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The aim of this work was to study the adaptation of enzymatic antioxidant cell defense to the nature of the membrane polyunsaturated fatty acids (PUFA). 3T3 Swiss fibroblasts were grown for 5 days in a medium supplemented with 50 microM linoleic acid (LA) or eicosapentaenoic acid (EPA) and compared to control cells (C). The phospholipid fatty acid content was evaluated: LA were enriched in n-6 PUFA (27.8%) in comparison to C (6.7%) or EPA (5.6%); EPA were enriched in n-3 PUFA (26.2%) in comparison to LA (4.4%) or C (4.6%). The fatty acid double bond index (DBI) increased from C to LA and EPA. The activities of the three key enzymatic antioxidant defenses, SOD, GPx and GST, increased with the degree of unsaturation of the phospholipid fatty acids. In the cells with fatty acids that are very sensitive to oxidative stress, the higher activities of SOD and GPx might act to limit the initiation of lipid peroxidation and the higher activities of GST and GPx to decrease the toxic effects of the various species produced from lipid degradation.

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Twenty-seven rats were divided into three groups and fed on diets containing 0.3, 6 or 60 RE (retinol equivalent) retinyl palmitate/g food. After 7 weeks, hepatic vitamin A uptake was found to be more efficient in vitamin A-deficient rats than in rats given adequate vitamin A. We showed that during the metabolic adaptation of the animals to the level of vitamin A in the diet, extensive modifications occur in the antioxidant defences of the organism. In parallel with the increase in the level of vitamin A, the decrease in the level of alpha-tocopherol in the plasma can bring about a greater susceptibility of the lipoproteins to oxidative stress. Similarly, the decrease in the hepatic alpha-tocopherol level and in glutathione peroxidase activity leads to the weakening of the liver's antioxidant defences.

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The effects of catechin, a well-known in vitro antioxidant, on 3T3 Swiss fibroblasts are studied under different conditions of oxidative stress leading to cell proliferation or cytotoxicity. Various levels of reactive oxygen species (ROS), generated extracellularly by the xanthine-xanthine oxidase (X-XO) system, are at the origin of the biphasic effect on DNA synthesis by 3T3 Swiss fibroblasts. The addition of 10(-2) U XO/mL, in the absence of exogenous X, catalyzes the production of low levels of O2-. and H2O2 in the extracellular medium, which stimulate DNA synthesis and cell division. The increase in the level of ROS, by addition of increasing X concentrations, did not enhance this effect proportionally. On the contrary, high levels of ROS inhibit DNA synthesis, the cytotoxicity induced being proportional to the level of H2O2 generated by the enzyme system. Catechin does not significantly modify DNA synthesis induced by low levels of ROS, but protects in a dose-dependent manner against the cytoxicity of high levels of ROS.

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Descriptions of the effects of paraquat (P2+) on the peroxidation of liver microsomes are very divergent. Therefore, the presence of ferric iron in the medium and the activity of microsomal mixed-function oxidase system are two factors that we have taken into consideration to explain the discrepancies. The results showed that 100 microM P2+ potentializes the slight production of MDA induced by low concentrations of Fe3+ (< or = 15 microM). In these conditions, P+., arising from the one-step reduction of P2+ by NADPH-cytochrome C reductase, could reduce Fe3+ and cause the formation of species that initiate peroxidation. However, unlike the results obtained with CBrCl3, for animals induced by phenobarbital (Ph), the production of MDA in the presence of FeCl3 and of P2+ was weaker than for the controls. The establishment of a new Fe3+/Fe2+ equilibrium owing to increased production of P+. could be responsible.

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Direct membrane injury by CCl4, in situations excluding metabolic activation, was evaluated in saponin-permeabilized hepatocytes and in microsomes by measuring immediate Ca2+ efflux. A good correlation appears between the Ca2+ efflux and the level of CCl4 in the membrane and also the variations in fluidity. Mixtures of CCl4 with water-soluble vehicles were used to improve the dispersion of CCl4 in the medium. The mixtures varied in their ability to elicit the membrane effects of CCl4. The performance of ethanol and, to a lesser degree, other alcohols, suggests the existence of a water stable structural organization between CCl4 and these amphiphilic vehicles, facilitating the transfer of CCl4 to the membrane.

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A polarographic method to assess the scavenging capacity of a molecule for O2-. is proposed. This method is based on the fact that O2-. is not detected by the Clark electrode and that a scavenger competes with spontaneous dismutation of O2-. So, the reduction of O2 into O2-. and the decomposition of H2O2 by catalase, releasing O2, show a biphasic kinetic. Various kinetic parameters can be used to calculate the nmol of O2-. scavenged and also supply data on the reaction mechanisms (oxidation or reduction of O2-.) involved in scavenging. This method presents several other advantages: scavenging capacity can be assayed without added indicators which themselves behave as scavengers (as demonstrated for NBT), the presence of scavengers which interfere with the O2-. generating system (xanthine-xanthine oxidase) does not invalidate the measurements made.

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Pesticides represent a good picture of the Risk-Profits equation. Owing to their pest-destroying properties they are required in the mondial food but they remain inevitably present as residues in foods from both animal and vegetal origins. Therefore due to their biological activity a problem arise in Public Health. Actually, the investigations carried out on food contamination show that--except in pinpoint cases--pesticides seem less worrying that other contaminant such as heavy metals or mycotoxins. However questions remain concerning the consequences of repeated ingestion at low levels, the extrapolation for human of experimental results from animals, the simultaneous presence of several active compounds, their consequences on the food-value and the relationship between toxicity and nutritional or pathological states.

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We have recently shown the kinetic behavior of liver retinyl esters in rats with adequate vitamin A levels receiving oral vitamin supplementation. In the present work we have studied the effects of intramuscular administration of a vitamin A preparation on the metabolism of vitamin A in the rat. Retinol administered intramuscularly to rats in the form of an emulsion brought about a significant increase in the serum and liver concentration of vitamin A; this increase was slightly less than in orally treated rats. In each group, retinyl palmitate constituted 80-85% of the total retinyl esters, followed by stearate (9-13%), laurate, palmitoleate, myristate, linoleate and pentadecanoate making up 3-10%. The subcellular localization of all retinyl esters is similar and dependent on age but not on the route of administration. These results indicate that although the best hepatic storage is achieved with an orally administered vitamin A emulsion, the intramuscular administration of a physiological dose might provide an effective supplementation method if oral vitamin A is contraindicated.

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Rehabilitation in the Philippines has in the past been based on the Western model, with an emphasis on hospital departments located in the major cities. This approach is inappropriate for the majority of disabled people in the Republic as 70% of the population live in rural areas. A community-based programme was devised using local volunteers who had simple training. These volunteers can identify and support disabled people in their own villages, avoiding long journeys and expensive institutional care.

PIP: Because 70% of the disabled people in the Philippines live in rural areas, a trial of community-based rehabilitation services (CBRS) according to the WHO model was conducted in Bacolod City, the capital of Negros Occidental province, in 1981. Until this time, rehabilitation could only be done in major centers with highly trained specialists and special equipment. Design of rehabilitation for people in rural areas is complicated by the fact that disabled people are isolated from each other and have varying types of disabilities; funds, personnel and equipment are scarce; Western-based methods may not apply in rural areas; rural people cannot afford to pay for rehabilitation; and they often have secondary disabilities due to poverty. Community-based services are provided by community workers, families, and volunteers, intermediate level medical personnel, and specialists after referral. This pilot project employed local supervisors trained in early detection and simple techniques. Local supervisors were chosen based on residence, willingness to work with the disabled, and English literacy. An expert committee followed the WHO manual, "Training the Disables in the Community," to train the local supervisors in the areas of mobility, speech, hearing, seeing, and learning. A local project committee was formed to implement and monitor the project. The local supervisors were trained at the Department of Rehabilitation Medicine of the Philippine General Hospital by a multi-disciplinary team. They were given specially prepared forms to document their work and they had at least 3 clients each. The major obstacles of the program were a lack of referral services, the low priority given to the project by some local agencies, difficulty gaining cooperation of some families, and a misunderstanding of the role of the local supervisors. Nevertheless, the CBRS is a cost-effective means of delivering rehabilitation services to rural areas.

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A column ion-exchange chromatographic method for the determination of 2-carboxy thiazolidine-4-carboxylic acid (TDCA) in blood and urine is described. After elimination of endogenous thiols, alkaline hydrolysis of the compound yields cysteine which is determined spectrophotometrically. The described method is specific for intact TDCA. Using a 3-ml aliquot of sample it allows the linear determination of this hepatoprotective drug from 5 micrograms TDCA ml-1 with a detection limit of 3 micrograms ml-1. The method was applied to study the time course of plasma levels and urinary excretion. An in vitro metabolism study showed that TDCA has a potential as a cysteine donor for glutathione synthesis.

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The effect of dietary essential fatty acid (EFA) deficiency on lipid composition, fluidity and important enzyme and transport activities of liver microsomal membrane was studied in weanling rats. After 133 d of EFA deficiency, no difference was noticed in membrane phospholipid, cholesterol and protein levels, but a significant change occurred in the fatty acid composition of bilayer phospholipids. In EFA-deficient rats, linoleic (18:2(n-6] and arachidonic (20:4(n-6] acids were both severely lower while oleic (18:1(n-9], palmitoleic (16:1(n-7] and particularly 5,8,11-eicosatrienoic (20:3(n-9] acids were significantly higher than in controls. The higher level of the latter tended to compensate for the lower level of 20:4(n-6). Membrane fluidity, as estimated by the reciprocal of the order parameter S, was lower in the deficient rats than in the controls, and all the measured microsomal enzyme activities were markedly affected. NADH-Cyt b5 electron transferring system, coupled with the fatty acid desaturation system, was higher than in controls. In contrast, the cytochrome P450 complex activity was lower and some of the important liver detoxifying enzyme activities were lower due to physical-chemical changes in the microsomal membrane. Calcium uptake and Ca2+-ATPase activity were also significantly lower in EFA-deficient rats than in controls. It was concluded that fatty acid composition may be the major factor contributing to membrane fluidity and function and that EFA might play a role in regulating the intrinsic membrane protein activities.

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The in vivo syntheses of two liver microsomal cytochromes P-450 PB3a, P-450 UT50 [(1987) Eur. J. Biochem., submitted] (Mr 50,000, 52,000) have been estimated by measuring the specific activity 2 h after i.p. administration of delta-[3H]aminolevulinic acid to male Sprague Dawley rats. The animals were fed either a standard rat chow (5% lard, 22% casein) or unbalanced diets (high lipid, 30% lard or low protein, 6% casein) with or without 50 ppm Phenoclor DP6. The high-lipid diet supported a more rapid body weight gain but had little impact on cytochrome P-450 content, expressed either per whole liver or per mg microsomal protein, and on the incorporation of the precursor into cytochrome P-450. The latter was determined by measuring the radioactivity incorporated into the cytochrome P-450 fraction, partially purified by affinity chromatography, as well as into two cytochrome P-450 isozymes (Mr 50,000 or 52,000) purified by DEAE-52 cellulose ion-exchange chromatography. The low-protein diet, on the other hand, severely depressed body weight gain and cytochrome P-450 content as well as incorporation of radioactivity, the lower-Mr cytochrome (Mr 50,000) being particularly affected. However, when a potent inducer, Phenoclor DP6, was added to the low-protein diet, cytochrome synthesis was restored indicating that the effect was reversible.

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The temporal effects of retinoic acid supplementation on hepatic cytochrome P-450-dependent enzymes were studied on the rat. Four groups of male weanling rats were fed semi synthetic diets: two groups containing 0 or 4.4 mg retinol equivalents per kg diet as retinyl palmitate (A- RA- and A+ RA- groups) and two similar groups supplemented with all trans retinoic acid (12 mg/kg diet) (A- RA+ and A+ RA+ groups). After five or ten weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for aniline hydroxylase, aminopyrine N demethylase activities and cytochrome P-450 levels. Whereas no change was observed between the four groups after 5 weeks, the following modifications appeared after 10 weeks: Vitamin A deficiency decreased hepatic drug metabolism by phase I enzymes (hydroxylase and N demethylase) but only when liver storage pool was not detectable. Vitamin A concentration as low as 4 micrograms/g is sufficient to avoid any perturbation of these enzymes. Parallel to a sparing effect on liver reserves of vitamin A, retinoic acid maintained a normal activity of enzymes of xenobiotic metabolism. However, retinoic acid treatment produced an alteration of phase I enzymes in vitamin A supplemented group (A+ RA+). As this was accompanied by a doubling of vitamin A liver reserves, compared to A+ RA- group, it is suggested that this might result from a liver vitamin A overloading, leading to membrane damage perturbing microsomal enzymes. These results indicate the need for a more careful use of retinoids as a therapeutic agent.

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In vitro effects of the ethylene bis-dithiocarbamate fungicides, nabam and zineb, on the hepatic microsomal monooxygenases of male rats were examined. Incubation of nabam and zineb with hepatic microsomes, without NADPH, leads to an inhibition of the metabolism of aminopyrine and aniline and to a denaturation of cytochrome P-450 into cytochrome P-420; in addition nabam causes the destruction of cytochrome P-450. Addition of NADPH into the incubation medium increases the inhibition of the monooxygenases, principally the inhibition of the metabolism of aniline induced by nabam. We studied the in vitro effects of three of the chief breakdown products of these fungicides: ethylene bis-isothiocyanate sulfide (EBIS), ethylene thiourea (ETU), and carbon disulfide (CS2). EBIS appears to be the only metabolite affecting directly (without NADPH) the hepatic monooxygenases activity. EBIS accounted partly for nabam-induced inhibition of the hepatic microsomal monooxygenases. The data suggest that the decrease of monooxygenases activity seen on incubation of nabam with hepatic microsomes may be due to the denaturation and destruction of cytochrome P-450 resulting from covalent binding of the compounds with cysteine sulfhydryl groups in cytochrome P-450. Inhibition of monooxygenase activity induced by zineb seems to be due to the reaction with the sulfhydryl groups of cytochrome P-450 and to another mechanism, probably related to its lipophilic character.

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Rats were fed vitamin A deficient diets (-A) or supplemented with vitamin A (+A) (4.4 mg retinol equivalents/kg diet), either without (-RA) or with retinoic acid (+RA) (12 mg/kg diet) supplementation for up to six weeks. Plasma and liver levels as well as the subcellular localization of vitamin A were determined. In rats reared on the vitamin A rich diet the localization of retinyl palmitate (principal reserve form) is shown to be dependent on age. Two pools exist, i.e. one consisting of the nuclear and mitochondrial-lysosomal fractions and the other containing the microsomal and cytosol fractions. A rapid replenishment of mitochondrial-lysosomal fractions occurs in the first weeks after the weaning. During six weeks of deficient diet an identical mobilization was seen from the different subcellular fractions. Supplementation with RA caused an immediate and sustained reduction of serum vitamin A levels but did not disturb the subcellular localization of retinyl palmitate. A relationship between these phenomena and the subcellular distribution of the retinyl palmitate hydrolase (RPH) and the cellular vitamin A binding proteins (CRBP) is likely to exist.

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Nabam (fungicide dithiocarbamate) has been incorporated with the diet of rats during six months (the doses were: 0, 10, 50, 100, 500, 1000 and 2000 ppm). It decreases significantly the hepatic microsomal enzymes activity (aniline hydroxylase and aminopyrine N-demethylase and the liver P 450 content, but the cytochrome b 5 concentration doesn't seem to be modified. The microsomal lipidic and proteic content is only modified with the highest Nabam dose. Several hypotheses may be proposed to explain the Nabam effects: one is the inhibition of the monooxygenases and their biosynthesis repression.

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