Suppression of tumour-promoting factors in fat-induced colon carcinogenesis by the antioxidants caroverine and ubiquinone.

Fatty acid hydroperoxides are produced from unsaturated fatty acids in the presence of oxygen at elevated temperatures during food processing. Their effects on gene expression in colorectal tumour cells were studied using linoleic acid hydroperoxide (LOOH) as a model compound. Addition of LOOH to the medium of LT97 adenoma and SW480 carcinoma cells enhanced the production of hydrogen peroxide. Both cell lines were observed to increase VEGF factors based on mRNA. High consumption of dietary fat promotes colon carcinogenesis in the long-term. While this effect is well known, the underlying mechanisms are not understood. An approach was made starting from the assumption that LOOH is present in dietary fats as a result of heating. LOOH undergoes homolytic cleavage in the presence of iron. Various radicals are formed on mixing LT97 or SW480 cells with LOOH. The expression of tumour-promoting factors was inhibited by caroverine and ubiquinone, which may be justified as active chemopreventive agents.

Antioxidant activity of bakuchiol: experimental evidences and theoretical treatments on the possible involvement of the terpenoid chain.

The protective activity of the plant-derived meroterpene, bakuchiol [1-(4-hydroxyphenyl)-3,7-dimethyl-3-vinyl-1,6-octadiene, 1], against oxidative damages to lipids and proteins has been investigated and rationalized based on the scavenging activity of 1 against various oxidizing radicals (Cl(3)CO(2)(*), linoleic acid peroxyl radicals, LOO(*), DPPH radicals, (*)OH, and glutathiyl radicals). The rate constants of the scavenging reactions, transients formed in these reactions, and their mechanistic pathways have been probed using optical pulse radiolysis technique. Besides 1, its methyl ether derivative 2 also could prevent lipid peroxidation in rat brain homogenate, indicating the probable participation of their terpenoid chains in scavenging LOO(*). This was further corroborated from the pulse radiolytic studies on the reaction between the glutathiyl radicals and the compounds 1 and 2 as well as two other congeners, 3 and 4, which showed transient absorptions at approximately 300 nm attributable to some C-centered allylic radicals. On the basis of the strong signals at approximately 300 nm with 1-3 as compared to compound 4, formation of the allylic radical adjacent to the trisubstituted olefin function in 1-3 was envisaged. This was confirmed by quantum chemical calculations of the relative energies of the probable radical species derivable from 2 using Hartree-Fock and density functional theory along with self-consistent reaction field model. In the case of 1, the allylic radical was found to be transformed into the phenoxyl radical at a later stage. All of these data revealed, for the first time, the importance of the terpenoid moiety of bakuchiol in controlling its antioxidant action via radical scavenging.

Kinetics of inhibition of leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.

Lipoxygenases (LOXs) are a ubiquitous family of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Their role in a diverse range of biological processes has prompted the development of a large number of lipoxygenase inhibitors of possible therapeutic and probative value. The isoform-selective inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was previously shown to inhibit leukocyte-type 12-LOX by a novel mechanism in which it binds to both the ferrous and ferric forms of the enzyme. The current study provides a detailed kinetic model of this inhibition. Nonlinear regression analysis of OPP's inhibition of arachidonic acid dioxygenation indicated mixed inhibition toward the ferric form of 12-LOX with apparent K(I) values in the low micromolar range: 2.0 +/- 0.2 microM for the free enzyme and 4.5 +/- 0.7 microM for the substrate-bound form of the enzyme. Rapid kinetic techniques allowed OPP's inhibition of the activation of the enzyme from the ferrous to the ferric form to be investigated. Titration of ferrous 12-LOX with OPP indicated that it bound to the ferrous form with an apparent K(I) value of 70 +/- 20 nM, suggesting a significantly higher affinity for the ferrous form than for the ferric form of the enzyme. Investigation of the LOX inhibitors nordihydroguaiaretic acid, N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, BWA137C, and eicosatetraynoic acid revealed that eicosatetraynoic acid also inhibited the activation of 12-LOX. These results demonstrate that LOX inhibitors are capable of binding to multiple forms of LOXs with high affinity and suggest that inhibition of enzyme activation may be an unrecognized mechanism of inhibition of additional LOX inhibitors.

Flavonoids suppress the cytotoxicity of linoleic acid hydroperoxide toward PC12 cells.

The suppressive effect of flavonoids on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat phenochromocytoma PC12 cells was examined. The extent of cytotoxicity was shown on the basis of % survival determined by the trypan blue exclusion test. On preincubation of cells with either 3-hydroxyflavone, quercetin, or luteolin prior to LOOH exposure, the cytotoxicity was considerably suppressed. In contrast, on coincubation of cells with either eriodictyol, quercetin, kaempherol, luteolin, or 3-hydroxyflavone and LOOH, it was markedly suppressed. Regardless of incubation conditions, quercetin, 3-hydroxyflavone, and luteolin were thus more effective as protective agents against the cytotoxicity than the other flavonoids. These flavonoids further showed a suppressive effect on coincubation rather than on preincubation. These results suggest that such flavonoids are beneficial for cells under oxidative stress.

Mechanism of inhibition of porcine leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.

The mechanism of inhibition of porcine leukocyte 12-lipoxygenase by 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was investigated. This compound is selective for the leukocyte form of the 12-lipoxygenase and inhibits the purified recombinant enzyme with an IC(50) value of approximately 2 microM. OPP induced a concentration-dependent lag phase in the oxygenation of arachidonic acid and decreased the maximal rate of reaction. Addition of the fatty acid hydroperoxide 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE) to the reaction greatly reduced the OPP-induced lag. Lineweaver-Burk analysis of the effect of OPP on 12-lipoxygenase kinetics with arachidonic acid indicated that it was a mixed-type inhibitor. OPP was not metabolized by 12-lipoxygenase as evidenced by its quantitative recovery from incubations with stoichiometric amounts of enzyme and 13-HPODE or arachidonic acid. OPP inhibited the pseudoperoxidase activity of the enzyme with 13-HPODE and the reducing agent, BWA137C. Lineweaver-Burk analysis of the effect of OPP on pseudoperoxidase kinetics suggested that OPP was competitive with 13-HPODE. Single-turnover experiments indicated that OPP inhibited the reduction of 13-HPODE by a stoichiometric amount of ferrous 12-lipoxygenase. Addition of 13-HPODE shortened the OPP-induced lag phase but did not affect the maximal rate of enzyme activity. In addition, OPP had no effect on total product formation in either the presence or the absence of 5 microM 13-HPODE when the reaction was allowed to go to completion. All of these observations are consistent with a model for inhibition of 12-lipoxygenase activity in which OPP slows the oxidation of the inactive ferrous enzyme to the active ferric enzyme and competes with arachidonic acid for the ferric enzyme.

Inhibition of linoleic acid hydroperoxide-induced toxicity in cultured human umbilical vein endothelial cells by catechins.

The protective effect of catechins, major components of green tea, was studied in cultured human umbilical vein endothelial cells exposed to toxicity induced by linoleic acid hydroperoxide (LOOH). In the case where cells were incubated in medium containing both LOOH and catechins, (+)-catechin (C) was effective in suppressing of LOOH-induced cytotoxicity, but (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) had no effect. EGCG monoglucoside (EGCG-G1) and EGCG diglucoside (EGCG-G2), apophilic derivatives of EGCG, show a protective effect on LOOH-induced cytotoxicity when present at the time of treatment with LOOH. On the other hand, when cells were incubated with catechins for 24 h before treatment with LOOH there was no protection against the oxidative damage by LOOH. Furthermore, the interaction between catechins and alpha-tocopherol was examined under these culture conditions. C showed a synergistic effect with alpha-tocopherol in protecting against LOOH-induced damage. These results suggest that catechins interact with LOOH present in the medium or near the surface of membranes, but not with LOOH incorporated into cellular membranes and that catechins are able to interact with alpha-tocopherol to provide synergistic protection against the cytotoxicity of LOOH.

Unsaturated fatty acids are required for continuous proliferation of transformed androgen-dependent cells by fibroblast growth factor family proteins.

Increase in dietary fat intake has been reported to be associated with progression of hormone-dependent cancers. To explore its mechanism, we examined the effects of fatty acids on the growth of androgen-dependent SC-3 cells cloned from mouse mammary cancer (Shionogi carcinoma 115). Their androgen-dependent growth was potentiated by linoleic acid in the defined medium. The effect of linoleic acid on fibroblast growth factor (FGF)-dependent growth was also addressed because androgen had been demonstrated to exert its mitogenic activity on SC-3 cells through an induction of the unique FGF family protein termed as androgen-induced growth factor. Exposure of SC-3 cells to basic FGF or androgen-induced growth factor exhibited only transient growth response. However, simultaneous addition of linoleic acid to the medium sustained the proliferation of FGF-stimulated, but not FGF-unstimulated, cells, although linoleic acid did not exert the significant effect on the process of S-phase entry of basic FGF-stimulated cells. Palmitoleic acid and oleic acid appeared to exert the actions similar to linoleic acid, while stearic acid was without any effect. Neither cyclooxygenase inhibitor nor 5-lipoxygenase inhibitor could block the growth-promoting ability of linoleic acid. Linoleic acid also enhanced their anchorage-independent growth in the presence of basic FGF. These results indicate that these unsaturated fatty acids play a role in sustaining the proliferation of FGF-stimulated SC-3 cells.

Protection of linoleic acid hydroperoxide-induced cytotoxicity by phenolic antioxidants.

The protective effects of phenolic antioxidants on linoleic acid hydroperoxide (LOOH)-induced toxicity to cultured human umbilical vein endothelial cells were examined. Our previous results were confirmed that for tocopherol homologs, lipophilicity and the presence of a phenolic hydroxyl group and two alkyl groups at its ortho positions are critical for protection against LOOH-induced cytotoxicity. Probucol and butylated hydroxytoluene (BHT) were more effective than other simple alkylated phenols. It was found that the protective effects of alkylated phenols were depended on by the presence of two alkyl groups; in particular, two tert-butyl groups, at positions ortho to a hydroxyl group and an alkyl group at the para position. Among alpha-tocopherol, 2,2,5,7,8-pentamethylchroman-6-ol, and BHT, the relative effectiveness of protection against the cytotoxicity (1.0:0.86:0.58, respectively) was inconsistent with the previously reported, relative antioxidant activity in homogeneous solution (1.0:1.2:0.004, respectively). Probably, the effectiveness of protection by phenolic antioxidants against the cytotoxicity depend primarily on their incorporation rate into cells due to their lipophilicity, secondly on their antioxidant activity, and thirdly on their orientation in biomembranes.

Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.

The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.

Injury induced by fatty acids or bile acid in isolated human colonocytes prevented by calcium.

Measurement of the modulation of the growth fraction of isolated normal colonocytes from adult subjects in primary monolayer culture was used as a sensitive quantitative assay to evaluate toxic effects of several endogenous compounds found within the colon. This assay was used to study the role of CaCl2 in blocking cell injury. When added simultaneously with the injurious agent, 5-10 mM CaCl2 blocked the toxicity of physiological concentrations of deoxycholic acid, oleic acid, palmitic acid and linoleic acid.

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets.

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.

[Protective effect of alpha-linolenic acid in encephalomalacia in chickens]. / L'effet protecteur de l'acide alpha-linolénique sur l'encéphalomalacie chez le poulet.

Encephalomacia is a vitamin E deficiency syndrome which affects the cerebellum of young chicks. The lesion includes degenerative alterations of cellular and fibrillar elements, apparently as the result of the ischaemia caused by thrombotic events in the microvascular system. A supply of linoleic acid, as fatty acid methyl esters prepared from safflower oil (Carthamus tinctorius), caused a high incidence of encephalomalacia. On the other hand, linseed oil esters, rich in alpha-linolenic acid, did not induce any symptoms and protected the chicks to a large extend against the development of signs produced by linoleic acid. Fatty acid esters of cod liver oil, rich in long-chain derivatives of alpha-linolenic acid, exerted a relatively weak protective effect. The analytical results show that a supply of alpha-linolenic acid led to an accumulation of eicosapentaenoic acid, 20:5 omega 3, and a reduced concentration of arachidonic acid in the phospholipds of liver and plasma. The results suggest that, under the conditions leading to encephalomalacia, the prostacyclin-thromboxane balance is shifted in direction of an excessive production of TXA2, causing thrombus formation in the capillaries of the cerebellum, alpha-linolenic acid, by modifying the PUFA profile, exerts a multiple action the main result of which appears to be an antithrombotic effect at the level of the microvascular system of the cerebellum.