Thyroid Function and Cognition during Aging.

We summarize here the studies examining the association between thyroid function and cognitive performance from an aging perspective. The available data suggest that there may be a continuum in which cognitive dysfunction can result from increased or decreased concentrations of thyroid hormones. Clinical and subclinical hypothyroidism as well as hyperthyroidism in middle-aged and elderly adults are both associated with decreased cognitive functioning, especially memory, visuospatial organization, attention, and reaction time. Mild variations of thyroid function, even within normal limits, can have significant consequences for cognitive function in the elderly. Different cognitive deficits possibly related to thyroid failure do not necessarily follow a consistent pattern, and L-thyroxine treatment may not always completely restore normal functioning in patients with hypothyroidism. There is little or no consensus in the literature regarding how thyroid function is associated with cognitive performance in the elderly.

A constructionist model predicting the emergence, complementarity and classification of the nucleotide bases.

We are proposing an analytical matrix that models a logical process simulating the emergence of the bases of vital nucleotides. The construction and properties of the matricial model are outlined. The Graph 1 matrix specifies a unique distribution pattern of eight terms coded in binary triplet configurations and obeys specific dynamic laws. The whole set of binary triplet configurations is in dynamic equilibrium. For that reason, it is possible to carry out an analysis by entering by any one of the eight terms on the condition that the operating mode obeys all the internal laws of orientation and symmetries inherent in the matrix. The four chemical elements at the origin of life are distributed following their atomic structures in the order hydrogen (H), carbon (C), nitrogen (N) and oxygen (O) and organized according to the model. The dynamic properties of the model necessitate the running of three successive circular periodic studies per analysis in order to show the emergence of the four bases--adenine, guanine, cytosine and thymine--precisely in that order. The fifth base of the nucleotides--uracil--shows up twice but always in an intermediate position, thus in transition, as it is the case for messenger ribonucleic acid (mRNA). We show also that the model provides for a logical explanation of the law of complementarity of the bases and their chemical classification. It is proposed that subsequent developments of the dynamic laws of this matrix may lead to the study of the logical operations for the formation of protein sequences and of their analysis and to genetic bioprogramming in general. Thus, a strictly logical and dynamic approach to molecular genetics is possible.

Fatty acid changes during the induction of differentiation of human promyelocytic leukemia (HL-60) cells by phorbolmyristate acetate.

We studied fatty acid changes that are likely to occur during phorbolmyristate acetate (PMA)-induced differentiation of HL-60 cells. It was observed that PMA-induced differentiation is associated with increased uptake, but not synthesis, of fatty acids. Fatty acid analysis revealed that arachidonic acid (AA), 20:5 n-3 and 22:6 n-3 levels are reduced with a concomitant increase in 22:5 n-6 in the phospholipid fraction. In the FFA fraction there are increases in free AA, free 20:5 n-3, 22:5 n-3 and 22:6 n-3, and a fall in free 22:5 n-6 in PMA-treated cells. PMA-induced differentiation and nitroblue tetrazolium reduction by PMA-treated cells was only partially inhibited (about 20-30%) by indomethacin and nordihydroguiaretic acid (cyclooxygenase and lipoxygenase inhibitors respectively), but not by superoxide dismutase, catalase or mannitol. These results indicate that PMA-induced differentiation of HL-60 cells is accompanied by specific changes in the fatty acid composition of the cells.

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.

Avarol restores the altered prostaglandin and leukotriene metabolism in monocytes infected with human immunodeficiency virus type 1.

Infection of monocytes with human immunodeficiency virus type 1 (HIV-1) (strain Ada-M) caused increased levels of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in vitro. These two products result from the activities of the two enzymes cyclooxygenase and 5-lipoxygenase. The addition of the sesquiterpenoid hydroquinone Avarol, an HIV inhibitor, strongly reduced the levels of LTB4 and PGE2 via inhibition of both cyclooxygenase and lipoxygenase in monocytes. The 50% inhibition concentrations (IC50) for the enzymes were determined to be 2.26 microM (cyclooxygenase) and 1.97 microM (lipoxygenase). A 50% reduction of the extent of PGE2 and LTB4 production in HIV-infected monocytes was measured at a concentration of 0.9 microM Avarol, a dose which caused an 80% anti-HIV effect in vitro (50% inhibition of virus release from infected cells: 0.3 microM). We conclude that Avarol inhibits the enzymes cyclooxygenase and lipoxygenase and suggest that, in general, inhibitors of these enzymes are promising anti-HIV compounds.

Effects of eicosapentaenoic acid and arachidonic acid on incorporation and metabolism of radioactive linoleic acid in cultured human fibroblasts.

Effects of exogenous eicosapentaenoic acid, arachidonic acid and oleic acid on incorporation and metabolism of [14C] linoleic acid were examined in cultured human fibroblasts obtained from three donors of different ages. Eicosapentaenoic acid treatment (40 microM) inhibited incorporation of radioactive linoleic acid and actively reduced radioactivity of desaturation-elongation metabolites in phospholipids, predominantly in the phosphatidylethanolamine fraction. In contrast, radioactivities of the metabolites in triacylglycerols were significantly increased with arachidonic acid treatment (40 microM): eicosapentaenoic acid had a smaller effect or none. Oleic acid had virtually no effect. These effects were consistent in the three cell lines, but responses to treatment with the acids differed considerably among individual cells. The pool of linoleic acid metabolites in triacylglycerols may not be negligible. The exogenous fatty acids may influence both the transfer of lipids between the major lipid pools as well as the activities of the desaturation-elongation system.

Transformation renders MDR cells more sensitive to polyunsaturated fatty acids.

A series of genetically related cell lines that express mdr genes but differ in their ability to form tumors has been challenged with gamma-linolenate and eicosapentaenoate to verify if the sensitivity of tumorigenic mdr cells to cytotoxic PUFAs differs from the sensitivity of non-tumorigenic mdr cells. The tumorigenic mdr cell lines were derived by transformation of their parental non-tumorigenic mdr cell line with myc and ras oncogenes. Four ras and five myc transformed cell lines were used for the estimation of clonal variability. The data as based on colony forming assays, showed that six out of nine of the tumorigenic mdr cell lines were more sensitive than the non-tumorigenic mdr cells. These results suggest that a tumorigenic phenotype renders mdr cells more sensitive to PUFAs and that PUFA supplementation either alone or in conjunction with existing forms of cancer therapy may have significant clinical implications.

Modulation of CD4 expression on lymphoma cells transplanted to mice fed (n - 3) polyunsaturated fatty acids.

Groups of adult AKR mice were fed well defined fats controlled diet regimens. These consisted of either saturated (beef tallow: 'BT') or (n - 3) polyunsaturated (fish oil: 'FO') fatty acids supplementation to basal mix mouse food. In other groups, the basal mix was given without any fat supplement ('NF'). Six weeks or more after the initiation of these diet regimens, mice received intraperitoneal injection of histocompatible RDM-4 lymphoma cells. Ascites RDM-4 tumors were harvested approximately two weeks later, and some of their physicochemical properties were studied. It was repeatedly found that: (1) the tumor grew considerably faster in the FO-fed donor than in the BT- or NF-fed donors; (2) cell membrane fluidity, content of C20(n - 3) and of C22(n - 3) fatty acids were significantly higher in the FO groups than in both BT and NF groups, while the content of C20(n - 6) and 22:4(n - 6) fatty acids was concomitantly decreased; (3) expression of the CD4 cell surface marker was always significantly diminished in the FO groups, whereas other markers such as CD8, H2K, Thy-1 and LFA-1 were not affected. Similar results were obtained, whether fats constituted from 1% to 16% by weight of the food intake. Use of a recently selected line of the RDM-4 lymphoma, exhibiting higher CD4 marker expression, resulted in similar observations. On the other hand, CD4 expression on cells from lymphoid organs of healthy adult AKR mice was not detectably modulated by the dietary fats.

Fatty acid and enzymatic compositional changes in the pancreas of rats fed dietary n-3 and n-6 polyunsaturated fatty acids.

The influence of n-3 and n-6 PUFA on the fatty acid composition and the enzyme content of zymogen granules of the normal exocrine pancreas was tested on rats. The animals were fed on different diets comprising 5% fish oil (FO), safflower oil (SFO), and evening primrose oil (EPO) used singly or in combination as dietary fats. The results were compared with those from animals fed 5% hydrogenated beef tallow (HBT). The fatty acid composition and digestive enzyme content were analyzed after a 6-wk feeding period. Differences in the pancreatic fatty acid profiles were related to the fatty acid composition of the ingested fats. Equivalent levels of n-3 fatty acids and 20:3n-6 were obtained with either EPO or FO fed singly or in combination. Similar results were observed with SFO/FO. Higher C20:3n-6/C20:4n-6 ratios were obtained with the oil mixtures. An increase in amylase levels, but a decrease in serine protease (Band 21 kdalton) levels, was associated with EPO. An elevation in procarboxypeptidase levels paralleled an increase in 18:0 levels, whereas the proportion of lipase (Band 49 kdalton) varied inversely with the proportion of C20:3n-6. The SFO/FO mixture elevated the proportions of protease II and proelastase. These results suggest that specific fatty acids influence the proportion of specific digestive enzymes in the zymogen granules.

Plasma fatty acid levels in patients with acquired immune deficiency syndrome and in controls.

Polyunsaturated fatty acids (PUFAs) are known to modulate the immune system in vivo and to inactivate envelope viruses in vitro. Patients with AIDS had low total plasma lipid levels and low levels of a number of individual fatty acids. However, the C20 and C22 essential fatty acids of the n-3 series were selectively and highly significantly reduced. Normalization of these fatty acid levels in AIDS patients may be a worthwhile therapeutic aim.

Differential sensitivity of tumorigenic and genetically related non-tumorigenic cells to cytotoxic polyunsaturated fatty acids.

A series of closely related rat brain cell lines that differ in their ability to form tumors has been used to investigate the selectivity of cytotoxic polyunsaturated fatty acids. The colony-formation ability of tumorigenic F4 cells was markedly reduced when the cells were challenged with GLA and EPA. In contrast, the non-tumorigenic revertants were less affected. All retransformed tumorigenic variants exposed to GLA were as sensitive as their parental tumorigenic cells and more sensitive than the non-tumorigenic clones. However, two out of three retransformed tumorigenic variants exposed to EPA were less sensitive than either the parental tumorigenic or non-tumorigenic clones. The addition of ferrous chloride to the culture medium increased the cytotoxicity of GLA in tumorigenic but not in non-tumorigenic variants. These results suggest that tumorigenicity per se is characterized by a high sensitivity to PUFAs exogenously administered at appropriate concentrations and that the sensitivity is fatty acid specific.

Type of dietary lipids exerts a major influence on the secretory activity of the exocrine pancreas: medium-term studies.

A possible influence of the type of dietary lipids on the pancreas exocrine function was tested on rats. For this purpose, four groups of rats were fed on different diets comprising 5% of different types of lipids: fish oil, evening primose oil, hydrogenated beef tallow, and a mixture of fish oil and evening primose oil. After a 6 week feeding period, the secretory activity of the pancreas was measured. Under resting conditions, rats fed hydrogenated beef tallow release different proportions of amylase, lipase, and serine proteases as compared to rats fed unsaturated lipids. In stimulated conditions, there was no significant difference in the relative proportions of enzymes secreted by the pancreas among the different groups of rats but the secretory response to cerulein stimulation from rats fed saturated lipids was increased by more than 40%. These results demonstrate that the type of dietary lipids exerts a major influence on the secretory activity of the pancreas.

Polyunsaturated fatty acid-induced cytotoxicity against tumor cells and its relationship to lipid peroxidation.

The contribution of lipid peroxidation to the killing of human breast cancer cells by gamma-linolenate (GLA) was examined. Other fatty acids of different cytotoxic potential containing 2, 4, 5, and 6 double bonds were also tested for comparison. It was found that the cytotoxic potential varied with the ability of the fatty acids to stimulate the production of superoxide radicals. Neither hydrogen peroxide nor hydroxy radicals are significantly involved in cell killing. As nonspecific indicators of lipid peroxidation, measurements of the loss of unsaturated fatty acid in the phospholipids together with the generation of hydroperoxide breakdown products were done with the use of the thiobarbituric acid test. The results of these experiments showed that the effectiveness of a given fatty acid in killing cancer cells correlated with the intracellular thiobarbituric acid-reactive material (TBARM) content: GLA and arachidonate with 3 and 4 double bonds generated the most TBARM and were the most cytotoxic fatty acids, whereas docosahexaenoate with 6 double bonds was the least effective either in raising TBARM or in killing the malignant cells. Iron and copper accelerated the rate of cell death, whereas antioxidants such as vitamin E and butylated hydroxyanisole inhibited the effect of GLA dose dependently. Indomethacin, an inhibitor of endoperoxide formation, did not reduce either cell kill or TBARM amounts. In contrast, the addition of vitamin E acetate to the cancer cell cultures challenged with eicosapentaenoate reduced both cell killing and TBARM content. These results suggest that the effectiveness of a given fatty acid in killing cancer cells correlated with the extent of lipid peroxidation of the added fatty acid in the cells.

Effects of polyunsaturated fatty acids and of their oxidation products on cell survival.

The stimulatory, cytostatic and cytotoxic effects of polyunsaturated fatty acids, prostaglandins, thromboxanes, hydroperoxy fatty acids, hydroxy fatty acids and leukotrienes on normal and tumor cells are described. Their effects are related to the ability of the cells to undergo lipid peroxidation. The significance of controlled peroxidation of selected polyunsaturated fatty acids in the control of tumor development is examined. It is suggested that selected polyunsaturated fatty acids if used at appropriate concentrations may have a protective role against cancer development by inducing and/or mediating cytotoxic reactions in malignant cells directly or indirectly through the intermediacy of immune cells.

Effect of different ratios of dietary N-6 and N-3 fatty acids on fatty acid composition, prostaglandin formation and platelet aggregation in the rat.

Five groups of male Sprague-Dawley rats (150 g) were fed a fat-free diet supplemented with 10% by weight of evening primrose oil (Efamol, rich in linoleic acid and gamma-linolenic acid) and/or marine oil (Polepa, rich in eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) combined in several ratios (Efamol/Polepa; 10.0%/0%; 7.5%/2.5%; 5.0%/5.0%; 2.5%/7.5%; 0%/10.0%). The n-6 fatty acid levels in aortic, platelet and plasma phospholipids decreased in proportion as Efamol was replaced with Polepa. The exception in phospholipids was dihomo-gamma-linolenic acid (20:3n-6), which increased when marine oil was provided in the diet with Efamol. The ratio of 20:3n-6 to arachidonic acid (20:4n-6) was positively correlated with 20:5n-3, docosapentaenoic acid (22:5n-3) or 22:6n-3 in aortic, platelet and plasma phospholipids under these dietary conditions. In contrast, 20:3n-6 in plasma cholesterol esters, triglycerides and free fatty acids did not show any increase in the presence of Polepa. Aortic prostaglandin (PG) production (6-keto-PGF1 alpha, PGE2 and PGE1) was reduced as Efamol was progressively replaced with Polepa. Aortic PG production was positively correlated with 20:4n-6 content in aortic phospholipids. Thrombin-induced thromboxane B2 production in whole blood was related to 20:4n-6 content in platelet phospholipids. However, ADP-induced platelet aggregation was significantly decreased only in the 7.5% Efamol/2.5% Polepa group as compared to the other 4 groups. These results suggest that combined treatment with Efamol and Polepa increases the ratio of 20:3n-6 to 20:4n-6 in tissue and plasma phospholipids. An appropriate ratio of these oils favorably affects aortic PG production and platelet ADP aggregation.

Polyunsaturated fatty acids augment free radical generation in tumor cells in vitro.

Polyunsaturated fatty acids (PUFAs) have been shown to inhibit both normal and tumor cell growth in vitro. As PUFAs are known to induce a respiratory burst and free radical generation in polymorphonuclear leukocytes and since free radicals are toxic to cells, we investigated the effect of PUFAs on a measure of free radical generation (nitroblue tetrazolium reduction) in normal human fibroblasts and breast cancer cells in vitro. Results suggested that linoleate (LA), gamma-linolenate (GLA), arachidonate (AA) and eicosapentaenoate (EPA) can enhance nitroblue tetrazolium reduction in tumor cells but not in normal cells. GLA, AA and EPA were 1 1/2 to 2 times more effective than LA in inducing free radical generation. This difference was not due to increased uptake of LA, AA and EPA by tumor cells. In fact, the uptake of LA was the same both in normal and tumor cells whereas that of AA and EPA occurred at approximately half the rate in the tumor cells compared to normal cells. This indicates that PUFA induced growth inhibition and cytotoxicity to tumor cells may, at least in part, be due to enhanced free radical generation.

Effects of C18 fatty acids on breast carcinoma cells in culture.

The relative cytotoxic potential of C18 carbon fatty acids on human breast tumor cells was determined. The saturated (18:0) and monounsaturated (cis C18:1n-9) fatty acids exhibited no cytotoxic effects, while cis polyunsaturated fatty acids (C18:2n-6, C18:3n-6, C18:3n-3, C20:4n-6, C20:5n-3 and C22:6n-3) were cytotoxic dose-dependently. Fatty acids with 3, 4 and 5 double bonds were more effective than those with 2 and 6 double bonds. Cis, but not trans, isomers of C18:2n-6 were cytotoxic. The cells did not demonstrate resistance to the cytotoxic effects induced by the fatty acids at any concentrations. It is concluded that in these cells the cytotoxic potential of the fatty acids depended on the number, the position and the geometric form of the double bond in the fatty acid carbon chain.