Belur R. Lokesh

Interleukin-1 and tumor necrosis factor synthesis by mouse peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids

The peritoneal macrophages from mice maintained for 16 days on a diet containing (10%) menhaden oil contained less arachidonic acid and more n-3 polyunsaturated fatty acids (n-3 PUFA) than those maintained on diets containing an equivalent amount of corn oil. Following stimulation with lipopolysaccharide, the production of PGE2, interleukin-1 (IL-1) and tumor necrosis factor (TNF) was 2.1 vs. 5.3 ng PGE2/micrograms DNA; 685 vs. 30 units IL-1/micrograms DNA and 14 vs. less than 4 units TNF by macrophages from mice consuming menhaden and corn oil, respectively. Macrophages from animals on diets containing olive oil generated intermediate amounts of PGE2 and equivalent amounts of IL-1 and TNF to those on corn oil. The data indicate that dietary n-3 PUFA at specific intake levels relative to n-6 PUFA may enhance cytokine generation by reducing PGE2 synthesis.

Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in rats fed diets enriched with docosahexaenoic (22:6n3) or eicosapentaenoic (20:5n3) acids

The objective of this experiment was to elucidate the effect(s) of eicosapentaenoic (20:5n3) vs docosahexaenoic (22:6n3) acid on prostaglandin biosynthesis and related thrombotic parameters. Diets were formulated to contain oils absent in (control) or enriched with either 20:5n3) EPA) or 22:6n3 (DHA). The diets were fed to rats for three weeks and the following evaluated: 1) bleeding time; 2) blood viscosity; 3) platelet aggregation; 4) tissue fatty acids; 5) serum thromboxane (TXB2), aortic prostacyclin (6-keto) ad 6) arachidonic acid conversion to eicosanoids by lung microsomes. There were no significant differences between treatments for bleeding time, red blood cell viscosity or platelet aggregation. In EPA fed rats 20:5n3 increased significantly in platelet and aorta phospholipids. In platelets and aorta 20:4n6 was slightly decreased in EPA and DHA animals. Platelet 22:6n3 levels were not altered by treatment, but 22:6n3 increased in the aorta of EPA and DHA fed rats. Similar changes were noted in lung and liver fatty acid composition. Serum TXB2 levels were significantly decreased only in the EPA vs control group. No differences were noted for aortic 6-keto levels or in the amount of hydroxy fatty acids, PGE, TXB2 or PGF2 alpha produced by lung microsomes. While fish oils have been shown to alter hematologic parameters in humans this study suggests that the rat is not similarly affected. Furthermore, it is evident that in the rat, 20:5n3 and not 22:6n3 is responsible for the alterations in platelet prostaglandin biosynthesis; however, these observations may not be directly applicable to other species.

Superoxide anion generated by potassium superoxide is cytotoxic and mutagenic to Chinese hamster ovary cells

Chinese hamster ovary cells in vitro were exposed to superoxide anion (O2-) generated by the addition of potassium superoxide (KO2) to the cell culture media. It was determined that 2 nmoles O2- per ml media resulted in approximately 50% cell mortality. The number of 6-thioguanine-resistant mutants was also increased in a dose-related fashion. Both these effects of KO2 were inhibitable by the prior addition of superoxide dismutase, indicating that superoxide anion is cytotoxic and genotoxic in mammalian cells.

Docosahexaenoic acid and other dietary polyunsaturated fatty acids suppress leukotriene synthesis by mouse peritoneal macrophages

The efficacy of individual ω-t-3 polyunsaturated fatty acids (PUFA) in altering eicosanoid synthesis in peritoneal macrophages was studied by feeding mice for 10 days a diet containing 2 wt% fat, which included 0.5 wt% ethyl esters of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linolenic acid (LNA). Upon stimulation with calcium ionophore A23187, macrophages from these animals produced significantly lower amounts of leukotriene C4, leukotriene B4 and 12-hydroxyeicosatetraenoic acid, prostaglandin E2 and 6-keto prostaglandin F1α compared with those obtained from animals on the diets containing olive oil or safflower oil. The decrease in leukotriene synthesis was similar in the animals fed DHA, EPA or LNA diets. This depression of eicosanoids by DHA and EPA was associated with decreased levels of arachidonic acid (AA); however, LA that altered eicosanoids did not have the same effect on AA levels.

Modulation of Prostaglandin Synthesis in Mouse Peritoneal Macrophages by Enrichment of Lipids with either Eicosapentaenoic or Docosahexaenoic Acids in vitro

The n-3 polyunsaturated fatty acids (PUFA) of fish oils alter arachidonic acid (AA) metabolism in macrophages. The present investigation studied the efficacy of eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), two n-3 PUFA of fish, to alter lipid composition and specific functions of mouse peritoneal macrophages. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were readily incorporated by macrophages in vitro and replaced 25-50% of AA in cellular lipids. The EPA- or DHA-enriched cells synthesized significantly less (50-65%) prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6 keto prostaglandin F1(1) alpha (6 keto PGF1 alpha) when stimulated with opsonized zymosan. The enrichment with EPA or DHA did not affect phagocytosis nor superoxide anion formation in macrophages. These studies demonstrated that EPA or DHA can be used to decrease prostaglandin synthesis selectively without affecting the other physiological functions of macrophages.

Differential effects of docosahexaenoic acid and eicosapentaenoic acid on suppression of lipoxygenase pathway in peritoneal macrophages

Docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) was facilely incorporated into phospholipids of mouse peritoneal macrophages following incubation with pure fatty acids complexed to bovine serum albumin. Following stimulation with calcium ionophore A23187, the DHA-enriched cells synthesized significantly smaller amounts of leukotriene C4 and leukotriene B4 compared to control or EPA-enriched cells. The EPA-enriched cells synthesized lower amounts of leukotriene C4 and leukotriene B4 compared to control cells. The stimulated macrophages utilized endogenously released arachidonic acid for leukotriene B4 and leukotriene C4 synthesis. Exogenous arachidonic acid increased the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE and macrophages enriched with DHA or EPA produced similar amounts of 12-HETE and 15-HETE compared to control cells. These studies demonstrated that the synthesis of leukotriene C4, leukotriene B4 and HETE in macrophages is differentially affected by DHA and EPA.

Inhibition of the genotoxicity of bleomycin by superoxide dismutase

Bleomycin was found to be cytotoxic and mutagenic in the CHO/HGPRT forward mutation assay. Approximately 50% cell mortality was achieved after a 1-h exposure to 10 micrograms/ml BLM. Bleomycin was also found to induce mutation to thioguanine resistance in a dose-dependent manner. Both the cyto- and geno-toxicity resulting from BLM exposure could be reduced by the prior addition of superoxide dismutase, implicating a role for activated oxygen metabolites in the mechanism of toxicity of bleomycin.

Alterations in the Lipids and Prostaglandins in Mouse Spleen following the Ingestion of Menhaden Oil

Two groups of male mice were fed for 2 weeks with a semisynthetic diet supplemented with either 10% hydrogenated coconut oil or 10% menhaden oil. The spleen from animals fed with menhaden oil contained significantly higher amounts of polyunsaturated n-3 fatty acids. The n-3 fatty acids reciprocally replaced arachidonic acid in the phospholipids. The synthesis of 6-keto prostaglandin F1 alpha and prostaglandin E2 by spleen tissues were significantly depressed (70-80%) in mice consuming menhaden oil. These studies indicated that n-3 fatty acids can effectively displace arachidonic acid from spleen lipids and thereby affect the synthesis of prostaglandins. The implications of these observations are discussed.

Further studies on the formation of oxygen radicals by potassium superoxide in aqueous medium for biochemical investigations.

Potassium superoxide (KO2) forms superoxide anion (O2-) in aqueous medium as measured by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay of McCord and Fridovich. The reduction of cytochrome c by O2- formed by KO2 was observed only above pH 7.0 and demonstrated a pH optimum at pH 9.6. SOD was an effective inhibitor of the cytochrome c reduction produced by KO2. Hydroxyl radical scavengers and singlet oxygen quenchers did not interfere with the reduction of cytochrome c by KO2. These data demonstrate that addition of KO2 to aqueous medium is an easy chemical method for the production of O2- in controlled amounts.

Modulation of zymosan stimulated leukotriene release by dietary unsaturated fatty acids

The quantity of leukotrienes produced in an inflammation model, the stimulated mouse peritoneum, was affected by dietary manipulation of tissue arachidonic acid, the immediate leukotriene (LT) precursor. Fifty ng of LTE4 was synthesized (after injection of zymosan) by the peritoneal cavity of mice maintained on olive oil as a dietary source of unsaturated fatty acids. Animals maintained on corn oil, exhibited significantly enhanced leukotriene biosynthesis upon stimulation by zymosan. Mice fed menhaden oil, a fat source rich in n-3 fatty acids produced 50% less leukotriene E4 than animals fed olive oil. The results indicated that production of leukotrienes, potent mediators of inflammatory reactions, are affected by the type of polyunsaturated fatty acids in the diet.