Richard W. Scholz

Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on in vitro lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it has no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.

Evidence for the occurrence of selenium-independent glutathione peroxidase activity in rat liver microsomes.

Abstract Rat liver microsomes exhibit selenium-independent glutathione peroxidase activity which is associated with glutathione S-transferase activity. The peroxidase activity is not due to contamination with either soluble selenium-dependent or selenium-independent glutathione peroxidase activities of the cytosol. N-Ethylmaleimide treatment which stimulates rat liver microsomal glutathione transferase activity concomitantly stimulates the glutathione peroxidase activity. In contrast, N-ethylmaleimide depresses both enzyme activities of the cytosol. A protein exhibiting both glutathione peroxidase and glutathione transferase activity was isolated from the microsomes and purified to homogeneity by DEAE cellulose ion-exchange and S-hexylglutathione Sepharose 6B affinity chromatography.

Mechanism of Interaction of Vitamin E and Glutathione in the Protection against Membrane Lipid Peroxidation

Although the function of vitamin E as the major, if not only, chain-breaking, lipidsoluble antioxidant in biological membranes has been established,’ its mode( s) of action has yet to be elucidated. One proposed mechanism of vitamin E function involves a possible glutathione-dependent system that inhibits in vitro lipid peroxidation. Previously, we have described a rat liver microsomal system that inhibited both enzymatic and nonenzymatic lipid peroxidation in the presence of both glutathione and microsoma1 vitamin E., The GSH-dependent inhibition of lipid peroxidation was not observed in microsomes obtained from vitamin E-deficient rats and was independent of the selenium status of the animal. This inhibition, however, was not seen in heatdenatured microsomes, despite the continued presence of vitamin E. We therefore suggested that a microsomal heat-labile protein was likely involved in the mediation of the vitamin E-dependent, glutathione inhibition of lipid peroxidation. Other groups have also recently reported the presence of a glutathione-dependent liver microsomal factor that inhibited lipid per~xidation.’-~ The experiments reported here were designed to investigate the nature of the GSH-dependent inhibition of lipid peroxidation in the presence or absence of vitamin E. Weanling male Long-Evans hooded rats were fed chemically defined diets either deficient or supplemented with 150 IU dl-a-tocopheryl acetate/kg diet for 7 weeks? In vitro lipid peroxidation of liver microsomes was monitored by the formation of thiobarbituric acid (TBA)-reactive products, using both NADPH-dependent enzymatic and ascorbate/ADP-dependent nonenzymatic procedures, and by the consumption of 0, from the reaction medium. As shown in FIGURE lA, 5 mM GSH inhibited lipid peroxidation in this system for approximately 45 minutes. Interestingly, the addition of both GSH and oxidized glutathione (GSSG) resulted in a synergistic interaction that inhibited lipid peroxidation to a greater extent than the addition of GSH alone. GSSG alone did not inhibit lipid peroxidation of rat liver microsomes, and additional experiments (data not shown) revealed that GSH was oxidized to a limited extent to GSSG, whereas the conversion of GSSG to GSH was not observed. A possible explanation for this effect

Cadmium, methylmercury, mercury, and lead inhibition of calf liver glutathione S-transferase exhibiting selenium-independent glutathione peroxidase activity

Abstract Non-selenium glutathione peroxidase was isolated from calf liver to approximately 70% of purity (by analytical and sodiumdodecyl sulfate-gel electrophoresis) by a simple purification procedure. The preparation had a specific activity of 74 units/mg with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate and 14 units/mg with cumene hydroperoxide (COP). Both the peroxidase and transferase activities of the enzyme were markedly inhibited by cadmium, methylmercury, mercury, and lead. Of the various metal ions tested, cadmium was the most potent inhibitor. The nature of the inhibition of the transferase activity by metal ions was noncompetitive with respect to glutathione and CDNB. The peroxidase activity also was inhibited noncompetitively with respect to glutathione and COP. The inhibition constants (Ki) for cadmium, methylmercury, mercury, and lead were calculated to be 2, 4, 6.5, and 9 × 10−5 m , respectively, for the transferase activity and 1.5, 7, 9, and 3 × 10−6 m , respectively, for the peroxidase activity. These findings suggest that metal ion-induced lipid peroxidation may be mediated in part via inhibition of non-selenium glutathione peroxidase activity. In addition, metal ion toxicity may be expressed in part through inhibition of glutathione transferase activity which plays an important role in the detoxification of a broad spectrum of compounds.

Antioxidant effects on cell-mediated immunity.

Experiments were performed to determine the effects of dietary selenium and/or vitamin E deficiency on cell‐mediated cytotoxicity in the mouse. Natural killer cell‐mediated cytotoxicity (NKCC) was depressed after 8 wk on diets deficient in selenium and/or vitamin E. In contrast, antibody‐dependent cell‐mediated cytotoxicity (ADCC) was not affected by 8 wk of dietary deficiency of selenium and/or vitamin E. T‐lymphocyte‐mediated cytotoxicity (TCMC) was found to be depressed by combined selenium‐vitamin E deficiency after 7 weeks on diets.

Reduced glutathione effects on α-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent lipid peroxidation

Factors involved in reduced glutathione (GSH) and vitamin E-mediated inhibition of NADPH-dependent rat liver microsomal lipid peroxidation were examined. Lipid peroxidation was monitored over a time-course of 180 min by thiobarbituric acid reactive product formation. The addition of 5 mM GSH to the reaction system containing microsomes from rats fed a diet supplemented with 150 IU/kg of α-tocopherol acetate for eight weeks produced a lag in peroxidation of >30 min. This effect was not observed for microsomes prepared from rats fed a diet deficient in vitamin E. Indeed, a prooxidant effect of 5 mM GSH was observed in assays containing microsomes from rats fed a diet deficient in vitamin E. The inhibition by GSH of lipid peroxidation in microsomes prepared from livers of vitamin E supplemented rats was not restricted by its availability, for it was found that approximately 92% of the GSH remained in the reduced form after 60 min. Additional experiments revealed that the α-tocopherol content of peroxidizing microsomes decreased rapidly in the absence of GSH. The addition of 5 mM GSH to the assay system markedly depressed the loss of microsomal α-tocopherol. The results ofin vivo labeling of liver microsomes with [14C] α-tocopherol demonstrated that i) GSH addition to thein vitro peroxidizing medium reduced the disappearance of α-tocopherol, and ii) a compound that interfered with the determination of α-tocopherol was separated by HPLC and was not an oxidation product of α-tocopherol. A portion of the microsomal14C-labeled α-tocopherol was converted to an unidentified product with HPLC retention characteristics that was similar, but not identical, to α-tocopherol quinone.

The effects of ozone inhalation on the immunological response of selenium- and vitamin E-deprived rats

Deficiencies in vitamin E (E) or Se result in immune alterations, possibly due to reduction of antioxidant activity. Such reductions might greatly compromise the ability of the immune system to deal with additional oxidant stress, as encountered during exposure to air pollutants such as ozone (O3). To study possible interactions of these oxidative stresses on immune function, male Long-Evans hooded rats were maintained 5 weeks on torula yeast-based diets, with or without the addition of E or Se. Each dietary group was subdivided into O3-exposed and nonexposed groups. Two different regimens of O3 exposure were used: continuous (1.0 ppm, 8 hr/day for 7 days) or intermittent (2.0 ppm, 8 hr/day for 4 days, 2-4 days in ambient air followed by 1 day of exposure prior to sacrifice). Exposure to O3 in either regimen resulted in increased numbers of cells recovered by pulmonary lavage. With continuous exposure this increase was due to macrophage influx and, with intermittent exposure, due to influx of both macrophages and neutrophils. Combined deficiency of E and Se led to an enhanced ability of spleen and lung cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCMC). In animals deficient in E, but not Se, O3 exposure depressed spleen cell ADCMC. Deficiencies of either E or Se also depressed lymphocyte response to mitogens. Although intermittent exposure to O3 caused no changes in mitogen response, in animals exposed continuously to O3 there was a significant enhancement of this response.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Effects of inadequate vitamin E and/or selenium nutrition on the release of arachidonic acid metabolites in rat alveolar macrophages

Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg). Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hanks balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 micrograms/ml). The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay. Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways. Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandin E2 and thromboxane B2. Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B4 were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E. Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites.

Dietary selenium effects on milk eicosanoid concentration in dairy cows during coliform mastitis

The effect of selenium deficiency on the product profile of arachidonic acid oxidation by enzymatic pathways in Holstein cows with experimentally-induced coliform mastitis was investigated. The animals were fed dairy rations containing 0.05 mg Se/kg dry matter, with the supplemented group receiving additional Se to increase the dietary concentration to approximately 0.35 mg Se/kg dry matter. Cows were inoculated intracisternally with 30 colony-forming-units of Escherichia coli at 14-16 weeks of lactation. Eicosanoids and bacteria numbers were recorded at various intervals of time for 60 h postinoculation. Milk from cows fed the Se-depleted diet had significantly higher (p less than 0.05) concentrations of TXB2 between 24 and 48 h and 6-keto-PGF1 alpha between 24 and 60 h postinoculation. Milk PGE2 concentration was significantly higher in the Se-deficient group at 24 h, whereas LTB4 was higher between 36 and 60 h postinoculation in the Se-deficient cows (p less than 0.05). Milk bacteria numbers were significantly higher between 16 and 24 h postinoculation in the Se-deficient group and three of the four cows in this group required euthanasia, whereas all four cows in the Se-supplemented group recovered without therapeutic intervention. These data indicate marked effects of dietary Se on milk eicosanoid concentrations in response to an E. coli infection. The changes in eicosanoid concentrations may be associated with the altered pathogenesis and outcome of mastitis in a Se-deficient state.