Endotoxin and lipid peroxidation in vivo in selenium- and vitamin E-deficient and -adequate rats.

The effect of Salmonella typhimurium endotoxin injected intraperitoneally (0.5 mg/kg body weight) on lipid peroxidation in vivo was assessed. Peroxidation was monitored by measuring ethane production, an autoxidation product of (n-3) unsaturated fatty acids. Weanling rats were fed a selenium- and vitamin E-deficient basal diet or one supplemented with 0.2 mg Se/kg and/or 200 mg vitamin E/kg. After 11 to 13 wk of feeding, ethane production was tripled in LPS-treated Se- and vitamin E-deficient rats compared to saline-treated deficient rats. In both doubly deficient and adequate rats, LPS increased ethane production, but it did so to a greater extent in Se- and vitamin E-deficient rats. Dietary Se or vitamin E supplementation alone significantly reduced ethane production from LPS-treated rats. Vitamin E was more protective than Se against LPS-induced lipid peroxidation. Escherichia coli and Salmonella minnesota LPS also increased ethane production in Se- and vitamin E-deficient rats. These results show that low doses of LPS stimulate lipid peroxidation in vivo in Se- and vitamin E-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.

The metabolism of 75Se-selenomethionine in sheep given supplementary copper and molybdenum.

Four groups of three ram lambs were fed, in group pens, the following diets from 4 to 19 wk of age: 1) control (6 mg Cu/kg and 1 mg Mo/kg); 2) control plus 10 mg Cu/kg; 3) control plus 10 mg Mo/kg and 4) control plus 10 mg/kg of both Cu and Mo. Copper and Mo were added to the diet as copper sulfate and sodium molybdate. The main ingredients of the diets were alfalfa hay (20%), oats (20%) and corn (59%). At 19 wk, the animals were allocated randomly to individual metabolism cages and received a single oral dose of 75Se-selenomethionine. Liver Cu concentrations at slaughter (22 wk) were 77, 259, 68 and 316 mg/kg fresh weight for treatments 1 through 4. There was clinical evidence of Cu poisoning in lambs on treatment 2. Sheep given Cu supplements without additional Mo had reduced (P less than .05) levels of 75Se activity in muscle compared with control animals. This decrease in muscle 75Se in Cu-supplemented lambs was associated with a nonsignificant increase in 75Se content of other tissues and a nonsignificant increase in fecal excretion of 75Se. Apparent absorption and net retention of 75Se was 80% and 74%, respectively. Long-term ingestion of moderate levels of Cu influenced the metabolism of Se fed as selenomethionine, possibly through effects of chronic Cu toxicity on liver function.

Effects of copper and molybdenum supplements on the copper and selenium status of pregnant ewes and lambs.

The aim of this work was to investigate whether Cu, alone or in combination with Mo, reduces the Se status of sheep. Thirty-six Hampshire ewes were separated prior to mating into four treatment groups of nine sheep each. The four groups consisted of 1) basal (alfalfa hay or legume-grass low-moisture silage, corn, oats), 2) basal plus 10 mg Cu/kg diet, 3) basal plus 10 mg Mo/kg and 4) basal plus Cu and Mo (each 10 mg/kg). Copper and Mo were added to both ewe and lamb diets. Treatments continued until lambs were weaned at 10 wk postpartum. The basal ewe diet contained, in mg/kg, 5.6 Cu, .05 Se and 1.0 Mo. The basal lamb diet contained, in mg/kg, 6.1 Cu, .07 Se and less than 1.0 Mo. Sulfur concentrations in the ewe and lamb diets were .24% and .20%, respectively. Copper supplements increased, and Mo supplements decreased, the concentration of Cu in the livers of ewes and lambs (P less than .05). Despite this, there were no effects of treatment on Se concentration or glutathione peroxidase activity in blood or tissues. Selenium levels in plasma of all ewes declined throughout pregnancy and lactation, indicating that .05 mg/kg Se was insufficient for the maintenance of Se status during pregnancy in Hampshire ewes. Copper and Se levels (mg/kg fresh weight) in the liver of lambs at weaning for Treatments 1 through 4 were 48 and .13, 158 and .10, 11 and .11, and 136 and .13, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 3-methylindole on respiratory ethane production in selenium and vitamin E deficient rats.

Lipid peroxidation has been proposed as a mechanism of 3-methylindole pneumotoxicity. In this report, lipid peroxidation was measured over 16 h in awake rats given 400 mg/kg i.p. 3-methylindole or its carrier, Cremophore EL. Rats were studied after 8 weeks of feeding a diet either adequate or deficient in vitamin E and selenium. Respiratory ethane production was used as the index of lipid peroxidation. 3-methylindole had no effect on lipid peroxidation for rats fed the adequate diet. For rats on the deficient diet, 3-methylindole suppressed lipid peroxidation by 50% of control. These results indicate that lipid peroxidation is not a mechanism of 3-methylindole pneumotoxicity and support the conclusion that 3-methylindole may act as an antioxidant.

Effects of dietary selenium concentration on the development of enzyme-altered liver foci and hepatocellular carcinoma induced by diethylnitrosamine or N-acetylaminofluorene in rats.

Three protocols were used to determine the effects of dietary selenium concentration on the development of gamma-glutamyl-transpeptidase (GGT)-positive foci and hepatocellular carcinoma induced by either diethylnitrosamine (DEN) or N-acetylaminofluorene in rats. In the first experiment, foci were induced by a carcinogenic dose of DEN (100 mg/kg body weight, p.o.) at 20-22 h after two-thirds partial hepatectomy. One wk after DEN administration, during which time 0.1 ppm (representing a control level), 3.0, or 6.0 ppm selenium as Na2SeO3 was fed for 8 or 16 wk, at which time focal analysis was conducted using quantitative stereology. The results demonstrated that 3.0 and 6.0 ppm dietary selenium, initiated 1 wk following carcinogen administration, decreased focal growth rate without affecting the number of GGT foci compared to 0.1 ppm selenium. Decreased focal growth was temporary and reversible with 6.0 ppm selenium which may be related to chronic selenosis observed after 16 wk of 6.0 ppm selenium feeding. A second experiment involved a noncarcinogenic dose of DEN (25 mg/kg body weight, p.o.), then 0.1 or 6.0 ppm selenium feeding for 8 wk, followed by 0.05% phenobarbital (PB), a liver tumor promoter in a diet containing 0.1 ppm selenium. Analysis of GGT foci at 5 or 8 wk of PB feeding indicated that 6.0 ppm selenium caused a trend towards an increase in the number of foci/cm3 of liver and mean focal volume and a significant increase in GGT focal volume as a percentage of liver volume by 8 wk of PB feeding. Thus, high dietary selenium concentrations prior to PB enhance the tumor-promoting ability of PB. In a third experiment, using male Fischer 344 rats (150 g), 0.1 or 6.0 ppm selenium was fed concurrently with 0.02% AAF which was fed in a cyclic regimen. After 4 cycles, where 1 cycle equalled 4 wk of AAF, followed by 1 wk of control diet (0.1 ppm selenium), 6.0 ppm selenium significantly decreased the mean focal volume and focal volume as a percentage of liver volume, while not affecting the number of foci/cm3 of liver, again indicating a selenium effect on focal growth while not affecting the number of "preneoplastic" lesions in the liver. Six ppm selenium feeding after AAF treatment had no effect on the percentage of incidence of hepatocellular carcinoma (100%) but did cause a significant decrease in the percentage of liver volume occupied by macroscopic subcapsular liver lesions compared to 0.1 ppm selenium.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of dietary selenium concentration and duration of selenium feeding on hepatic glutathione concentrations in rats.

Studies were conducted in rats to determine the effect of dietary selenium (Se) concentration on hepatic glutathione concentrations and enzyme activities associated with the maintenance of the cellular glutathione status. Male rats were fed 0.1, 3.0, or 6.0 ppm Se as Na2SeO3 for 2, 4, or 6 weeks at which time they were killed and analyses were performed. Both 3.0 and 6.0 ppm Se caused a significant dose-dependent increase in hepatic-reduced glutathione (GSH) by 4 weeks of feeding compared to 0.1 ppm Se. The increase in GSH was preceded by significant, dose-dependent increases in oxidized glutathione (GSSG) as well as the GSSG to GSH ratio. Increases in GSSG and the GSSG to GSH ratio as well as in glutathione reductase and glucose-6-phosphate dehydrogenase activities were observed by 2 weeks of high Se feeding. The current findings substantiate previous results demonstrating effects of high Se on hepatic glutathione concentrations (R. A. LeBoeuf and W. G. Hoekstra, J. Nutr. 113:845-854, 1983) and further suggest that increased cellular GSSG concentrations or the GSSG to GSH ratio caused by 3.0 and 6.0 ppm dietary Se signals for "adaptive" changes in hepatic glutathione metabolism.

Effects of selenium on cell proliferation in rat liver and mammalian cells as indicated by cytokinetic and biochemical analysis.

Studies were conducted in vivo with regenerating liver and in vitro with mammalian cells to determine the effects of selenium on cell proliferation and the stages of the cell cycle affected by selenium. Six ppm selenium as Na2SeO3 fed to weanling male F344 rats for 6 wk significantly reduced the percentage of 3H-labeled hepatocyte nuclei by one-half compared to 0.1 ppm selenium when [methyl-3H]thymidine was injected at 23 h post-two-thirds hepatectomy. Sampling was done at 30 h post-hepatectomy. A trend towards decreased 3H per DNA per labeled cell was also observed, suggesting that selenium decreased the rate of DNA synthesis as well as delaying the entry of cells into S phase (i.e., increasing the duration of G0-G1). Studies in vitro with H-4 "minimal deviation" hepatomas and 3T3 mouse fibroblasts demonstrated that selenium decreased the growth of these cells in a dose-dependent manner, and this inhibition was reversible upon removal of selenium from the growth medium. Cytokinetic analysis using fluorescence flow cytometry and microscopic techniques indicated that selenium treatment increased the duration of G1, S, and G2 phases of the cell cycle, while having no effect on mitosis under the conditions of our experiments. Biochemical analyses of H-4 cells demonstrated that selenium treatment caused a significant dose-dependent increase in oxidized and reduced glutathione (GSSG and GSH) as well as in the GSSG:GSH ratio as was previously observed in liver in vivo. In addition, glutathione reductase activity as well as the oxidized nicotinamide adenine dinucleotide phosphate:reduced nicotinamide adenine dinucleotide phosphate ratio was significantly increased with selenium treatment. These results indicate that selenium affects all "synthetic" stages of the cell cycle, and elevated GSSG or the GSSG:GSH ratio may explain the antiproliferative effects of selenium on cells.

Changes in cellular glutathione levels: possible relation to selenium-mediated anticarcinogenesis.

Mechanisms of Se-mediated anticarcinogenesis were investigated. Excess Se as Na2SeO3 reversibly decreased mammalian cell proliferation in vitro. This inhibitory effect was exerted primarily in G1 of the cell cycle; however, S and G2 were also affected. Cellular glutathione concentrations both in vitro and in vivo were altered by excess Se administration. These results are discussed in terms of altered cellular glutathione metabolism and its possible implications for cell proliferation.

The effects of zinc deficiency on turnover of cadmium-metallothionein in rat liver.

The object of this experiment was to determine the effects of Zn deficiency on the turnover of Cd-induced metallothionein (MT) in rat liver. Male rats were fed a purified Zn-deficient or Zn-adequate diet. After 13 days, the rats were given three daily injections of Cd2+ totaling 1.5 or 3.0 (Zn-deficient) and 3.0 or 6.0 (Zn-adequate) mg Cd/kg body weight. The MT was labeled by injecting the rats with [35S]cystine 2 hours after the final Cd injection. One, 3 or 5 days after labeling, the rats were killed, and their livers were assayed for MT 35S and metal content. The metal composition of MT (mole %) was 41-42% Cd, 51-54% Zn and 4-7% Cu in the Zn-adequate groups and 64% Cd, 27-31% Zn and 6-9% Cu in the Zn-deficient groups. The half-lives of Cd-induced MT in the Zn-deficient rats were 2.6 days (1.5 mg Cd/kg) and 2.8 days (3.0 mg Cd/kg). In the Zn-adequate rats, the half-lives were 3.6 days (3.0 mg Cd/kg) and 3.1 days (6.0 mg Cd/kg). The half-lives of general, soluble hepatic proteins were 4.1 to 4.3 days in all groups. Despite the stabilizing effect of the higher Cd content, the half-life of hepatic MT in the Zn-deficient rats was significantly shorter than in the Zn-adequate rats. These results indicate that hepatic MT degradation is faster in Zn-deficient animals.

Effect of calcium phosphates and zinc in salt-mineral mixtures on ad libitum salt-mix intake and on zinc and selenium status of sheep.

In two separate experiments, 72 crossbred ewes were fed hay, haylage (50% dry matter) and corn diets with ad libitum salt-mineral mixtures (SMM; Exp. 1) or salt (Exp. 2). Calcium phosphates (Ca X P) and(or) zinc (Zn) were added in a 2 X 2 factorial arrangement to salt + trace minerals for ewes 7 mo prepartum through lactation in Exp. 1 and to salt only for ewes 3 mo prepartum through lactation in Exp. 2. The diets fed were estimated to contain 23 and 28 mg Zn/kg dry diet (ppm), respectively, and .08 and .05 ppm Se. Large variations (up to fivefold) were found in SMM intake per month between replicates and from month-to-month within treatment; thus, monthly variations of up to sevenfold occurred in Zn and Se intakes of supplemented groups. There were no significant treatment effects on SMM intake. Small but significant Zn treatment effects were detected for plasma and wool Zn of ewes and lambs, but all values were in the normal range. There was no significant treatment effect on plasma alkaline phosphatase activity. In Exp. 2, erythrocyte glutathione peroxidase (GSH-Px) activity was significantly lower in all treatment groups compared with a Se-supplemented control group but only rare occurrences of subclinical muscular dystrophy were found. There was no significant treatment effect on GSH-Px activity, whole blood Se in ewes and lambs or plasma creatine phosphokinase activity in lambs. These results indicate large animal and seasonal variability in SMM intake and no significant treatment effects of Ca X P on SMM intake or on Zn and Se status. Zinc addition to SMM had no effect on Se status.

Investigation of putative androgen-like activity of alpha 2u-globulin in castrated and estrogen-treated male rats.

alpha 2u-Globulin, a male sex-dependent protein of the rat, was tested for its ability to produce androgenic or anabolic responses. Castrated rats were injected with alpha 2u-globulin isolated from urine or kidney and the effects on urinary nitrogen excretion, body weight, and weight of ventral prostate, seminal vesicles, and levator ani muscle were studied. alpha 2u-Globulin had no effect but testosterone propionate gave the expected stimulation of nitrogen retention, body growth, and growth of androgen-responsive tissues. alpha 2u-Globulin injected into estrogen-treated male rats (animals with suppressed serum levels of alpha 2u-globulin, gonadotropins, and testosterone) was also found to be without effect on body weight and on the weight of ventral prostate, seminal vesicles, levator ani muscle, and testes. We conclude that the alpha 2u-globulin molecule, by itself, is incapable of producing, directly or indirectly, androgenlike responses.

Adaptive changes in hepatic glutathione metabolism in response to excess selenium in rats.

Experiments were conducted with rats to determine if administration of excess selenium (Se) alters hepatic glutathione (GSH) metabolism. Se was fed, 6 ppm as Na2SeO3, for 6 weeks in a 15% casein, tocopherol-free diet and compared to 0.1 ppm Se. High Se significantly increased the concentrations of hepatic nonprotein sulfhydryls (NPSH) and oxidized glutathione (GSSG) as well as the GSSG:NPSH ratio. Of the NPSH, 94.1 +/- 1.7% was shown to be GSH. Similar increases in NPSH, GSSG and GSSG:NPSH due to high Se were seen when the diet was supplemented with methionine (0.3%) and/or vitamin E (100 IU/kg diet) although methionine independently increased NPSH and GSSG. Hepatic glucose-6-P dehydrogenase, glutathione reductase and gamma-glutamyl-transpeptidase activities were significantly increased by high Se but cysteine dioxygenase activity was unaffected. An i.p. injection of Na2SeO3 (15 mumol Se/kg body weight) significantly increased hepatic GSSG, which was followed by an increase in NPSH(GSH) compared to saline controls. These results indicate that excess Se causes a shift in hepatic GSH toward a more oxidized state. Increases in NPSH(GSH) and in the enzyme activities observed appeared to be adaptive changes initiated in an attempt to maintain a normal GSSG:GSH ratio. Elevated GSSG or the increased GSSG:GSH ratio caused by Se may initiate these adaptations.

Effects of ferrous chloride and iron dextran on lipid peroxidation in vivo in vitamin E and selenium adequate and deficient rats.

The effects of intraperitoneally injected ferrous chloride and iron-dextran on lipid peroxidation in vivo were assessed. Peroxidation was estimated by measuring ethane, a volatile autoxidation product of omega-3-unsaturated fatty acids. Rats supplemented with 0.1 ppm dietary selenium and rats supplemented with 0.1 ppm selenium and 200 IU vitamin E/kg diet were injected with ferrous chloride at 30 mg iron/kg, or with sodium chloride, or left uninjected. In both dietary groups ferrous chloride increased ethane production while sodium chloride did not, but the iron-caused ethane increase was 8 times greater in the low E group. Iron-dextran injected at 500 mg iron/kg was fatal to rats fed a basal diet deficient in selenium and vitamin E or diet supplemented with 0.5 ppm selenium; supplemental vitamin E at 200 IU/kg diet prevented this mortality. Iron-dextran quadrupled ethane production in rats fed the basal diet and tripled ethane production in rats fed the selenium-supplement diet. Vitamin E supplementation prevented the iron-dextran-caused rise in ethane production. A histological examination of rats killed by iron-dextran showed severe generalized necrosis of the diaphragm and severe focal necrosis of thigh muscle. Vitamin E protected more effectively than selenium against iron-dextran-caused peroxidation as well as against acute iron-dextran-caused mortality.

Effect of dietary methionine on the biopotency of selenite and selenomethionine in the rat.

The effect of dietary methionine (Met) on the Biopotency of selenium (Se) from selenite and selenomethionine (Se-Met) was studied in rats fed a 30% torula yeast-based diet containing 0.24% Met and less than 0.02 ppm Se. Biopotency was quantitated by assaying liver, plasma and heart for the glutathione peroxidase (GSH-Px) increase elicited by a given level of Se fed for 1 week to Se-deficient rats. At dietary Se levels up to 0.5 ppm Se, the level of dietary Met supplementation did not alter selenite biopotency. In contrast, dietary Met supplementation increased the biopotency of Se-Met. With basal Met intakes, the biopotency of Se-Met was 25% that of selenite below 0.5 ppm Se. The addition of 0.4% Met to the diet made Se-Met biopotency equivalent to selenite biopotency in one experiment, but in a second experiment with younger, faster growing rats, 0.4% Met did not completely restore the biopotency of Se-Met. These results indicate that low dietary Met decreases the biopotency of Se-Met but not of selenite. Altered Se metabolism at suboptimal dietary Met may occur because more Se-Met is incorporated into protein and thus less Se is available for GSH-Px synthesis. These results suggest that adequate dietary Met is required for optimal utilization of the Se in feedstuffs of plant origin, as Se-Met is presumably a major form of plant selenium.