W. G. Hoekstra

Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake

Abstract Glutathione peroxidase (E.C. 1.11.1.9: glutathione: H 2 O 2 oxidoreductase) activity and selenium concentration were measured in lenses of female rats and their offspring after long-term feeding of either a selenium-supplemented ( 0·1 parts 10 6 ) or selenium-deficient ( 0·02 parts 10 6 ) diet. Long-term selenium deficiency decreased lens glutathione peroxidase activity in parent rats and their offspring to 15 and 14% respectively of supplemented controls. For comparison to lens, glutathione peroxidase was also measured in liver, heart, lung, erythrocytes, kidney, adrenal, testis, and brain of the offspring. Selenium deficiency caused the enzyme to decrease most dramatically in liver (to 0) and least in brain (to 62% of selenium supplemented controls). Although glutathione peroxidase in lens was lower than that in the other organs assayed, it was among the organs more sensitive to depletion caused by selenium deficiency. A short-term selenium deficiency of 8 weeks in newborn lambs had no effect on lens glutathione peroxidase, but the enzyme in organs such as liver was dramatically decreased. Therefore, an extensive period of selenium deficiency appears necessary to affect lens glutathione peroxidase activity, which probably relates to the relatively slow turnover and slow growth of the lens. The possible role of the seleno-enzyme, glutathione peroxidase, in the prevention of cataracts and the relationship of selenium to vitamin E and sulfur-containing amino acids in this regard are discussed.

Incorporation of selenium from selenite and selenocystine into glutathione peroxidase in the isolated perfused rat liver

Abstract The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated 75 Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced 75 Se incorporation from L[ 75 Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting 75 Se incorporation from [ 75 Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.

Glutathione peroxidase: Inhibition by cyanide and release of selenium☆

Abstract Treatment of highly purified ovine erythrocyte glutathione peroxidase with KCN resulted in loss of enzyme activity and release of selenium from the enzyme. The inactivation by cyanide was time-dependent and the rate was strongly influenced by temperature, concentration of cyanide, oxidation state of the enzyme, and pH. The pH effect could be explained on the basis of the increasing proportion of cyanide ion with increasing pH. Inhibition could be prevented by prior reduction of the purified enzyme with glutathione, dithiothreitol, or dithionite. Oxidation with cumene hydroperoxide was necessary to demonstrate cyanide inhibition of glutathione peroxidase activity in rat liver cytosol. These observations explain why cyanide inhibition of glutathione peroxidase has not been noted previously and provide new approaches for studying the chemical nature of the enzyme-selenium.

Studies on the binding of methylmercury by thionein

Abstract The 113,000 × g supernatant fraction from liver and kidney of rats and Japanese quail fed toxic levels of methylmercury for prolonged periods was fractionated on Sephadex G-75. Only a few percent of the total Hg was accounted for in the fractions corresponding to metallothionein. Methylmercury, in contrast to Hg++ and Cd++, was found to have a low affinity for the metal-free form of metallothionein isolated from horse kidney. Metallothionein appears to have no significant role in the detoxification or body distribution of methylmercury except to scavenge Hg++ formed by the slow biotransformation of methylmercury in the body.

Alleviation of silver toxicity by selenite in the rat in relation to tissue glutathione peroxidase.

Summary Dietary Se (0.5 ppm Se supplied as sodium selenite to a casein-based diet containing 0.02 ppm Se and lacking in vitamin E) prevented the growth depression observed in rats receiving 76 ppm Ag in the water supply and markedly improved growth and survival of those given 751 ppm Ag. The Ag concentration of liver and possibly of kidney was increased by Se. Liver glutathione peroxidase activities from rats fed 0.5 ppm Se and given 76 and 751 ppm Ag for 52 days in their water were, respectively, 30% and 4% of those from control rats fed 0.5 ppm Se without Ag. In rats fed a diet, adequate in vitamin E (100 IU/kg) and Se (0.5 ppm as sodium selenite), administration of 751 ppm Ag in the water for 15 wk reduced liver GSH-Px activity to 5% of that from control rats receiving no Ag. GSH-Px activity of erythro-cytes and kidney was decreased by Ag to 37% and 38%, respectively, of control values. It is concluded that in vivo administration of Ag dramatically decreased liver GSH-Px in rats fed Se-supplemented diets with or without vitamin E. Furthermore, supplemental Se (0.5 ppm) prevented the growth depression and mortality caused by Ag in rats fed a diet lacking vitamin E, while increasing the Ag concentration of liver and kidney. We wish to thank Mr. S. H. Oh for determining the Se content of the diets.

Trialkyllead metabolism and lipid peroxidation in vivo in vitamin E- and selenium-deficient rats, as measured by ethane production.

Abstract The ability of trialkyllead compounds to induce lipid peroxidation (measured by ethane production in vivo) was tested in rats fed a diet deficient in vitamin E and selenium or supplemented with 200 IU of dl -α-tocopherol or 0.6 ppm selenium supplied as sodium selenite. Trimethyllead induced lipid peroxidation in vivo to the greatest extent in vitamin E and selenium-deficient animals. Vitamin E and, to a lesser extent selenium, offered protection against this effect of trimethyllead. In the case of triethyllead, metabolism of the chemical caused the release of large amounts of ethane and ethylene, thereby preventing the use of ethane production as an index of lipid peroxidation. Such decomposition of triethyllead was lower in vitamin E and selenium-deficient animals compared to supplemented animals, and selenium in the diet promoted increased release of ethane and ethylene from triethyllead.

Stimulation of Lipid Peroxidation in Vivo by Injected Selenite and Lack of Stimulation by Selenate

Abstract Male weanling rats were raised on diets based on torula yeast which were deficient in vitamin E and selenium, or supplemented with these substances. They were injected intraperitoneally with sodium selenite at 2 mg Se/kg or with sodium sulfite to give the same level of sodium. Following injection, lipid peroxidation in vivo was estimated by monitoring the production of ethane, a volatile product formed on the peroxidation of ω-3-unsaturated fatty acids. In the hour following injection, vitamin E- and selenium-deficient rats injected with selenite produced 15 times as much ethane as did controls injected with sulfite. All rats in this group died from 1 to 4 hr after injection. Rats fed diets supplemented with selenite showed only a two- to threefold stimulation of ethane production by selenite and 3/4 survived. Rats fed diets supplemented with vitamin E did not produce more ethane in response to selenite injection and 3/4 survived. All four rats supplemented with both vitamin E and selenium survived without showing increased ethane production. Thus, the increased vulnerability of vitamin E- and selenium-deficient rats to acute selenite toxicity may involve peroxidation in vivo. Rats fed diets supplemented with vitamin E could survive at least twice as much selenite as rats deficient in selenium and vitamin E. Seven-day survival figures for rats fed the basal diet and injected with selenite were: 1 mg Se/kg, 7/8; 2 mg Se/kg, 0/8. For rats supplemented with vitamin E the figures were 2 mg Se/kg, 7/8; 4 mg Se/kg, 2/8. When sodium selenate was injected into rats deficient in vitamin E and selenium at 3 mg Se/kg it caused acute mortality without increasing peroxidation in vivo.

Effects of Vitamin E and Selenium on Copper-Induced Lipid Peroxidation in Vivo and on Acute Copper Toxicity

Abstract Copper sulfate injected intraperitoneally at a dose of 2 mg Cu/kg into vitamin E-and selenium-deficient rats caused a sixfold increase in the formation in vivo of the lipid peroxidation product ethane, and caused acute mortality in 4/5 rats. Selenium supplementation of the diet at 0.5 ppm Se largely prevented the increase in ethane production caused by copper injection and reduced mortality to 1/5 rats. Vitamin E supplementation of the diet at 200 IU/kg fully eliminated the increase in ethane production caused by copper injection, and completely prevented mortality. Vitamin E-deficient rats injected with copper sulfate at 5 mg Cu/kg produced over 10 times the ethane produced by rats injected with sodium sulfate or left uninjected. The ethylene produced by the rats injected with copper sulfate was 5% of the ethane produced, and did not differ significantly from the ethylene produced by the controls. Adding copper sulfate at 5 ppm Cu to a liver homogenate stimulated the production of ethane but not of ethylene. The correlation of increased ethane production with increased mortality suggests that lipid peroxidation may be important in the increased toxicity of copper in vitamin E- and selenium-deficient rats.

ENDOTOXIN AND LIPID PEROXIDATION IN VITRO IN SELENIUM- AND VITAMIN E-DEFICIENT AND -ADEQUATE RAT TISSUES

The effect of Salmonella typhimurium endotoxin injected intraperitoneally into rats (0.5 mg/kg of body weight) on subsequent lipid peroxidation in vitro was assessed. Peroxidation was monitored by measuring ethane production from tissue slices, as well as thiobarbituric acid-reactive substances and conjugated dienes in tissue homogenates. Weanling rats were fed a selenium- and vitamin E-deficient basal diet or one supplemented with 0.2 mg of Se/kg of diet and 200 mg of vitamin E/kg. After 9 to 16 wk, ethane production and thiobarbituric acid-reactive substances in liver and lung generally were increased by LPS treatment of Se- and vitamin E-deficient rats. Conjugated dienes were increased by LPS treatment in liver of Se- and vitamin E-deficient rats, but paradoxically, were higher in Se- and vitamin E-adequate liver tissue. Daily injections of 1 g of hydroxyurea/kg of body weight, a cell proliferation inhibitor, for 2 d prior to LPS injection significantly decreased the LPS-induced ethane production in Se- and vitamin E-deficient rat liver and lung. These results show that low doses of LPS injected into rats stimulated lipid peroxidation in vitro in Se- and vitamin E-deficient rat liver tissue. Hydroxyurea decreased LPS-induced lipid peroxidation in vitro; this suggests that neutrophils or macrophages are involved in LPS-induced lipid peroxidation.

Effects of diethyl ether and thymol on the ultrastructural and biochemical properties of purified sarcoplasmic reticulum fragments from skeletal muscle

1. 1. Studies were conducted to determine if the biochemical alterations of sarcoplasmic reticulum fragments produced by diethyl ether or thymol treatment were related to the membrane ultrastructure. 2. 2. Concentrations of diethyl ether or thymol that abolished Ca2+-accumulating ability but did not reduce the Ca2+-activated ATPase activity caused the formation of transparent patches on the surface of negatively stained vesicles. 3. 3. Higher concentrations of diethyl ether or thymol caused a decrease in the Ca2+-activated ATPase activity, an apparent loss of the 40-A subunits and an increased irregularity in the vesicle surface structure. The vesicle shapes observed in sectioned preparations were also altered.