Carla Cristine Kanunfre

Ranking the toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis

The fatty acids have an important role in the control of leukocyte metabolism and function. Higher concentrations of certain fatty acids, particularly polyunsaturated fatty acids (PUFAs) and volatile fatty acids, can cause cell death via apoptosis or, when concentrations are greater, necrosis. In this study, we determined the highest concentrations of various fatty acids that are non-toxic to two human leukemic cell lines, Jurkat (T-lymphocyte) and Raji (B-lymphocyte). Toxicity was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. There were no remarkable differences for the toxicity of the fatty acids between B and T cell lines. The cytotoxicity of the fatty acids was related to the carbon chain length and number of double bonds: docosahexaenoic acid=eicosapentaenoic acid=arachidonic acid=gamma-linolenic acid=stearic acid=palmitic acid > linoleic acid=palmitoleic acid > vacenic acid=lauric acid > oleic acid > elaidic acid > capric acid > butyric acid > caprylic acid=caproic acid=propionic acid. The proportion of cells undergoing apoptosis or necrosis, induced by the fatty acids tested, remains to be investigated.

Effects of EPA and DHA on proliferation, cytokine production, and gene expression in Raji cells

The effects of EPA and DHA on the function and gene expression of a B-lymphocyte cell line (Raji) were investigated. Proliferation; production of interleukin-10 (IL-10), tumor necrosis factor (TNF)-α, and interferon (INF)-γ; and expression of pleiotropic genes were evaluated. Cell proliferation was increased in the presence of 12.5 μM EPA (approximately twofold) and 12.5 μM DHA (approximately 1.5-fold). EPA and DHA (25 μM) also decreased production of the key immunoregulatory cytokines IL-10, TNF-α, and INF-γ. EPA and DHA changed the expression of specific genes, but this effect was more marked for EPA (25.9% of genes investigated) compared with DHA (8.4% of genes investigated). EPA and DHA affected the expression of genes clustered as: cytokines, signal transduction, transcription, cell cycle, defense and repair, apoptosis, cell adhesion, cytoskeleton, and hormones. The most remarkable changes were observed in the genes of signal transduction and transcription. These results led us to conclude that the mechanism of DHA and EPA effects on B-lymphocyte functions includes regulation of gene expression. Thus, the ingestion of fish oil, a rich source of EPA and DHA, may have a strong effect on B-lymphocyte function in vivo. However, remarkable differences were observed between DHA and EPA, demonstrating that specific effects of these FA may be responsible for the marked differences in edible oil effects on immune function in vivo reported by others.

Effect of fish oil supplementation for two generations on changes of lymphocyte function induced by Walker 256 cancer cachexia in rats.

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-α and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-γ plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

Genes regulated by arachidonic and oleic acids in Raji cells.

FA are known to modulate immune function in conditions such as arthritis and lupus erythematosus. The effects of arachidonic (AA) and oleic acids (OA) on function and pleiotropic gene expression of Raji cells were investigated. The following parameters were evaluated: cytotoxicity as assessed by loss of membrane integrity and DNA fragmentation; proliferation as measured by [14C]thymidine incorporation; production of interleukin (IL)-10, interferon (INF)-γ, and tumor necrosis factor (TNF)-α; and expression of pleiotropic genes by a macroarray technique (83 genes in total). AA was more toxic to Raji cells than OA. Both FA promoted an increase in Raji cell proliferation at 75 μM, whereas OA at high concentrations (200 μM) decreased proliferation. AA reduced the production of IL-10, TNF-α, and INF-γ. On the other hand, OA provoked an increase of INF-γ production but did not affect the production of IL-10 and TNF-α. The proportions of genes with altered expression were 27% for AA and 35% for OA. The FA affected the expression of genes clustered as: cytokines, signal transduction pathways, transcription factors, cell cycle, defense and repair, apoptosis, DNA synthesis, cell adhesion, cytoskeleton, and hormone receptors. The most remarkable changes were observed in the genes of signal transduction pathways. These results led us to conclude that the effect of these FA on B-lymphocytes includes regulation of gene expression. Thus, diets enriched with fat containing OA or AA may affect B lymphocyte function in vivo.