Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes

Selenium deficiency and vitamin E deficiency both affect xenobiotic metabolism and toxicity. In addition, selenium deficiency causes changes in the activity of some glutathione-requiring enzymes. We have studied glutathione metabolism in isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats. Cell viability, as measured by trypan blue exclusion, was comparable for all groups during the 5-h incubation. Freshly isolated hepatocytes had the same glutathione concentration regardless of diet group. During the incubation, however, the glutathione concentration in selenium-deficient hepatocytes rose to 1.4 times that in control hepatocytes. The selenium-deficient cells also released twice as much glutathione into the incubation medium as did the control cells. Total glutathione (intracellular plus extracellular) in the incubation flask increased from 47.7 +/- 8.9 to 152 +/- 16.5 nmol/10(6) selenium-deficient cells over 5 h compared with an increase from 46.7 +/- 7.1 to 92.0 +/- 17.4 nmol/10(6) control cells and from 47.7 +/- 11.7 to 79.5 +/- 24.9 nmol/10(6) vitamin E-deficient cells. This overall increase in glutathione concentration suggested that glutathione synthesis was accelerated by selenium deficiency. The activity of gamma-glutamylcysteine synthetase was twice as great in selenium-deficient liver supernatant (105,000 X g) as in vitamin E-deficient or control liver supernatant (105,000 X g). Hemoglobin-free perfused livers were used to determine the form of glutathione released and its route. Selenium-deficient livers released 4 times as much GSH into the caval perfusate as did control livers. Plasma glutathione concentration in selenium-deficient rats was found to be 2-fold that in control rats, suggesting that increased GSH synthesis and release is an in vivo phenomenon associated with selenium deficiency.     

Effect of vitamin A deficiency on pulmonary and hepatic in vitro metabolism of benzo(a)pyrene in rat

The metabolism of (3H)-benzo(a)pyrene and the activities of enzymes involved in its metabolism were studied in rat lung and liver in vitamin A deficiency. Deficiency of vitamin A resulted a significant decrease in the overall metabolism of benzo(a)pyrene in the liver in vitro, whereas no significant difference was evident in the lung. The ethyl acetate-soluble metabolites of benzo(a)pyrene formed by lung and liver preparations were unaltered qualitatively by vitamin A deficiency. However, quantitative analysis revealed that vitamin A deficiency decreased the yield of dihydrodiols, quinones and phenols in liver, and dihydrodiols in lung. The hepatic cytochrome P-450 content, arylhydrocarbon hydroxylase and uridine diphosphate-glucuronosyl transferase activities were reduced, whereas glutathione S-transferase activity was increased in the vitamin A deficient animals. Contrary to this, pulmonary cytochrome P-450 content was above the control values (p less than 0.01) and no alteration in pulmonary arylhydrocarbon hydroxylase activity was observed in vitamin A deficient rats. Uridine diphosphate-glucuronosyltransferase and glutathione S-transferase activities were impaired in lung by inducing vitamin A deficiency. However, no significant difference was evident in the overall metabolism of benzo(a)pyrene by lung supernatants from the two groups.     

Vitamin C partially reversed some biochemical changes produced by vitamin E deficiency

Feeding a basal diet free of vitamins E and C to weanling male rats for 8 months resulted in biochemical changes characteristic of vitamin E deficiency. These included increased liver thiobarbituric acid values; decreased blood GSH levels, plasma vitamin E levels, and glutathione peroxidase activities; and increased activities of plasma pyruvate kinase, glutamic-oxaloacetic transaminase, creatine kinase, lactic dehydrogenase, and malic dehydrogenase. Tube-feeding vitamin C for 21 days resulted in partial reversal effects on the above parameters except activities of glutathione peroxidase, lactic dehydrogenase, and malic dehydrogenase. The results suggest that vitamin C may spare in part the metabolism of vitamin E through its antioxidant property.     

Vitamin E supplementation and glutathione peroxidase activity.

Both excess dietary vitamin E and vitamin E deficiency in rats can significantly depress the activity of GSH peroxidase in liver and plasma of rats. Of all the six levels of vitamin E tested in this study, the dietary level of vitamin E found to maintain the maximum activity of GSH peroxidase in tissues of rats was somewhere between 25 and 250 IU/kg diet. This study conclusively indicates that the excess dietary vitamin E represses GSH peroxidase activity.     

Lipid peroxidation and benzo[a]pyrene activation to mutagenic metabolites: in vivo influence of vitamins A, E and C and glutathione in both dietary vitamin A sufficiency and deficiency

Rats fed with either a sufficient-vitamin A or a vitamin A-free diet were pretreated with 750 mg/kg body weight of retinyl palmitate, alpha-tocopherol acetate, ascorbic acid or glutathione. Benzo[a]pyrene (BaP) metabolism and BaP-induced mutagenesis in Salmonella typhimurium TA98 were investigated and related to lipid peroxidation activities in postmitochondrial (S9) liver fraction. The microsomal mixed-function oxidase activities were decreased by vitamin A deficiency and weakly affected by scavenger treatment. The rate of lipid peroxidation of microsomal membranes was unaffected by vitamin A deficiency because of decreased polyunsaturated fatty acids and increased vitamin E contents. However, lipid peroxidation was decreased by pretreatment with fat-soluble vitamins (chiefly vitamin E) and increased by ascorbic acid. Within each experimental group both BaP metabolism and BaP mutagenic activity were closely correlated with the rate of lipid peroxidation. In vitamin A deficiency, the increased BaP metabolism and mutagenicity could be related to a decrease in cytosolic contents of scavengers (vitamin A and glutathione). In Ames test conditions, the free radical pathway became a route for BaP metabolism and thus the BaP activation to mutagenic metabolites is related to the cellular status in free radical scavengers.     

Modification of hepatic vitamin E stores in vivo. III. Vitamin E depletion by 1,2-dibromoethane may be related to initial conjugation with glutathione

In the companion paper we demonstrated that hepatic vitamin E in rats becomes depleted and extrahepatic pools of vitamin E are altered by treatment with 1,2-dibromoethane (DBE). Vitamin E depletion may be dependent upon initial steps of DBE metabolism that are either oxidative (cytochrome P450 dependent) or conjugative (glutathione transferase dependent). That the liver content of glutathione (GSH) and vitamin E, the plasma concentration of vitamin E, and the serum activities of AST and ALT may be influenced by cytosolic metabolism of DBE was assessed by comparison of findings from rats treated with either 1,2-dichloroethane (DCE) or 1-bromo-2-chloroethane (BCE). The extent of oxidative metabolism was diminished by the use of tetradeutero-DBE (d4-DBE), and the availability of GSH for conjugative metabolism was diminished by pretreatment of rats with L-buthionine-S,R-sulfoximine (BSO) prior to treatment with DBE. Our results indicate that neither DCE nor BCE provokes a liver vitamin E depletion in rats, that d4-DBE treatment hastens but does not enhance the observed hepatic vitamin E depletion by comparison to animals treated with an equimolar dose of DBE, and that BSO pretreatment prevented the hepatic vitamin E depletion observed from animals treated with DBE alone. These results indicate that hepatic vitamin E depletion is the unique sequelae to conjugation of GSH with DBE, and we suggest the reactive episulfonium ion intermediate or a macromolecular adduct of this ion derived from DBE may play a role in liver vitamin E depletion associated with exposure to DBE.     

Effect of excess and deficiency of vitamin A on the utilization of FFA by liver and skeletal muscle.

Fatty acid metabolism in liver and skeletal muscle has been studied in rats treated with high doses of vitamin A and in those made vitamin A-deficient. Ingestion of 30,000 IU of vitamin A for two days resulted in increased incorporation of palmitate-1-14C into triglycerides but not into phospholipids. Accumulation of hepatic triglycerides was observed in vitamin A-fed rats. Deficiency of vitamin A did not cause any change in the triglyceride or phospholipid content of the liver. The rate of hepatic fatty acid oxidation and ketogenesis was markedly increased in vitamin A-fed rats. The experimental evidence indicated that vitamin A may have a stimulatory effect on these processes apart from that exerted by the high plasma FFA level in vitamin A-fed rats. Oxidation of palmitate-1-14C into C32 by skeletal muscle (latissimus dorsi) was also increased as a result of vitamin A administration. Vitamin A deficiency did not cause any change in fatty acid oxidation by liver and skeletal muscle. Hepatic palmitoyl-CoA synthetase activity was decreased in vitamin A-deficient rats. The results presented suggest that vitamin A may be required for the uptake and utilization of fatty acids by liver and akeletal muscle.     

Vitamin E and neurologic function in man

Despite the well-known detrimental effect of vitamin E deficiency on the nervous system of many experimental animal models for decades, only over the past decade has vitamin E become recognized as essential for the maintenance of the structure and function of the human nervous system. This discovery of the neurologic role of vitamin E in man is due primarily to the identification of a degenerative neurologic syndrome in children and adults with chronic vitamin E deficiency caused by gastrointestinal diseases impairing fat and vitamin E absorption. A compelling body of clinical, neuropathologic, and therapeutic response evidence conclusively demonstrates that vitamin E deficiency is responsible for the neurologic disorder seen in such patients. In addition, an inborn error in vitamin E metabolism, the Isolated Vitamin E Deficiency Syndrome, causes vitamin E deficiency and similar neurologic degeneration in the absence of fat malabsorption. Guidelines for the evaluation and treatment of vitamin E deficiency in relevant clinical circumstances are provided. The possible role of vitamin E in treating other neurologic diseases is discussed.     

Free Radical-Induced Liver Injury. I. Effects of Dietary Vitamin E Deficiency on Triacylglycerol Level and its Fatty Acid Profile in Rat Liver

Effects of dietary vitamin E deficiency on the fatty acid compositions of total lipids and phospholipids were studied in several tissues of rats fed a vitamin E-deficient diet for 4, 6, and 9 months. No significant differences were observed between the vitamin E deficiency and controls except in the fatty acid profiles of liver total lipids. Triacylglycerol (TAG) accumulation was found in the liver of rats fed a vitamin E-deficient diet. The levels of TAG-palmitate and -oleate increased particularly in the liver from such animals. The fatty acid compositions of hepatic phospholipids were not affected by the diet. Increased TAG observed in the liver of rats fed a vitamin E-deficient diet was restored to normal when the diet was supplemented with 20 mg α-tocopheryl acetate/kg diet. These findings indicate that dietary vitamin E deficiency causes TAG accumulation in the liver and that the antioxidant, vitamin E, is capable of preventing free radical-induced liver injury.     

Effect of iron and/or vitamin A re-supplementation on vitamin A and iron status of rats after a dietary deficiency of both components

Iron and vitamin A deficiency are common nutritional problems in developing countries. From animal experiments and intervention studies, growing evidence is pointing to a possible influence of iron on vitamin A metabolism. We assessed the affects of an oral supplementation of vitamin A and/or iron on the recovery of rats from vitamin A and iron deficiency. Weanling male Wistar rats were kept for four weeks on an iron and vitamin A deficient diet. Thereafter, rats were repleted with iron 35 mg/kg feed, with vitamin A 4500 IU/kg feed both, or with iron 35 mg/kg and vitamin A 4500 IU/kg for five weeks. Retinol and retinyl esters in plasma and tissues were determined by HPLC. Iron was determined by atomic absorption spectrophotometry. The determination of haematological parameters showed that rats developed an anaemia during depletion. This was reversed by the re-supplementation with iron but not vitamin A alone. The simultaneous supplementation of vitamin A was of no additional benefit. When rats were resupplemented with iron alone a substantial further decrease in plasma retinol (P < 0.002) and liver vitamin A (P < 0.05) was observed. A similar but less pronounced decrease in plasma retinol was observed in the rats re-supplemented with vitamin A alone, despite a substantial increase in liver vitamin A (P < 0.002). Despite lower liver vitamin A levels compared to the group re-supplemented with vitamin A lone, the group re-supplemented with iron and vitamin A had substantial higher plasma levels compared to the one supplemented with iron alone (P < 0.002). In conclusion, the study supports an interaction of iron and vitamin A on the level of retinol transport in plasma. Despite a comparable availability of vitamin A as indicated by the comparable liver levels only the re-supplementation of both iron and vitamin A can normalize the retinol level in plasma. This might be of nutritional consequence in developing countries with regard to the supplementation regime of both nutrients iron and vitamin A to prevent a functional deficiency of vitamin A despite sufficient dietary availability.     

Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia.

Dietary copper deficiency causes hypercholesterolemia and increased hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (MHG-CoA) reductase activity and increased hepatic glutathione (GSH) in rats. We hypothesized that inhibition of GSH production by L-buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor, would abolish the cholesterolemia and increased HMG-CoA reductase activity of copper deficiency. In two experiments, two groups of 20 weanling male rats were fed diets providing 0.4 and 5.8 micrograms Cu/g, copper-deficient (Cu-D) and copper-adequate (Cu-A), respectively. At 35 days plasma cholesterol was significantly elevated by 30 to 43% in Cu-D and 10 animals in each of the Cu-D and Cu-A groups were randomly assigned to receive 10 mM BSO solution in place of drinking water and continued on the same diets for another 2 wk. At necropsy Cu-D animals had a significant 52 to 58% increase in plasma cholesterol. BSO administration abolished the cholesterolemia in Cu-D rats, but had no influence on plasma cholesterol of Cu-A rats. Hepatic GSH was increased 39 to 82% in Cu-D rats and BSO abolished this increase. BSO was without effect on cardiac hypertrophy, plasma and liver copper, and hematocrit indices of copper status. Liver microsome HMG-CoA reductase activity was significantly increased 85 to 288% in Cu-D rats and BSO administration abolished this increase in activity in Cu-D rats. The results suggest that copper deficiency cholesterolemia and elevated HMG-CoA reductase activity are a consequence of elevated hepatic GSH, and provide evidence for GSH regulation of cholesterol metabolism in intact animals.     

Selenium deficiency activates mouse liver Nrf2-ARE but vitamin E deficiency does not

Selenium (Se) and vitamin E are antioxidant micronutrients. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2-antioxidant response element (ARE) pathway was investigated using ARE-reporter mice and Nrf2-/- mice. Weanling males were fed Se-deficient (0 Se), vitamin E-deficient (0 E), or control diet for 16 or 22 weeks. The ARE reporter was elevated 450-fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2-ARE (glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase-1 (HO-1)) were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO-1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2-ARE and other stress-response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. This suggests that the oxidant defense mechanisms in which these antioxidant nutrients function are independent of one another.     

The importance of selenium in the prenatal and postnatal development of calves and lambs.

Selenium deficiency is responsible for Zenker type muscle degeneration in calves, lambs, and foals in the prenatal and postnatal stages of development. Investigations have shown that the selenium GSH Px, and vitamin E content of the maternal and fetal parts of the placenta in cattle are different. Similarly, low concentrations of selenium are present in milk from cows and sheep. In addition to an inadequate supply of selenium and vitamin E as a contributory cause of fetal nutritive muscular dystrophy (FNMD), it is assumed that a placental transport block and/or impaired selenium metabolism in the placenta are also responsible. Postnatal nutritive muscular dystrophy, however, is attributed to either acute selenium and vitamin E deficiency in basic feed or impaired plant absorption of selenium as a result of antagonistic elements, such as sulphur.     

The importance of selenium in the prenatal and postnatal development of calves and lambs

Selenium deficiency is responsible for Zenker type muscle degeneration in calves, lambs, and foals in the prenatal and postnatal stages of development. Investigations have shown that the selenium GSH P_x, and vitamin E content of the maternal and fetal parts of the placenta in cattle are different. Similarly, low concentrations of selenium are present in milk from cows and sheep. In addition to an inadquate supply of selenium and vitamin E as a contributory cause of fetal nutritive muscular dystrophy (FNMD), it is assumed that a placental transport block and/or impaired selenium metabolism in the placenta are also responsible. Postnatal nutritive muscular dystrophy, however, is attributed to either acute selenium and vitamin E deficiency in basic feed or impaired plant absorption of selenium as a result of antagonistic elements, such as sulphur.     

Carnitine metabolism in the vitamin B-12-deficient rat.

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.     

Effect of vitamin A deficiency on hepatic and intestinal drug metabolizing enzymes in rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant reduction in the hepatic cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase activities. Contrary to this, the levels of these Phase I enzymes were found to be significantly elevated in all the 3 portions (proximal, middle and distal) of the intestine in deficient animals as compared to corresponding pair-fed controls. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed a significant decrease whereas glutathione S-transferase showed a significant increase in vitamin A-deficient rat liver and small intestine. The study suggests that vitamin A deficiency causes an imbalance between the Phase I and phase II drug metabolizing enzyme systems which may decrease the capacity of the organism to withstand the neoplastic effects of chemical carcinogens in vitamin A deficiency.     

Features of poly-ADP-ribosylation and chromatin structure under conditions of varying NAD content in the rat liver

It is shown that nicotinamide-induced in vivo stimulation of NAD biosynthesis in the liver nuclei of rats causes a decrease of the DNA sensitivity to treatment with DNAse I under conditions of weak hydrolysis. When rats are given synthetic vitamin PP-deprived food, the NAD level in the liver falls down to 40% and a great number of DNAse I-hypersensitive chromatin sites appear. A 24% decrease in the level of poly-ADP-ribosylation of total histones in comparison with the control has been observed with hypovitaminosis. Under conditions of vitamin PP deficiency nicotinamide administered to animals has increased the 14C NAD incorporation into histones 2 times (as compared with the control). These variations occur primarily due to increase of the label incorporation to histone H1. Fractionation of chromatin by solutions of different ionic strength has confirmed that vitamin PP deficiency and NAD amount decrease in the liver are accompanied by a relative increase of the NAD-pyrophosphorylase and poly-ADP-ribose polymerase activities in the fraction extracted by the low ionic solution.     

Modification of hepatic vitamin E stores in vivo. I. Alterations in plasma and liver vitamin E content by methyl ethyl ketone peroxide

Since experiments with freshly isolated rat hepatocytes have shown that cellular vitamin E is consumed in response to insult by compounds that induce an oxidative stress only after cellular glutathione (GSH) concentrations have been substantially depleted, experiments were performed to determine whether this sequence of events occurred in response to oxidative insult in vivo. The role that plasma vitamin E plays in the response to chemically induced oxidative injury in vivo was also assessed. Treatments with 40 mg/kg of methyl ethyl ketone peroxide (MEKP) quickly induced lipid peroxidation in vivo and from one to 4 h after treatment caused a depression in the plasma content of vitamin E and the liver content of GSH, as well as signs of toxicity (elevations in serum activities of alanine and aspartate aminotransferases). At these time points however, the liver content of vitamin E was either indistinguishable from or slightly elevated from controls. By 12 to 24 h after treatment the liver content of vitamin E was reduced by 20-25% whereas values for all other indicators had returned toward control levels. Pretreatment of rats with L-buthionine-S,R-sulfoximine, an inhibitor of GSH by 4 or 24 h after treatment, did not alter the time course or extent of hepatic vitamin E depletion that was observed after treatment with MEKP. Other compounds that induce oxidative stress and lipid peroxidation to the liver, carbon tetrachloride and menadione, did not provoke an alteration in hepatic vitamin E levels as compared to controls 1 day after treatment. These findings indicate that depletion of hepatic vitamin E may not occur as an immediate consequence of oxidative insult to the liver and that the depletion of hepatic vitamin E levels may not be related to the extent of prior GSH depletion. Moreover, these findings suggest that alterations in the plasma concentration of vitamin E may not reflect concurrent alterations in hepatic vitamin E levels. A mechanism whereby liver vitamin E stores are mobilized for the maintenance of plasma vitamin E levels is proposed.     

Vitamin A deficiency modulates iron metabolism via ineffective erythropoiesis

Vitamin A modulates inflammatory status, iron metabolism and erythropoiesis. Given that these factors modulate the expression of the hormone hepcidin (Hamp), we investigated the effect of vitamin A deficiency on molecular biomarkers of iron metabolism, the inflammatory response and the erythropoietic system. Five groups of male Wistar rats were treated: control (AIN-93G), the vitamin A-deficient (VAD) diet, the iron-deficient (FeD) diet, the vitamin A- and iron-deficient (VAFeD) diet or the diet with 12 mg atRA/kg diet replacing all-trans-retinyl palmitate by all-trans retinoic acid (atRA). Vitamin A deficiency reduced serum iron and transferrin saturation levels, increased spleen iron concentrations, reduced hepatic Hamp and kidney erythropoietin messenger RNA (mRNA) levels and up-regulated hepatic and spleen heme oxygenase-1 gene expression while reducing the liver HO-1 specific activity compared with the control. The FeD and VAFeD rats exhibited lower levels of serum iron and transferrin saturation, lower iron concentrations in tissues and lower hepatic Hamp mRNA levels compared with the control. The treatment with atRA resulted in lower serum iron and transferrin concentrations, an increased iron concentration in the liver, a decreased iron concentration in the spleen and in the gut, and decreased hepatic Hamp mRNA levels. In summary, these findings suggest that vitamin A deficiency leads to ineffective erythropoiesis by the down-regulation of renal erythropoietin expression in the kidney, resulting in erythrocyte malformation and the consequent accumulation of the heme group in the spleen. Vitamin A deficiency indirectly modulates systemic iron homeostasis by enhancing erythrophagocytosis of undifferentiated erythrocytes.