Influence of fatty acids and bovine serum albumin on the growth of human hepatoma and immortalized human kidney epithelial cells

Summary The protective influence of bovine serum albumin against growth inhibition caused by fatty acids was studied in human hepatoma (HepG2) and immortalized human kidney epithelial (IHKE) cells. In general, growth inhibition by unsaturated fatty acids (0.15 mmol/liter) increased with increasing number of double bonds. For HepG2 cells crude albumin (1g/100 ml) did not greatly modify growth inhibition by arachidonic, eicosapentaenoic, and docosahexaenoic acid. With oleic, linoleic, and linolenic acids, crude and defatted albumin stimulated cell growth. In contrast, for IHKE cells both albumins counteracted growth inhibition by unsaturated fatty acids to approximately the same extent. When HepG2 cells were cultured in the presence of saturated fatty acids (0.3 mmol/liter), C2, C6, and C8 had no or little inhibitory effect. C10 and C12 inhibited cell growth appreciably, whereas C14, and especially C16, had poor inhibitory effects. Crude albumin counteracted growth inhibition by all these fatty acids. In contrast, defatted albumin had little or no effect (except against C10 and C12), and even increased the growth inhibition by C14 and C16. With unsaturated fatty acids there seemed to be an inverse relationship between cell growth and the concentration of thiobarbituric acid reactive substances (TBARS) in media. Vitamin E abolished growth inhibition (and the increase in TBARS concentration) by unsaturated fatty acids. The complex interaction between fatty acids and albumins calls for great caution when interpreting data on growth effects.     

UV-C irradiation-induced peroxidative degradation of microsomal fatty acids and proteins: protection by an extract of Ginkgo biloba (EGb 761).

After exposure of rat liver microsomes to UV-C irradiation, analysis of membrane fatty acids by gas chromatography confirmed that EGb 761, a drug containing a dosed and standardized extract of Ginkgo biloba, provides effective protection against free radical attack in vitro. This analysis, coupled with thiobarbituric acid (TBA) reaction, permitted qualitative and overall quantitative evaluation of radical-induced damage to polyunsaturated fatty acids (PUFA), as well as evidence of the antioxidant properties of the Ginkgo biloba extract. Assay of thiobarbituric acid reactive substances (TBARS) showed a correlation between TBARS concentration and the state of degradation of the polyunsaturated fatty acids. Mannitol (5.5 mM) did not prevent degradation of microsomal PUFA or malondialdehyde (MDA) production, nor did it prevent polymerization of membrane proteins. Low doses of EGb 761 were found to provide efficient protection of membrane PUFA regardless of individual susceptibility to peroxidation. This protection was accompanied by a decrease in the production of TBARS. EGb 761 also protected membrane proteins from the irreversible polymerization induced by these degradation products, but did not appear to prevent thiols oxidation into disulfide bonds.     

Shewanella sp. GA-22, a psychrophilic hydrocarbonoclastic antarctic bacterium producing polyunsaturated fatty acids

AIMS: The effects of different growth media and temperature on production of polyunsaturated fatty acids (PUFA) by Shewanella sp. GA-22 were investigated. The attempts to characterize the GA-22 genes, homologous to those of PUFA biosynthesis gene cluster, was performed. METHODS AND RESULTS: Physiological and phylogenetic characterization of new Antarctic isolate GA-22 was performed. Total fatty acids were isolated from the cells growing under different conditions and analysed by gas chromatography-mass spectrometry (GC-MS). Using degenerated primers derived from the conserved regions within PUFA fatty acid synthase operons, five fragments of homological genes were amplified from GA-22 DNA, and two of them corresponding to pfaA and pfaC synthase subunits were sequenced. CONCLUSIONS: Strain GA-22 was shown to be able to produce three different PUFA: linoleic, arachidonic and eicosapentaenoic acids. The PUFA production was temperature- and carbon source-dependent. The deduced gene products exhibited high similarity to corresponding fatty acid synthases PfaA and PfaC. SIGNIFICANCE AND IMPACT OF STUDY: The PUFA production was detected on media supplemented with crude oil, gasoline and n-tetradecane. The apparent conservation of PUFA genes may point to the potential utilization of designed primers as functional markers in culture-independent ecological studies, and for initial screening in biotechnological fields.     

Effects of polyunsaturated fatty acids on the growth and differentiation of the cellular slime mould, Dictyostelium discoideum

Cells of Dictyostelium discoideum grown on media containing polyunsaturated fatty acid (PUFA) exhibit impaired differentiation when placed on a solid surface in the absence of all nutrients. Differentiation is inhibited at all temperatures, and although the inhibition is somewhat less pronounced at low temperatures, there is neither a lowering of the optimum temperature of aggregation nor a reversal of inhibition at low temperatures. Furthermore, although the in vitro aggregation of vegetative cells and the reaggregation of dispersed aggregation-phase cells are markedly temperature dependent, PUFA supplementation does not markedly influence this dependence. These data are not consistent with the hypothesis that impaired differentiation is due to increased plasma membrane fluidity. PUFA had no adverse effect on cell growth at temperatures at or below the optimum growth temperature, 22 °C. At 25 °C, however, there was considerable inhibition and at 27 °C growth was completely eliminated in the presence of PUFA.     

Flavonoids and their oxidation products protect efficiently albumin-bound linoleic acid in a model of plasma oxidation

Although LDL esterified polyunsaturated fatty acids (PUFA) contribute largely to the pool of oxidizable lipids in plasma, they coexist with a non-negligible content of free PUFA. In some pathological conditions, the free PUFA/albumin ratio becomes abnormally elevated. Modeling was performed in a system constituted of linoleic acid bound to human serum albumin (HSA) in which oxidation was initiated by hydrophilic AAPH. Inhibition of lipid peroxidation was evaluated for various flavonoids. The accumulations of hydroperoxyoctadecadienoic acids (HPODE), hydroxyoctadecadienoic acids (HODE) and ketooctadecadienoic acids (KODE) were similarly inhibited: isoquercitrin>quercetin>catechin=isorhamnetin>kaempferol>quercetin-4'-beta-D-glucoside=quercetin-3,4'-di-beta-D-glucoside. Surprisingly, quercetin and isorhamnetin afforded a protection to linoleic acid long after their consumption. Elucidation by mass spectrometry and NMR of the quercetin oxidation products and assessment of their antioxidant capacity pointed out that 3,4-dihydroxybenzoic acid and 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one are major contributors to the apparent quercetin antioxidant capacity.     

Modification of phospholipids fatty acid composition in reuber H35 hepatoma cells: effect on HMG-CoA reductase activity

There is controversy about the effect of saturated and polyunsaturated fats on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the main regulatory enzyme of cholesterogenic pathway. Results from dietary studies are difficult to interpret because diets normally contain a mixture of fatty acids. Therefore, we have used Reuber H35 hepatoma cells whose phospholipids were enriched in different individual fatty acids and have studied their effects on the cellular reductase activity. Lauric, myristic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids were supplemented to the culture medium coupled to bovine serum albumin. The four fatty acids were incorporated into phospholipids from cells grown in media containing whole serum or lipoprotein-poor serum (LPPS). Reductase activity of cells cultivated in a medium with LPPS was three to four times higher than those cultivated in medium with whole serum. Saturated fatty acids increased reductase activity of cells grown in medium with whole serum, whereas n-3 polyunsaturated fatty acids (PUFA) decreased it. However, both saturated and polyunsaturated fatty acids increased reductase activity when serum lipoproteins were removed. In conclusion, this is one of the first reports demonstrating that saturated and n-3 PUFA only show differential effects on HMG-CoA reductase activity in the presence of lipoproteins.     

Changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in Daphnia magna during the aging process

Age-related changes in the balance between endogenous pro-oxidative and antioxidative processes in the freshwater cladoceran Daphnia magna (Crustacea) were assessed. The activities of key antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase and levels of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) were determined in eight age classes, covering juvenile, young and senescent adults. Age-related changes in fatty acid composition were also measured to examine the contribution of polyunsaturated fatty acids (PUFA) in the peroxidation status of animals. Biochemical responses depicted in this study demonstrated that age-related decline in survival was accompanied by increasing oxidative stress and oxidative damage. Enhanced oxidative stress in aging D. magna was suggested by the significant increase in the formation of lipid peroxides, and a concomitant reduction of unsaturated fatty acids of 20 or more carbon atoms. Because aging was accompanied by selective loss of key antioxidant enzymes and small changes in the amount of PUFA, the breakdown of antioxidant defences might have directly contributed to oxidative stress, membrane lipid peroxide and a decline of survival. Indeed, the results reported here, indicate that age-related increases of lipid peroxides were at least partially due to the functional imbalance of enzymatic antioxidant defences.     

Effects of fatty acids on the growth of Caco-2 cells

Epidemiological studies suggest that polyunsaturated fatty acids may protect against colorectal neoplasia. In order to explore this observation, cell proliferation and viability, lipid composition, membrane fluidity, and lipid peroxidation were measured in Caco-2 cells after 48h incubation with various fatty acids. Saturated and monounsaturated fatty acids incorporated less well in the membranes than polyunsaturated fatty acids (PUFAs). All of the PUFAs tested had an inhibitory effect on cell proliferation/viability whereas the saturated and monounsaturated fatty acids did not. Addition of palmitic acid had no significant effect on membrane fluidity whereas unsaturated fatty acids increased membrane fluidity in a dose-dependent manner. PUFAs strongly increased tumor cell lipid peroxidation in a dose-dependent manner. Saturated and monounsaturated fatty acids increased lipid peroxidation in this cell line only at high concentration. Preincubation of Caco-2 cells with vitamin E prevented the inhibition of proliferation/viability, the elevation of the MDA concentration and the increased membrane fluidity induced by PUFAs. Our data indicate that PUFAs are potent inhibitors of the growth of colon cancer cells in vitro.     

Albumin bound nonesterified fatty acids inhibit in vitro lipid peroxidation

Individual nonesterified fatty acids were bound to albumin in vitro and these fatty acid albumin complexes were used to study their effect on lipid peroxidation in liver microsomes. Peroxidation was induced by various methods and malondialdehyde (MDA) was measured as an index of peroxidation. Among the fatty acids tested, albumin-bound monounsaturated fatty acids showed more inhibition of peroxidation as compared to other fatty acids. Increasing the concentration of iron in the peroxidizing system, partially reversed the inhibition by fatty acids. Moreover, albumin-bound fatty acid did not inhibit iron independent peroxidation. This suggests that, like nonesterified fatty acids, albumin-bound fatty acids inhibit peroxidation by chelating the iron. Albumin fatty acid complex, similar to the fatty acid composition present in the circulating albumin, also showed inhibition of peroxidation. These data indicate that nonesterified fatty acids even when bound to albumin are capable of inhibiting peroxidation and circulating albumin, which contains various fatty acids bound to it, may impart some antioxidant effect in addition to other plasma antioxidants.     

UTILIZATION OF POLYUNSATURATED FATTY ACID SUPPLEMENTS BY CULTURED NEUROBLASTOMA CELLS1

Abstract— Brain phosphoglycerides are known to contain large amounts of polyunsaturated fatty acids (PUFA). However, neuroblastoma cells contain very low amounts of PUFA. Since the serum used for cell culture has been shown to be deficient in PUFA, a supplementation of this serum with various PUFA was undertaken. Linoleic, arachidonic and docosahexaenoic acids were incorporated in cell phosphoglycerides, mainly at the expense of oieic acid, while linolenic acid was only poorly incorporated. Linoleic. linolenic and arachidonic acids were transformed to their elongation and desaturation products; but the last step of transformation, which involves the action of a Δ 4 desaturase, was never observed. The levels of incorporation and transformation of exogenous PUFA could vary strikingly in different lines of neuroblastoma cells. The simultaneous addition of two PUFA (linoleic and linolenic acids) was followed by a reduction in the amount of their respective derivatives in cell phosphoglycerides. compared to that obtained when only one PUFA was given to the cells, suggesting a competitive inhibition of the desaturation of each PUFA. Such alterations in membrane lipids may provide a useful model for the study of membrane structure-function relationships.     

High affinity of alpha-foetoprotein for arachidonate and other fatty acids.

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.     

In vivo breath alkane as an index of lipid peroxidation

Interaction of active oxygen species with polyunsaturated fatty acids (PUFA) results in a series of reactions called lipid peroxidation. During the process of peroxidation of polyunsaturated fatty acids there is a scission of an alkane fragment extending from the methyl end of the fatty acid to the double bond. Thus, with a w-6 polyunsaturated fatty acid pentane is released, and with a w-3 polyunsaturated fatty acid ethane is released. These hydrocarbons are distributed in the body, partly metabolized, and excreted in the breath, making it possible to estimate the magnitude of in vivo lipid peroxidation by measuring pentane and ethane exhaled in breath. Advantages of this method are discussed as well as limitations and possible sources of error.     

Phospholipid hydroperoxide glutathione peroxidase activity and vitamin E level in the liver microsomal membrane: effects of age and dietary alpha-linolenic acid deficiency

Age and diet-induced variations of phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and alpha-tocopherol concentration in the liver microsomal membrane were studied in male Wistar rats fed a semipurified diet either balanced in n-6 and n-3 polyunsaturated fatty acids (PUFA) (Control) or deprived of alpha-linolenic acid, i.e. n-3 PUFA (Deficient) over two generations. The animals were studied at the age of 6 months (adult) or 24 months (old). Both PHGPx activity and vitamin E level were significantly higher in 24-month old rats as compared to 6-month old rats. By contrast, the thiobarbituric acid reactive substances (TBARS) following stimulated in vitro peroxidation of membrane lipids were markedly lower (P < 0.01) with aging. The fatty acid composition of microsomal membrane phospholipids (PL) was also considerably modified by age. In particular, the levels of arachidonic acid and total n-6 PUFA were lower (P < 0.001) whereas n-3 PUFA levels were higher (P < 0.001) in most PL main classes. The alpha-linolenic acid deficiency markedly influenced these age-related changes. The higher PHGPx activity in the old rats as compared to the adult rats was only significant in those fed the control diet. In the 6-month old rats (but not in the 24-month old rats), the deficient diet led to a higher membrane vitamin E level and to lower TBARS production than the control diet. The results suggest that the nature of dietary PUFA may influence the age-related variations in this pair of membrane antioxidants and also in the fatty acid composition of microsomes.     

The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

Mitochondria: omega-3 in the route of mitochondrial reactive oxygen species

Mitochondria are the main organelles that produce reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage to macromolecules, including lipids, and can damage cellular membrane structure and functions. Mitochondria, the main target of ROS-induced damage, are equipped with a network of antioxidants that control ROS production. Dietary intake of omega-3 polyunsaturated fatty acids (ω3PUFAs) and consequently the increase in ω3PUFA content of membrane lipids may be disadvantageous to the health because ROS-induced oxidative peroxidation of ω3PUFAs within membrane phospholipids can lead to the formation of toxic products. Mitochondrial control of lipid peroxidation is one of the mechanisms that protect cell against oxidative damage. This review discusses the role of mitochondria in ROS generation and the mechanisms by which it regulates ROS production. The susceptibility to peroxidation of PUFAs by ROS raises the question of the adverse effects of ω3PUFA dietary supplementation on embryonic development and prenatal developmental outcomes.     

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast

To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.     

Lipid peroxidation and the thiobarbituric acid assay: standardization of the assay when using saturated and unsaturated fatty acids

Saturated fatty acids are less vulnerable to lipid peroxidation than their unsaturated counterparts. In this investigation, individual fatty acids of the C(16), C(18) and (20) families were subjected to the thiobarbituric (TBA) assay. These fatty acids were chosen based on their degree of saturation and configuration of double bonds. Interestingly, an assay threshold was reached where increasing the fatty acid concentration resulted in no additional decrease in the TBARS concentrations. Therefore, the linear range of TBARS inhibition was determined for fatty acids in the C(16) and C(20) families. The rate of TBARS inhibition was greater for the saturated than for unsaturated fatty acids, as measured from the slope of the linear range. These findings demonstrate the need to standardize the TBARS assay using multiple fatty acid concentrations when using this assay for measuring in vitro lipid peroxidation.     

Peroxisome proliferator-activated receptor alpha agonist, clofibrate, has profound influence on myocardial fatty acid composition

The hypolipidemic fibrates have been identified as agonists of the peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a critical role in the regulation of cardiac fatty acid metabolism. Despite the widespread clinical use of fibrates, their role in myocardial oxidative stress and fatty acid composition is less known. In this study, male Sprague-Dawley rats were treated with either vehicle (olive oil, 1 ml/kg) or clofibrate (300 mg/kgday i.p.) for 1-14 days. Lipid peroxidation in heart homogenate was determined by thiobarbituric acid reactive substance (TBARS) assay. Results show that hearts from clofibrate-treated rats are more susceptible to FeSO(4)-induced TBARS production. The antioxidants including catalase and glutathione-related enzymes were marginally affected. We demonstrated that myocardial fatty acid composition was dramatically altered by clofibrate treatment. In hearts from clofibrate-treated rats, the principal n-6 polyunsaturated fatty acids (PUFAs), linoleic acid (C18:2 n-6) and arachidonic acid (C20:4 n-6), was significantly reduced, while the content of the principal n-3 PUFA, docosahexaenoic acid (C22:6 n-3), was markedly increased. The overall effect was to reduce n-6/n-3 ratio and increase the unsaturation extent of myocardial fatty acids. Functional study showed that hearts from clofibrate-treated rats had an improved recovery of post-ischemic contractile function and reduced ischemia/reperfusion (I/R)-induced infarct size. The data shows that clofibrate has a profound impact on cardiac fatty acid composition, which may contribute to its cardioprotective effect.     

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.