C.K. Miyasaka

Effect of fatty acids on leukocyte function

Fatty acids have various effects on immune and inflammatory responses, acting as intracellular and intercellular mediators. Polyunsaturated fatty acids (PUFAs) of the omega-3 family have overall suppressive effects, inhibiting lymphocyte proliferation, antibody and cytokine production, adhesion molecule expression, natural killer cell activity and triggering cell death. The omega-6 PUFAs have both inhibitory and stimulatory effects. The most studied of these is arachidonic acid that can be oxidized to eicosanoids, such as prostaglandins, leukotrienes and thromboxanes, all of which are potent mediators of inflammation. Nevertheless, it has been found that many of the effects of PUFA on immune and inflammatory responses are not dependent on eicosanoid generation. Fatty acids have also been found to modulate phagocytosis, reactive oxygen species production, cytokine production and leukocyte migration, also interfering with antigen presentation by macrophages. The importance of fatty acids in immune function has been corroborated by many clinical trials in which patients show improvement when submitted to fatty acid supplementation. Several mechanisms have been proposed to explain fatty acid modulation of immune response, such as changes in membrane fluidity and signal transduction pathways, regulation of gene transcription, protein acylation, and calcium release. In this review, evidence is presented to support the proposition that changes in cell metabolism also play an important role in the effect of fatty acids on leukocyte functioning, as fatty acids regulate glucose and glutamine metabolism and mitochondrial depolarization.

Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids

Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180%). Removal of the gonads in both males and females (comparison between castrated groups) increased the difference in SOD activity from 83 to 138% and reduced the difference in CAT activity from 180 to 86%. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48%) CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle.

Walker‐256 tumor growth causes oxidative stress in rat brain

The elevated rate of oxygen consumption and high amount of polyunsaturated fatty acids make the central nervous system vulnerable to oxidative stress. The effect of Walker‐256 tumor growth on oxi–reduction indexes in the hypothalamus (HT), cortex (CT), hippocampus (HC) and cerebellum (CB) of male Wistar rats was investigated. The presence of the tumor caused an increase in thiobarbituric acid reactant substances (TBARs) in the HT, CB and HC. Due to tumor growth, the activity of glucose‐6‐phosphate dehydrogenase increased in the HT and CB, whereas citrate synthase activity was reduced in the HT, CT and CB. Therefore, the potential for generation of reducing power is increased in the cytosol and decreased in the mitochondria of various brain regions of Walker‐256 tumor‐bearing rats. These changes occurred concomitantly with an unbalance in the brain enzymatic antioxidant system. The tumor decreased the activities of catalase in the HT and CB and of glutathione peroxidase in the HT, CB and HC, and raised the CuZn‐superoxide dismutase activity in the HT, CB and HC. These combined findings indicate that Walker‐256 tumor growth causes oxidative stress in the brain.

Percentage of phagocytosis, production of O2·−, H2O2 and NO, and antioxidant enzyme activities of rat neutrophils in culture

Changes in the integrity, ultrastructure, phagocytosis capacity, and production of H2O2, O2· −and NO2− were evaluated in cultured neutrophils. The activities of the antioxidant enzymes (catalase—CAT, superoxide dismutase—SOD and glutathione‐dependent peroxidase—GSH‐Px) were measured under similar conditions. The integrity of the cells remained unchanged up to 18 h. After 24 h, the number of viable cells in culture dropped by 16 per cent. The percentage of viable cells in culture was of 72 per cent even after 72 h. An ultrastructural analysis of the cells was carried out after 3, 6, 12, 24, 48, and 72 h in culture. Neutrophils started developing morphologic changes after 24 h: decreased cell volume, abundant vacuoles (mainly around the nucleus), and also the presence of autophagic vacuoles. This period was then chosen for the study of neutrophil function and antioxidant enzyme activities. Neutrophils cultured for 24 h presented reduced phagocytosis capacity. The rates of production of H2O2 and O2· − remained unchanged after 24 h in culture. Concomitantly, these cells were also able to produce NO in significant amounts. The production of O2·− in response to PMA stimulus was lowered in 24‐h cultured cells. Possibly, the production of oxygen and nitrogen reactive species accomplished with a decrease in the activities of CAT and GSH‐Px play a key role for the process of apoptosis which takes place in neutrophils under these conditions.

Comparative effects of fish oil given by gavage and fish oil-enriched diet on leukocytes.

The comparative effects of fish oil given by gavage and fish oil enriched diet on metabolism and function of lymphocytes and macrophages were investigated. For this purpose, the following parameters were examined: 1) phagocytosis capacity, production of superoxide (O2*-) and hydrogen peroxide (H2O2) by macrophages, 2) lymphocytes proliferation capacity, 3) antioxidant enzyme activities in the mesenteric lymph nodes (MEN) and liver, 4) Thiobarbituric Acid Reactive Substances (TBARS) content in MLN, liver, and plasma, 5) total antioxidant capacity of the plasma, and 6) fatty acid composition of macrophages, MLN, liver and plasma. Both FO treatments did not affect phagocytosis capacity but increased hydrogen peroxide production by macrophages in the presence of PMA. FO given by gavage markedly increased lymphocytes proliferation both in the absence (5.8-fold) and in the presence (16.7-fold) of Con A, whereas FO-rich diet showed an increase in the presence of Con A only (53.3%). FO given by gavage raised the proliferation index by 2.9-fold and FO-rich diet increased by 29% only as compared to controls. Concomitantly, FO given by gavage was more effective to increase TBARS content in plasma. The proportion of some fatty acids in the tissues and cells was also differently changed depending on the way FO was administered to rats: in particular: myristic, arachidonic, and eicosapentaenoic acids. This fact may partially explain the differences between both FO treatments.

Glutathione metabolism and glutathione S‐conjugate export ATPase (MRP1/GS‐X pump) activity in cancer

Mg2+‐dependent vanadate‐sensitive glutathione S‐conjugate ATPase (GS‐X pump) activity is a common feature of some ATP‐binding cassette (ABC) transporters, such as the multidrug resistance‐associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S‐transferase catalyzed reaction and be eliminated through GS‐X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S‐(2,4‐dinitrophenyl)‐glutathione (DNP‐SG) conjugate through the GS‐X pump, using whole cells and inside‐out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS‐X pump activity. In particular, HEp‐2 larynx carcinoma cells possess an elevated DNP‐SG export rate through the GS‐X pump compared with HeLa, K562, U937 or HL‐60 cells, which exhibit the lowest activity. The differences in DNP‐SG export rates are not due to decreased glutathione S‐transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS‐X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS‐X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.

Effects of the antiproliferative cyclopentenone prostaglandin A1 on glutathione metabolism in human cancer cells in culture

Homeostatic mechanisms for the maintenance of glutathione (GSH) are fundamental in the provision of a cellular defense against electrophilic/oxidant challenges. Cyclopentenone prostaglandins (CP‐PGs) are powerful antiproliferative endogenous substances that may act as electrophilic regulating compounds, by virtue of the presence of an α,β‐unsaturated carbonyl group in the cyclopentane ring. Nevertheless, differential resistance to CP‐PG cytotoxic/cytostatic effect has been reported in different cell types. It is reported that the activity/expression of γ‐glutamylcysteine synthetase (γ‐GCS, the rate‐limiting enzyme in GSH biosynthesis) can be inducibly activated by electrophiles, including CP‐PGs. The response of the human cancer strains HEp‐2 (larynx carcinoma) and HL‐60 (promyelocytic leukemia) cells to treatment with the CP‐PG PGA1 in culture was investigated by evaluating the time‐course of GSH synthesis and activity of enzymes of GSH metabolism, other than γ‐GCS, after PGA1 addition. HEp‐2 cells, being more resistant to PGA1 cytotoxic and cytostatic effects, have basal GSH levels that were 2.4‐fold higher than that of HL‐60 cells. The activities of GSH S‐transferase (GST), glutathione reductase (GSRd) and glutathione peroxidase (GSPx) are constitutively higher in HL‐60 cells than in HEp‐2 cells (respectively, 17.0‐, 28.5‐ and 12.3‐fold). When challenged with PGA1, both cell types exhibited a dose‐dependent rise in GSH content that was maximal 18 h after PGA1 addition and was preceded by a rise in GST and GSRd activities in both cell types (at 12 h). GSPx activity increased only in HEp‐2 (PGA1 evoked a 93.4%‐inhibition in HL‐60 cells). Moreover only HEp‐2 cells exhibited early capacity to enhance GSH content (1‐2 h just after PGA1 addition). These results and earlier data showing that leukemia cells are sensitive to CP‐PG treatment suggest that deficiencies in GSH metabolism may be strategic in therapeutic approaches to the treatment of human leukemias.

Fish oil given by gavage increases lymphocyte proliferation and production of hydrogen peroxide by rat macrophages

1. The effect of fish oil (FO) administration by gavage (0.4% body weight) on macrophage and lymphocyte function was investigated in young male rats. The results were compared with those obtained by administration of soybean oil (SB) and cocoa butter (CB). 2. Lymphocyte proliferation was markedly increased by FO administration compared with control and other oils. 3. Macrophage phagocytosis capacity was not affected by FO, but it was increased by CB and SB. 4. The oils did not affect the production of O2.- but increased the production of H2O2 in the presence of PMA. 5. The administration of the oils did not markedly affect the activity of antioxidant enzymes in macrophages, except for a decrease in superoxide dismutase activity by FO.

MODULATION OF HYPERSENSITIVITY REACTION BY LIPIDS GIVEN ORALLY

The effect of lipids administration by gavage (0.4% body weight) given daily during four weeks on the hypersensitivity reaction in trachea, upper and lower bronchi, liver, kidney, mesentery, and pancreas was investigated in male rats. The plasma exudation was assessed by using Evans blue (EB) dye extravasation method. There was a significant difference in the permeability of the organs in nonimmunized rats. The immunization increased the vascular permeability and the response with the organs varied greatly. The effect of lipids on anaphylactic reaction was compared to those of untreated rats (control group). The EB extravasation was significantly increased in the trachea obtained from rats treated with cocoa butter and soybean oil. In the upper bronchi of rats treated with soybean oil, the EB extravasation was increased. However, in the lower bronchi, none of the treatments with lipids changed the extravasation of EB. The same was observed in the liver and kidney. The animals treated with lipids by gavage did not present differences in EB extravasation in the mesentery. However, in the pancreas and duodenum, the treatment with fish and soybean oils and cocoa butter markedly lowered EB extravasation.

Cholesterol changes the fatty acid composition of rat enterocytes

The effect of free cholesterol on the fatty acid composition and growth of rat fetal enterocytes was investigated in the absence and presence of 10% (v/v) fetal calf serum. Cholesterol caused a significant reduction of cell number after 6 and 12 h in culture. The fatty acid composition of enterocytes cultured in the presence of serum was also changed by the presence of 20 microM cholesterol. The fatty acid profile was determined by HPLC using fluorescence detection (325 nm excitation and 395 nm emission). Cholesterol (20 microM) increased the proportion (given in percentage of the total fatty acids) of the following fatty acids in cultured cells: lauric (by 42%), oleic (by 34%), linoleic (by 44%) and gamma-linolenic (by 20%) acids and reduced the proportion of palmitic (by 12%), stearic (by 20%), arachidonic (by 21%) and docosahexaenoic (by 44%) acids. In addition to modifying the content of individual fatty acids, cholesterol increased the polyunsaturated/saturated fatty acid ratio from 0.48 to 0.67 and the unsaturation index from 67.12 to 75.30. This is the first evidence that cholesterol modifies fatty acid composition possibly via de novo fatty acid synthesis and desaturation.