Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells.

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Down-regulation of GPx1 mRNA and the loss of GPx1 activity causes cellular damage in the liver of selenium-deficient rabbits.

The effects of 10 weeks of dietary selenium and/or vitamin E deficiency (< 0.03 mg Se and 1.5 mg vitamin E per kg diet) on body Se and vitamin E stores and on the down-regulation of liver cellular glutathione peroxidase (GPx1) and plasma glutathione peroxidase (GPx3) were examined in growing female New Zealand White rabbits in comparison to Se (+ 0.40 mg Se/kg diet) and/or vitamin E (+ 150 I.U./kg diet) supplemented controls. Additionally plasma lactate dehydrogenase (LDH) activity, liver thiobarbituric acid-reactive substances (TBA-RS) and liver protein carbonyls were measured to assess the development of oxidative stress during an alimentary Se and/or vitamin E deficiency. Significantly decreased concentrations of Se and vitamin E in plasma (Se: - 70%; vitamin E: - 87%) and liver (Se: - 90%; vitamin E: - 95%) indicated an efficacious Se and vitamin E depletion of the rabbits within 10 weeks. GPx1 messenger RNA levels (GPx1 mRNA) in the livers of Se-depleted rabbits were down-regulated to 1/3-1/8 of the Se supplemented controls. GPx1 enzyme activity in the livers of Se-deficient rabbits declined to 10% of the Se-supplied control rabbits. A significantly elevated LDH activity in the blood plasma of Se- and vitamin E-deficient rabbits indicated a general impairment of tissues. Markedly increased TBA-RS concentrations and protein carbonyl contents in the livers of Se- and vitamin E-deficient rabbits gave further evidence for severe oxidative damage of cellular lipids and proteins during an alimentary Se and/or vitamin E deficiency. Both a full expresssion of GPx1 attained by dietary Se supplementation and dietary vitamin E supply effected an almost complete protection against oxidative cellular damage of the liver.

Prevention of peroxidative stress in rats fed on a low vitamin E-containing diet by supplementing with a fermented bovine milk whey preparation: effect of lactic acid and beta-lactoglobulin on the antiperoxidative action.

We examined the antiperoxidative properties of a fermented bovine milk whey preparation in rats fed on a low vitamin E-containing diet and identified the active principle in the preparation. An exogenous supply of either lactic acid or an amino acid mixture simulated the unfermented whey proteins to prevent red blood cell (RBC) hemolysis and to lower liver thiobarbituric acid reactive substances (TBARS). The supply of either whey proteins or beta-lactoglobulin resulted in an increase in liver GSH and prevented iron-mediated lipoprotein peroxidation. These protein effects were reproduced in rats orally administered with either GSH or its precursor, gamma-glutamylcysteine. The amount of TBARS formed during in vitro lipoprotein peroxidation were positively correlated with liver TBARS. These results suggest that fermented milk products containing lactic acid and bovine milk whey proteins can ameliorate peroxidative stress in tissues subjected to vitamin E deficiency.

Deficiency of vitamin E and selenium enhances calcium-independent phospholipase A2 activity in rat lung and liver.

Conditions promoting oxidative stress, which is implicated in many diseases, activate phospholipases A2, a family of enzymes central to phospholipid metabolism and signal transduction. Little is known about isozyme specificity with respect to this activation process. Accordingly, a dietary deficiency model known to induce oxidative stress was used to investigate phospholipase A2 isozyme activity in rat tissues. Long-Evans hooded rats were fed purified diets for 6 wk with or without the addition of vitamin E and selenium in a 2 x 2 factorial design. Phospholipase A2 activity was assessed in lung, liver, kidney and heart cytosol and microsomes in the presence (5 mmol/L CaCl2) or absence (5 mmol/L EGTA) of calcium with dipalmitoylphosphatidylcholine at pH 6.5. Lung phospholipase A2 activity was also assessed with 1-stearoyl-2-arachidonoylphosphatidylcholine as substrate at pH 8.5. Organ samples from rats deficient in both nutrients showed two- to tenfold higher calcium-independent phospholipase A2 activity in lung cytosol and microsomes, and in liver cytosol compared with samples from control and single nutrient-deficient rats. In contrast, the calcium-dependent activity was affected only slightly. The malondialdehyde concentration of the organs was measured and the pattern obtained mirrored that of enhanced phospholipase A2 activity for lung but not for liver. The enhanced phospholipase A2 activity in the lung cytosol and microsomes from rats deficient in both nutrients was partially blocked by p-bromophenacylbromide, further enhanced by dithiothreitol and unaffected by treatment with diisopropylfluorophosphate. These results suggest that deficiency of both vitamin E and selenium activates and/or induces unique calcium-independent forms of phospholipase A2 markedly in rat lung, and to a lesser extent in liver.

Biochemical and morphological changes in the lenses of selenium and/or vitamin E deficient rats.

The activities of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), superoxide dismutase (SOD) and the contents of malondialdehyde (MDA) and free radicals were measured, and the morphological changes were observed in the lens of control rats, selenium-deficient (SeD) and/or vitamin E deficient (VED) rats. The activities of GSH-Px in the lens of SeD rats decreased significantly. The GSH-Px activities of lens were positively related to erythrocytes selenium level. There was a free radical at g = 2.0015 in the rat lens of all groups, but the content of free radicals in the lens of SeD group was significantly higher than that of the control group. The free radical content of lens was negatively related to erythrocytes selenium level, as well as the GSH-Px activities in the lens. In vitro, ultraviolet radiation caused the generation of another kind of free radical (g = 2.0097) in the lens of all groups, but the amount of the free radical in the lens of the SeD group was also significantly higher than that of the control group. The activities of SOD and GSSG-R in VED rat lens were significantly decreased. The amount of MDA in the lens of SeD and/or VED rats were significantly increased. The results showed that the decrease of antioxidative capability in the lenses of SeD and/or VED rats accelerated the lipid peroxidation and generation of free radicals. Although only early morphological changes in SeD and/or VED rat lens were observed, it is considered that selenium and vitamin E deficiency may be involved in the occurrence of cataract.

Antioxidant enzyme activity in the muscles of calves depleted of vitamin E or selenium or both.

Feeding diets depleted of vitamin E and Se to cattle can induce a disease known as nutritional degenerative myopathy. It is believed that an increased peroxidative challenge in muscle is involved in the pathogenesis of this disease. A number of species can up-regulate the activity of some antioxidant enzymes, including glutathione reductase (EC 1.6.4.2), glutathione transferase (EC 2.5.1.18), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), catalase (EC 1.11.1.6), and superoxide dismutase (EC 1.15.1.1), in an attempt to mitigate the effects of a peroxidative challenge. A 2 x 2 factorial study was set up to examine possible changes in the activities of these antioxidant enzymes in muscles of ruminant calves fed on diets low in either vitamin E or Se. Four groups of four calves each were fed on a basal diet of NaOH-treated barley which was supplemented with alpha-tocopherol or Se or both for a total of 50 weeks. Calves fed on diets depleted of vitamin E, but not those fed on diets low in Se, developed subclinical myopathy, as judged by increases in the activity of plasma creatinine kinase (EC 2.7.3.2), and had increased muscle concentrations of two indices of lipid peroxidation, namely thiobarbituric acid-reactive substances, with and without ascorbate activation. Feeding diets depleted of vitamin E and diets low in Se both increased muscle activities of glucose-6-phosphate dehydrogenase in heart, biceps and supraspinatus. This change may have occurred in an attempt to maintain intracellular pools of reduced glutathione. No other changes in antioxidant enzyme activity were observed.

Glucose-6-phosphate dehydrogenase activity in dorsal root ganglia of vitamin E-deficient rats.

The effect of dietary vitamin E on the activity of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) was studied in the dorsal root ganglia of rat. One-month-old male Sprague-Dawley rats were randomly assigned to two dietary treatment groups for 2 months. The first received a standard diet supplemented with vitamin E, the second was fed a basal vitamin E-deficient diet. The activity of G6PD was markedly decreased in ganglia of the deficient animals with respect to the controls. On the other hand, the activity of the 6PGD was not significantly altered in the deficient animals. In the red cells the two enzyme activities presented a similar situation and the level of the reduced glutathione in the red cells was not significantly altered by the status of dietary vitamin E. Kinetic analysis with crude extracts of ganglia or partially purified G6PD demonstrated that there was no direct modulatory effect of the vitamin on the enzyme activity. Moreover, nondenaturing gel electrophoresis performed in this study revealed that none of the three G6PD activity bands which appeared on the acrylamide gel were significantly altered in the deficient animals. At present, the mechanism linking the G6PD activity with the status of dietary vitamin E remains unknown. Our results suggest, however, that a reduced NADPH generation produced by a decay of G6PD activity may limit the glutathione peroxidase, a very active enzyme in detoxifying peroxides, and may predispose the nervous tissue to oxidant injury.

[Latent antioxidant deficiency in the East German population--causes and clinical significance. II]. / Latenter Antioxidantienmangel in der DDR-Population--Uraschen und klinische Bedeutung. II. Mitteilung.

The hitherto yielded results of the research groups for arteriosclerosis and trace elements of the Medical Clinic of Leipzig University were summarized and valuated. The most important results were than in 800 healthy persons of three counties of the GDR who were examined depending on sex and age a serum selenium deficiency with regional differences was stated and this deficiency could be increased by an antiatherogenic mode of nutrition (n = 94, p less than 0.001). A consecutive depletion of vitamin E out of normal values was to be explained as a reference of an insufficient status of antioxidants. Analyses on nearly 1,000 foodstuffs and sorts of coffee, tea, tobacco, alcohol, etc. showed as cause a high-degree depletion of vegetable foodstuffs in selenium. The established daily minimum uptake of selenium of 12 to 15 micrograms by cooked foods was covered by above all cholesterol-rich animal foodstuffs and was further lowered by their withdrawal. In a double blind study first results of a supplementation of sodium selenite referred to a favourable influence on the immune status. After explanation of the reasons which may lead to an additional insufficiency of antioxidants was postulated that the protection from activated oxygen species is indicated both for the primary prevention of autoimmunigenic, atherogenic and cancerogenic diseases and for the curative medicine by a supplementation of selenium.

Vitamin E. deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes.

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6 min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129-240, 131-227 and 248-354 pmol/10(6) cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [3H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [3H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28 +/- 0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06 +/- 0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26 +/- 0.71 and 4.26 +/- 0.06 nmol/min/mg protein, respectively), measured as degradation of [3H]PAF to [3H]lysoPAF. In vitro addition of alpha-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, indicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by alpha-tocopherol. The acetyl-transferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 microM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 microM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.

[Effect of vitamin E from by-products of cotton oil processing on the glutathione peroxidase system in rats]. / Vliianie vitamina E iz otkhodov pererabotki khlopkovogo masla na glutationperoksidaznuiu sistemu krys.

The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione peroxidase from liver, skeletal muscles and erythrocytes of rats fed a vitamin E-deficient, or supplemented, diet were studied. Vitamin E was added in the diet either as a pure pharmacy form of alpha-tocopherol or as a tocopherol mixture derived from oil wastes. The deficiency of vitamin E caused an increase in the activity of the above mentioned enzymes. Both alpha-tocopherol and the tocopherol mixture were found to influence the glutathione peroxidase system. The dose-dependent response of the glutathione peroxidase system was revealed. Possible mechanisms of the changes in the antioxidizing enzymes induced by vitamin E are discussed.

Vitamin E and selenium participation in fatty acid desaturation. A proposal for an enzymatic function of these nutrients.

A critical review of the literature on the effects of vitamin E and selenium deficiences on unsaturated fatty acid metabolism reveals that some of these effects are inconsistent with the antioxidant hypothesis of these nutrients as their only biological function. On the basis of these data it is proposed that vitamin E and selenium play a role in the desaturation of n-3 and n-6 polyunsaturated fatty acids by participating in the microsomal electron transport chain and in a proposed peroxidase moiety of the desaturase complex, respectively. A re-interpretation of the experimental literature in terms of the proposed hypothesis is provided, with some suggestions to test its main tenets.

Enzymatic defense against radiation damage in mice. Effect of selenium and vitamin E depletion.

Radiation effects are mediated in part by the generation of oxygen-derived free radicals and hydrogen peroxide. Membrane polyunsaturated fatty acids are important biological targets of these toxic molecules which cause lipid peroxidation. Radiation damage to DNA is also known to result in base hydroperoxides, especially thymidine hydroperoxide. Glutathione (GSH) is known to inhibit lipid peroxidation both chemically and through its interaction with the selenium-dependent glutathione peroxidase (GSH-Px). Although cytosolic GSH-Px can metabolize organic lipid peroxides in solution, it cannot metabolize phospholipid peroxides in micelles. This may be due to the interference of phase differences between the aqueous cytosol and the membrane, or the result of steric hindrance. Recent studies have suggested the presence of a membrane-bound GSH-dependent peroxidase system. We examined the cytosolic versus membrane-associated GSH-Px, in various tissues of mice on a selenium and vitamin E deficient diet, and found significant differences among organs in the distribution of enzyme activity in these two subcellular fractions. The effect of single high-dose whole body irradiation did not appear to be related to the activity of these enzymes.

Use of sodium hydroxide treated selenium deficient barley to induce vitamin E and selenium deficiency in yearling cattle.

Selenium deficient barley grown in Northern Ireland was treated with sodium hydroxide to deplete it of vitamin E. Housed cattle fed a complete diet based on this treated barley developed nutritional degenerative myopathy, showing that spontaneous myopathy in yearling cattle can be the result of vitamin E and selenium deficiency alone. The diet used is as effective and cheaper than others presently in use for inducing degenerative myopathy.

The glutathione S-transferases in selenium and vitamin E deficiency.

Preliminary experiments confirmed the work of others showing that the total glutathione peroxidase (GSH-px) activity of rat liver supernatant fraction may be resolved into two peaks of activity (peaks I and II) by gel filtration, and that peak I is the selenium-containing enzyme and peak II is another peroxidase indistinguishable from glutathione S-transferase (GST). In selenium and vitamin E deficiency, the total activity of the GSH-px became very low, and the total activity of GST with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was enhanced. Study of the time course of these changes as deficiency progressed indicated that the stimulus for the rise in GST (CDNB) activity was the fall in GSH-px activity which preceded it. The peroxidase activity of GST was found to reside only in the GST AA, B and B2 forms of the enzyme, which were shown to be respectively a homodimer of the Yc subunit, a homodimer of the Ya subunit and a heterodimer of the YaYc subunit. As vitamin E and selenium deficiency progressed, the B2 and AA forms of the enzyme showed enhanced activity, which was interpreted as implying that the Yc subunit of the enzyme becomes enriched as a consequence of the withdrawal of selenium from the animal's diet. Densitometric measurements of the Yc and Ya subunits confirmed that the amount of the Yc subunit was nearly doubled in selenium deficiency, relative to the Ya subunit.

Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri).

Duplicate groups of rainbow trout (Salmo gairdneri) (mean weight 11 g) were given for 40 weeks one of four partially purified diets that were either adequate or low in selenium or vitamin E or both. Weight gains of trout given the dually deficient diet were significantly lower than those of trout given a complete diet or a diet deficient in Se. No mortalities occurred and the only pathology seen was exudative diathesis in the dually deficient trout. There was significant interaction between the two nutrients both with respect to packed cell volume and to malondialdehyde formation in the in vitro NADPH-dependent microsomal lipid peroxidation system. Tissue levels of vitamin E and Se decreased to very low levels in trout given diets lacking these nutrients. For plasma there was a significant effect of dietary vitamin E on Se concentration. Glutathione (GSH) peroxidase (EC 1.11.1.9) activity in liver and plasma was significantly lower in trout receiving low dietary Se but was independent of vitamin E intake. The ratios of hepatic GSH peroxidase activity measured with cumene hydroperoxide and hydrogen peroxide were the same for all treatments. This confirms the absence of a Se-independent GSH peroxidase activity in trout liver. Se deficiency did not lead to any compensatory increase in hepatic GSH transferase (EC 2.5.1.18) activity; values were essentially the same in all treatments. Plasma pyruvate kinase (EC 2.7.1.40) activity increased significantly in the trout deficient in both nutrients. This was thought to be due to leakage of the enzyme from the muscle and may be indicative of incipient (subclinical) muscle damage.

Differential changes of glutathione S-transferase activity by dietary selenium.

Dietary selenium deficiency produced increased activity of the glutathione S-transferases in the liver, kidney and duodenal mucosa. In these tissues, the residual activity of total glutathione peroxidase that included selenium-independent activity was considerably higher than that of selenium-dependent glutathione peroxidase. The enhanced activity of glutathione S-transferases was restored to control level 48 hr after an injection of selenite equivalent to the amount of daily selenium intake. Under the same conditions, selenium-dependent glutathione peroxidase activity increased with time and reached 11.9, 11.6 and 46.2% of the activity in the liver, kidney and duodenal mucosa of selenium-supplemented rats, respectively, 48 hr after selenite injection, whereas total glutathione peroxidase activity was not altered except in the kidney. These differential changes of glutathione S-transferase activity were intimately related to those of selenium-dependent glutathione peroxidase activity produced by selenium depletion and repletion, suggesting that the glutathione S-transferase activity was regulated by dietary selenium. Present findings support the idea that glutathione S-transferases having selenium-independent glutathione peroxidase activity function as a substitute for selenium-dependent glutathione peroxidase in selenium-deficient rats.

Effects of long-term vitamin E deficiency and restoration on rat hepatic peroxisomes.

Effects of vitamin E deficiency and its restoration on biochemical characteristics of hepatic peroxisomes were studied. Rats were maintained on the vitamin E-deficient diet for 25 weeks and then on a diet supplemented with vitamin E for 5 weeks. Blood hemolysis by hydrogen peroxide and lipid peroxidation in the liver increased markedly in vitamin E-deficient rats. The former returned to the control level after the resupplying of vitamin E, but the latter did not. Of liver peroxisomal enzymes, the activities of catalase, D-amino-acid oxidase and urate oxidase decreased in vitamin E-deficient rats. On the other hand, activities of fatty acyl-CoA oxidase and carnitine acetyltransferase increased significantly in vitamin E-deficient rats. All activities of these peroxisomal enzymes were restored to the control levels in vitamin E-supplemented rats. The activities of the mitochondrial, lysosomal and microsomal enzymes tested showed no apparent change except that the change of mitochondrial palmitoyltransferase was shown to be similar to that of peroxisomal fatty acid oxidation. These results were also supported by cell fractionation techniques. Following the methods of aqueous polymer two-phase systems, the characteristics of peroxisomal surface membranes altered in respect of their hydrophobicity, but not in respect of the surface charge of peroxisomal membranes. These results indicate that peroxisomal functions, especially those of the fatty acid oxidation system, change their activities more sensitively than other intracellular organelles in response to the condition of vitamin E deficiency.

Effects of vitamin E depletion and repletion on renin release from renin granules.

The present study was carried out to investigate the effects of vitamin E-deficiency and supplementation of alpha-tocopheryl acetate (TOCA) on renin release from renin granules. Male Wistar rats were fed either a control or a vitamin E-deficient diet for 4 weeks. Subsequently, the vitamin E-deficient rats received dietary supplementation of TOCA (40 mg/100 g diet) for 5 d. The renin granule fraction was prepared from the kidney cortex homogenate by a discontinuous sucrose density gradient centrifugation. The intake of vitamin E-deficient diet for 4 weeks resulted in an increased level of endogenous lipid peroxides in the renin granule fraction, accompanied by a marked decrease in alpha-tocopherol content, and led to a significant increase in the rate of renin release from the granules during incubation at 37 degrees C. These changes in alpha-tocopherol content, lipid peroxide level and renin release in the renin granule fraction were restored to the control values by dietary TOCA supplementation. Similarly, dietary supplementation of N,N'-diphenyl-p-phenylenediamine (80 mg/100 g diet), which has an antioxidative ability, suppressed the increases in lipid peroxidation and renin release due to vitamin E-deficiency, although this compound was ineffective in restoring alpha-tocopherol levels. These results suggest that vitamin E functions in maintenance of membrane integrity of renin granules by inhibiting the lipid peroxidation.