proliferating tissue characteristic of psoriasis and wound healing. In this study, we found decreased expression of the differentiation-specific proteins keratin 1, involucrin, and loricrin in E-FABP(-/-) keratinocytes relative to E-FABP(+/+) keratinocytes. We also determined that incorporation of linoleic acid was significantly reduced in E-FABP(-/-) keratinocytes. Although linoleic acid did not directly affect keratinocyte differentiation, keratin 1 expression was induced by the linoleic acid derivative 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), and this induction was concomitant with increased NF-κB activity. In E-FABP(-/-) keratinocytes, the expression of 13(S)-HODE and the subsequent induction of NF-κB activity was lower than in wild-type keratinocytes. The reduction of linoleic acid in E-FABP(-/-) keratinocytes led to decreased cellular 13(S)-HODE content, resulting in decreased keratin 1 expression through downregulation of NF-κB activity. The regulation of fatty acid metabolism by E-FABP during keratinocyte differentiation suggests that E-FABP may have a role in the pathogenesis of psoriasis.

Topics: Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Epidermis; Fatty Acid-Binding Proteins; Fatty Acids; Keratin-1; Keratinocytes; Linoleic Acid; Linoleic Acids; Membrane Proteins; Mice; Mice, Knockout; Neoplasm Proteins; NF-kappa B; Protein Precursors; Psoriasis; Signal Transduction

Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k(cat)/K(M) and k(cat) for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k(cat)/K(M) KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k(cat)/K(M) SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K(M)[O(2)] for AA to 15 microM but increases the K(M)[O(2)] for linoleic acid (LA) to 22 microM, such that the k(cat)/K(M)[O(2)] values become similar for both substrates (approximately 0.3 s(-1) microM(-1)). Considering that the oxygen concentration in cancerous tissue can be less than 5 microM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Allosteric Regulation; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Biocatalysis; Blood Platelets; Carbon Isotopes; Humans; Kinetics; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Recombinant Proteins; Reticulocytes; Solvents; Temperature

The circadian production of melatonin by the pineal gland during the night provides an inhibitory signal to tissue-isolated steroid receptor SR+ and - MCF-7 human breast cancer xenografts in female nude rats. A pivotal mechanism for melatonin's anticancer effects in vivo involves a melatonin receptor-mediated inhibition of linoleic acid (LA) uptake and its metabolism to mitogenically active 13-hydroxyoctadecadienoic acid (13-HODE). Exposure of (SR-) xenograft-bearing rats to increasing intensities of polychromatic white light at night suppresses melatonin while increasing tumor growth rates, DNA content, [3H]thymidine incorporation into DNA, LA uptake, 13-HODE formation, cAMP levels and ERK1/2 activation a dose-dependent manner. Similar effects occur in SR- human breast cancer xenografts perfused in situ with melatonin-depleted blood from healthy female subjects after their exposure to a single bright intensity (2800 lux) of polychromatic light at night. Additionally, SR- human breast cancer xenografts exhibit robust circadian rhythms of LA uptake, 13-HODE formation and proliferative activity. Exposure of xenograft-bearing rats to dim light at night results in the complete elimination of these rhythms which culminates in unfettered, high rates of tumor metabolism and growth. The organization of tumor metabolism and growth within circadian time structure by the oncostatic melatonin signal helps create a balance between the cancer and its host that is disrupted by host exposure to light at night. This biological mechanism may partially explain the higher risk of breast and other cancers in women working rotating night shifts and possibly others who also experience prolonged exposure to light at night.

Topics: Animals; Anticarcinogenic Agents; Breast Neoplasms; Cell Growth Processes; Cell Proliferation; Circadian Rhythm; Female; Humans; Light; Linoleic Acid; Linoleic Acids; Melatonin; Neoplasm Transplantation; Photoperiod; Rats; Rats, Nude; Receptors, Melatonin; Signal Transduction; Transplantation, Heterologous

The ligand activated nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) induces transcriptional repression of pro-inflammatory factors. Activation of PPARgamma is followed by amelioration of colitis in animal models of inflammatory bowel disease (IBD). A reduced expression of PPARgamma was found in epithelial cells of patients with ulcerative colitis. The eicosanoids 13-HODE and 15-HETE are products of 12/15-lipoxygenase (LOX) and endogenous ligands for PPARgamma. Dehydrogenation of 13-HODE by 13-HODE dehydrogenase results in formation of the 13-Oxo-ODE. Highest activity of 13-HODE dehydrogenase is found in colonic epithelial cells (CECs). We therefore investigated whether 13-Oxo-ODE is a new endogenous ligand of PPARgamma in CECs.. LOX activity and 13-HODE dehydrogenase in CECs were investigated after stimulation with arachidonic or linoleic acid. LOX metabolites were identified by RP-18 reversed-phase HPLC. Binding of (14)C-labelled 13-Oxo-ODE was demonstrated using a His-tagged PPARgamma.. Stimulation of HT-29 and primary CECs homogenates with and without Ca-ionophor was followed by the formation of high amounts of the linoleic acid metabolite 13-Oxo-ODE (155 and 85 ng/ml). The decrease of IL-8 secretion from IEC was more pronounced after pre-incubation with 13-Oxo-ODE compared to the PPARgamma agonist troglitazone and higher as with the known PPARgamma ligands 13-HODE and 15-HETE. Binding assays with (14)C-labelled 13-Oxo-ODE clearly demonstrated a direct interaction.. High amounts of 13-Oxo-ODE can be induced in CECs by stimulation of linoleic acid metabolism. 13-Oxo-ODE binds to PPARgamma and has anti-inflammatory effects. 13-HODE dehydrogenase might be a therapeutic target in IBD.

Topics: Animals; Arachidonic Acid; Blotting, Western; Carbon Radioisotopes; Cattle; Chromatography, High Pressure Liquid; Colon; Epithelial Cells; HT29 Cells; Humans; Hydroxyeicosatetraenoic Acids; Immunoprecipitation; Interleukin-8; Ligands; Linoleic Acid; Linoleic Acids; Linolenic Acids; Molecular Structure; PPAR gamma; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Mass, Electrospray Ionization; Transfection

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that strongly influence molecular events in normal and cancer cells. PPAR-beta/delta (PPAR-b/d) overexpression suppresses the activity of PPAR-gamma (PPAR-g) and PPAR-alpha. This interaction has been questioned, however, by studies with synthetic ligands of PPARs in PPAR-b/d-null cells, and it is not known whether an interaction between PPAR-b/d and PPAR-g exists, especially in relation to the signaling by natural PPAR ligands. Oxidative metabolites of linoleic and arachidonic acids are natural ligands of PPARs. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), the main product of 15-lipoxygenase-1 (15-LOX-1) metabolism of linoleic acid, downregulates PPAR-b/d. We tested (a) whether PPAR-b/d expression modulates PPAR-g activity in experimental models of the loss and gain of PPAR-b/d function in colon cancer cells and (b) whether 15-LOX-1 formation of 13-S-HODE influences the interaction between PPAR-b/d and PPAR-g. We found that (a) 15-LOX-1 formation of 13-S-HODE promoted PPAR-g activity, (b) PPAR-b/d expression suppressed PPAR-g activity in models of both loss and gain of PPAR-b/d function, (c) 15-LOX-1 activated PPAR-g by downregulating PPAR-b/d, and (d) 15-LOX-1 expression induced apoptosis in colon cancer cells via modulating PPAR-b/d suppression of PPAR-g. These findings elucidate a novel mechanism of the signaling by natural ligands of PPARs, which involves modulating the interaction between PPAR-b/d and PPAR-g.

Topics: Adenoviridae; Arachidonate 15-Lipoxygenase; Colorectal Neoplasms; Down-Regulation; Humans; Linoleic Acid; Linoleic Acids; Oxidation-Reduction; PPAR delta; PPAR gamma; PPAR-beta

The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.

Topics: Acetonitriles; Cell Line, Tumor; Dinitrochlorobenzene; Dose-Response Relationship, Drug; Glutathione; Humans; Kinetics; Linoleic Acid; Linoleic Acids; Linolenic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Peroxidase

Both physiological and pharmacological levels of the pineal hormone melatonin exhibit substantial anticancer activity in tissue-isolated rat hepatoma 7288CTC via melatonin receptor-mediated blockade of tumor uptake of linoleic acid (LA) and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Melatonin is also present in significant amounts in edible plants and is supplied in nutritional supplements. We confirmed the presence of significant quantities of melatonin in 20 varieties of edible plants. In pinealectomized tumor-free rats, 3 weeks of ingestion of either 5 or 50 microg/day of melatonin contained in a semi-purified diet resulted in a dose-dependent elevation in steady-state plasma melatonin levels within the nocturnal physiological range. In pineal-intact tumor-bearing rats, the daily intake of 5 microg/day of melatonin for 3 weeks resulted in an enhanced amplitude and duration of the nocturnal melatonin levels within physiological circulating limits. The nocturnal melatonin amplitude in rats ingesting 500 ng of melatonin/day remained within the physiological range. A dose-related increase in tumor concentrations of melatonin occurred in animals ingesting melatonin from the diet. Perfusion of tumors in situ with physiological, nocturnal blood levels of melatonin resulted in a mean 31% uptake and retention of the melatonin. Chronic ingestion of 50 ng, 500 ng or 5 microg of melatonin/day supplied in a semi-purified 5% corn oil diet led to a significant dose-dependent reduction in the rates of tumor total fatty acid uptake, LA uptake, 13-HODE production and tumor growth. The co-ingestion of melatonin receptor antagonist S20928 completely blocked the effects and prevented the intra-tumoral accumulation of melatonin. Melatonin receptor-mediated suppression of tumor growth, LA uptake and metabolism, and stimulation of tumor melatonin uptake and retention in response to the dietary intake of phytomelatonin from edible plants or melatonin from nutritional supplements, could play an important role in cancer growth prevention.

Topics: Animals; Diet; Dose-Response Relationship, Drug; Linoleic Acid; Linoleic Acids; Liver Neoplasms, Experimental; Male; Melatonin; Plants; Rats; Rats, Inbred BUF; Receptors, Melatonin; Signal Transduction

In established rodent tumors and human cancer cell lines, conjugated dienoic isomers of linoleic acid (CLA) suppress the growth-stimulating effects of linoleic acid (LA) and its metabolism to the mitogenic agent, 13-hydroxyoctadecadienoic acid (13-HODE). Here, we compared the effects of three CLA isomers on LA uptake and metabolism, and growth in human breast xenografts perfused in situ in female nude rats. The results demonstrated that two CLA isomers [10t, 12c-CLA>9t, 11t-CLA] caused a dose-dependent inhibition of LA uptake, cAMP content, 13-HODE formation, Erk 1/2 activity, and [(3)H]thymidine incorporation into tumor DNA; 9c, 11t-CLA showed no effect. The inhibitory effect is reversible with either pertussis toxin (PTX) or 8-Br-cAMP suggesting that CLA isomers differentially inhibit LA uptake and metabolism and cell proliferation in human breast cancer in vivo via a receptor-mediated, PTX-sensitive pathway.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Antithrombins; Biological Transport; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Fatty Acids; Female; Humans; Kinetics; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Male; Neoplasm Transplantation; Pertussis Toxin; Protein Isoforms; Rats; Rats, Nude; Rats, Sprague-Dawley

The relationship between 15(S)-HETE and 13(S)-HODE from different human tumor cells exposed to n-6 and n-3 essential fatty acids (EFAs) and E-cadherin expression was studied. Colon cancer cells (HRT-18) exposed to gamma linoleic acid (18:3n-6, GLA) and eicosapentaenoic (20:5n-3, EPA) (50microM) showed an increased expression of E-cadherin. Breast cancer (MCF-7) exposed to EPA showed an increment whereas GLA had no effect on E-cadherin expression. No expression of E-cadherin was observed for urothelial cancer (T-24) after GLA or EPA treatment. Significant levels of 15(S)-HETE and 13(S)-HODE were detected after GLA or EPA treatment for all tumor lines. E-cadherin expression was inversely proportional to the 13(S)-HODE:15(S)-HETE ratio when cells were pretreated with GLA or EPA. Nevertheless, the liberation of these metabolites seems to be independent of the E-cadherin expression. The increase in the13(S)-HODE:15(S)-HETE correlates to a decrease in the expression of E-cadherin. Both factors may play a role in metastasis development.

Topics: Arachidonic Acid; Breast Neoplasms; Cadherins; Cell Differentiation; Colonic Neoplasms; Female; Humans; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Linoleic Acid; Linoleic Acids; Neoplasm Metastasis; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urothelium

The activation of peroxisome proliferator activated receptor gamma (PPARgamma) may play a role in the control of colorectal carcinogenesis. The expression of PPARgamma was examined by Western blotting in human colorectal tumors and matched normal adjacent tissues, as well as in various colorectal carcinoma cell lines. In the tissues, the expression of PPARgamma was elevated in tumors relative to the adjacent normal tissues. Each colorectal carcinoma cell line expressed PPARgamma. The ability of various eicosanoids to bind PPARgamma in colorectal carcinoma cells was investigated using luciferase reporter assays. The well-known PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) strongly induced PPARgamma binding activity. Products of lipoxygenases displayed moderate binding activity, while other prostaglandins and fatty acids displayed little or no reporter activation. The activation of PPARgamma by 13(S)-HODE, the major metabolite of 15-lipoxygenase-1 from linoleic acid, was concentration dependent reaching maximum at 10 micro M (35-fold activation). The endogenous production of 13(S)-HODE by expression of 15-LO-1 did not activate PPARgamma. The ability of various nonsteroidal anti-inflammatory drugs (NSAIDs) to induce PPARgamma activation was also evaluated. The conventional NSAIDs that inhibit both cyclooxygenases (COX-1 and COX-2) also induced PPARgamma binding activity. In general, however, neither COX-1- nor COX-2-specific inhibitors induced the activation of PPARgamma. Taken together, the metabolites of 15-lipoxygenase and the conventional NSAIDs were confirmed as exogenous ligands for PPARgamma in colorectal carcinoma cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Caco-2 Cells; Cell Line, Tumor; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Eicosanoids; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Humans; Ligands; Linoleic Acid; Linoleic Acids; Luciferases; Protein Binding; Receptors, Cytoplasmic and Nuclear; Transcription Factors

A major bioactive metabolite of linoleic acid formed by the action of 15-lipoxygenase-1 is 13(S)-hydroxy-cis-9, trans-11-octadecadienoic acid (13(S)-HODE). 13(S)-HODE is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Separation and quantification of 13(S)-HODE from biological materials has previously been achieved only by using radiolabeled linoleic acid as the substrate and two serially connected or two separate HPLC columns to achieve separation of 13(S)-HODE. In the current method, separation and quantification of 13(S)-HODE was achieved by use of a normal-phase HPLC and a solvent system containing hexane/isopropanol/acetonitrile/acetic acid (800/8/30/1, v/v) using isocratic elution with detection at 235 nm. With the currently described method, good separation from unreacted interfering compounds and quantification for 13(S)-HODE were achieved within 35 min with a minimum detection limit of 0.5 ng per injection.

Topics: 2-Propanol; Acetic Acid; Acetonitriles; Animals; Chromatography, High Pressure Liquid; Hexanes; Linoleic Acid; Linoleic Acids; Liver; Lung; Rats; Rats, Sprague-Dawley

The nocturnal melatonin (MLT) surge is a relevant oncostatic signal for a variety of experimental malignancies. Population studies support the hypothesis that exposure to light at night may represent a new risk factor for breast cancer possibly through the suppression of pineal MLT production and/or circadian disruption. We tested the ability of constant light exposure to suppress MLT production in female nude rats and stimulate the growth of tissue-isolated MCF-7 human breast cancer xenografts via increased tumor linoleic acid (LA) metabolism. Rats maintained on an alternating light/dark cycle (L:D group) exhibited a robust circadian MLT rhythm that was abolished following constant light exposure. During the exposure of animals bearing tissue-isolated human MCF-7 breast cancer xenografts to constant light, the rate of tumor growth markedly increased relative to the L:D group. Tumor LA uptake and its metabolism to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) were also substantially higher under constant light conditions. This is the first biological evidence for a potential link between constant light exposure and increased human breast oncogenesis involving MLT suppression and stimulation of tumor LA metabolism.

Topics: Animals; Antioxidants; Antithrombins; Breast Neoplasms; Cell Transformation, Neoplastic; Circadian Rhythm; Female; Humans; Light; Linoleic Acid; Linoleic Acids; Melatonin; Mice; Mice, Nude; Pineal Gland; Transplantation, Heterologous

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.

Topics: Animals; Arteries; Cell Division; Cell Membrane; Diabetes Mellitus, Experimental; DNA; Endothelium, Vascular; Immunohistochemistry; Insulin-Like Growth Factor I; Linoleic Acid; Linoleic Acids; Lipid Metabolism; Models, Biological; Muscle, Smooth; Oleic Acid; Phospholipase D; Phosphorylation; Swine; Thymidine; Time Factors; Triglycerides

Increased reactive oxygen species generation by the leukocytes of the obese may be responsible for increased oxidative injury to lipids and proteins and, hence, atherosclerosis. We have investigated whether reactive oxygen species generation by leukocytes and other indexes of oxidative damage in the body fall with short-term dietary restriction and weight loss. Nine nondiabetic obese subjects (body mass index, 32.5-64.4 kg/m(2)), not taking any antioxidants, were put on a 1000-Cal diet. Fasting blood samples were taken at 0, 1, 2, 3, and 4 weeks and at 12 weeks after the cessation of dietary restriction. Blood samples were also obtained at 1 and 2 h after administration of 75 g oral glucose at 0 and 4 weeks. Mononuclear cells (MNC) and polymorphonuclear leukocytes (PMN) were isolated, and reactive oxygen species generation was measured. Plasma concentrations of thiobarbituric acid-reactive species (TBARS), 13-hydroxyoctadecadienoic acid (13-HODE), 9-hydroxyoctadecadienoic acid (9-HODE), carbonylated proteins, o-tyrosine, and m-tyrosine as indexes of oxidative damage to lipids, proteins and amino acids, respectively, were measured. Antioxidant vitamins were measured as indexes of antioxidant reserves. Plasma tumor necrosis factor-alpha concentrations were also measured. Mean weight loss was 2.4 +/- 0.6 kg at week 1, 2.5 +/- 1.7 kg at week 2, 3.9 +/- 0.8 kg at week 3, and 4.5 +/- 2.8 kg at week 4 (P < 0.05). Reactive oxygen species generation by PMN fell from 236.4 +/- 95.8 to 150.9 +/- 69.0, 125.9 +/- 24.3, 96.0 +/- 39.9, and 103.1 +/- 35.7 mV at weeks 1, 2, 3, and 4, respectively (P < 0.001). It increased 3 months after the cessation of dietary restriction to 270.0 +/- 274.3 mV. Reactive oxygen species generation by MNC fell from 187.8 +/- 75.0 to 101.7 +/- 64.5, 86.9 +/- 42.8, 63.8 +/- 14.3, and 75.1 +/- 32.2 mV and increased thereafter to 302.0 +/- 175.5 mV at 1, 2, 3, 4, and 16 weeks, respectively (P < 0.005). Reactive oxygen species generation by PMN and MNC increased in response to glucose; the relative increase was greater at 4 weeks than that at week 0 due to a fall in the basal levels of reactive oxygen species generation. Consistent with the fall in reactive oxygen species generation, there was a reduction in plasma TBARS from 1.68 +/- 0.17 micromol/L at week 0 to 1.47 micromol/L at 4 weeks (P < 0.05). The 13-HODE to linoleic acid ratio fell from a baseline of 100% to 56.4 +/- 36.1% at 4 weeks (P < 0.05), and the 9-HODE to linoleic acid ratio fel

Topics: Adult; Aged; Diet, Reducing; Female; Glucose Tolerance Test; Humans; Leukocytes; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxides; Male; Middle Aged; Obesity; Phenylalanine; Proteins; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Tyrosine; Weight Loss

The effect of overexpression of 15-lipoxygenase-1 (15-LO-1) was studied in the human prostate cancer cell line, PC-3. Stable PC-3 cell lines were generated by transfection with 15-LO-1-sense (15-LOS), 15-LO-1-antisense (15-LOAS) or vector (Zeo) and selection with Zeocin. After characterization by RT-PCR, western and HPLC, a PC3-15LOS clone was selected that possessed 10-fold 15-LO-1 enzyme activity compared with parental PC-3 cells. The PC3-15LOAS clone displayed little or no 15-LO-1 activity. These PC-3 cell lines were characterized for properties of tumorigenesis. The proliferation rates of the cell lines were as follows: PC3-15LOS > PC-3 = PC3-Zeo > PC3-15LOAS. Addition of a specific 15-LO-1 inhibitor, PD146176, caused a dose-dependent inhibition of proliferation in vitro. Overexpression of 15-LO-1 also caused [(3)H]thymidine incorporation to increase by 4.0-fold (P < 0.01). Compared with parental and PC-3-Zeo cells, PC3-15LOS enhanced whereas PC3-15LOAS reduced the ability of PC-3 cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. These data suggested a pro-tumorigenic role for 15-LO-1 in PC-3 cells in vitro. Therefore, to clarify the role of 15-LO-1 in vivo, the effect of 15-LO-1 expression on the growth of tumors in nude mice was investigated. The PC-3 cell lines were inoculated subcutaneously into athymic nude mice. The frequency of tumor formation was increased and the sizes of the tumors formed were much larger in the PC3-15LOS compared with PC3-15LOAS, parental PC-3 and PC-3-Zeo cells. Immunohistochemistry for 15-LO-1 confirmed expression throughout the duration of the experiment. The expression of factor VIII, an angiogenesis marker, in tumor sections was increased in tumors derived from PC3-15LOS cells and decreased in those from PC3-15LOAS cells compared with tumors from parental or Zeo cells. These data further supported the evaluation by ELISA of vascular endothelial growth factor (VEGF) secretion by PC-3 cells in culture. Secretion of this angiogenic factor was elevated in PC3-15LOS cells compared with the other cell lines. These results support a role for 15-LO-1 in a novel growth-promoting pathway in the prostate.

Topics: Adenocarcinoma; Animals; Arachidonate 15-Lipoxygenase; Blotting, Western; Cell Division; Chromatography, High Pressure Liquid; Colony-Forming Units Assay; Electrophoresis, Polyacrylamide Gel; Endothelial Growth Factors; Enzyme Induction; Factor VIII; Humans; Immunoenzyme Techniques; Linoleic Acid; Linoleic Acids; Lymphokines; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H(2)O(2)). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H(2)O(2) after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress.

Topics: Animals; Catalase; Cells, Cultured; Endothelium, Vascular; Fatty Acids; Femoral Artery; Gene Expression; Humans; Hydrogen Peroxide; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Muscle, Smooth, Vascular; Oxidants; Oxidation-Reduction; Rabbits; RNA, Messenger; Transfection; Umbilical Veins

The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator-activated receptor gamma (PPARgamma). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARgamma. Modified apoB had no such effect. Consistent with a participation of the PPARgamma signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARgamma in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.

Topics: Animals; Apolipoproteins B; Arteriosclerosis; Cells, Cultured; Down-Regulation; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoproteins, LDL; Mice; Monocytes; Muscle, Smooth, Vascular; Phospholipids; Receptors, CCR2; Receptors, Chemokine; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Rosiglitazone; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors

We recently reported that the mutant form of the tumor-suppressor gene p53 up-regulates 15-LO-1 gene expression in a murine cell line. Here, we examine the expression of 15-lipoxygenase (LO)-1 and mutant p53 (mtp53) in human prostatic tissues and 15-LO-1 in the human prostate adenocarcinoma cell line PC-3. Reverse transcription-PCR and western analyses conclusively demonstrated expression of 15-LO-1 in PC-3 cells. Western blotting for 15-LO-1 in freshly resected 'normal' and prostate adenocarcinoma specimens showed 15-LO-1 expression in normal tissue, but significantly higher levels were detected in prostate adenocarcinomas. Prostate adenocarcinoma tissues generated chirally pure 13-S-hydroxyoctadecadienoic acid from exogenous linoleic acid, a preferred substrate of 15-LO-1. To study the correlation of 15-LO-1 expression with mtp53 in prostate cancer, we immunostained 48 prostatectomy specimens obtained by transurethral resection of the prostate and needle biopsy (median age 68 years, range 52-93) of different Gleason grades (n = 48), using antibodies specific for 15-LO-1, mtp53 and MIB-1 (a proliferation marker). We compared staining in cancerous foci with adjacent normal appearing prostate tissues. In only 5 of 48 patients did 'normal' tissue adjacent to cancerous foci display staining for 15-LO-1. However, no staining for mtp53 was observed in any of the normal tissues. In cancer foci, robust staining was observed for both 15-LO-1 (36 of 48, 75%) and mtp53 (19 of 48, 39%). Furthermore, the intensities of expression of 15-LO-1 and mtp53 correlated positively with each other (P < 0.001) and with the degree of malignancy, as assessed by Gleason grading (P < 0.01). By immunohistochemistry, 15-LO-1 was located in secretory cells of peripheral zone glands, prostatic ducts and seminal vesicles, but not in the basal cell layer or stroma. Based on these and other studies, we propose a model describing a possible role for 15-LO-1 expression in influencing the malignant potential and pathobiological behavior of adenocarcinomas.

Topics: Adenocarcinoma; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Blotting, Western; Enzyme Induction; Humans; Immunohistochemistry; Linoleic Acid; Linoleic Acids; Male; Mutation; Neoplasm Staging; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Stereoisomerism; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We previously found (I. Shureiqi et al., Carcinogenesis (Lond.), 20: 1985-1995, 1999; I. Shureiqi et al, J. Natl. Cancer Inst., 92: 1136-1142, 2000) that (a) 15-lipoxygenase-1 (15-LOX-1) protein and its product 13-S-hydroxyoctadecadienoic acid (13-S-HODE) are decreased; and (b) nonsteroidal anti-inflammatory drug (NSAID)-induced 15-LOX-1 expression is critical to NSAID-induced apoptosis in colorectal cancer cells expressing cyclooxygenase-2 (COX-2). We used the NSAIDs sulindac sulfone (COX-2-independent) and NS-398 (a COX-2 inhibitor) to assess NSAID upregulation of 15-LOX-1 in relation to COX-2 inhibition during NSAID-induced apoptosis in the DLD-1 (COX-2-negative) colon cancer cell line. We found that: (a) NSAIDs up-regulated 15-LOX-1, which preceded apoptosis; and (b) 15-LOX-1 inhibition blocked NSAID-induced apoptosis, which was restored by 13-S-HODE but not by its parent, linoleic acid. NSAIDs can induce apoptosis in colon cancer cells via up-regulation of 15-LOX-1 in the absence of COX-2.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Antioxidants; Antithrombins; Apoptosis; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Blotting, Western; Caffeic Acids; Cell Line; Colonic Neoplasms; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Linoleic Acid; Linoleic Acids; Membrane Proteins; Nitrobenzenes; Prostaglandin-Endoperoxide Synthases; Recombinant Proteins; Sulfonamides; Sulindac; Time Factors; Tumor Cells, Cultured; Up-Regulation

During our ongoing project on the biosynthesis of R-(+)-octane-1,3-diol the metabolism of linoleic acid was investigated in stored apples after injection of [1-14C]-, [9,10,12,13-3H]-,13C18- and unlabeled substrates. After different incubation periods the products were analyzed by gas chromatography-mass spectroscopy (MS), high-performance liquid chromatography-MS/MS, and HPLC-radiodetection. Water-soluble compounds and CO2 were the major products whereas 13(R)-hydroxy- and 13-keto-9(Z),11(E)-octadecadienoic acid, 9(S)-hydroxy- and 9-keto-10(E),12(Z)-octadecadienoic acid, and the stereoisomers of the 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids were identified as the major metabolites found in the diethyl ether extracts. Hydroperoxides were not detected. The ratio of 9/13-hydroxy- and 9/13-keto-octadecadienoic acid was 1:4 and 1:10, respectively. Chiral phase HPLC of the methyl ester derivatives showed enantiomeric excesses of 75% (R) and 65% (S) for 13-hydroxy-9(Z),11(E)-octadecadienoic acid and 9-hydroxy-10(E),12(Z)-octadecadienoic acid, respectively. Enzymatically active homogenates from apples were able to convert unlabeled linoleic acid into the metabolites. Radiotracer experiments showed that the transformation products of linoleic acid were converted into (R)-octane-1,3-diol. 13(R)-Hydroxy-9(Z),11(E)-octadecadienoic acid is probably formed in stored apples from 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid. It is possible that the S-enantiomer of the hydroperoxide is primarily degraded by enzymatic side reactions, resulting in an enrichment of the R-enantiomer and thus leading to the formation of 13(R)-hydroxy-9(Z),11(E)-octadecadienoic acid.

Topics: Chromatography, High Pressure Liquid; Fruit; Linoleic Acid; Linoleic Acids; Spectrum Analysis

The growth of rat hepatoma 7288CTC in vivo is stimulated by the uptake of linoleic acid (LA) and its metabolism to 13-hydroxyoctadecadienoic acid (13-HODE), an important mitogenic signaling molecule within this tumor. Conversely, the growth of a variety of experimental cancers in vivo is inhibited by either physiological or pharmacological levels of the pineal gland hormone melatonin, although the mechanism(s) are unknown. We tested the hypothesis that the mechanism of melatonin's anticancer action in vivo involves the inhibition of tumor LA uptake and metabolism to 13-HODE in hepatoma 7288CTC. Tumor uptake of LA and release of 13-HODE, measured in tissue-isolated rat hepatoma 7288CTC at 4-h intervals over a 24-h period, were highest during the light phase and lowest during the mid-dark phase, when plasma melatonin levels were lowest and highest, respectively. Pinealectomy eliminated this rhythm of tumor LA uptake and 13-HODE production, indicating that it was driven by the circadian melatonin rhythm. Perfusion of tissue-isolated tumors in situ with melatonin (1 nM) rapidly and reversibly inhibited the uptake of plasma fatty acids (FAs), including LA, and its metabolism to 13-HODE. These inhibitory effects of melatonin on tumor FA uptake and 13-HODE release were completely reversed by perfusion of tumors in situ with melatonin receptor antagonist S-20928, pertussis toxin, forskolin, or 8-bromo-cAMP. Perfusion of tumors in situ with melatonin also decreased tumor [3H]thymidine incorporation and DNA content; these effects on DNA synthesis were also prevented by the coperfusion of tumors with melatonin and S-20928, pertussis toxin, forskolin, 8-Br-cAMP, or 13-HODE. Pinealectomy stimulated tumor growth, LA uptake and metabolism to 13-HODE, and FA storage in hepatoma 7288CTC, whereas melatonin administration (200 microg/day) was inhibitory in vivo. Northern blot analysis revealed that, compared with normal liver tissue, hepatoma 7288CTC overexpressed mRNA transcripts for a plasma membrane-associated FA transport protein (FATP). FATP mRNA expression was unaffected by the treatment of tumor-bearing rats with daily afternoon melatonin injections or exposure to constant light. These results support a novel mechanism of tumor growth inhibition by melatonin involving a melatonin receptor-mediated suppression of cAMP levels, resulting in diminished tumor FA transport, possibly via decreased FATP function. The inhibition of these signal transduction events by melatoni

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Biological Transport; Cell Division; Circadian Rhythm; Colforsin; Linoleic Acid; Linoleic Acids; Liver Neoplasms, Experimental; Male; Melatonin; Models, Biological; Naphthalenes; Perfusion; Pertussis Toxin; Rats; Rats, Inbred BUF; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Signal Transduction; Virulence Factors, Bordetella

Growth of hepatoma 7288CTC in male Buffalo rats is directly dependent on uptake of linoleic acid (LA) from the arterial blood. One to 5% of the LA taken up is converted to 13-hydroxyoctadecadienoic acid (HODE), an agent that enhances epidermal growth factor-dependent mitogenesis. The role of 13-HODE in LA-dependent growth of solid tumors is not known. In this study, we examined LA uptake and 13-HODE formation on growth of tissue-isolated hepatoma 7288CTC in vivo and on [3H]thymidine incorporation and DNA content during perfusion in situ. Fatty acid uptake and metabolite release were determined from arteriovenous difference measurements. Tumor-bearing and blood donor rats were fed either LA-sufficient or -deficient diets. Hepatoma 7288CTC removed LA from the arterial blood and released 13-HODE [and a small amount of 13-ketooctadecadienoic acid (KODE)] into the venous blood both in vivo and during perfusion. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) did not affect tumor LA uptake, but inhibited release of 13-HODE and 13-KODE in vivo and during perfusion, suppressed growth in vivo, and inhibited [3H]thymidine incorporation during perfusion. The addition of 13-HODE to the nordihydroguaiaretic acid-containing whole blood perfusate increased the rate of [3H]thymidine incorporation 10 times and nearly doubled tumor DNA content; the addition of 13-KODE or 9-HODE had no effect. 13-HODE and 13-KODE were not released from tumors growing in rats fed a LA-deficient diet, and the rates of tumor growth in vivo and [3H]thymidine incorporation during perfusion were decreased. The addition of 13-HODE to the LA-deficient blood perfusate promoted tumor 13-HODE uptake and a dose-dependent increase in [3H]thymidine incorporation and tumor DNA content. These results provide strong evidence that 13-HODE is the mitogenic signal responsible for LA-dependent growth in hepatoma 7288CTC in vivo.

Topics: Animals; Biological Transport; Cell Division; DNA, Neoplasm; Linoleic Acid; Linoleic Acids; Liver Neoplasms, Experimental; Male; Masoprocol; Rats; Rats, Inbred BUF; Thymidine

Tumor linoleic acid uptake and metabolism, and growth are suppressed by melatonin, the synthesis of which is inhibited by light. Linoleic acid, via its mitogenic metabolite 13-hydroxyoctadecadienoic acid (13-HODE) is an important growth stimulant of rat hepatoma 7288CTC. Here we compared the effects of an alternating light:dark cycle (12L:12D), dim light (0.25 lux) present during the dark phase of a diurnal light cycle, and constant light on growth and fatty acid metabolism in hepatoma 7288CTC. Our results show that dim light suppressed melatonin release by the pineal gland, increased tumor linoleic acid uptake and 13-HODE production, and promoted tumor growth as effectively as did constant light.

Topics: Animals; Cell Division; Darkness; Disease Progression; Light; Linoleic Acid; Linoleic Acids; Male; Melatonin; Rats; Rats, Inbred BUF

Topics: Animals; Carcinoma 256, Walker; Cell Communication; Dietary Fats; Epoprostenol; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Linoleic Acid; Linoleic Acids; Neoplasm Metastasis; Rats; Rats, Wistar; Thrombosis

Topics: Adult; Aged; Aged, 80 and over; Aging; Arteriosclerosis; Arthritis, Rheumatoid; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxidation; Lipoproteins, LDL; Middle Aged

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.

Topics: Animals; Coronary Vessels; Electrophysiology; Fatty Acids; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Membrane Potentials; Swine; Vasodilation

The purpose of our studies was to examine differentiation-dependent expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase (PGHS) isoforms in cultured normal human tracheobronchial epithelial cells. In the presence of retinoic acid (RA) the cultures differentiated into a mucociliary epithelium. When cultured in RA-depleted media, the cultures differentiated into a squamous epithelium. In the absence of RA the cultures did not express 15-LO or either of the PGHS isoforms. The PGHS-1 isoform was not expressed in RA-sufficient cultures, but both PGHS-2 messenger RNA (mRNA) and protein were strongly expressed, and prostaglandin E2 (PGE2) was produced during the predifferentiation phase. No PGHS-2 expression or PGE2 could be detected in fully differentiated mucociliary cultures. 15-LO showed the opposite expression pattern: neither mRNA nor protein were detected during the predifferentiation stage, but both were strongly expressed once mucous differentiation had occurred. Cytosolic phospholipase A2 protein was expressed throughout all stages of growth and differentiation. The cultures generated no 15-LO metabolites when incubated with 10 microM to 50 microM arachidonic acid (AA) and stimulated with ionophore. However, lysates prepared from such cultures generated 15-hydroxyeicosatetraenoic acid (15-HETE) and 12-HETE from AA, indicating that the cells contained active enzyme. When cultures expressing 15-LO protein were incubated with 10 microM linoleic acid (LA) instead of AA, and were stimulated with ionophore, they generated 13-hydroxy-9,11-octadecadienoic acid. LA rather than AA appeared to be the preferred substrate for the 15-LO enzyme. Our studies indicated that the expression of 15-LO and PGHS-2 is differentiation dependent in airway epithelial cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antithrombins; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Bronchi; Cell Differentiation; Cells, Cultured; Chromatography, High Pressure Liquid; Cilia; Cytosol; Epithelial Cells; Gene Expression Regulation, Enzymologic; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Linoleic Acid; Linoleic Acids; Mucous Membrane; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Retinoids; Trachea

Oxidized lipids present in atherogenic lipoproteins are derived, in part, from the diet. To address the effects of an oxidized lipid on intestinal lipoprotein assembly and secretion, CaCo-2 cells were incubated with 13-HODE or its native fatty acid, linoleic acid, and triacylglycerol-rich lipoprotein synthesis and secretion were investigated. 13-HODE was readily taken up by cells and esterified to lipids. Although both fatty acids were largely esterified to neutral lipids, in comparison to neutral lipids containing linoleic acid, a greater proportion of cellular neutral lipids containing 13-HODE and/or its metabolites was secreted. Compared to linoleic acid, however, 13-HODE caused less triacylglycerol, derived from de novo synthesis, and less triacylglycerol mass to be secreted. Cells incubated with both linoleic acid and 13-HODE together secreted less triacylglycerol mass than did cells incubated with linoleic acid alone. Less newly synthesized apoB and apoB mass were secreted by cells incubated with 13-HODE without altering the abundance of apoB mRNA. The fraction of newly synthesized apoB translocated into the secretory pathway of cells exposed to 13-HODE was significantly less than that observed in cells incubated with linolenic acid, suggesting that 13-HODE interfered with the assembly and secretion of triacylglycerol-rich lipoprotein particles.

Topics: Apolipoproteins B; Biological Transport; Caco-2 Cells; Cell Division; Cell Survival; Humans; Kinetics; Linoleic Acid; Linoleic Acids; Lipid Metabolism; Lipoproteins; Lipoproteins, LDL; Transcription, Genetic; Triglycerides

The ubiquitous hydroxylated fatty acids derived from arachidonic acid (HETEs) or linoleic acid (HODEs) exhibit diverse biological effects including chemotaxis, cell proliferation, and modulation of several enzymatic pathways, including the 5-lipoxygenase leading to the inflammatory leukotrienes. It was observed that 12(S)- and 15(S)-HETE and 13(S)-HODE (12- and 15-lipoxygenase-derived metabolites, respectively) inhibited the 5-lipoxygenase present in rat basophilic leukemia (RBL-1) cell homogenates whereas the 15(R) chiral enantiomer and the nonhydroxylated linoleic, oleic, and stearic acids were either less potent or ineffective. In examining the mechanism of this inhibition, the relative effectiveness of several fatty acids in displacing [3H]15-HETE bound to cytosol preparations were compared and the results indicated that these (S) hydroxy fatty acids and 5(S)-HETE were significantly more potent than either the 15(R) enantiomer, 15(S)-HETE methyl ester, arachidonic acid, or prostaglandin F2alpha. In order to identify the protein(s) that specifically binds HETEs, 15(S)-HETE biotin hydrazide was used as a probe to detect any HETE-protein complexes as this compound both inhibited the 5-lipoxygenase and interfered with the binding of [3H]15-HETE to cytosol preparations. SDS-PAGE analysis and chemiluminescent detection revealed that the major cytosolic proteins that bound this biotinylated probe had molecular masses of 43 and 51 kD. Fatty acid competition experiments indicated that the order of effectiveness in displacing this probe from these proteins was 13(S)-HODE > 5(S)-HETE approximately equal to 15(S)-HETE > > stearic acid approximately equal to arachidonic acid approximately equal to 15(R)-HETE. Amino acid sequence analysis showed that the 43 kD protein was actin. These findings suggest the possibility that actin may play a major role in the biological effects of monohydroxylated metabolites derived from cellular 5-, 12-, and 15-lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actins; Amino Acid Sequence; Animals; Arachidonate 15-Lipoxygenase; Biotinylation; Carrier Proteins; Cytosol; Dinoprost; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Ligands; Linoleic Acid; Linoleic Acids; Molecular Sequence Data; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Rats; Stearic Acids; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

Oxidative modification of low density lipoprotein has been suggested as patho-physiologically relevant process in atherogenesis and the lipid peroxidizing enzyme 15-lipoxygenase may be involved. For experimental evidence on the in vivo action of this enzyme in the time course of plaque formation we analyzed the lipid extracts of lesional areas representing various stages of human atherogenesis for the occurrence of specific 15-lipoxygenase products. In advanced human lesions the degree of oxygenation of the lesion lipids measured as hydroxy linoleic acid/linoleic acid ratio varied between 0.2 and 3.2%. Here an unspecific pattern of oxygenated lipids that did not differ from the pattern formed during copper-catalyzed LDL oxidation was detected. In both cases an enantiomer ratio (S/R-ratio) of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) of approximately 1:1 was found. In young human lesions which were obtained from the collection of the pathological determinants of atherosclerosis in youth (PDAY) program the hydroxy linoleic acid/linoleic acid ratio was much smaller (variation between 0.05 and 0.6%), and a significant share of specific 15-lipoxygenase products was detected (S/R-ratio of 13-hydroxy linoleic acid of 54 +/- 3.1/46 +/- 3.1 [mean +/- SD]). These data suggest that the 15-lipoxygenase is enzymatically active on endogenous substrates in young human lesions and thus, may be of patho-physiological importance for early atherogenesis. In advanced human plaques the 15-lipoxygenase may be functionally silent and specific lipoxygenase products formed in earlier stages may be decomposed or superimposed by large amounts of nonenzymatic lipid peroxidation products.

Topics: Adolescent; Adult; Age Factors; Aged; Arachidonate 15-Lipoxygenase; Arteriosclerosis; Cholesterol, LDL; Chromatography, High Pressure Liquid; Copper; Fatty Acids; Female; Humans; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Male; Middle Aged; Oxidation-Reduction

Epidemiological and experimental data suggest a role for polyunsaturated fatty acids in the etiology of breast cancer. In this report we have studied arachidonic acid and linoleic acid metabolism in the human breast carcinoma cell line BT-20 which overexpresses both EGF receptor and the homologous erbB-2 oncogene product. EGF and TGF alpha stimulated DNA synthesis in these cells which was attenuated by the addition of a lipoxygenase inhibitor, NDGA. The addition of a prostaglandin H synthase inhibitor did not alter DNA synthesis. Analytical studies reveal little arachidonic acid metabolism while linoleic acid was metabolized to 13-hydroxyoctadecadienoic acid (13-HODE). The formation of 13-HODE was inhibited by the addition of NDGA and was dependent on EGF or TGF alpha. These results suggest the metabolism of linoleic acid by a n-6 or 15-lipoxygenase regulated by EGF/TGF alpha, RT-PCR was used to isolate a clone, and sequenced the cDNA for this enzyme and it was found to be identical to the human 15-lipoxygenase previously characterized from human pulmonary tissue. EGF/TGF alpha did not alter the expression of this enzyme suggesting a potential post-translational regulation of activity. This study provides a link between metabolism of linoleic acid and growth factor regulation of cell proliferation in a human breast carcinoma cell line.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acid; Breast Neoplasms; Cyclooxygenase Inhibitors; DNA; Epidermal Growth Factor; Female; Humans; Indomethacin; Linoleic Acid; Linoleic Acids; Lipoxygenase Inhibitors; Masoprocol; Polymerase Chain Reaction; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

9-Hydroxy-10,12-octadecadienoic acid (9-HODE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODE) are accumulated in the low density lipoproteins of patients suffering from rheumatoid arthritis for a factor of 20-50 compared to healthy individuals of the same age. Both acids, derived by lipid peroxidation of linoleic acid, induce the release of interleukin 1 beta. The latter induces bone degression. The genesis of 9- and 13-HODE seems therefore to be an important factor in the development and progression of rheuma; in addition 9-HODE was reported to be a stimulus of inflammation, comparable to leukotrienes.

Topics: Adult; Aged; Aldehydes; Arthritis, Rheumatoid; Chromatography, Gas; Female; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxidation; Lipid Peroxides; Lipoproteins, LDL; Male; Mass Spectrometry; Middle Aged

Lipid peroxidation is initiated by cell damage. After homogenisation of porcine heart tissue in aqueous solution we observed the same lipid peroxidation products as detected after heart infarction. We used this observation to study the influence of ebselen (2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) on the generation of oxidatively derived monohydroxy fatty acids and alpha-hydroxyaldehydes, typical lipid peroxidation (LPO) products. Heart tissue was homogenised before and after enzyme destruction and with addition of ebselen. The obtained LPO products were analysed by GC/MS after appropriate derivatisation and quantified by using internal standards. The amount of monohydroxy fatty acids and alpha-hydroxyaldehydes increased considerably in the porcine heart homogenates in which the enzymes were kept active. Addition of ebselen caused an additional significant increase of hydroxy fatty acids, while the increase of aldehydic compounds was less. These results confirm the glutathione peroxidase-like activity of ebselen but demonstrate also that it does not prevent lipid peroxidation.

Topics: Acetamides; Aldehydes; Animals; Antioxidants; Azoles; Cell Extracts; Fatty Acids; Fluoroacetates; Gas Chromatography-Mass Spectrometry; Isoindoles; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxidation; Lipid Peroxides; Molecular Structure; Myocardial Infarction; Myocardium; Organoselenium Compounds; Plasmalogens; Swine; Trifluoroacetic Acid; Trimethylsilyl Compounds

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Embryo, Mammalian; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Hybrid Cells; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Mesocricetus; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; Stimulation, Chemical

Topics: Animals; Arachidonic Acid; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Cyclooxygenase Inhibitors; Dexamethasone; DNA Replication; Epidermal Growth Factor; Fibroblasts; Hydroxyeicosatetraenoic Acids; Indomethacin; Linoleic Acid; Linoleic Acids; Lipoxygenase; Masoprocol; Mesocricetus; Receptor, ErbB-2; Recombinant Fusion Proteins; Transfection

Oxidative modification of LDL is believed to play a major role in atherogenesis. As major lipid peroxidation products oxygenated linoleic acid derivatives and oxysterols have been described in human atherosclerotic lesions. Here we report that human lesions contain isoprostanes as peroxidation products of arachidonic acid at a level of 27.1 +/- 21.2 pg/mg wet weight (n = 10), which corresponds to 75.9 +/- 59.3 pg/mg dry weight, n contrast, human umbilical veins (n = 10), which were used as nonatherosclerotic control vessels, contain much smaller amounts of isoprostanes (1.4 +/- 0.7 pg/mg wet weight, which corresponds to 11.7 +/- 6.2 pg/mg dry weight), and there are significant differences between the two types of vessels. As major products of linoleic acid oxidation, racemic hydroxy linoleate isomers were detected in the lesional ester lipids. In human lesions, the hydroxy linoleic acid/linoleic acid ratio was about 0.5%, a result indicating that 5 out of 1000 linoleate residues are present as hydroxylated derivatives. In umbilical veins, no hydroxy linoleic acid could be detected. These data show that human atherosclerotic lesions contain increased amounts of hydroxy linoleic acid isomers and isoprostanes when compared with nonatherosclerotic vessel wall and suggest a link between local lipid peroxidation and progression of atherosclerosis. For evaluation of the degree of lipid peroxidation, the determination of the hydroxy linoleic acid/linoleic acid ratio appears to be more suitable than the isoprostane content.

Topics: Aged; Arachidonic Acid; Arteries; Arteriosclerosis; Chromatography, High Pressure Liquid; Dinoprost; Female; Humans; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxidation; Lipoproteins, LDL; Male; Middle Aged; Oxidation-Reduction; Umbilical Veins

Topics: Animals; Arachidonic Acid; Cell Division; Cells, Cultured; Cricetinae; Cyclooxygenase 1; Cyclooxygenase 2; Embryo, Mammalian; Epidermal Growth Factor; Fibroblasts; Isoenzymes; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Mesocricetus; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Transcription, Genetic

Topics: Carbon Radioisotopes; Cells, Cultured; Endothelium, Vascular; Humans; Hydroxylation; Indomethacin; Interleukin-1; Kinetics; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Phospholipids; Radioisotope Dilution Technique; Umbilical Veins

Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Female; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Mice; Phospholipases A; Protein Kinase C; Skin Neoplasms

This study was focused on the characterization of the metabolism of linoleic acid by human dermal fibroblasts and the effect of interleukin-1 on the biosynthesis of octadecanoids. Dermal fibroblasts untreated and treated with recombinant IL-1beta were incubated with exogenous labeled linoleic acid. A combination of high performance liquid chromatography and gas chromatography-mass spectrometry was used as the analytic technique. We found that dermal fibroblasts convert linoleic acid mainly into 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) and 9-hydroxy-10-trans,12-cis-octadecadienoic acid (9-HODE), 13(S)-HODE and 9(R)-HODE being the predominant enantiomers. IL-1beta increased the formation of both 13-HODE and 9-HODE in a concentration-dependent manner with similar EC50 values as for prostanoid formation. This effect of IL-1beta on HODEs formation was concomitant with the expression of prostaglandin H-synthase-2. Formation of octadecanoids was inhibited in a concentration-dependent manner by acetylsalicylic acid and indomethacin. Dexamethasone, actinomycin D, and cycloheximide abolished the effect of IL-1beta on HODEs biosynthesis. Octadecanoid biosynthetic activity was associated with the microsomal fraction. Dermal fibroblasts incorporated [14C]-9-HODE and [14C]-13-HODE into phospholipids, mainly into phosphatidylcholine. IL-1beta increased significantly the esterification of 13-HODE in all glycerophospholipids, the major increase being observed in phosphatidylinositol. These results indicate that prostaglandin H-synthase-2 is the enzyme responsible for the increase in the ability to form HODEs of dermal fibroblasts stimulated with IL-1beta.

Topics: Cells, Cultured; Female; Fibroblasts; Humans; Interleukin-1; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Prostaglandin-Endoperoxide Synthases; Skin

Lipid mediators released by inflammatory and immune cells play an important role in inflammatory and immune processes. Most attention has been focussed on arachidonic-derived mediators, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Literature data, however, suggest that also metabolites of the unsaturated fatty acid linoleic acid may be important in this respect. We have studied the formation and release of 9-hydroxy- and 13-hydroxy-linoleic acid (9-HODE and 13-HODE) by enriched populations of human peripheral blood neutrophils, eosinophils, basophils, monocytes, and lymphocytes. We demonstrate that the eosinophil preferentially produces 13-HODE, whereas the other cell types produce equal amounts of 9-HODE and 13-HODE. The biological significance of these findings is discussed.

Topics: Arachidonic Acid; Basophils; Eosinophils; Humans; Leukocytes; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lymphocytes; Monocytes; Neutrophils

In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-beta) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-alpha and beta-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-beta expression and activity and the reversal of hyperproliferation. In contrast, although, the expression and activity of PKC-alpha was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-alpha expression was not down regulated after refeeding the safflower oil diet to the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animal Nutritional Physiological Phenomena; Animals; Blotting, Western; Cell Division; Coconut Oil; Cocos; Diet; Dietary Fats; Diglycerides; Down-Regulation; Fatty Acids, Essential; Guinea Pigs; Isoenzymes; Linoleic Acid; Linoleic Acids; Male; Plant Oils; Protein Kinase C; Safflower Oil; Signal Transduction; Skin

Topics: Animals; Cell Division; Cell Line, Transformed; Cricetinae; Embryo, Mammalian; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Linoleic Acid; Linoleic Acids; Mesocricetus; Phosphorylation; Phosphotyrosine; Signal Transduction; Tyrosine

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 determines, 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.

Topics: Adult; Amidines; Chromatography, High Pressure Liquid; Erythrocyte Membrane; Fatty Acids, Unsaturated; Humans; Hydrogen Peroxide; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxidation; Phosphatidylcholines; Phosphatidylethanolamines

The fruit body of a Basidiomycete Agaricus blazei, Jun-17 (Himematsutake) was extracted with hexane and chloroform-methanol (2:1, v/v), and the antimutagenic effect of the extracts was examined using an Ames/Salmonella/microsome assay. Both extracts of Agaricus inhibited the mutagenicity of benzo[a]pyrene(B[a]P). The hexane extract was purified by silica gel column chromatography and high performance liquid chromatography (HPLC), and linoleic acid was isolated as a main substance having antimutagenic activity. Fr. IIa, IIb, IIc and IIb, which reduced the number of His+ revertant colonies induced by B[a]P, were separated from the chloroform-methanol extract by silica gel column chromatography and HPLC. An antimutagenic substance in Fr. IIa was linoleic acid. From Fr. IIb, a bactericidal, not antimutagenic, substance was isolated and identified as 13-hydroxy cis-9, trans-11-octadecadienoic acid (13ZE-LOH). Antimutagenic substances in Fr. IIc and IId were not purified. The possible source and mechanism of formation of 13ZE-LOH are discussed.

Topics: Agaricus; Antimutagenic Agents; Drug Resistance, Microbial; Linoleic Acid; Linoleic Acids; Salmonella typhimurium

Ionizing radiation can induce the production of tumor necrosis factor (TNF-alpha) in a variety of cell types, although the signal transduction pathways that are involved have not been fully elucidated. Recently hydroxy lipids have been implicated in lipopolysaccharide (LPS)-induced expression of TNF-alpha by macrophages. We hypothesized that irradiation may act through a similar pathway. The effect of irradiation on the production of the linoleic acid derivative 13-hydroxyoctadecadienoate (13-HODE) by murine peritoneal macrophages was therefore examined and correlated with radiation-induced production of TNF-alpha. We have shown that low to intermediate doses of radiation (0.5-5 Gy) increase levels of 13-HODE, and in particular the free rather than the ester form. Irradiation also "primed" macrophages for elevated production of TNF-alpha and 13-HODE in response to LPS. Linoleate treatment in vitro and in vivo similarly enhanced the ability of macrophages to make TNF-alpha in response to LPS. Radiation-induced oxidized derivatives of linoleate may mediate many inflammatory and noninflammatory effects of irradiation. Although the mechanism by which radiation leads to production of oxidized lipid derivatives and how they interact with other elements in the TNF-alpha pathway have yet to be elucidated fully, our findings suggest an important role for lipid metabolites in radiation-induced signal transduction.

Topics: Animals; Cells, Cultured; Cobalt Radioisotopes; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gamma Rays; Linoleic Acid; Linoleic Acids; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred C3H; Tumor Necrosis Factor-alpha

Ceramides and phospholipids constitute two important structural lipids of normal skin that are notably rich in polyunsaturated fatty acids. Although linoleic acid (LA) is high in the ceramides, the localization of its 15-lipoxygenase product, 13-hydroxyoctadecadienoic acid (13-HODE) in the epidermis is unknown. In this study, we investigated the relative incorporation of [14C]LA and [14C]13-HODE into ceramides and phospholipids in isolated epidermal slices. Our data revealed minor incorporation of [14C]LA and [14C]13-HODE into ceramides. In contrast, both [14C]LA and [14C]13-HODE are markedly incorporated into phospholipids, particularly, phosphatidylcholine (PC) and phosphatidylinositol (PtdIns). The incorporation of 13-HODE into the PtdIns pool in particular prompted us to investigate into its fate in the signal transduction process and its possible incorporation into diacylglycerol. Our data revealed that 13-HODE is incorporated into epidermal phosphatidylinositol 4,5-bisphosphate (PtdIns4,5-P2) resulting in epidermal phospholipase C-catalyzed release into a novel 13-HODE-containing diacylglycerol (1-acyl-2-13-HODE-glycerol). The possibility now exists that this novel 13-HODE-containing diacylglycerol could function to modulate the activity of epidermal protein kinase C and hyperproliferation/differentiation.

Topics: Animals; Carbon Radioisotopes; Ceramides; Diglycerides; Epidermis; Glycine max; Guinea Pigs; Inositol; Linoleic Acid; Linoleic Acids; Lipoxygenase; Male; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipases A; Phospholipids; Snake Venoms; Tritium; Type C Phospholipases

Epidermal growth factor induces the oxygenation of linoleic acid in Syrian hamster embryo fibroblasts, and the lipoxygenase-derived products potentiate the mitogenic signal. We have further characterized the linoleate metabolites of growth factor-activated cells by chiral phase HPLC analysis. The primary product was identified as the pure (S) enantiomer of 13-hydroxyoctadecadienoic acid (HODE). In comparison to 13(R)-HODE isomer, only the biologically derived 13(S)-HODE was active in augmenting DNA synthesis as assessed by [3H]thymidine incorporation. To extend these investigations, we defined the structural requirements of analogous lipid compounds necessary for stimulation of mitogenesis in these cells. Carbon-chain length, degree of unsaturation, type of oxidized functionality, position of oxygenated moiety, double-bond geometry, and chirality were all identified as factors that modulate the mitogenic activity of related compounds. The results demonstrate a high degree of specificity for (S)-isomer hydro(pero)xylinoleic acid metabolites in stimulating DNA synthesis and further define the relationship between linoleic acid metabolism and growth-factor-dependent cell growth.

Topics: Animals; Cell Division; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; DNA; Dose-Response Relationship, Drug; Embryo, Mammalian; Epidermal Growth Factor; Linoleic Acid; Linoleic Acids; Mesocricetus; Stereoisomerism; Structure-Activity Relationship; Thymidine; Tritium

We have investigated whether cellular metabolism of linoleic acid (18:2) can influence prostacyclin (PGI2) production by cultured rat aortic smooth muscle cells (SMC) and tissues. Incubation of rat SMC homogenates with [1-14C]18:2 results in the enzymatic synthesis of [14C]13-HODE (hydroxyoctadecadienoic acid) and to a lesser extent [14C]9-HODE as defined by gas-liquid chromatography-mass spectrometry (GLC-MS). The observed changes, in percent enzymatically synthesized 13-HODE in the presence of indomethacin, aspirin, metyrapone, 15-HPETE (hydroperoxyeicosatetraenoic acid), and NDGA, suggest that it is formed from the PGH (prostaglandin endoperoxide) synthase pathway. Incubation of intact adherent SMC with [14C]linoleic acid demonstrates that the monohydroxylated compounds are predominantly esterified within the membrane phospholipids and not released into the incubation medium. The simultaneous incubation or a short-term preincubation of 18:2 and arachidonic acid (20:4) do not modify the enzymatic profile of 20:4 transformation. By contrast, long-term preincubation of cells with 18:2 or 13-HODE stimulates the transformation of exogenously added [14C]20:4 to [14C]6-keto PGF1 alpha. However, exogenous 13-HODE does not enhance [14C]6-keto PGF1 alpha recovery from [14C]20:4 prelabeled SMCs. Our results demonstrate that 18:2 is a substrate for PGH-synthase in rat aortic SMC and tissues. The 13-HODE formed is essentially esterified in cell phospholipids and remains without any significant effects on the release of [14C]6-keto PGF1 alpha from [14C]20:4 prelabeled SMC.

Topics: Animals; Antithrombins; Aorta; Arachidonic Acid; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Epoprostenol; Fatty Acids; Gas Chromatography-Mass Spectrometry; Linoleic Acid; Linoleic Acids; Lipoxygenase; Male; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Substrate Specificity

1-Deamino-8 D-arginine vasopressin (DDAVP) has been used effectively to normalize the bleeding time in various hemostatic disorders. In von Willebrand disease the reduction in bleeding time is due to the preferential release of large multimers of von Willebrand factor from endothelial cells. However, since the bleeding time correction in patients with uremia and liver disease is independent of the release of von Willebrand antigen and activity, other mechanisms of action of DDAVP need to be considered. Endothelial cells generate several thromborepellant factors including 13-hydroxyoctadecadienoic acid (13-HODE), an inhibitor of platelet adhesion to subendothelium. Using cultured fetal bovine aortic endothelial cells (FBAECs), we have investigated whether DDAVP modulates the production of 13-HODE. We have demonstrated that 14C-linoleic acid labeled FBAECs release several oxygenated derivatives of linoleic acid following a 120 min incubation in the presence of serum. One of these products was identified by chromatographic procedures as 13-HODE. The production of 13-HODE was decreased significantly by DDAVP (1-100 ng/ml) with maximal reduction (approx. 25%) seen at 1 ng/ml of DDAVP. While vehicle treated control FBAECs generated 6780 +/- 690 cpm of 13-HODE per 10(6) cells (mean +/- SE, n = 8), DDAVP treated FBAECs produced 4950 +/- 310 (P < 0.01), 5390 +/- 390 (P < 0.01), and 5720 +/- 410 cpm (P < 0.05) of 13-HODE at 1, 10, and 100 ng/ml DDAVP respectively. Our findings of a decrease in 13-HODE would explain the previously observed morphologic changes of increased platelet adhesion to subendothelium following DDAVP infusion and contributes to our understanding of the mode of action of this therapeutic agent in hemostatic disorders.

Topics: Animals; Aorta; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Deamino Arginine Vasopressin; Dose-Response Relationship, Drug; Endothelium, Vascular; Hemostasis; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Mass Spectrometry; Platelet Adhesiveness

Recent data indicate that platelets may play an important role in the host defence against Toxoplasma gondii infections. T. gondii-stimulated human platelets release thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE) from arachidonic acid and 13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid (Yong et al. 1991; Henderson et al. 1992). We have previously demonstrated that the eicosanoid TXA2 has potent cytotoxic activity against T. gondii trophozoites (Yong et al. 1991). In this study, we examined whether 12-HETE, 13-HODE, and linoleic acid also have toxoplasmacidal activity. 13-HODE at concentrations > or = 10(-8) M rapidly induced cytotoxic changes in T. gondii. Ultrastructural changes induced by 13-HODE in T. gondii included an initial leakage of cytoplasmic contents into a space between the inner and outer parasite bilayer membrane units which was followed by intracellular vacuolation and loss of cytoplasmic contents. In contrast, linoleic acid and 12-HETE lacked toxoplasmacidal activity at 10(-10)-10(-6) M concentrations. These data indicate that 13-HODE, a product of linoleic acid metabolism, has potent cytotoxic activity against T. gondii; this toxoplasmacidal activity may be important in the inflammatory response to this pathogen.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antithrombins; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Microscopy, Electron; Toxoplasma

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12 + 15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Aorta; Arachidonate 15-Lipoxygenase; Benzyl Compounds; Cells, Cultured; Copper; Copper Sulfate; Endothelium, Vascular; Enzyme Activation; Hydroxamic Acids; Hydroxyeicosatetraenoic Acids; Indomethacin; Linoleic Acid; Linoleic Acids; Lipoproteins, LDL; Lipoxygenase Inhibitors; Masoprocol; Oxidation-Reduction; Quinolines; Swine; Umbilical Veins

We have recently demonstrated a novel cytotoxic effect of human platelets against Toxoplasma gondii and a role for thromboxane (TX) in this process (Yong et al., 1991). We now report on the spectrum of lipid mediators released by human platelets after interaction with T. gondii. In addition to TXB2, human platelets after incubation with T. gondii for 90 min released 12-hydroxyheptadecatrienoic acid (12-HHT), 12-hydroxyeicosatetraenoic acid (12-HETE), and an unidentified peak (UVmax 234 nm) as determined by reverse-phase high-performance liquid chromatography. Thermospray-liquid chromatography/mass spectrometry analysis and straight-phase HPLC identified the unknown peak as a mixture of 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE. Radiolabeling studies with [14C]linoleic acid indicated that the platelets were the cellular source of the octadecanoids with 13-HODE (87.7%) greater than 9-HODE (12.3%). Inhibitor studies with indomethacin indicated that 13-HODE was a lipoxygenase product and 9-HODE was a cyclooxygenase product of linoleic acid. Thus, Toxoplasma-stimulated platelets release oxygenated products of both arachidonic acid and linoleic acid which may be important in the host response to T. gondii infection.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Blood Platelets; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Indomethacin; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Platelet Activation; Stereoisomerism; Toxoplasma

Apart from the generation of potent inflammatory mediators, the effects of fatty acid oxygenase activation, per se, on the host cell have not been well-delineated. Fatty acid oxygenases were activated in rat basophilic leukemia cells (RBL-1) by incubating them for 2-4 hr with 33-300 microM of arachidonic acid (AA) or linoleic acid (LA). As a control, the cells were incubated with one of two analogs of these fatty acids which are not oxygenase substrates: eicosatetraynoic acid or linoelaidic acid. Effects of oxygenase activation on cell viability were monitored by an assay for mitochondrial function. Cytotoxicity occurred in incubations with exogenous AA or LA in direct proportion to the substrate concentration but was not found in the control incubations or in incubations with the principal monohydroxylated AA products, 5-, 15-, and 12-HETE. Nordihydroguaiaretic acid (80 microM) and alpha-tocopherol (100 microM) significantly decreased the cell death observed during incubations with AA or LA. It is concluded that extensive oxygenase activation can result in cell death from intermediates produced proximal to the stable monohydroxylated derivatives.

Topics: Animals; Arachidonic Acid; Cell Survival; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Linoleic Acid; Linoleic Acids; Lipoxygenase; Masoprocol; Rats; Tumor Cells, Cultured; Vitamin E

Enzymatic transformation of the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of other n-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if these n-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 microM) and different concentrations (0-100 microM) of the n-6 fatty acids linoleic acid (LA) and dihomo-gamma-linolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50 = 45 microM) and DGLA (IC50 = 40 microM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50 = 7.5 microM and IC50 = 0.2 microM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50 = 0.75 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8,11,14-Eicosatrienoic Acid; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 15-Lipoxygenase; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; L-Lactate Dehydrogenase; Leukotriene B4; Linoleic Acid; Linoleic Acids; Mass Spectrometry; Neutrophils

Fatty acid-derived inflammatory mediators are considered to play an important role in airway hyperresponsiveness of asthmatic patients. The pulmonary macrophage may be an important source for these mediators in airway tissue. We investigated the metabolism of arachidonic acid and linoleic acid by human bronchoalveolar lavage cells, mainly comprising pulmonary macrophages. Arachidonic was mainly metabolized by 5-lipoxygenase, giving rise to the formation of leukotriene B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). Linoleic acid was converted to 5 major metabolites, including the 9-hydroxy and 13-hydroxy derivatives, 9- and 13-hydroxy-octadecadienoic acid (9- and 13-HODE). The formation of HODEs could be inhibited by cyclooxygenase inhibitors as well as lipoxygenase inhibitors, indicating that both enzymic species play a role in the generation of HODEs.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase Inhibitors; Macrophages

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Cell Adhesion; Down-Regulation; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Linoleic Acid; Linoleic Acids; Lipoxygenase; Protein Kinase C; Tumor Cells, Cultured

Topics: Animals; Cell Division; Cell Line; DNA; Epidermal Growth Factor; Fibroblasts; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Mice; Mice, Inbred BALB C; Signal Transduction

The principal in vivo oxygenase products of arachidonic acid and linoleic acid in psoriatic skin scales are 12-hydroxyeicosatetraenoic acid (R/S ratio = 5.7), 13-hydroxyoctadecadienoic acid (S/R = 1.9), and 9-hydroxyoctadecadienoic acid (R/S = 2.4). Definition of the enzymatic origin of these fatty acid derivatives is an important step in assessing their possible role in the pathogenesis of psoriasis. Psoriatic skin scales were incubated with radiolabeled arachidonic acid and linoleic acid and the monohydroxylated derivatives produced in vitro were characterized. The products of incubation with [3H]arachidonic acid were an enantiopure 15(S)-[3H]hydroxyeicosatetraenoic acid and a nonracemic mixture of the 12-[3H]hydroxyeicosatetraenoic acid steroisomers (R/S ratio = 4.5). An enantiopure 13(S)-[14C]hydroxyoctadecadienoic acid was produced from [14C]linoleic acid. No radiolabeled products were derived from incubations with heat-denatured scales. These results provide evidence for two distinct oxygenase activities that are preserved in psoriatic skin scales. One is that of an omega-6 oxygenase with strict (S) stereospecificity, consistent with the activity of a lipoxygenase. This enzyme activity appears to be similar to that of the 15-lipoxygenase which has been described in cultured human keratinocytes. The second activity is that of an arachidonic acid 12(R)-oxygenase that has not been observed in normal human epidermis but which appears to be expressed in psoriatic epidermis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Oxidation-Reduction; Oxygenases; Psoriasis; Skin; Stereoisomerism

An oxidized derivative of linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE), is dehydrogenated by an NAD+ dependent dehydrogenase present in rat colon mucosa. The product of the reaction is the 2,4-dienone, 13-oxooctadecadienoic acid. Enzyme activity was determined by HPLC analysis of incubation mixtures as well as by measuring the increase in absorbance at 285 nm, which represents formation of the 2,4-dienone chromophore. Characteristics of the reaction with respect to protein concentration, time of incubation and substrate dependence were investigated. Several inhibitors of known dehydrogenases had no effect on the 13-HODE dehydrogenase. These include, ethanol, indomethacin, 6-methyl-17-hydroxyprogesterone acetate, 4-(diethylamino)-benzaldehyde, and aspirin. The enzyme was mildly inhibited by pyrazole, 4-methylpyrazole and ibuprofen. Disulfiram was found to be a potent inhibitor of enzyme activity with an IC50 of 200 microM. Inhibitor specificity, and other characteristics of the reaction suggest the enzyme is neither alcohol dehydrogenase, diol dehydrogenase, nor a prostaglandin dehydrogenase. It is possible this enzyme plays an important role in the response of the colonic mucosa to the mitogenic effect of oxidized fatty acids.

Topics: Alcohol Oxidoreductases; Animals; Chromatography, High Pressure Liquid; Colon; Ethanol; Hydrogen-Ion Concentration; Intestinal Mucosa; Linoleic Acid; Linoleic Acids; Male; Oxidation-Reduction; Rats; Rats, Inbred Strains

Oxidation products of linoleic acid, including hydroperoxy- and hydroxyoctadecadienoic acids have been shown to possess biological activities in a number of different systems. In this work we describe an enzymatic activity which catalyzes the conversion of 13-hydroxyoctadecadienoic acid to a 2,4-dienone product, 13-oxooctadecadienoic acid. The enzyme activity is widely distributed, with the highest activity in the colon and the liver. The distribution of activity among various tissues is distinct from other dehydrogenases known to use oxygenated unsaturated fatty acids as substrates. This enzyme may play a key role in the metabolism of 13-hydroxyoctadecadienoic acid in epithelial tissues.

Topics: Alcohol Oxidoreductases; Animals; Colon; Linoleic Acid; Linoleic Acids; Linolenic Acids; Liver; Male; Organ Specificity; Oxidation-Reduction; Rats; Rats, Inbred Strains; Subcellular Fractions

Cultured epithelial cells obtained from guinea pig tracheal preparations metabolized arachidonic acid into 5- and 15-hydroxy-eicosatetraenoic acid and into the prostaglandins E2 and F2 alpha. Linoleic acid was converted by the epithelial cells into 9-hydroxy-octadecadienoic acid (9-HODE) and smaller amounts of 13-HODE. It was further investigated whether linoleic acid metabolites are of importance for the regulation of airway smooth muscle function. 13-HODE caused an increase of maximal contraction of tracheal rings to histamine, while 9-HODE had no effect.

Topics: Animals; Epithelial Cells; Epithelium; Histamine; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Muscle Contraction; Muscle, Smooth; Trachea

Reversal of essential fatty acid deficiency (EFA) induced epidermal hyperproliferation was recently suggested to require linoleic acid and an active lipoxygenase product. Because the nature of this lipoxygenase product is unknown, we employed a model of n-3 polyunsaturated fatty acid (PUFA) induced hyperproliferation in guinea pig skin to test a possible reversal of the hyperproliferation by an oxidative metabolite of linoleic acid. Topical applications of two n-3 PUFA: 0.5% of eicosapentaenoic acid (20:5n-3) and/or of docosahexaenoic acid (22:6n-3) for 5 d induced severe epidermal hyperproliferation. Development of the epidermal hyperproliferation paralleled a marked decrease in the major epidermal linoleic acid lipoxygenase product (13-hydroxyoctadecadienoic acid; 13-HODE). The application of 0.1% of 13-HODE to the n-3 PUFA-induced guinea pig hyperproliferative skin resulted in the restoration of normal epidermal histology and reversal of hyperproliferation as determined by epidermal uptake of 3H-thymidine. These data support the view that 13-HODE may represent the endogenous cutaneous mediator necessary for full restoration of cutaneous symptoms of essential fatty acid deficiency. Furthermore, the topical use of n-3 PUFA for the disruption of normal metabolism of skin n-6 EFA (linoleic acid) does serve as a useful tool for further investigations into the regulatory mechanisms of in vivo epidermal proliferation/differentiation.

Topics: Administration, Topical; Animals; Arachidonic Acid; Arachidonic Acids; Cell Division; Chemical Phenomena; Chemistry; DNA; Epidermis; Fatty Acids; Fatty Acids, Unsaturated; Guinea Pigs; Linoleic Acid; Linoleic Acids; Skin

Antibodies against 13-hydroxyoctadecadienoic acid (13-HODE) were produced in rabbits by immunizing the animal with 13-HODE-thyroglobulin conjugate. The antibodies appeared to be rather specific for 13-HODE since other hydroxy fatty acids showed minimal crossreaction. The radioimmunoassay was capable of detecting 50 pg per assay tube and was applied to the study of the biosynthesis of 13-HODE in platelets and leukocytes. In contrast to reported findings from endothelial cells, A-23187, thrombin and collagen stimulated synthesis and release of 13-HODE from platelets. However, insignificant synthesis of 13-HODE was found in leukocytes following A-23187 stimulation. Exogenous addition of linoleic acid stimulated the synthesis of 13-HODE from both platelets and leukocytes. The majority of 13-HODE synthesized was found in the medium. These studies suggest that both types of blood cells possess active (omega-6) lipoxygenase. Platelets may use endogenously released linoleic acid to synthesize 13-HODE, whereas leukocytes may utilize linoleic acid released from other cell types for 13-HODE synthesis.

Topics: Animals; Antibodies; Antibody Specificity; Antigens; Antithrombins; Blood Platelets; Calcimycin; Collagen; Leukocytes; Linoleic Acid; Linoleic Acids; Rabbits; Radioimmunoassay; Thrombin

The 15-omega-lipoxygenase enzyme in endothelial cells metabolizes endogenous linoleic acid (18:2) into 13-hydroxyoctadecadienoic acid (13-HODE) under basal conditions, i.e., in unstimulated endothelial cells. 13-HODE is thought to regulate the non-adhesivity of the endothelium, contributing to vessel wall/blood cell biocompatibility. We performed experiments, therefore, to determine the relationship between basal levels of cAMP, 13-HODE synthesis, and platelet/endothelial cell adhesion. We found that 13-HODE synthesis increased with elevated cAMP levels and that the elevated 13-HODE levels correlated with increased 18:2 turnover in the triacylglycerol pool. In contrast, neither 18:2 nor arachidonic acid (20:4) turnover in the phospholipid nor prostacyclin (PGI2) production were changed with elevated cAMP levels. Platelet/endothelial cell adhesion was inversely proportional to 13-HODE synthesis. We conclude that intracellular 13-HODE influences platelet/vessel wall interactions, is synthesized from 18:2 released from the endogenous triacylglycerol pool, and that this pathway is modulated by intracellular cAMP levels.

Topics: Arachidonic Acid; Arachidonic Acids; Cell Adhesion; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cyclic AMP; Endothelium, Vascular; Humans; Linoleic Acid; Linoleic Acids; Phospholipids; Platelet Adhesiveness; Thrombosis; Triglycerides

Locally administered 9Ds-hydroxy-10,12(E,Z)-octadecadienoic acid (9-HODE) caused a drastic inflammatory response in the experimental model of granulation tissue formation of the rat according to RUDAS (Arzneimittelforsch. 10,226-229, 1960). Three days after implantation of the polyvinyl chloride rings the granulation tissue became inhomogeneous with proliferation islets surrounded by edematous regions containing a diminished number of cells. The number of polymorphonuclear leukocytes and of macrophages was greatly enhanced in the whole tissue, whereas the number of lymphocytes was reduced. After seven days the whole granulation tissue was loosened, and its mass was twice as high as in the control animals. The number of fibroblasts per area unit and the hydroxyproline content were diminished. Linoleic acid and 13Ls-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) caused also some changes in the formation of granulation tissue, but in a different manner, in particular, without accumulation of polymorphonuclear leukocytes and macrophages, indicating the specificity of the effect of 9-HODE. The recruitment of leukocytes was not due to a direct chemotactic action of 9-HODE as shown in an agarose diffusion test comparing the effects of 9-HODE and leukotriene B4. The possible biological importance of the proinflammatory effect of 9-HODE is discussed.

Topics: Animals; Chemotaxis, Leukocyte; Fibroblasts; Foreign-Body Reaction; Granulation Tissue; Inflammation; Leukotriene B4; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Macrophages; Male; Neutrophils; Prostheses and Implants; Rats; Wound Healing

Lewis lung carcinoma cells express a plasma membrane receptor (i.e., IRGpIIb/IIIa) which is immunologically and functionally related to the platelet aggregation receptor complex (i.e., GpIIb/IIIa). Both fluorescence microscopy and flow cytometric analysis reveal that surface expression and/or activation of this tumor cell receptor is enhanced by a phorbol ester [i.e., 12-O-tetradecanoylphorbol-13-acetate (TPA)] and a lipoxygenase metabolite of arachidonic acid; 12-hydroxyeicosatetraenoic acid (i.e., 12-HETE). TPA-enhanced expression appears to be mediated by a lipoxygenase metabolite, as this effect can be reversed by lipoxygenase inhibitors but not by cyclooxygenase inhibitors. In parallel with these results both TPA and 12(S)-HETE [but not 12(R)-HETE] enhance tumor cell adhesion to endothelial cells, subendothelial matrix and fibronectin, but not to type IV collagen. TPA-enhanced adhesion can be reduced by lipoxygenase inhibitors but not by cyclooxygenase inhibitors and in addition, stimulated adhesion can be blocked by pretreatment of tumor cells with specific polyclonal or monoclonal antibodies which react against IRGpIIb/IIIa. 12(S)-HETE-enhanced adhesion can also be inhibited by these same antibodies. In contrast, a lipoxygenase product of linoleic acid, 13(S)-hydroxyoctadecadienoic acid, inhibited TPA and 12(S)-HETE-enhanced tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin. These results suggest that (a) IRGpIIb/IIIa is a multifunctional receptor which mediates tumor cell adhesion to a variety of biological substrata, (b) TPA enhances surface expression and/or activation of this receptor possibly via a lipoxygenase metabolite of arachidonic acid, and (c) these effects are opposed by a lipoxygenase metabolite of linoleic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antibodies; Antibodies, Monoclonal; Antigen-Antibody Complex; Arachidonic Acids; Cell Adhesion; Cell Membrane; Collagen; Endothelium, Vascular; Flow Cytometry; Fluorescent Antibody Technique; Hydroxyeicosatetraenoic Acids; Isomerism; Linoleic Acid; Linoleic Acids; Lipoxygenase; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Platelet Membrane Glycoproteins; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Topics: Blood Platelets; Cell Communication; Cells, Cultured; Endothelium, Vascular; Homeostasis; Humans; Linoleic Acid; Linoleic Acids; Platelet Aggregation

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.

Topics: Chromatography, High Pressure Liquid; Humans; In Vitro Techniques; Kinetics; Leukocytes; Linoleic Acid; Linoleic Acids; Oxidation-Reduction

The effect of 13-hydroxyoctadecadienoic acid (13-HODE), an endogenous lipoxygenase metabolite of linoleic acid, on prostacyclin production by fetal bovine aortic endothelial cells was evaluated. Time-dependent release of radioimmunoassayable 6KPGF1 alpha in the presence of 13-HODE (10 uM) was stimulated by 39%, 27%, and 34% at 10, 30 and 120 min respectively. 13-HODE (10 uM) had no effect on the conversion of exogenous [14C] arachidonic acid (AA) to prostacyclin. When the effect on AA release was evaluated in [14C] AA prelabeled cells, 13-HODE (10nM) stimulated the release of AA from membrane phospholipids. Analysis of cellular phospholipids revealed a significant decrease in phosphatidylethanolamine. Our results demonstrate that 13-HODE stimulates prostacyclin production by enhancing AA release from phospholipids.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Arachidonic Acids; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelium; Epoprostenol; Linoleic Acid; Linoleic Acids; Lipoxygenase; Membrane Lipids; Phospholipids

Two different lipoxygenases have been identified in human and rat epidermis. One lipoxygenase has a (n-9)-specificity, converts arachidonic acid into 12-hydroxyeicosatetraenoic acid (12-HETE), and has been described by several investigators. Linoleic acid is not a substrate for this enzyme. The other lipoxygenase, with (n-6)-specificity, converts arachidonic acid into 15-HETE and linoleic acid into 13-hydroxyoctadecadienoic acid (13-HOD). Especially the latter lipoxygenase is thought to be involved in the regulation of the differentiation of the skin cells into a proper water-barrier layer. Linoleate is supposed to be the physiological substrate; this fatty acid is especially present in characteristic sphingolipids with unique structures.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Psoriasis; Skin

Reticulocytes from various species (rat, mouse, rhesus monkey) obtained by phenylhydrazine treatment of the animals metabolized polyenoic fatty acids via a lipoxygenase pathway. Linoleic acid was converted to 13-hydro(pero)xy-9,11(Z,E)octadecadienoic acid [13-H(P)ODE] and 9-hydro(pero)xy-10,12(E,Z)octadecadienoic acid [9-H(P)ODE], whereas arachidonic acid was oxygenated to 15-hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid (15-HETE) as shown by straight-phase high-pressure liquid chromatography (SP-HPLC). Addition of calcium and ionophore A 23,187 strongly enhanced the formation of lipoxygenase products, whereas 5,8,11,14eicosatetraenoic acid (ETYA) completely inhibited their formation. Estimates of the specific radioactivities of the lipoxygenase products indicate differences in the metabolization of externally added and endogenously released polyenoic fatty acids. These results strongly suggest that lipoxygenases generally occur in immature red blood cells.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Macaca; Mice; Phenylhydrazines; Rats; Reticulocytes; Species Specificity

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Leukotriene B4; Linoleic Acid; Linoleic Acids; Lipoxygenase; Neutrophils; Phospholipids; Substrate Specificity; Swine

We have previously reported that endothelial cells synthesize a cytosol-associated, lipoxygenase-derived metabolite, LOX, which acts as a chemorepellant and, in so doing, maintains the vessel wall thromboresistance. In this study we demonstrate that LOX is a 13-hydroxylinoleic acid (13-OH-18:2) derived from linoleic acid and identical to 13-hydroxy-9-cis,11-trans-octadecadienoic acid, as measured by both reverse phase high pressure liquid chromatography and gas chromatography/mass spectrometry. In addition, we demonstrate that 13-OH-18:2 is produced in significantly greater quantities by endothelial cells than by smooth muscle cels or by fibroblasts. Furthermore, we demonstrate that 13-OH-18:2 is produced in microgram amounts under basal conditions and is decreased by thrombin, calcium ionophore, and trypsin stimulation. And finally, we demonstrate that endothelial cells do not synthesize any significant amounts of lipoxygenase-derived arachidonic acid metabolites either under basal or stimulated conditions unless exogenous arachidonic acid is added. These observations indicate that the major lipoxygenase-derived, chemorepellant metabolite produced by the endothelial is 13-hydroxy-9-cis,11-trans-octadecadienoic acid.

Topics: Antithrombins; Arachidonic Acid; Arachidonic Acids; Carbon Radioisotopes; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Female; Fibroblasts; Gas Chromatography-Mass Spectrometry; Humans; Linoleic Acid; Linoleic Acids; Lipoxygenase; Muscle, Smooth, Vascular; Pregnancy; Umbilical Veins

The breakage of double-strand (ds) DNA by 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3'-end and 5'-end-labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13-L-hydroxy-cis-9,trans-11-octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site-specific cleavage of dsDNA.

Topics: Animals; DNA, Superhelical; Drosophila melanogaster; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Piperidines; Plasmids

Rabbit peritoneal tissue contains a lipoxygenase which converts arachidonic acid preferentially into 15-hydroxy-5,8,11,13-eicosatetraenoic acid. Stereochemical analysis of the menthyloxycarbonyl derivative of this metabolite by means of a high-pressure liquid chromatography method, involving the use of a Ag+ -loaded cation-exchange column, indicated that it has mainly the 15-Ls-hydroxy configuration. The biosynthesis of 15-hydroxy-5,8,11,13-eicosatetraenoic acid could be confirmed during examination of the monohydroxy acids obtained without addition of fatty acids, thus formed from endogenously released substrate. However, the 9-and 13-hydroxy derivatives of linoleic acid were also formed and in quantities exceeding those of 15-hydroxy-5,8,11,13-eicosatetraenoic acid.

Topics: Animals; Arachidonic Acids; Chemical Phenomena; Chemistry; Hydroxyeicosatetraenoic Acids; Isomerism; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Peritoneum; Rabbits