N-oxidative metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans: excretion of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx in urine.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a major heterocyclic aromatic amine (HAA) formed in cooked meats, is metabolically transformed to mutagenic/carcinogenic intermediates. Cytochrome P4501A2 (CYP1A2)-mediated N-hydroxylation followed by phase II O-esterification by N-acetyltransferase (NAT2) are generally regarded as activation processes in which MeIQx and other HAAs are converted to genotoxic species. In this study, we determined the relationship between the activities of these two enzymes and the urinary excretion level of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx--N2-(beta-1-glucosiduronyl)-2-hydroxyam ino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx-N2-glucuronide)--among healthy subjects fed a uniform diet containing high-temperature cooked meat. The individuals (n = 66) in the study ate meat containing known amounts of MeIQx, and urine was collected from 0 to 12 h after the meal. After addition of the deuterium-labeled internal standard to urine, N-OH-MeIQx-N2-glucuronide was isolated using solid-phase extraction and immunoaffinity separation. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline (2-OH-MeIQx) by heating with acetic acid. 2-OH-MeIQx and its deuterated analogue were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry using selected ion monitoring procedures. The subjects in the study excreted an average of 9.4 +/- 3.0% (+/-SD) of an ingested dose of MeIQx as N-OH-MeIQx-N2-glucuronide in urine; the range varied from 2.2 to 17.1%. A significant correlation was found between the level of N-OH-MeIQx-N2-glucuronide in urine and the amount of MeIQx ingested (r(s) = 0.44; P = 0.0002). The excretion level of N-OH-MeIQx-N2-glucuronide in urine was not associated with the enzyme activities of NAT2 or CYP1A2. This is expected with the latter enzyme because the metabolism of MeIQx is first order and very rapid at the amounts ingested. The amount of N-OH-MeIQx-N2-glucuronide in urine was not correlated with the age or sex of the individuals. Our results indicate that biotransformation of MeIQx via CYP1A2 oxidation to form the N-hydroxylamine followed by N2-glucuronidation is a general pathway of MeIQx metabolism in humans; the variability in the excreted levels of N-OH-MeIQx-N2-glucuronide is probably due to interindividual differences in UDP-glucuronosyltransferase activity and/or excretion pathways.

Urinary excretion of unmetabolized and phase II conjugates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans: relationship to cytochrome P4501A2 and N-acetyltransferase activity.

Cooking meat, fish, or poultry at high temperature gives rise to heterocyclic aromatic amines (HAAs), which may be metabolically activated to mutagenic or carcinogenic intermediates. The enzymes cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2) are principally implicated in such biotransformations. We have determined the relationship between the activity of these two enzymes and the urinary excretion of unmetabolized and Phase II conjugates of the two HAAs MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in individuals fed a uniform diet containing high-temperature cooked meat. The subjects in the study ate meat containing known amounts of MeIQx and PhIP, and urine collections were made 0-12 and 12-24 h after a meal. MeIQx and PhIP were measured in urine after acid treatment that quantitatively hydrolyzes the Phase II conjugates to the respective parent amine. The extracts containing the HAAs were purified by immunoaffinity chromatography and analyzed by liquid chromatography using electrospray ionization-tandem mass spectrometry. The MeIQx content in the 0-12 h urine increased after acid hydrolysis by a factor of 3-21-fold. After acid treatment, the total amount of MelQx (unmetabolized plus the N2-glucuronide and sulfamate metabolites) excreted in the 0-12 h urine was 10.5 +/- 3.5% (mean +/- SD) of the dose, whereas the total amount of PhIP [unmetabolized plus acid-labile conjugate(s)] in the 0-12 h period was 4.3 +/- 1.7% (mean +/- SD) of the dose. The total amount of PhIP in the 12-24 h urine after acid treatment was 0.9 +/- 0.4% (mean +/- SD) of the dose. Linear regression analysis of the amounts of MeIQx and PhIP excreted in the 0-12 h period expressed as a percentage of the ingested dose, for all subjects, gave a low but significant correlation (r = 0.37, P = 0.005). Linear regression analyses showed that lower total MeIQx (unmetabolized plus the N2-glucuronide and sulfamate metabolites) in urine was associated with higher CYP1A2 activity, whereas total PhIP (unmetabolized plus conjugated) in urine showed no association to CYP1A2 activity. These results indicate that in humans, MeIQx metabolism and disposition are more strongly influenced by CYP1A2 activity than are those of PhIP. Linear regression analysis found no association between NAT2 activity and the levels (unmetabolized plus acid-labile conjugates) of MeIQx or PhIP excreted in urine.

Urinary excretion of nitrate, N-nitrosoproline, 3-methyladenine, and 7-methylguanine in a Colombian population at high risk for stomach cancer.

Urinary excretion levels of nitrate and N-nitrosoproline were determined in 160 individuals in a Colombian population at high risk for gastric cancer. In 156 of these subjects urinary levels of 3-methyladenine and 7-methylguanine were determined. Gastric biopsy specimens were obtained from 118 individuals and were histologically characterized according to pathological criteria into the following groups: normal, superficial gastritis, chronic atrophic gastritis, chronic atrophic gastritis with intestinal metaplasia, and dysplastic. The histological changes were correlated with the four variables listed above. There were no significant differences in the excretion of nitrate, N-nitrosoproline, 3-methyladenine, or 7-methylguanine in subjects with different pathological changes. A statistically significant correlation was present between nitrate and N-nitrosoproline excretion in the total population group (r = 0.297, P = 0.0001). A highly significant correlation (r = 0.56, P = 0.0002) was noted for urinary nitrate and N-nitrosoproline excretion in individuals with intestinal metaplasia and dysplasia. An increase in the urinary excretion of 3-methyladenine and 7-methylguanine was associated with tobacco smoking in the total population group.

Comparison of phosphatidylcholines containing one or two docosahexaenoic acyl chains on properties of phospholipid monolayers and bilayers.

Docosahexaenoic acid (DHA) is the longest and most unsaturated of the n - 3 fatty acids found in membranes. Although a number of membrane properties have been demonstrated to be affected by the presence of this fatty acid, its mode of action has yet to be clearly elucidated. Prior reports on biological membranes have not distinguished the effect of mono-docosahexaenoyl phospholipids from those caused by phospholipids containing docosahexaenoic acid in both chains. This report compares properties of monolayers and bilayers composed of either 1-stearoyl-2-linolenoyl-sn-glycero-3-phosphocholine (as a control), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine or 1,2-di-docosahexaenoyl-sn-glycero-3-phosphocholine. When compared to the mono-DHA phosphatidylcholine (PC), the di-DHA PC occupies a much larger area/molecule, supports a more fluid and permeable bilayer, and is less susceptible to peroxidation. Monolayers made from either phospholipid are not condensable by cholesterol. We suggest many of the membrane properties linked to the presence of DHA may be the result of phospholipids which have lost their normal positional selectivity and have incorporated DHA into both positions.

Docosahexaenoic acid (DHA) alters the structure and composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.

Membrane lipid microdomains are regions of the membrane thought to be functionally important, but which have remained poorly characterized because they have proven to be difficult to isolate. The exfoliation of small membranous vesicles from the cell surface is a continuous and normal activity in many cells. If microdomains are relatively large or stable, they may influence the structure and composition of exfoliated vesicles, which are easy to isolate. We tested the ability of docosahexaenoic acid (DHA), a fatty acid proposed to alter the structure of microdomains, to change the structure and composition of vesicles exfoliated from a murine leukemia cell line. Cells were cultured in normal and DHA-enriched media for 72 h, then washed and given a 15-h exfoliation period. Afterwards, the pooled vesicles and their parent plasma membrane were collected and analyzed. Vesicles and plasma membrane from cells grown in normal culture medium had similar fatty acid compositions, including equal, and low, proportions of DHA, but the vesicles had much more cholesterol and displayed higher anisotropy than the plasma membrane. When cells were grown in DHA-enriched medium, both the plasma membrane and exfoliated vesicles had 10-fold elevated levels of DHA in their phospholipids, with the DHA displacing other polyunsaturates. These cells released vesicles having significantly reduced levels of cholesterol and monoenoic fatty acids than those in normal culture. The anisotropy of these vesicles was also dramatically reduced. These data are consistent with DHA altering the structure and composition of membrane microdomains on the cell surface, and suggest that exfoliated vesicles may prove useful in the further study of membrane microdomains.

Abscisic acid increases lipid bilayer permeability to cations as studied by phosphorus-31 NMR.

Using a 31P-NMR lanthanide shift technique, abscisic acid is shown to enhance the permeability to praeseodymium of lipid bilayers composed of 80 mol% phosphatidylcholine and 20 mol% phosphatidylethanolamine. Praeseodymium permeability is immeasurably slow in the absence of the hormone whether or not phosphatidylethanolamine is present in the bilayers. Only in the presence of abscisic acid is praeseodymium permeability observed, the effect being significantly greater when phosphatidylethanolamine is present. These results substantiate prior reports from nonelectrolyte permeability studies that abscisic acid interacts with phosphatidylethanolamine in lipid bilayers.

Effect of retinol and retinoic acid on permeability, electrical resistance and phase transition of lipid bilayers.

Retinol and retinoic acid have been incorporated into the artificial membrane systems, planar bimolecular lipid membranes and liposomes, and their effects on several membrane parameters have been measured. 1. Retinol and retinoic acid increased the permeability of egg lecithin liposomes to K+, I- and glucose when incorporated into the membranes at levels as low as 0.5 membrane mol%. Retinoic acid influenced permeability more than did retinol for each of the solutes tested. 2. Retinol and retinoic acid both decreased the electrical resistance of egg lecithin-planar bimolecular lipid membranes from 0.5 to 8 membrane mol%. Retinoic acid effected a larger change than did retinol. 3. Retinol and retinoic acid increased the permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine liposomes to water at 1.0 and 3.0 membrane mol%. A larger effect on water permeability was measured for retinoic acid than for retinol. 4. Retinol and retinoic acid at 1.0 and 3.0 membrane mol% were shown to lower the phase-transition temperature of liposomes composed of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. Phase-transition temperatures were monitored by abrupt changes in water permeability and liposome size associated with the transition. Retinoic acid lowered the phase-transition temperature of dimyristoylphosphatidylcholine liposomes more than did retinol, while both retinoids had almost the same effect on dipalmitoylphosphatidylcholine liposomes.

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers.

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.

Plant sterol inhibition of abscisic acid-induced perturbations in phospholipid bilayers.

Abscisic acid (ABA)-induced phospholipid bilayer perturbations (permeability and lipid vesicle aggregation) are shown to be reversed by incorporation of a commercially available mixture of plant sterols (60% beta-sitosterol, 27% campesterol and 13% dihydrobrassicasterol) into the membranes. As little and 5 membrane mol% plant sterol inhibits ABA-stimulated permeability of both saturated and unsaturated mixed phosphatidylcholine/phosphatidylethanolamine bilayers to the fluorescent anion carboxyfluorescein by more than 50%. The same conclusion was reached by an osmotic swelling technique for the uncharged permeant solute erythritol. Hormone-induced carboxyfluorescein permeability to mixed acyl chain phosphatidylcholine bilayers was similarly inhibited by the sterols, but only if the membranes were tested at a temperature where liquid crystal and gel states coexist. The plant sterols were also shown to prevent the ABA-induced fusion of mixed phosphatidylcholine/phosphatidylethanolamine bilayers. The ABA effect on membranes is inhibited equally by plant sterols as well as cholesterol. From these experiments a possible role is suggested for plant sterols in controlling the mode of action of ABA.

Interaction of alpha-tocopherol with fatty acids in membranes and ethanol.

The techniques of fluorescence polarization, ultraviolet light absorbance and fluorescence quenching by acrylamide are used to probe the structural role of alpha-tocopherol in phospholipid bilayers. Using 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of (anthroyloxy)stearic acid (AS) fluorescence probes, alpha-tocopherol is shown to increase fluidity and decrease order of gel state bilayers, and to decrease fluidity and increase order of bilayers in the liquid crystalline state. More complex behavior is noted for bilayers made from mixed acyl chain phosphatidylcholines (PCs) where the sn-1 position is saturated and the sn-2 position unsaturated compared to bilayers composed of PCs where both acyl chains are either saturated or unsaturated. Complexation between alpha-tocopherol and either free fatty acids or fatty acids esterified to the sn-2 position of PCs is indicated by ultraviolet light absorbance in both organic solution and in lipid bilayers. The strength of the complexes, expressed as interaction constants, are dependent upon the number of acyl chain unsaturations from 0 (stearic acid), to 6 (docosahexaenoic acid). Relation of the strength of these complexes to the degree of acyl chain unsaturation is confirmed by monitoring the fatty acid protection from acrylamide bleaching of alpha-tocopherol. These experiments suggest that the extent of acrylamide bleaching is related to the extent of association with the fatty acids.

Cholesterol condensation of alpha-linolenic and gamma-linolenic acid-containing phosphatidylcholine monolayers and bilayers.

Cholesterol is demonstrated to condense phosphatidylcholine (PC) monolayers and bilayers containing stearic acid in the sn-1 position and alpha-linolenic acid in the sn-2 position (18:0, alpha-18:3 PC) but has no effect when gamma-linolenic acid occupies the sn-2 position (18:0,gamma-18:3 PC). Cholesterol-induced condensation is measured by area/molecule determinations made on monolayers using a Langmuir trough, while condensation in bilayers is followed by the fluorescent dyes merocyanine (MC540) and dansyllysine. Permeability to erythritol is also demonstrated to be diminished by cholesterol for the condensable 18:0,alpha-18:3 PC bilayer membranes but not the 18:0,gamma-18:3 PC membranes. alpha- and gamma-linolenic acid are isomers containing 18 carbons and three unsaturations. Both fatty acids have unsaturations at positions 9 and 12 and differ only in the location of the third unsaturation, at either position 6 for gamma-linolenic acid (an omega-6 fatty acid) and at position 15 for alpha-linolenic acid (an omega-3 fatty acid). Here lipid-cholesterol interaction is used to distinguish the effect of position of unsaturation on membrane structure.

Abscisic acid-lipid interactions: a phospholipid monolayer study.

Lipid monolayer studies were performed on a Langmuir trough in the absence and in the presence of the plant hormone abscisic acid (ABA). The ABA-induced effects on the lipid monolayers can be summarized as follows: (i) ABA as the free acid (pH below 5.3) increased the molecular area and slightly decreased the surface pressure in the collapse points of monolayers made of saturated, unsaturated and of mixed lipids; ABA as the anion showed only minor effects. (ii) The ABA-induced area increase of the lipid monolayers decreased when the surface pressure increased, but some ABA remained in the monolayers made of unsaturated phospholipids even at collapse pressure. (iii) The incorporation of ABA into the monolayers could be inhibited by adding the plant sterol beta-sitosterol to the monolayer forming phospholipids. (iv) There was no substantial difference of ABA action on plant phospholipids as compared with other phospholipids. (v) ABA had a much stronger influence on unsaturated phospholipids than on saturated ones. (vi) ABA decreased the phase-transition temperature of saturated phospholipids. These results, which agree with those obtained from phospholipid vesicle studies, indicate that the physical state of the lipid is important for the ability of ABA penetrating into the lipid monolayer. Finally, a possible relevance of these results is discussed in terms of the action of ABA on guard cell membranes of plants.

Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane.

It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techniques are employed, pressure-area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At X(SDPE)> or =0.15 in SM, a more horizontal region emerges in the pressure-area isotherms indicating 'squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of 'squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase. This conclusion is corroborated by DSC studies where as little as X(Chol)=0.0025 induces a phase separation between the two lipids.

Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.

Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes.

Docosahexaenoic acid-containing phosphatidylcholine affects the binding of monoclonal antibodies to purified Kb reconstituted into liposomes.

Class I major histocompatibility complex (MHC I) molecules are transmembrane proteins that bind and present peptides to T-cell antigen receptors. The role of membrane lipids in controlling MHC I structure and function is not understood, although membrane lipid composition influences cell surface expression of MHC I. We reconstituted liposomes with purified MHC I (Kb) and probed the effect of lipid composition on MHC I structure (monoclonal anti-MHC I antibody binding). Four phospholipids were compared; each had a phosphocholine head group, stearic acid in the sn-1 position, and either oleic, alpha-linolenic, arachidonic, or docosahexaenoic acid (DHA) in the sn-2 position. The greatest binding of monoclonal antibody AF6-88.5, which detects a conformationally sensitive epitope in the extracellular region of the MHC I alpha-chain, was achieved with DHA-containing proteoliposomes. Other epitopes (CTKb, 5041.16.1) showed some sensitivity to lipid composition. The addition of beta2-microglobulin, which associates non-covalently with the alpha-chain and prevents alpha-chain aggregation, did not equalize antibody binding to proteoliposomes of different lipid composition, suggesting that free alpha-chain aggregation was not responsible for disparate antibody binding. Thus, DHA-containing membrane lipids may facilitate conformational change in the extracellular domains of the alpha-chain, thereby modulating MHC I function through effects on that protein's structure.

Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells.

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.

Docosahexaenoic acid-induced alteration of Thy-1 and CD8 expression on murine splenocytes.

Here we test whether the incorporation of docosahexaenoic acid (DHA, 22:6), an (n-3) fatty acid, into lymphocyte membranes affects the expression of the surface proteins Thy-1.2 and CD8. DHA was incorporated into splenocytes by three methods: feeding mice diets containing menhaden (fish) oil, fusing splenocytes with DHA-containing phosphatidylcholine vesicles, and culturing splenocytes with DHA. Thy-1.2 and CD8 expression were measured by flow cytometry and complement-mediated lysis using a panel of monoclonal antibodies. As (n-3) fatty acid incorporation into the lymphocytes increased, the expression of one Thy-1.2 epitope and one CD8 epitope decreased; the expression of two CD8 epitopes increased. Although diet-induced changes in surface protein expression may result from selective migration of cell populations or the diet's effect on protein biosynthesis, fusion with lipid vesicles demonstrated that DHA-containing phospholipids can mediate a direct and immediate effect. The decrease in Thy-1.2 expression was sustained for more than a week after removal of (n-3) fatty acids from the diet, most likely due to retention of membrane-bound (n-3) fatty acids. Because Thy-1.2 and CD8 participate in T cell activation, modulation of their expression by DHA suggests that DHA, when serving as a membrane structural element, may alter immune function.

Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1.

The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose-response effect demonstrated that 5 or 10 microM DHA has no effect on Jurkat cell survival; however, 15 microM DHA rapidly decreased cell survival to 40% within 2 h of treatment. Cytotoxicity of 15 microM DHA was prevented by PA. Structurally similar phospholipids (lysophosphatidic acid, sphingosine 1-phosphate, sphingosine, and sphingosine phosphocholine) or metabolites of PA (lyso-PA and diacylglycerol) did not prevent DHA-induced cytotoxicity. PA did not produce micelles alone or in combination with DHA as examined spectrophotometrically, indicating that PA did not entrap DHA and therefore did not affect the amount of DHA available to the cells. Supporting this observation, the uptake or incorporation of [1-14C]DHA in Jurkat cells was not affected by the presence of PA. However, PA treatment reduced the amount of DHA-induced inorganic phosphate released from Jurkat leukemic cells and also inhibited DHA-induced dephosphorylation of cellular proteins. These observations indicate that PA has exerted its anti-cytotoxic effects by causing inhibition of protein phosphatase activities. Cytotoxicity of DHA on Jurkat cells was also blocked by the use of a highly specific caspase-3 inhibitor (N-acetyl-ala-ala-val-ala-leu-leu-pro-ala-val-leu-leu-ala-leu-leu-ala-pro-asp-glu-val-asp-CHO), indicating that the cytotoxic effects of DHA were due to the induction of apoptosis though activation of caspase-3. Consistent with these data, proteolytic activation of procaspase-3 was also evident when examined by immunoblotting. PA prevented procaspase-3 degradation in DHA-treated cells, indicating that PA causes inhibition of DHA-induced apoptosis in Jurkat leukemic cells. Since DHA-induced apoptosis can be inhibited by PA, we conclude that the process is mediated through activation of PP1.

Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 microM) of arachidonic acid and oleic acid had little effect. To explore the mechanism of inhibition, we used several measures of apoptosis to determine whether this process was involved in DHA-induced cell death in Jurkat cells. Caspase-3, an important cytosolic downstream regulator of apoptosis, is activated by death signals through proteolytic cleavage. Incubation of Jurkat cells with 60 and 90 microM DHA caused proteolysis of caspase-3 within 48 and 24 h, respectively. DHA treatment also caused the degradation of poly-ADP-ribose polymerase and DNA fragmentation as assayed by flow cytometric TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. These results indicate that DHA induces apoptosis in Jurkat leukemic cells. DHA-induced apoptosis was effectively inhibited by tautomycin and cypermethrin at concentrations that affect protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) activities, respectively, implying a role for these phosphatases in the apoptotic pathway. Okadaic acid, an inhibitor of protein phosphatase 2A, had no effect on DHA-induced apoptosis. These results suggest that one mechanism through which DHA may control cancer cell growth is through apoptosis involving PP1/PP2B protein phosphatase activities.

Electron spin resonance study of the interactions of retinoids with a phospholipid model membrane.

The effects of up to 20 mol% incorporation of all-trans-retinol (vitamin A), retinal (vitamin A aldehyde) and retinoic acid (vitamin A acid) on acyl chain order and dynamics in liquid crystalline dipalmitoylphosphatidylcholine membranes at pH 7.5 were studied by electron spin resonance (ESR) of 5-, 7-, 10-, 12- and 16-doxyl spin-labelled stearic acids intercalated into the membrane. Order parameters S and correlation times tau c determined from the ESR spectra demonstrate that the influence of retinoic acid differs from retinol or retinal. Whereas the latter two retinoids have negligible effect (less than 1%) on acyl chain order towards the membrane surface (5 position), retinoic acid reduces the order parameter by as much as 8% at 20 mol% incorporation. All three retinoids restrict acyl chain motion to a similar extent approaching the center of membrane (10, 12 and 16 positions), where up to 22% increases in order parameter and correlation time were observed. Complementary osmotic swelling and carboxyfluorescein release measurements show that the enhancement in permeability of egg phosphatidylcholine membranes to erythritol and carboxyfluorescein is greater with all-trans-retinoic acid than all-trans-retinol or retinal.