Combination of Lutein and Zeaxanthin, and DHA Regulated Polyunsaturated Fatty Acid Oxidation in H2O2-Stressed Retinal Cells.

Photochemical and oxidative damages in retinal pigment epithelial (RPE) cells are key events in the pathogenesis of age-related macular degeneration. Polyunsaturated fatty acids (PUFA) and carotenoids are rich in retinal cells, and under oxidative stress leads to oxidation and release lipid mediators. We evaluated the impact of carotenoids (lutein, zeaxanthin) and docosahexaenoic acid (DHA) supplementation on RPE cells under oxidative stress. ARPE-19 cells were exposed to H2O2 after pre-treatment with lutein, zeaxanthin, DHA, lutein + zeaxanthin or lutein + zeaxanthin with DHA. The data showed H2O2 reduced cell viability and DHA content, while promoted catalase activity and certain oxidized PUFA products. Treatment with DHA enhanced omega-3 PUFA enzymatic oxidation namely, anti-inflammatory mediators such as hydroxy-DHA, resolvins and neuroprotection compared to control; the effects were not influenced by the carotenoids. Omega-6 PUFA oxidation, namely pro-inflammatory HETE (5-, 9-, 12 and 20-HETE), and isoprostanes (5- and 15-F2t-IsoP and 4-F3t-IsoP) were reduced by lutein + zeaxanthin while the addition of DHA did not further reduce these effects. We observed transcriptional regulation of 5-lipoxygenase by DHA and GPx1 and NEFEL2 by the carotenoids that potentially resulted in decreased HETEs and glutathione respectively. 4-HNE was not affected by the treatments but 4-HHE was reduced by lutein + zeaxanthin with and without DHA. To conclude, carotenoids and DHA appeared to regulate inflammatory lipid mediators while the carotenoids also showed benefits in reducing non-enzymatic oxidation of omega-6 PUFA.

NGF blocks polyunsaturated fatty acids biosynthesis in n-3 fatty acid-supplemented PC12 cells.

Regulation of polyunsaturated fatty acid (PUFA) biosynthesis in proliferating and NGF-differentiated PC12 pheochromocytoma cells deficient in n-3 docosahexaenoic acid (DHA 22:6n-3) was studied. A dose- and time-dependent increase in eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and DHA in phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) glycerophospholipids (GPL) via the elongation/desaturation pathway following alpha-linolenic acid (ALA, 18:3n-3) supplements was observed. That was accompanied by a marked reduction of eicosatrienoic acid (Mead acid 20:3n-9), an index of PUFA deficiency. EPA supplements were equally effective converted to 22:5n-3 and 22:6n-3. On the other hand, supplements of linoleic acid (LNA, 18:2n-6) were not effectively converted into higher n-6 PUFA intermediates nor did they impair elongation/desaturation of ALA. Co-supplements of DHA along with ALA did not interfere with 20:5n-3 biosynthesis but reduced further elongation to 22-hydrocarbon PUFA intermediates. A marked decrease in the newly synthesized 22:5n-3 and 22:6n-3 following ALA or EPA supplements was observed after nerve growth factor (NGF)-induced differentiation. NGF also inhibited the last step in 22:5n-6 formation from LNA. These results emphasize the importance of overcoming n-3 PUFA deficiency and raise the possibility that growth factor regulation of the last step in PUFA biosynthesis may constitute an important feature of neuronal phenotype acquisition.

Synthesis of long-chain polyunsaturated fatty acids is inhibited in vivo in hypercholesterolemic rabbits and in vitro by oxysterols.

Plasma total lipids, total cholesterol (cholesterol esters and free cholesterol) and oxysterol (mainly 7 beta-hydroxycholesterol (7 beta OH)) concentrations were significantly elevated in New Zealand rabbits fed a 2% cholesterol-containing diet with respect to controls fed the same diet without cholesterol. In addition, linoleic (18:2 n-6) and alpha-linolenic acid (18:3 n-3) plasma concentrations were significantly elevated in hypercholesterolemic rabbits, while concentrations of long-chain n-6 and n-3 derivatives were reduced. Studies in monocytic cell line THP-1 revealed that 7 beta OH markedly inhibited the conversion of 18:2 to 20:4 n-6 and of 18:3 to 22:6 n-3, indicating depression of the desaturation steps; in particular the inhibition was greater for the Delta 5 desaturation step. Furthermore, experiments of Real-Time PCR showed that 5-10 microM 7 beta OH decreased the Delta 5 gene expression. In conclusion, atherogenic oxysterols interfere with the production of long-chain polyunsaturated fatty acids from their precursors both in hypercholesterolemic rabbits and in cultured cells.

Hepatoprotective role of ferulic acid: a dose-dependent study.

Alcohol use is contributing to an unprecedented decline in life expectancy. Damage to the liver after ethanol administration is a well-known phenomenon. Free radical mechanisms have been proposed to play a part in ethanol-induced liver toxicity. Ingestion of diets rich in polyunsaturated fatty acids (PUFAs) along with alcohol is known to result in enhanced liver damage. The present work is aimed at evaluating the protective role of ferulic acid, a naturally occurring plant component, on alcohol- and PUFA-induced liver toxicity. Three different doses of ferulic acid (10, 20, and 40 mg/kg of body weight) were administered to rats given alcohol, heated PUFA (DeltaPUFA), and alcohol + DeltaPUFA. Influence of ferulic acid on alcohol-and PUFA-induced liver damage was evaluated by analyzing the activities of the liver marker enzymes alkaline phosphatase, gamma-glutamyl transferase, alanine transaminase, and aspartate transaminase. The activities of these liver marker enzymes were increased in the alcohol, DeltaPUFA, and alcohol + DeltaPUFA groups but were decreased significantly on treatment with ferulic acid. The low dose (10 mg/kg of body weight) was not effective, but both 20 mg and 40 mg/kg of body weight were found to be effective. The 20 mg/kg of body weight dose was found to be more effective than 40 mg/kg of body weight (the high dose). The administration of ferulic acid to normal rats did not produce any harmful effects. Thus our results show that ferulic acid is an effective anti-hepatotoxic agent without side effects and may be a good candidate in the current search for a natural hepatoprotective agent.

[The endocannabinoid system as a target for the development of new drugs for cancer therapy]. / Il sistema endocannabinoide quale bersaglio di terapie anti-tumorali. Conoscenze attuali e prospettive.

Studies on the main bioactive components of Cannabis sativa, the cannabinoids, and particularly delta 9-tetrahydrocannabinol (THC), led to the discovery of a new endogenous signalling system that controls several physiological and pathological conditions: the endocannabinoid system. This comprises the cannabinlid receptors, their endogenous agonists--the endocannabinoids--and proteins for endocannabinoid biosynthesis and inactivation. Recently, evidence has accumulated indicating that stimulation of cannabinoid receptors by either THC or the endocannabinoids influence the intracellular events controlling the proliferation and apoptosis of numerous types of cancer cells, thereby leading to anti-tumour effects both in vitro and in vivo. This evidence is reviewed here and suggests that future anti-cancer therapy might be developed from our knowledge of how the endocannabinoid system controls the growth and metastasis of malignant cells.

Therapeutic potential of cannabinoids in CNS disease.

The major psychoactive constituent of Cannabis sativa, delta(9)-tetrahydrocannabinol (delta(9)-THC), and endogenous cannabinoid ligands, such as anandamide, signal through G-protein-coupled cannabinoid receptors localised to regions of the brain associated with important neurological processes. Signalling is mostly inhibitory and suggests a role for cannabinoids as therapeutic agents in CNS disease where inhibition of neurotransmitter release would be beneficial. Anecdotal evidence suggests that patients with disorders such as multiple sclerosis smoke cannabis to relieve disease-related symptoms. Cannabinoids can alleviate tremor and spasticity in animal models of multiple sclerosis, and clinical trials of the use of these compounds for these symptoms are in progress. The cannabinoid nabilone is currently licensed for use as an antiemetic agent in chemotherapy-induced emesis. Evidence suggests that cannabinoids may prove useful in Parkinson's disease by inhibiting the excitotoxic neurotransmitter glutamate and counteracting oxidative damage to dopaminergic neurons. The inhibitory effect of cannabinoids on reactive oxygen species, glutamate and tumour necrosis factor suggests that they may be potent neuroprotective agents. Dexanabinol (HU-211), a synthetic cannabinoid, is currently being assessed in clinical trials for traumatic brain injury and stroke. Animal models of mechanical, thermal and noxious pain suggest that cannabinoids may be effective analgesics. Indeed, in clinical trials of postoperative and cancer pain and pain associated with spinal cord injury, cannabinoids have proven more effective than placebo but may be less effective than existing therapies. Dronabinol, a commercially available form of delta(9)-THC, has been used successfully for increasing appetite in patients with HIV wasting disease, and cannabinoid receptor antagonists may reduce obesity. Acute adverse effects following cannabis usage include sedation and anxiety. These effects are usually transient and may be less severe than those that occur with existing therapeutic agents. The use of nonpsychoactive cannabinoids such as cannabidiol and dexanabinol may allow the dissociation of unwanted psychoactive effects from potential therapeutic benefits. The existence of other cannabinoid receptors may provide novel therapeutic targets that are independent of CB(1) receptors (at which most currently available cannabinoids act) and the development of compounds that are not associated with CB(1) receptor-mediated adverse effects. Further understanding of the most appropriate route of delivery and the pharmacokinetics of agents that act via the endocannabinoid system may also reduce adverse effects and increase the efficacy of cannabinoid treatment. This review highlights recent advances in understanding of the endocannabinoid system and indicates CNS disorders that may benefit from the therapeutic effects of cannabinoid treatment. Where applicable, reference is made to ongoing clinical trials of cannabinoids to alleviate symptoms of these disorders.

Differential induction of plant volatile biosynthesis in the lima bean by early and late intermediates of the octadecanoid-signaling pathway.

Plants are able to respond to herbivore damage with de novo biosynthesis of an herbivore-characteristic blend of volatiles. The signal transduction initiating volatile biosynthesis may involve the activation of the octadecanoid pathway, as exemplified by the transient increase of endogenous jasmonic acid (JA) in leaves of lima bean (Phaseolus lunatus) after treatment with the macromolecular elicitor cellulysin. Within this pathway lima bean possesses at least two different biologically active signals that trigger different biosynthetic activities. Early intermediates of the pathway, especially 12-oxo-phytodienoic acid (PDA), are able to induce the biosynthesis of the diterpenoid-derived 4,8, 12-trimethyltrideca-1,3,7,11-tetraene. High concentrations of PDA result in more complex patterns of additional volatiles. JA, the last compound in the sequence, lacks the ability to induce diterpenoid-derived compounds, but is highly effective at triggering the biosynthesis of other volatiles. The phytotoxin coronatine and amino acid conjugates of linolenic acid (e.g. linolenoyl-L-glutamine) mimic the action of PDA, but coronatine does not increase the level of endogenous JA. The structural analog of coronatine, the isoleucine conjugate of 1-oxo-indanoyl-4-carboxylic acid, effectively mimics the action of JA, but does not increase the level of endogenous JA. The differential induction of volatiles resembles previous findings on signal transduction in mechanically stimulated tendrils of Bryonia dioica.

Cytotoxic action of cis-unsaturated fatty acids on human cervical carcinoma (HeLa) cells in vitro.

The effect of n-3 and n-6 fatty acids (FAs) on the growth of human cervical carcinoma (HeLa) cells was studied. Of all the FAs tested, docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) were found to be the most potent in their cytotoxic action on HeLa cells and the potency of various fatty acids with regard to their cytotoxic action was as follows: DHA > EPA > dihomo-gamma-linolenic acid (DGLA) = gamma-linolenic acid (GLA) > linoleic acid (LA) > arachidonic acid (AA) > alpha-linolenic acid (ALA). The cycloxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguaretic acid (NDGA), the antioxidants vitamin E, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), the superoxide anion quencher superoxide dismutase (SOD), the hydroxyl and hydrogen peroxide quenchers mannitol and catalase, respectively, and the calmodulin antagonists trifluoperazine (TFP) and chlorpromazine (CPZ) could all block the cytotoxic action of GLA, which was used as a representative cytotoxic FA, on HeLa cells. On the other hand, copper and iron salts and buthionine sulfoxamine, a glutathione (GSH) depletor, potentiated the cytotoxic action of suboptimal doses of GLA. GLA-induced radical generation and lipid peroxidation in HeLa cells could be blocked by indomethacin, NDGA and calmodulin antagonists. The cytotoxic action of cis-unsaturated fatty acids (c-UFAs) is not dependent on the alteration in the protein kinase C levels since no alteration in the diacylglycerol levels was observed. Hydroxy and hydroperoxy products of GLA were found to be toxic to HeLa cells, whereas prostaglandin (PG)E1, PGF2 alpha, and prostacyclin stimulated cell growth. From these results, it is evident that radicals are the modulators of the cytotoxic action of c-UFAs, that their formation is a calmodulin-dependent process, and that lipoxygenase products may mediate the tumoricidal action of FAs.

Dietary polyunsaturated fatty acid regulation of gene transcription.

We have known for nearly 30 years that dietary polyenoic (n-6) and (n-3) fatty acids potentially inhibit hepatic fatty acid biosynthesis. The teleological explanation for this unique action of PUFAs resides in their ability to suppress the synthesis of (n-9) fatty acids. By inhibiting fatty acid biosynthesis, dietary PUFAs reduce the availability of substrate for delta 9 desaturase (7, 22, 34, 36) and in turn reduce the availability of (n-9) fatty acids for incorporation into plasma membranes. In this way, essential biological processes dependent on essential fatty acids (e.g. reproduction and trans-dermal water loss) continue to operate normally. Therefore, if essential fatty acid intake did not regulate (n-9) fatty acid synthesis, the survival of the organism would be threatened. During the past 20 years, we have gradually elucidated the cellular and molecular mechanisms by which dietary PUFAs modulate fatty acid biosynthesis and (n-9) fatty acid availability. Central to this mechanism has been our ability to determine that dietary PUFAs regulate the transcription of genes coding for lipogenic enzymes (12, 40). The potential mechanisms by which PUFAs govern gene transcription are numerous, and it is unlikely that any one mechanism can fully elucidate the nuclear actions of PUFA. The difficulty in providing a unifying hypothesis at this time stems from: (a) the many metabolic routes taken by PUFAs upon entering the hepatocyte (Figure 1); and (b) the lack of identity of a specific PUFA-regulated trans-acting factor. However, the studies described above indicate that macronutrients, like PUFA, are not only utilized as fuel and structural components of cells, but also serve as important mediators of gene expression (12, 14, 40). As regulators of gene expression, PUFAs (or metabolites) are thought to affect the activity of transcription factors, which in turn target key cis-linked elements associated with specific genes. Whether this targeting involves DNA-protein interaction or the interaction of PUFA-regulated factors is unclear. A better understanding of the nuclear actions of PUFA will clarify the role of these compounds in lipid metabolism and lead to a better understanding of the role of PUFAs in disease processes such as insulin-resistant diabetes and certain forms of cancer.

Polyunsaturated fatty acids in the low-birth-weight infant.

The essentiality of certain PUFAs is related to their capability to be incorporated into lipids and to act as precursor in the formation of prostaglandins. Via phospholipids the EFA's influence the physico-chemical characteristics of biomembranes. EFAs are metabolized differently from nonessential PUFAs. While the nonessential fatty acids are metabolized rapidly, the organism tends to conserve the stores of EFAs. Inhibitions and competitions among the EFAs of the three series (oleic, linoleic, and alpha-linolenic) have been demonstrated. Apparently, for any given chain length the more unsaturated fatty acid has a greater affinity for the enzyme system responsible for further elongation and desaturation. EFAs are also necessary for the proper utilization of the saturated fatty acids. Vitamin E and pyridoxine seem to be involved in EFA metabolism. Normal growth of infants is dependent upon an adequate supply of EFA. The human fetus, like the adult, is unable to synthesize the EFAs, which must therefore be derived from the maternal circulation and pass through the placenta. In the fetus, increased concentration of the polyenoic fatty acids with advanced gestational age may result from increased activity of the fetomaternal unit by preferential transfer of these FAs. Enzymatic activity in the placenta or the fetus may also be responsible for desaturation and elongation of these EFAs. Several clinical manifestations have been ascribed in the human infant to prolonged EFA deficiency; however, none of these findings was noted in a group of sick newborn infants with very rapid onset of deficiency. Platelet dysfunction, decreased prostaglandin biosynthesis and turnover and altered pulmonary surfactant are among the effects of EFA deficiency on infants. Supplementation of the EFAs by the diet, parenterally or by the inunction of oil rich in linoleic acid, were reported to alleviate the symptoms of EFA deficiency. The minimal estimated requirement of linoleic acid is 1% of calories and 4% is an optimal intake. Most diets, including human breast milk, infant formulas and parenteral fat emulsions, far exceed the optimal intake of linoleic acid. Relatively little is known about the possible effects of high levels of linoleate in the diet; however, early studies suggest an adverse effect on platelet function, prostaglandin biosynthesis, pulmonary gas exchange and immune function.