Pro-apoptotic effects of low doses of dimethoate in rat brain.

Dimethoate (DMT), a widely used Organophosphorous insecticide, was administered for 5 weeks (sub-chronic) at low dose (15 mg/kg b.w.) to male Wistar rats with the aim to simulate potential exposure to pesticide residues in food and water. The induction of cell death programs was investigated in two brain regions, cortex (Cx) and substantia nigra (SN), after the exposure period. We found that DMT increased cytochrome C (CytC) release from mitochondria, the Bax/Bcl-2 ratio, the activity of caspase-3 and calpains, in both brain regions compared to VEH injected ones. DMT treatment induced oxidative damage of lipids with a consequent enrichment in saturated over unsaturated fatty acids. However, the activity of mitochondrial respiratory complexes was not affected by DMT treatment. The activation of the pro-apoptotic pathway can be correlated with a decrease of TH-immunoreactive neurons in SN, comparable to the reduction observed in this cell population by aging. The results of this work contribute to understand the toxic mechanism of DMT and the possible etiological role that residues of this insecticide, might play in neurodegenerative diseases.

Cytotoxic effects of copper overload on human-derived lung and liver cells in culture.

BACKGROUND: Copper (Cu) is an essential trace metal used as a catalytic cofactor for many enzymes. However, it can have nocive effects when it participates in the Fenton reaction, producing reactive oxygen species (ROS). Excess Cu is present in the plasma of patients with diseases in which cell survival is crucial. In order to investigate the effect of Cu overload on the induction of cellular damage we chose two human cell lines derived from liver (HepG2) and lung (A-549) as representative cells exposed to exogenous (polluted air) and/or endogenous (systemic) Cu overload. METHODS: We studied ROS production using thiobarbituric acid reactive substances (TBARS) and fluorimetric measurements with dichlorofluorescein, cell viability by the trypan dye exclusion test, the methyltetrazolium (MTT) and lactate dehydrogenase leakage (LDH) assays, various cytotoxic indexes, and caspasa-3 and calpain-dependent activation as the main signals involved in the apoptosis pathway. RESULTS: Cu overload induces cell death by a differential activation of calpains (m- and µ-) and caspase-3, and modifies various proliferative indexes in a cell-type and concentration-dependent manner. The involvement of these two protease systems and the response of the two main Cu homoestatic proteins ceruloplasmin and metallothioneins are specific to each cell type. We demonstrated that Cu can trigger cell death by activation of specific protease systems and modify various proliferative indexes in a cell-type and concentration-dependent manner. GENERAL SIGNIFICANCE: These findings contribute to understanding the diverse effects of Cu overload on the pathogenesis of human diseases like cancer, cirrhosis and degenerative disorders.

Antioxidant treatment prevents the development of fructose-induced abdominal adipose tissue dysfunction.

In the present study, we tested the effect of OS (oxidative stress) inhibition in rats fed on an FRD [fructose-rich diet; 10% (w/v) in drinking water] for 3 weeks. Normal adult male rats received a standard CD (commercial diet) or an FRD without or with an inhibitor of NADPH oxidase, APO (apocynin; 5 mM in drinking water; CD-APO and FRD-APO). We thereafter measured plasma OS and metabolic-endocrine markers, AAT (abdominal adipose tissue) mass and cell size, FA (fatty acid) composition (content and release), OS status, LEP (leptin) and IRS (insulin receptor substrate)-1/IRS-2 mRNAs, ROS (reactive oxygen species) production, NADPH oxidase activity and LEP release by isolated AAT adipocytes. FRD-fed rats had larger AAT mass without changes in body weight, and higher plasma levels of TAG (triacylglycerol), FAs, TBARS (thiobarbituric acid-reactive substance) and LEP. Although no significant changes in glucose and insulin plasma levels were observed in these animals, their HOMA-IR (homoeostasis model assessment of insulin resistance) values were significantly higher than those of CD. The AAT from FRD-fed rats had larger adipocytes, higher saturated FA content, higher NADPH oxidase activity, greater ROS production, a distorted FA content/release pattern, lower insulin sensitivity together with higher and lower mRNA content of LEP and IRS-1-/2 respectively, and released a larger amount of LEP. The development of all the clinical, OS, metabolic, endocrine and molecular changes induced by the FRD were significantly prevented by APO co-administration. The fact that APO treatment prevented both changes in NADPH oxidase activity and the development of all the FRD-induced AAT dysfunctions in normal rats strongly suggests that OS plays an important role in the FRD-induced MS (metabolic syndrome) phenotype.

Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol.

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3ß- and 17ß-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

Clinical parameters and biomarkers of oxidative stress in agricultural workers who applied copper-based pesticides.

Copper based-pesticides are widely used in agricultural practice throughout the world. We studied the (i) concentration of Cu and proteins involved in Cu homeostasis, (ii) plasma redox status, and (iii) biomarkers of exposure in Cu-based pesticide applicators in order to compare them with clinical biochemical tests. Thirty-one professional applicators and 32 control subjects were recruited. Oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), copper, hematological parameters, and biochemical markers for pancreatic, hepatic and renal function were measured in plasma. Copper was increased in the exposed group compared to the control group concomitantly with TBARS, protein carbonyls, and nitrate+nitrite levels. In the exposed group, α-tocopherol and the FRAP assay were lower and LDH, transaminases, GGT, ALP, urea, creatinine, CRP and MTs were higher than in the control group. The relative leukocyte subclasses were also different between the two groups. Clinical chemistry tests did not surpass the upper reference limit. Our results suggest that the incorporation of oxidative stress biomarkers to biochemical/clinical tests should be considered for validation and included in the human health surveillance protocols.

Early alterations in vascular contractility associated to changes in fatty acid composition and oxidative stress markers in perivascular adipose tissue.

AIM: To test the early effect of fructose-induced changes in fatty acid composition and oxidative stress markers in perivascular adipose tissue (PVAT) upon vascular contractility. METHODS: Adult male Wistar rats were fed a commercial diet without (CD) or with 10% fructose (FRD) in the drinking water for 3 weeks. We measured plasma metabolic parameters, lipid composition and oxidative stress markers in aortic PVAT. Vascular contractility was measured in aortic rings sequentially, stimulated with serotonin (5-HT) and high K+-induced depolarization using intact and thereafter PVAT-deprived rings. RESULTS: Comparable body weights were recorded in both groups. FRD rats had increased plasma triglyceride and fructosamine levels. Their PVAT had an increased saturated to mono- or poly-unsaturated fatty acid ratio, a significant decrease in total superoxide dismutase and glutathione peroxidase activities and in the total content of glutathione. Conversely, lipid peroxidation (TBARS), nitric oxide content, and gluthathione reductase activity were significantly higher, indicating an increase in oxidative stress. In aortic rings, removal of PVAT increased serotonin-induced contractions, but the effect was significantly lower in rings from FRD rats. This effect was no longer observed when the two contractions were performed in PVAT-deprived rings. PVAT did not affect the contractions triggered by high K+-induced depolarization either in CD or FRD rats. CONCLUSIONS: FRD induces multiple metabolic and endocrine systemic alterations which also alter PVAT and the vascular relaxant properties of this tissue. The changes in PVAT would affect its paracrine modulation of vascular function.

Microtubule depolymerization modifies the incorporation of fatty acids into glycerolipids.

BACKGROUND: The influence of cytoskeletal integrity on fatty-acid (FA) metabolism is an almost unexplored field of biochemical research. This study therefore investigated the influence of cytoskeletal integrity on the incorporation of palmitate and eicosa-8,11,14-trienoate into glycerolipids of Hep G2 human hepatoma cells. MATERIAL/METHODS: Attached cultures and suspended cells were exposed to colchicine (COL, 10 microM) or dihydrocytochalasin B (DHCB, 20 microM) and supplemented with [14C]FAs bound to delipidated BSA or [14C]glycerol during 0-300 min of incubation. Various key enzymes of lipid metabolism were also determined after COL or DHCB treatment. RESULTS: Incorporation of both FAs into phospholipids (PLs) was strongly reduced by COL treatment especially in the PE and PC subfractions at short incubation times and in PS and SM for 300 min. COL also produced increased incorporation of both FAs into neutral lipids (NL), especially in TG and its precursors (MG and DG). DHCB increased the labeling into lyso-PL and reduced incorporation into PE and SM. However, this drug did not modify the [14C]NL to [14C]PL ratio. DG-acyltransferase and phosphatidate phosphohydrolase were stimulated by COL treatment. Phospholipase A2 activity was reduced significantly by COL and stimulated by DHCB treatment. CONCLUSIONS: It was demonstrated that the microtubule and microfilament network is involved in the incorporation of FAs and in its channeling to neutral lipids and phospholipids. These effects had differential characteristics depending on the type of FA involved and may have potential significance in the understanding of physiological and/or pathological processes.

Effect of pesticides on cell survival in liver and brain rat tissues.

Pesticides are the main environmental factor associated with the etiology of human neurodegenerative disorders such as Parkinson's disease. Our laboratory has previously demonstrated that the treatment of rats with low doses of dimethoate, zineb or glyphosate alone or in combination induces oxidative stress (OS) in liver and brain. The aim of the present work was to investigate if the pesticide-induced OS was able to affect brain and liver cell survival. The treatment of Wistar rats with the pesticides (i.p. 1/250 LD50, three times a week for 5 weeks) caused loss of mitochondrial transmembrane potential and cardiolipin content, especially in substantia nigra (SN), with a concomitant increase of fatty acid peroxidation. The activation of calpain apoptotic cascade (instead of the caspase-dependent pathway) would be responsible for the DNA fragmentation pattern observed. Thus, these results may contribute to understand the effect(s) of chronic and simultaneous exposure to pesticides on cell survival.

Hepatic recovery after damage produced by sub-chronic intoxication with the cyanotoxin microcystin LR.

The effect of sub-chronic exposure of intraperitoneal (i.p.) injections of microcystin-LR (MC-LR) on microscopic tissue architecture, hepatic function and lipid peroxidation has been studied in liver and kidney of mice. Mice were treated i.p. with 25 microg of pure MC-LR/kg body weight or saline solution for 1 month (every 2 days) with the aim of producing an inflictive stage with evident damage. Histopathological analysis of dissected livers of mice showed a disrupted lobar architecture and the development of cytoplasmatic vacuoles. According to this, a significant increase in hepatic lipid content and in lipid peroxidation levels in liver and kidney was found in MC-LR-treated animals when compared with controls. Moreover, serum alkaline phosphatase and aspartate aminotransferase activities showed a significant alteration in MC-LR-treated animals. After damage, progression or recovery was studied for 1 and 2 months of wash-out. The recovery from liver damage was evident at the cytological and physiological level, only the recovery of lobar architecture was incomplete along the period investigated. In conclusion, the present study demonstrates the ability of hepatic tissue to recover from damage produced by sub-chronic MC-LR administration. The dynamic interplay between damage and tissue-repairing response in determining the ultimate outcome of toxicity should be considered in risk-assessment studies.

Dietary lipids modify redox homeostasis and steroidogenic status in rat testis.

OBJECTIVE: The present study explored the effect of dietary oils on lipid composition, antioxidant status, and the activity of the main steroidogenic enzymes in the testis. METHODS: Forty Wistar rats were randomly assigned to one of four groups (n = 10) fed for 60 d on the same basal diet plus different lipid sources as commercial oils: soybean, olive, coconut, or grapeseed. After sacrifice, testicular lipids and fatty acid composition, free radical biomarkers, antioxidant levels, hormones, and steroidogenic enzymes were determined. RESULTS: The lipid composition of diets produced significant changes in neutral/phospholipids, free/esterified cholesterol, and plasmalogen proportion. Fatty acid patterns of these lipids were also strongly modified, influencing the double bond index. We also found a close correlation between the type of diet and the generation of free radicals. The oxidative stress in testes was higher with the grapeseed oil-supplemented diet and decreased with the other diets in this order: soybean oil > olive oil > coconut oil. Animals fed with the olive oil and coconut oil diets showed the highest testicular levels of antioxidants in addition to significantly high levels of testosterone and 3beta- or 17beta-hydroxysteroid dehydrogenase enzymes. CONCLUSION: Different oils in the diets strongly modified the homeostasis of the testicular antioxidant defense system and, in consequence, affected steroidogenic function, showing a clear correlation with the damage induced. According to our results, an appropriate mixture of olive and soybean oils could be a healthy recommendation.

A reliable biomarker derived from plasmalogens to evaluate malignancy and metastatic capacity of human cancers.

Antigen tumor markers employed in monitoring therapeutical approaches are limited by their specificity (Sp) and sensitivity (Se). The aim of this study was to investigate the suitability of a lipid tumor marker derived from ether-linked phospholipids and to compare it with others usually assayed in clinical practice. Complex lipids from normal and pathological breast, lung, and prostate tissue were isolated and analyzed by TLC and c-GLC methods. Results were compared as pooled samples, or by means of the averaged percent changes with respect to the composition observed in the normal tissue of the same patient. Sp, Se, negative-predictive (NPV) and positive- predictive values (PPV) were established for conventional markers and for the proposed lipid-derived marker. Results demonstrated that the content of monoenoic fatty acyl chains was significantly increased in total lipids, phosphatidylethanolamine, and especially in ethanolamine-containing ether lipids of neoplastic tissues with respect to their corresponding normal ones. Major changes were observed in the plasmalogen sub-fraction where the ratio monoenoic/saturated fatty acids can distinguish with high Se normal tissues from either benign or neoplastic tissues from breast, lung, or prostate lesions. Analyses of fatty acyl chains from ethanolamine-containing plasmalogens provided a reliable tumor marker that correlated with high Se and linearity with metastases spreading. This fact may be useful in prognosis of the most frequently observed human cancers.

Lipid metabolism in rats is modified by nitric oxide availability through a Ca++-dependent mechanism.

We studied lipid metabolism and the antioxidant defense system in plasma and liver of rats fed diets supplemented with L(omega)-nitro-L-arginine methyl ester (L-NAME), isosorbide dinitrate (DIS), L-arginine (Arg), or the associations of these drugs. Liver hydroperoxide and thiobarbituric-acid-reactive substance (TBARS) levels were decreased by Arg and increased by L-NAME or DIS treatments. Oxidized glutathione and conjugated dienes were increased by DIS. Nitrate + nitrite levels and serum calcium ([Ca(++)]) were incremented by Arg or DIS and reduced by L-NAME. Superoxide dismutase and catalase activities decreased under Arg treatment, while L-NAME or DIS caused stimulation. Liver high-density lipoprotein (HDL) cholesterol was increased by DIS or NAME (alone or associated with Arg). Free fatty acids and neutral and polar lipids were increased by Arg, L: -NAME, and DIS. However, predominating phospholipid synthesis increased the neutral/polar ratio. Decreased levels of nitric oxide (NO) (low [Ca(++)]) was directly associated with increased fatty acid synthetase, decreased phospholipase A(2), carnitine-palmitoyl transferase, and fatty acid desaturase activities. Raised NO (high [Ca(++)]) inversely correlated with increased phospholipase-A(2) and acyl-coenzyme A (CoA) synthetase and decreased fatty acid synthetase and beta-oxidation rate. Arg or DIS produced changes that were partially reverted by association with L-NAME. Based on these observations, prolonged therapeutical approaches using drugs that modify NO availability should be carefully considered.

Trifluralin toxicity in a Chagas disease mouse model.

Even though trifluralin (alpha,alpha,alpha-2,6-dinitro-N-N-dipropyl-p-toluidine) is effective for the treatment of experimental Chagas disease, more preclinical toxicity studies need to be performed. Cell toxicity of trifluralin was studied in Hep-G2 and Vero C76 cells treated with 50 and 150 microM trifluralin. The results show that duplication time, amount of cellular protein and cell protein/DNA values were normal. Histological, haematological and chemical parameters were measured in CF1 mice after oral trifluralin administration. Acute toxic effects were assayed by administration of 50 or 200 mg/kg body weight daily for 30 days, and chronic effects by administration of 200 mg/kg body weight once a week for 90 days (n = 20). In the acute scheme treatment, hepatic (glutamic-pyruvic, glutamic-oxalacetic and alkaline phosphatase activities; proteins and albumin plasma concentrations) and pancreatic (amylase, glycaemia) functions were normal. Mean corpuscular volume, haemoglobin and haematocrit decreased. Creatine phosphokinase, lactate dehydrogenase and glutamic-oxalacetic activity increased, suggesting lesion in myocardial tissue. Histology was normal, excepting for the heart (mild myocarditis). Similar results were observed in acutely treated animals. There were no differences in body weight gain for treated mice compared to controls. In view of the published therapeutic effects of trifluralin on CF1 Chagas disease model and considering the present results, trifluralin seems to be a moderately toxic drug with a potential selective effect on the myocardium.

Neutral and polar lipid metabolism in liver microsomes of growing rats fed a calcium-deficient diet.

We studied the incorporation of (14)C-labeled fatty acids and glycerol into different classes of glycerolipids in an in vitro system containing liver microsomes from growing Wistar rats fed a calcium-deficient (CaD; 0.5 g/kg) diet for a 60-day period. Desaturase activities and incorporation of the elongation-desaturation metabolites into specific neutral and polar glycerolipids were also studied and correlated with the activities of various enzymes involved in complex lipid metabolism (acyl-CoA synthase, acyl-CoA hydrolase, DAG-acyltransferase, DAG-kinase, lysophospatidate-acyl-CoA transferase, phosphatidate-phosphohydrolase and phospholipase A(2)). Low calcium condition led to a significant increase in the incorporation (relative amounts and specific activities) of both labeled fatty acids and glycerol with a preferential increase of labeling in neutral lipids rather than in phospholipids. Acyl-CoA synthetase, diacylglycerol acyltransferase and diacylglycerol-3-P acyltransferase activities were increased in low calcium microsomes while diacylglycerol kinase, phospholipase A(2) and palmitoyl-, stearoyl-, linoleyl-, alpha-linolenyl, and eicosatrienoyl-desaturases were decreased. The modifications observed in the interlipid and lipid/protein relationships, enzyme activities, and pattern of incorporation of labeled precursors into each glycerolipid class, suggest that decreased intake of calcium should be considered as a harmful risk factor for the development of cardiovascular diseases.

Microtubular integrity differentially modifies the saturated and unsaturated fatty acid metabolism in cultured Hep G2 human hepatoma cells.

The influence of cytoskeleton integrity on the metabolism of saturated and unsaturated FA was studied in surface cultures and cell suspensions of human Hep G2 hepatoma cells. We found that colchicine (COL), nocodazol, and vinblastin produced a significant inhibition in the incorporation of labeled saturated FA, whereas incorporation of the unsaturated FA remained unaltered. These microtubule-disrupting drugs also diminished Delta9-, Delta5-, and Delta6-desaturase capacities. The effects produced by COL were dose (0-50 microM) and time (0-300 min) dependent, and were antagonized by stabilizing agents (phalloidin and DMSO). Dihydrocytochalasin B (20 microM) was tested as a microfilament-disrupting drug and produced no changes in either the incorporation of [14C] FA or the desaturase conversion of the substrates. We hypothesized that the interactions between cytoskeleton and membrane proteins such as FA desaturases may explain the functional organization, facilitating both substrate channeling and regulation of unsaturated FA biosynthesis.

Hepatic and intestine alterations in mice after prolonged exposure to low oral doses of Microcystin-LR.

Oral intake of Microcystin-LR (MC-LR) is the principal route of exposure to this toxin, with prolonged exposure leading to liver damage of unspecific symptomatology. The aim of the present paper was therefore to investigate the liver and intestine damage generated by prolonged oral exposure to low MC-LR doses (50 and 100 µg MC-LR/kg body weight, administrated every 48 h during a month) in a murine model. We found alterations in TBARS, SOD activity and glutathione content in liver and intestine of mice exposed to both doses of MC-LR. Furthermore, the presence of MC-LR was detected in both organs. We also found hepatic steatosis (3.6 ± 0.6% and 15.3 ± 1.6%) and a decrease in intraepithelial lymphocytes (28.7 ± 5.0% and 44.2 ± 8.7%) in intestine of 50- and 100-µg MC-LR/kg treated animals, respectively. This result could have important implications for mucosal immunity, since intraepithelial lymphocytes are the principal effectors of this system. Our results indicate that prolonged oral exposure at 50 µg MC-LR/kg every 48 h generates significant damage not only in liver but also in intestine. This finding calls for a re-appraisal of the currently accepted NOAEL (No Observed Adverse Effect Level), 40 µg MC-LR/kg body weight, used to derive the guideline value for MC-LR in drinking water.

Dietary fats significantly influence the survival of penumbral neurons in a rat model of chronic ischemic by modifying lipid mediators, inflammatory biomarkers, NOS production, and redox-dependent apoptotic signals.

OBJECTIVE: Brain stroke is the third most important cause of death in developed countries. We studied the effect of different dietary lipids on the outcome of a permanent ischemic stroke rat model. METHODS: Wistar rats were fed diets containing 7% commercial oils (S, soybean; O, olive; C, coconut; G, grape seed) for 35 d. Stroke was induced by permanent middle cerebral artery occlusion. Coronal slices from ischemic brains and sham-operated animals were supravitally stained. Penumbra and core volumes were calculated by image digitalization after 24, 48, and 72 h poststroke. Homogenates and mitochondrial fractions were prepared from different zones and analyzed by redox status, inflammatory markers, ceramide, and arachidonate content, phospholipase A2, NOS, and proteases. RESULTS: Soybean (S) and G diets were mainly prooxidative and proinflammatory by increasing the liberation of arachidonate and its transformation into prostaglandins. O was protective in terms of redox homeostatic balance, minor increases in lipid and protein damage, conservation of reduced glutathione, protective activation of NOS in penumbra, and net ratio of anti-to proinflammatory cytokines. Apoptosis (caspase-3, milli- and microcalpains) was less activated by O than by any other diet. CONCLUSION: Dietary lipids modulate NOS and PLA2 activities, ceramide production, and glutathione import into the mitochondrial matrix, finally determining the activation of the two main protease systems involved in programmed cell death. Olive oil appears to be a biological source for the isolation of protective agents that block the expansion of brain core at the expense of penumbral neurons.

Evidence in favor of a facilitated transport system for FA uptake in cultured L6 cells.

In this manuscript we report a study of the transport of FA in L6 muscle cells. Cultured L6 cells took up labeled FA (C10 to C20) as a linear function of time up to 15 min. Thereafter, the rate of uptake gradually declined although it persisted for at least 12 h after the addition of the substrate. Kinetic parameters (Km, Vm, and k(o)) were determined from a fitted Michaelis-Menten-type equation modified by a term for a saturable (linear) component of the measured total uptake. Vm values were different for some of the FA studied, and Km data showed significant differences between saturated and unsaturated FA. The maximal rate of uptake was observed at pH 7.40 for decanoate, palmitate, and eicosatrienoate. Uptake was significantly influenced when the pH of the incubation medium was changed. Experiments designed to study the influence of FA/albumin molar ratio indicated that Vm was dependent on the total (bound and free) concentration of the FA. A concentrative uptake was demonstrated in short-term experiments with an apparent plateau of 20 and 40 microM for palmitate and eicosatrienoate, respectively. A competitive inhibition was also observed between palmitate as substrate and the other FA. From our results we can postulate that the uptake of FA in L6 cells is the sum of passive diffusion plus a saturable component and that the rate of uptake is dependent on one (or more) protein structures, although their precise characteristics and functions remain to be elucidated.

Correlation between fatty acyl composition in neutral and polar lipids and enzyme activities from various tissues of calcium-deficient rats.

In this study we investigated the changes induced by feeding rats a calcium-deficient diet (0.5 g Ca/kg diet) during 65 d after weaning. Phospholipase A2, acyl-Co synthetase and FA delta9-, delta6-, and delta5-desaturase activities were also determined. Calcium deficiency evoked a general alteration in the quality and proportion of the FA chains acylated to neutral and polar lipids from liver, lungs, spleen, brain, kidneys, fat, articular cartilage, erythrocyte ghosts, and plasmas, characterized by an increment of saturated FA and a significant depletion of polyunsaturated acids derived from linoleate and alpha-linolenate. Several interlipid and lipid/protein relationships were also modified in microsomes from calcium-deprived rats, with a concomitant reduction in the rotational mobility of the probe diphenylhexatriene. Phospholipase A2 and acyl-CoA synthetase activities were also decreased and increased, respectively, in some tissues from calcium-deficient rats, whereas delta9-, delta6- and delta5-desaturases were significantly depressed. We conclude that changes in tissue fatty acyl composition evoked by calcium deprivation are due to alterations in the acylation/deacylation cycles via inhibition of the phospholipase A2. These changes were reflected in the physicochemical properties of the membranes, which in turn inhibits desaturase activities. A possible failure in the transcriptional rate for desaturase-mRNA was also discussed.