Linolenic acid-modified methoxy poly (ethylene glycol)-oligochitosan conjugate micelles for encapsulation of amphotericin B.

Introduction of linolenic acid (LNA) and methoxy poly (ethylene glycol) (MPEG) to the backbone of oligochitosan (CS) afforded LNA-modified MPEG-CS conjugate (MPEG-CS-LNA). Amphotericin B-loaded MPEG-CS-LNA micelles (AmB-M) were prepared via dialysis method with 82.27 ± 1.96% of drug encapsulation efficiency and 10.52 ± 0.22% of drug loading capacity. The AmB-M enhanced AmB's water-solubility to 1.64 mg/mL, being 1640-folds higher than native AmB. The AmB-M obviously reduced hemolytic effect and renal toxicity of AmB when compared to marketed AmB injection (AmB-I). Its antifungal activity against Candida albicans was equivalent to AmB-I although AmB's release from AmB-M was significantly retarded. According to fluorescence microscopy test, the unchanged activity should be attributed to enhanced fungal cellular uptake of AmB-M caused by combined inducement of LNA and CS. The pharmacokinetic studies demonstrated that AmB-M also improved the pharmacokinetic parameters of AmB with AmB-I as control. Conclusively, developed LNA-modified MPEG-CS micellar system could be a viable alternative to the current toxic commercial AmB-I as a highly efficacious drug delivery system.

Chemical communication between the endophytic fungus Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum.

Paraconiothyrium variabile, one of the specific endophytic fungi isolated from the host plant Cephalotaxus harringtonia, possesses the faculty to inhibit the growth of common phytopathogens, thus suggesting a role in its host protection. A strong antagonism between the endophyte P. variabile and Fusarium oxysporum was observed and studied using optic and electronic microscopies. A disorganization of the mycelium of F. oxysporum was thus noticed. Interestingly, the biological effect of the main secondary metabolites isolated from P. variabile against F. oxysporum did not account for this strong antagonism. However, a metabolomic approach of pure fungal strains and confrontation zones using the data analysis tool XCMS were analyzed and pointed out a competition-induced metabolite production by the endophyte in the presence of the phytopathogen. Subsequent MS/MS fragmentations permitted to identify one of the induced metabolites as 13-oxo-9,11-octadecadienoic acid and highlighted a negative modulation of the biosynthesis of beauvericin, one of the most potent mycotoxin of F. oxysporum, during the competition with the endophyte.

Protection of coumarins against linoleic acid hydroperoxide-induced cytotoxicity.

Coumarins comprise a group of natural phenolic compounds found in a variety of plant sources. Protective effects of coumarins against cytotoxicity induced by linoleic acid hydroperoxide were examined in cultured human umbilical vein endothelial cells. When the cells were incubated in medium supplemented with linoleic acid hydroperoxide and coumarins, esculetin (6,7-dihydroxycoumarin) and 4-methylesculetin protected cells from injury by linoleic acid hydroperoxide. Fraxetin and caffeic acid showed weak, but significant, protection. Esculin as well as esculetin and 4-methylesculetin were effective for protecting cells against linoleic acid hydroperoxide-induced cytotoxicity in the case of pretreatment for 24 h, however fraxetin and caffeic acid showed no protection. Since esculetin was detected after 24 h treatment with esculin, a sugar moiety in the esculin molecule appears to be hydrolyzed during pretreatment. Coumarins such as umbelliferone containing only one hydroxyl group showed no protective effect in pretreatment or concurrent treatment. Esculetin and 4-methylesculetin provided synergistic protection against cytotoxicity induced by linoleic acid hydroperoxide with alpha-tocopherol. Furthermore, the radical-scavenging ability of coumarins was examined in electron spin resonance spectrometry. Esucletin, 4-methylesculetin, fraxetin, and caffeic acid showed the quenching effect on the 1,1-diphenyl-2-picrylhydrazyl radical. These results indicate that the presence of an ortho catechol moiety in the coumarin molecules plays an important role in the protective activities against linoleic acid hydroperoixde-induced cytotoxicity.

Newly recognized cytotoxic effect of conjugated trienoic fatty acids on cultured human tumor cells.

We investigated the cytotoxic effect of conjugated trienoic fatty acids on various human tumor cell lines: DLD- 1, colorectal; HepG2, hepatoma; A549, lung; MCF-7, breast; and MKN-7, stomach. Conjugated linoleic acid (CLA) and conjugated linolenic acid were prepared from linoleic acid (18:2, n-6) and alpha-linolenic acid (18:3, n-3), respectively, by treatment with 6.6% or 21% potassium hydroxide. Spectrophotometric readings at 235 nm for the conjugated diene formation, and at 268 nm for the conjugated triene, were confirmed for the respective conjugated fatty acids. In addition, tung oil (Aleurites fordii) fatty acids consisting principally of a conjugated triene (eleostearic acid, approximately 80% of total fatty acids) were prepared using an alkaline saponification procedure. All tumor cells were incubated for 24 h with 5-100 microM of the conjugated fatty acids, and MTT dye reduction was measured to verify the cell viability. Among the conjugated fatty acids examined, conjugated linolenic acid and tung oil fatty acids exhibited the most intense cytotoxic effects on DLD-1, HepG2, A549, MCF-7 and MKN-7 cells, while CLA was not cytotoxic to the tumor cells. These results demonstrate that conjugated trienoic fatty acids are more cytotoxic to human tumor cells than the conjugated dienoic fatty acid, CLA.

A study on lipid peroxide-induced lens damage in vitro.

The effects of linolenic acid hydroperoxide (LAPO) on isolated rat lenses were investigated, because they are believed to be cataractogenic in vivo. They were also compared with the effect of linolenic acid (LA), source of LAPO. Rat lenses, which were exposed for 5 hr to either 210 microM or 420 microM LAPO, became cloudy and this change was associated with increases in the level of malondialdehyde (MDA), a breakdown product of lipid peroxide, and decreases in non-protein thiol (NP-SH) content. Concomitantly, there were changes in cation contents: Na+ and Ca2+ were increased whereas K+ and Mg2+ were decreased. The changes in the levels of the above parameters, correlated with the concentration and the treatment time of LAPO to which the lenses were exposed. The increase of MDA was 2-4-fold over normal level and was consistent with those in cataractous lenses of the human and experimental animal models. On the other hand, if the lenses were exposed to LA, the only change observed was a small alteration of cationic content. If the lenses were cultured for an additional 24 hr in the absence of either LA or LAPO, the cation content continued to change only in those lenses which were previously exposed to LAPO. These results show that at concentrations of lipid peroxides, which are associated with the development of cataract in the human and animal models, there are changes in vitro in cation content, MDA and NP-SH levels, which accompany the development of a lens opacity.

Genotoxicity of heated cooking oil vapors.

Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.

Levels of thiobarbituric acid reactive substances and the cytocidal potential of gammalinolenic and docosahexaenoic acids on ZR-75-1 and CV-1 cells.

To clarify the mechanism by which gammalinolenic acid (GLA) is more tumoricidal than docosahexaenoic acid (DHA), we have compared the incorporation of the respective exogenously added ethyl esters GLAe and DHAe into the phospholipids of tumorigenic ZR-75-1 and non-tumorigenic CV-1 cells relative to the ability of the cells to survive and to accumulate thiobarbituric acid reactive substances (TBARS). GLA and DHA were incorporated in the phospholipids to the same extent, but GLA disappeared more rapidly than DHA in both cell lines. GLAe induced about twice as much intracellular TBARS as DHAe in both cell lines, but killed ZR-75-1 cells four times more effectively than DHAe. DHAe induced 11-15 fmoles malondialdehyde-equivalents (MDA-eq)/cell in both ZR-75-1 and CV-1 cells, whereas GLAe induced 5-6 times more TBARS in ZR-75-1 cells (26-30 fmoles MDA-eq/cell) than in CV-1 cells (5-6 fmoles MDA-eq/cell). The results show that there is no difference in GLA and DHA incorporation into phospholipids, but that their metabolism differs in the two cell types. The data also suggest that the cytocidal potential is related to TBARS levels in a nonlinear fashion. The relationship between excess prostaglandin production and excessive cell death due to GLA is discussed.

Effect of black-pigmented Bacteroides gingivalis on cytotoxic activity of linolenic acid against mouse macrophages.

The effects of sonicates of black-pigmented Bacteroides gingivalis (Bg-sonicates) on the cytotoxicity of linolenic acid (LA) against mouse macrophages were examined. Treatment of macrophages with LA alone or in combination with Bg-sonicates resulted in the release of lactate dehydrogenase (LDH) from the cells. This observation suggests the cytotoxicity of LA. Also, an increase in thiobarbituric acid-reactive materials was observed in experimental systems of the addition of Bg-sonicates to LA. The cytoplasm of macrophages treated with LA was not stained with May-Grünwald-Giemsa solution, while neither the nucleus nor the cytoplasm was stained when the cells were treated with LA and Bg-sonicates. The above cytotoxicity was nearly abolished when LA and Bg-sonicates were preincubated before the addition to macrophages. B. gingivalis is considered to increase the extent of the cytotoxic effects of LA to the cell nucleus by promoting peroxidation of LA.

The effects of polyunsaturated fatty acids (PUFAs) on the sleep parameters of the rat pups.

In order to verify the hypothesis that at least during perinatal life the amount of active sleep (AS) is dependent on the level of the brain maturation, newborn rats received by an i.p. route for 6 days, a polyunsaturated fatty acid (PUFAs) preparation (Vitamin F) in doses of 1, 10 and 100 mg/day. In comparison with diluent alone injected controls, the PUFAs-injected animals presented a decrease in the AS percentage, decrease which was proportional with the dose. In parallel, it was noted a dose-dependent increase in mortality from 10% for 1 to 90% for 100 mg of PUFAs. We concluded that the PUFAs which greatly stimulate the synaptogenesis, enhance the release of a membrane component with a strong AS inhibiting effect, and, at least during perinatal life, the AS could be modulated by this factors and not by the classical neurotransmitters.

Antitumor effect of gamma-linolenic acid on cultured human neuroblastoma cells.

gamma-linolenic acid (GLA) was found to suppress the cell growth of 4 human neuroblastoma cell lines. GOTO was the most sensitive, followed by SK-N-DZ and NKP, while NCG was much less sensitive to GLA. In terms of GLA cytotoxicity, neither cyclo-oxygenase inhibitors nor a lipoxygenase inhibitor showed any effect. On the other hand, 4 antioxidants (Coenzyme Q, (D) alpha-tocopherol, (DL) alpha-tocopherol, butylated hydroxytoluene) reduced the growth inhibitory effect of GLA, but not in proportion to the decrease of GLA-stimulated lipid peroxidation. Accordingly, prostaglandins and leukotrienes probably do not play a role, and lipid peroxide may only be partly involved in the GLA effect.

Inhibition of sperm motility and agglutination of sperm cells by free fatty acids in whole semen.

The effects of a serial dilution of linoleic acid on human spermatozoa in whole semen was tested on 21 semen samples obtained from 11 normal volunteers. The minimal concentration of linoleic acid required to stop the movement of at least 75% of the moving sperm ranged from 1 to greater than 100 mg/dl. Fifteen of 21 (71%) of the semen samples were inhibited by added free fatty acids (FFA) concentrations that were less than or close to the physiologic concentration ranges of FFA in blood plasma (1 to 30 mg/dl). The immobilized sperm often formed aggregates similar to those formed by the action of autoantibodies against sperm cells. Preliminary studies conducted on a variety of other FFA have indicated that oleic acid (18/1) was less toxic than linoleic acid (18/2) and that linolenic acid (18/3) was more toxic than linoleic acid. The saturated FFA palmitic acid (16/0) and stearic acid (18/0) at concentrations up to 100 mg/dl showed little or no toxicity to sperm cells. It is suggested that FFA toxicity be included among physiologic factors that affect the motility and spontaneous aggregation of sperm cells.

Mutagenicity tests of lipid oxidation products in Salmonella typhimurium: monohydroperoxides and secondary oxidation products of methyl linoleate and methyl linolenate.

Nine hydroperoxy and hydroperoxy-epidioxy oxidation products derived from either autoxidation (AO) or photosensitized oxidation (PO) of methyl linoleate (MLo) or methyl linolenate (MLn) were tested for mutagenic activity by the Salmonella typhimurium his+ reversion assay using strains TA100, TA98, TA102, TA97 and TA1537. All nine oxidation products, monohydroperoxides from AO-MLn (I) or from PO-MLn (II), dihydroperoxides from PO-MLo (III), AO-MLn (IV) or PO-MLn (V), hydroperoxy epidioxides from PO-MLo (VI), AO-MLn (VII) or PO-MLn (VIII) and hydroperoxy bis-epidioxides from PO-MLn (IX), were weakly mutagenic in strains TA97 and/or TA100. The hydroperoxy epidioxides (VI-IX) exhibited significantly greater activity in strain TA97 than did the monohydroperoxides (I, II) or the dihydroperoxides (III-V). In strain TA100, all of the oxidation products tested exhibited similar activity. No major differences between products derived from autoxidized and photooxidized MLn (I v. II, IV v. V, VII v. VIII) were obtained. Rat-liver S-9 reduced the toxicity of all oxidation products to the tester strains. The greatest mutant yields were usually obtained in the presence of S-9, but mutagenic potency was sometimes greater without S-9. The structural feature common to all of the mutagenic oxidation products was the presence of a hydroperoxy group, suggesting that this characteristic is responsible for the observed mutagenicity, either directly or through a common degradative pathway to reactive products of lower molecular weight.

Toxicity of free fatty acids for cultured rat heart muscle and endothelioid cells. II. Unsaturated long-chain fatty acids.

Oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and arachidonic (C20:4) acids were compared for their toxic effects upon cultured rat heart muscle and endothelioid cells. The free fatty acids (FFA) were bound to albumin (6:1) and tested at concentrations from 5 x 10(-5)M to 5 x 10(-4)M. Reduction of cell viability (51Cr release) and in situ mitochondrial and lysosomal labilization were used as indices of injury. Oleic acids was non-toxic at all times and concentrations tested while linoleic acid increased cell death only in muscle cells after 32 h. Arachidonic acid, by contrast, demonstrated significant toxicity as early as 2 h while both linolenic and arachidonic acids produced major injury at longer durations. A detergent effect was excluded as the injury mechanism because of marked differences in the toxicities of the individual FFA. The similarity in the effects of linolenic and arachidonic acids would appear to exclude prostaglandins as responsible toxic products.

[Highly-sensitive method of studying the toxicity of lipid peroxides]. / Vysokochuvstvitel'nyi metod issledovaniia toksichnosti perekisei lipidov

A highly sensitive and simple method is developed for study of lipid peroxides toxicity with the use of quail developing embryos as a model. Toxicity of linolenic acid hydroperoxides, obtained by the lipoxygenase catalysis, was distinctly higher than the toxicity of the peroxides, produced alter autooxidation (LD50 = 0.12 and 0.38 mc-eq/embryo, respectively). Occurrence of toxic properties was observed in lipids, isolated from tissues of mice subjected to the oxygen stress. The advantages of the method described are discussed as compared with the published data.