GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis.

Ferroptosis, a newly defined mode of regulated cell death caused by unbalanced lipid redox metabolism, is implicated in various tissue injuries and tumorigenesis. However, the role of ferroptosis in stem cells has not yet been investigated. Glutathione peroxidase 4 (GPX4) is a critical suppressor of lipid peroxidation and ferroptosis. Here, we study the function of GPX4 and ferroptosis in hematopoietic stem and progenitor cells (HSPCs) in mice with Gpx4 deficiency in the hematopoietic system. We find that Gpx4 deletion solely in the hematopoietic system has no significant effect on the number and function of HSPCs in mice. Notably, hematopoietic stem cells (HSCs) and hematopoietic progenitor cells lacking Gpx4 accumulated lipid peroxidation and underwent ferroptosis in vitro. α-Tocopherol, the main component of vitamin E, was shown to rescue the Gpx4-deficient HSPCs from ferroptosis in vitro. When Gpx4 knockout mice were fed a vitamin E-depleted diet, a reduced number of HSPCs and impaired function of HSCs were found. Furthermore, increased levels of lipid peroxidation and cell death indicated that HSPCs undergo ferroptosis. Collectively, we demonstrate that GPX4 and vitamin E cooperatively maintain lipid redox balance and prevent ferroptosis in HSPCs.

Mitochondrial respiratory chain dysfunction in ageing; influence of vitamin E deficiency.

The causes and consequences of ageing are likely to be complex and involve the interaction of many processes. It has been proposed that the decline in mitochondrial function caused by the accumulation of oxidatively damaged molecules plays a significant role in the ageing process. In agreement with previous reports we have shown that the activities of NADH CoQ1 reductase and cytochrome oxidase declined with increasing age in both rat liver and gastrocnemius muscle mitochondria. However, only in the liver were the changes in lipid peroxidation and membrane fluidity suggestive of an age-related increase in oxidative stress. After 12 weeks on a vitamin E deficient diet, vitamin E levels were undetectable in both gastrocnemius muscle and liver. In skeletal muscle, this was associated with a statistically significant increase in lipid peroxidation, a decrease in cytochrome oxidase activity after 48 weeks, and an exacerbation in the age-related rate of decline of NADH CoQ1 reductase activity. This was consistent with the suggestion that an imbalance between free radical generation and antioxidant defence may contribute to the mitochondrial dysfunction with age. In contrast to this, vitamin E deficiency in the liver caused a significant increase in mitochondrial respiratory chain activities with increasing age despite evidence of increased lipid peroxidation. Comparison of other features in these samples suggested vitamin E deficiency; did not have a significant impact upon mtDNA translation; induced a compensatory increase in glutathione levels in muscle, which was less marked in the liver, but probably most interestingly caused a significant decrease in the mitochondrial membrane fluidity in muscle but not in liver mitochondria. These data suggest that while increased lipid peroxidation exacerbated the age-related decline in muscle respiratory chain function this relationship was not observed in liver. Consequently other factors are likely to be contributing to the age-related decline in mitochondrial function and specific stimuli may influence or even reverse these age-related effects as observed with vitamin E deficiency in the liver.

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.

Effect of vitamin E deficiency on rat brain monoamine metabolism.

We investigated the effects of vitamin E deficiency on the monoamine metabolism in the rat brain. Male Wistar rats fed on the vitamin E deficient diet for 24 weeks were analyzed. At 28 weeks, they showed a reduced growth rate (52% of reduction), muscle atrophy, a motor weakness of hind limbs and disturbance of gait. The concentrations of monoamines, their precursors and metabolites in the brain were simultaneously determined using high performance liquid chromatography (HPLC) coupled with a coulometric detection with electrode array system. In addition, tryptophan hydroxylase activity was measured. The dopamine (p = 0.009) and serotonin (p = 0.04) levels in the brain stem of vitamin E deficient rats were significantly lower than in the controls, whereas their precursors tyrosine (p = 0.0009) and tryptophan (p = 0.0065) levels in the brain stem were significantly higher than in the controls. Moreover, tryptophan hydroxylase activity (p = 0.0005) in the brain stem of vitamin E deficient brains was significantly lower than in the controls. All statistical comparisons were done using non-parametric tests (Mann-Whitney U test). These results suggest that vitamin E deficiency may play a role in the disturbance of monoamine metabolism in rat brain.

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.

Tocopherol modulates the effects of A23187, verapamil, and phorbol myristate acetate on RNA-polymerase activity of isolated rat liver nuclei.

Preincubation of rat liver nuclei with tocopherol decreased inhibition of RNA-polymerase activity of isolated rat liver nuclei by A23187, verapamil, and phorbol myristate acetate (PMA). In nuclei of vitamin E-deficient rats, A23187, verapamil, and PMA did not inhibit label incorporation into RNA with and without tocopherol. A23187, verapamil, and PMA did not inhibit the activity of nuclei treated with 1% Triton X-100; tocopherol activated RNA synthesis but co-administration of A23187 or verapamil and tocopherol had no effect and the combination of tocopherol and PMA inhibited RNA-polymerase activity by 25%. The effect of the combination of verapamil and PMA in the presence of free tocopherol was different from the effect of these compounds assayed in the presence of the complex of tocopherol with tocopherol-binding protein.

Vitamins C, E and A and heme oxygenase in rats fed methyl/folate-deficient diets.

There is evidence that the development of hepatocarcinoma in rats fed a methyl-deficient diet is associated with oxidative stress. We investigated, therefore, whether the tissue concentrations of the antioxidant vitamins ascorbic acid (AA) and alpha- and gamma-tocopherol (T) are altered in methyl/folate deficiency. We also measured retinol concentrations in tissues and hepatic mRNA expression of heme oxygenase (HO1). A 6% gelatin, 6% casein diet, devoid of choline and folate (CFD) was selected based on the high rate of tumor development in rats fed this diet. Spectrophotometric measurement of AA and HPLC determination of tissue T and retinol showed decreased concentrations of AA in blood; alpha- and gamma-T in lung, heart and plasma, alpha-T and retinol in liver; retinol in lung; and increased expression of hepatic HO1 mRNA. Similar alterations in tissue vitamin concentrations were found when the CFD diet devoid of niacin (CFND) was fed. Reducing alpha-T in the CFND diet (CFNED) further decreased hepatic alpha-T concentrations. These results show that chronic methyl/folate deficiency is associated with a compromised antioxidant defense system.

[Superoxide dismutase activity in microsomes of functionally different rat tissues in vitamin E deficiency]. / Aktivnost' superoksiddismutazy v mikrosomakh funktsional'no razlichnykh tkanei krys pri nedostatochnosti vitamina E.

Comparative investigation of microsome superoxide dismutase activity in functionally different rat tissues such as lung and liver has been carried out. Modification of superoxide dismutase fraction isolated from lung and liver microsomes and SOD-activity evaluation by means of superoxide radical-dependent pyrogallol autooxidation method have been described in detail. SOD-activity has been shown to be connected with the degree of lipid peroxidative oxidation (LPO) and total condition of tissue antioxidative system in case of different E-vitamin security in various functional types of cells (alveolocytes and hepatocytes).

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.

Effects of vitamin E deficiency on hepatic microsomal cytochrome P450 and phase II enzymes in male and female rats.

The effects of a vitamin E (VE) deficient diet given for 120 days on total cytochrome P450 (P450) contents and UDP-glucuronyl transferase (UDPGT) and sulfotransferase (ST) activities were investigated in rat liver of both sexes. The VE deficient regimen lowered the serum VE levels, increasing the hepatic malondialdehyde concentration regardless of animal sex, which shows an elevation of the tissue peroxi-disability. Hepatic total P450 contents were significantly decreased in male rats given the VE deficient diet, whereas the effects on female rats were less obvious. Concerning phase II enzymes, VE deficiency had no effect on the activities of UDPGT and ST in rats of both sexes. The data suggest that overall capacity of detoxification of xenobiotics may be impaired by VE deficiency to a much greater extent in male than in female rat liver.

Myopathy in vitamin E deficient rats: muscle fibre necrosis associated with disturbances of mitochondrial function.

Vitamin E deficiency in rats gives rise to a neuromuscular syndrome that includes a peripheral neuropathy as well as generalised muscle wasting and weakness. This is probably related to damage by oxygen-derived free radicals. In the present study, histological examination of lower limb muscles showed widespread myopathic changes which included the presence of amorphous electron-dense inclusions and tubular aggregates in muscle fibres and muscle fibre necrosis. Histochemical observations suggested a reduction in the activity of oxidative enzymes. The mitochondria showed nonspecific degenerative changes on electron microscopy; no paracrystalline inclusions were observed. Polarographic analysis of isolated muscle mitochondria revealed statistically significant decreases in oxygen utilisation rates with both NADH and FADH2-linked substrates. In confirmation of a generalised respiratory chain abnormality, enzymatic analyses revealed decreases in the activities of complexes I, II/III and IV, although only the decreases in complexes I and IV activities were statistically significant. Measurements of membrane fluidity showed that this is reduced in mitochondria from vitamin E deficient rats, indicating reduced stability of their membranes. The respiratory control ratio, derived from the polarographic results, was also reduced in mitochondria from vitamin E deficient animals, suggesting membrane damage. An altered lipid environment, possibly secondary to a higher level of lipid peroxidation, could result in the inhibition of complexes I and IV. This could also be caused by oxidative damage to the complexes or to mitochondrial DNA. The preservation of citrate synthase activity is against any generalised defect of mitochondrial function. The question as to whether these defects of mitochondrial respiratory chain function are responsible for the muscle fibre damage and necrosis requires further investigation.

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.

Alpha-tocopherol deficiency fails to aggravate toxic liver injury but liver injury causes alpha-tocopherol retention.

The possible aggravation of liver injury by impaired cellular antioxidant function was investigated. A vitamin E-deficient diet (0.5 mg/kg alpha-tocopherol; control 100 mg/kg) significantly reduced rat liver alpha-tocopherol concentrations after 4 weeks (1.8 +/- 1.7 micrograms/g; control 34.4 +/- 2.4 micrograms/g, p < 0.001). The effects of copper loading (Cu, 3 g/kg diet); galactosamine (GalN, 0.85 g/kg i.p.); or carbon tetrachloride (CCl4, 10 mmol/kg i.p.) were examined. Serum aspartate transaminase activity was elevated slightly by vitamin E deficiency but not by hepatic copper accumulation. In vitamin E-replete (E+) and vitamin E-deficient (E-) rats, GalN or CCl4 caused a large and comparable elevation in serum AST and OCT activity. This effect on AST was markedly reduced by copper loading in vitamin E replete (E+) rats, but in E(-) rats copper had significantly less protective effect. Copper also diminished the OCT response to GalN in E+, though not E-, rats. A significant rise in total hepatic alpha-tocopherol content followed administration of GalN or CCl4 in both normocupric and copper-laden E(-) rats. Thus alpha-tocopherol deficiency (a) was not hepatotoxic per se; (b) failed to potentiate the toxicity of copper, GalN or CCL4; but (c) partially abolished the protection by copper against toxin-induced liver injury. Retention of hepatic alpha-tocopherol after liver damage may partly explain low serum vitamin E levels seen in clinical liver disease.

Exercise-induced muscle damage in the rat: the effect of vitamin E deficiency.

Rats, fed a vitamin-E-deficient diet for 6 weeks, performed treadmill exercise for 2 h. Muscle damage was assessed by measuring the creatine kinase (CK) activity in plasma before and after exercise, and by studying semi-thin longitudinal sections of the soleus muscle 48 h after running. Vitamin-E-deficient male and female rats showed an increased post-exercise CK activity when compared to matched controls, but male rats showed a larger CK response than females. This rise in plasma CK activity was caused mainly by an increased activity of the muscle-specific CK-isoenzyme, CK-MM (males + 1238%; females + 540%, P less than 0.05). In a parallel histological study we observed in vitamin-E-deficient male rats a dramatic and significant disturbance of the normal cyto-architecture of the muscle fibres after exercise (focal necrosis, phagocytosis and cellular infiltrates), whereas in females only minor, non-significant, changes were seen. We conclude that vitamin E deficiency enhances the susceptibility to exercise-induced muscle damage in male rats more than in female rats. This difference between the sexes is attributed to the protective effect of oestradiol that remains operative in female rats when the vitamin E status is disturbed: male rats lack such hormonal protection.

Effect of vitamin E-deficiency on the activity of some lysosomal and non-lysosomal proteases in rabbit muscles.

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.

[The induction of the monooxygenase system and the incorporation of the radioactive label from 2-14C-lysine into the liver microsome fraction of rats exposed to phenobarbital against a background of deficiencies in lysine, methionine, threonine and vitamins A, C and E]. / Induktsiia monooksigenaznoi sistemy i vkliuchenie radioaktivnoi metki iz 2-14C-lizina vo fraktsiiu mikrosom pecheni krys pri vozdeistvii fenobarbitala na fone defitsita lizina, metionina, treonina i vitaminov A, C i E.

The effect of diet on induction of monooxygenases and distribution of label from 2-14C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital (80 mg/kg, three days) was studied. 2-14C-lysine was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monoxygenase induction, increase of relative liver weight and incorporation of label from 2-14C-lysine into fractions of liver homogenate in phenobarbital-treated rats were more pronounced as compared with the similarly treated rats that were fed a balanced diet. The possibility of mobilization of deficient essential components to liver from other organs and tissues for maintenance of monoxygenase induction is discussed.

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.