Mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase

Mercury compounds exert toxic effects via interaction with many vital enzymes involved in antioxidant regulation, such as selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). Selenium supplementation can reactivate the mercury-inhibited TrxR and recover the cell viability in vitro. To gain an insight on how selenium supplementation affects mercury toxicity in vertebrates, we investigated the effects of selenium on the mercury accumulation and TrxR and GPx activities in a fish model. Juvenile zebra-seabreams were exposed either to methylmercury (MeHg) or inorganic mercury (Hg(2+)) in the presence or absence of sodium selenite (Se) for 28 days followed by 14 days of depuration. Mercury accumulation was found to be 10-fold higher under MeHg exposure than under Hg(2+) exposure. Selenium supplementation caused a half decrease of the accumulation of MeHg but did not influence Hg(2+) accumulation. Exposure to both mercurials led to a decrease of the activity of TrxR (<50% of control) in all organs. Se supplementation coincident with Hg(2+) exposure protected the thioredoxin system in fish liver. However, supplementation of Se during the depuration phase had no effects. The activity of GPx was only affected in the brain of fishes upon the exposure to MeHg and coexposure to MeHg and Se. Selenium supplementation has a limited capacity to prevent mercury effects in brain and kidney. These results demonstrate that Se supplementation plays a protective role in a tissue-specific manner and also highlight the importance of TrxR as a main target for mercurials in vivo.     

In vivo and in vitro inhibition of mice thioredoxin reductase by methylmercury

The thioredoxin (Trx) system, involving redox active Trxs and thioredoxin reductases (TrxRs), sustain a number of important Trx-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense, and redox-regulated signaling cascades. Methylmercury (MeHg) is an important environmental toxicant that has a high affinity for thiol groups and can cause oxidative stress. The Trx system is the major system responsible for maintaining the redox state of cells and this function involves thiol reduction mediated by selenol groups in TrxRs. MeHg has a great affinity to thiols and selenols, thus the potential toxic effects of MeHg on TrxR inhibition were determined in the current study. A single administration of MeHg (1, 5, and 10 mg/Kg) caused a marked inhibition of kidney TrxR activity, while significant inhibition was observed in the liver after exposure to 5 and 10 mg/Kg of MeHg. TrxR activity was determined 24 h after MeHg. In the brain, MeHg did not inhibit TrxR activity. In vitro exposure to MeHg indicated that MeHg inhibits cerebral (IC₅₀, 0.158 μM), hepatic (IC₅₀, 0.071 μM), and renal TrxR activity (IC₅₀, 0.078 μM). The results presented herein demonstrated for the first time that renal and hepatic TrxRs can serve as an in vivo target for MeHg. This study suggests that MeHg can bind to selenocysteine residues present in the catalytic site of TrxR, in turn causing enzyme inhibition that can compromise the redox state of cells.     

Cyclophosphamide suppresses thioredoxin reductase in bladder tissue and its adaptive response via inductions of thioredoxin reductase and glutathione peroxidase

Mammalian thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin in a NADPH-dependent manner, and contains a selenocysteine residue near the C-terminus. Glutathione peroxidase (GPx) is one of the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Both TrxR and GPx play an important role in protecting against oxidative stress. Cyclophosphamide (CTX), one of the most widely prescribed antineoplastic drugs, could cause cystitis. We found that 4 h after a bolus dose of CTX (30, 90, 150, 300 and 450 mg/kg) were administrated intraperitoneally, TrxR activity was significantly decreased in a dose-dependent manner, by 32%, 44%, 68%, 87% and 99%, respectively, in comparison with control group. When fixing CTX dose at 150 mg/kg, TrxR activity changed over time, significantly reduced to 68% of the activity in comparison with control tissue at 2 h, and gradually recovered to normal level within 24 h. In addition, we found that GPx activity was induced significantly after 4h. The results of the present study suggest that marked suppression of TrxR activity could be involved in the mechanism of CTX-induced cystitis, bladder may have a protective system against tissue damage by CTX via upregulation of TrxR and GPx, which is an adaptive response to oxidative stress.     

Developmental study of rat brain glutathione peroxidase and glutathione reductase

Glutathione peroxidase and glutathione reductase activities were measured in whole rat brains at selected ages from birth to adulthood. On a wet weight basis glutathione peroxidase activity increased 70% during development and glutathione reductase activity increased 160%. On a protein basis glutathione peroxidase declined slightly in activity during the first two weeks of life and then maintained the 14-day activity into adulthood while glutathione reductase showed a 30% increase in activity. While less than the developmental changes in many enzymes involved in aerobic glycolysis or catecholamine metabolism, these increases do suggest a role in CNS metabolism.     

Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase

Singlet oxygen ((1)O(2)) is a reactive oxygen species generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g., myeloperoxidase and eosinophil peroxidase). (1)O(2) oxidizes the free amino acids Trp, Tyr, His, Cys, and Met, and those species present on peptides/proteins, with this resulting in modulation of protein structure and function. Impairment of the activity of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathologies involving either light exposure or inflammation. In this study, the effects of (1)O(2) on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (an established source of (1)O(2)) resulted in significant, photolysis time-dependent reductions in enzyme activity (10-40%, P<0.05). More extensive inhibition (ca. 2-fold) was detected when the reactions were carried out in D(2)O, consistent with the intermediacy of (1)O(2). No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts rapidly with (1)O(2) (k～10(7)M(-1) s(-1))(,) significantly reduced photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH(4), DTT, GSH, or NADPH) added after the cessation of (1)O(2) formation were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of nonreducible protein aggregates and/or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes GPx and TrxR can be irreversibly inactivated by (1)O(2).     

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.     

Effects of long-term selenium deficiency on glutathione peroxidase and thioredoxin reductase activities and expressions in rat aorta

The objective of this work was to determine whether long-term selenium (Se) deficiency might affect the antioxidant capacity of rat aorta, and the activities and expressions of glutathione peroxidase (GPx) and thioredoxin reductase (TR) in rat arterial walls. Weanling male Wister rats were fed Se-deficient or Se-adequate diets for 12 months. For the Se supplementation, sodium selenite was supplemented in drinking water (1 microg Se/ml) for 1 month. The aorta isolated from these groups were used to determine activities and mRNA levels. In comparison with the control, the activity and expression of GPx, superoxide dismutase activity and the total antioxidant capacity were significantly decreased in Se-deficient rats arterial walls. Following Se supplementation, they were restored to different extents. The content of malondialdehyde was increased markedly in Se-deficient rats. There seems an inverse relationship between the dietary Se and the activity and expression of TR. A positive relationship exists between dietary Se and the antioxidant capacity of rat arterial walls. The activities and expressions of GPx and TR in arterial walls were regulated by selenium by different mechanisms. Regulation of the expression of TR was mediated by reactive oxygen species, but of GPx by selenium status. The thioredoxin system may be the major cellular redox signaling system in rat arteries, rather than the glutathione system.     

A study on the inhibition of rat myocardium glutathione peroxidase and glutathione reductase by moniliformin

The yeasts of patients with oral cancer has been studied before and during Xr-therapy. Gram and PAS smears revealed an increase of yeast-like structures, during treatment, from 56% to 66% of the cases. Before radiotherapy oral yeasts were isolated from 56% of the patients with cancer represented by Candida albicans (30%); C. tropicalis (12%); C. glabrata and C. krusei (4%), besides six other different species (2%). During radiotherapy yeasts were isolated in 72% of the cases, as follow: C. albicans (36%); C. tropicalis (16%); Rhodotorula rubra (6%); C. kefyr; C. krusei and Pichia farinosa (4%), besides other nine species (2%). C. albicans serotype A represented 93% of the isolated samples, before treatment and 88,8% during Xr-therapy.     

Effects of dietary selenium on glutathione peroxidase and thioredoxin reductase activity and recovery from cardiac ischemia-reperfusion.

Glutathione peroxidase and thioredoxin reductase are selenocysteine-dependent enzymes that protect against oxidative injury. This study examined the effects of dietary selenium on the activity of these two enzymes in rats, and investigated the ability of selenium to modulate myocardial function post ischemia-reperfusion. Male wistar rats were fed diets containing 0, 50, 240 and 1000 microg/kg sodium selenite for 5 weeks. Langendorff perfused hearts isolated from these rats were subjected to 22.5 min global ischemia and 45 min reperfusion, with functional recovery assessed. Liver samples were collected at the time of sacrifice, and heart and liver tissues assayed for thioredoxin reductase and glutathione peroxidase activity. Selenium deficiency reduced the activity of both glutathione peroxidase and thioredoxin reductase systemically. Hearts from selenium deficient animals were more susceptible to ischemia-reperfusion injury when compared to normal controls (38% recovery of rate pressure product (RPP) vs. 47% recovery of RPP). Selenium supplementation increased the endogenous activity of thioredoxin reductase and glutathione peroxidase and resulted in improved recovery of cardiac function post ischemia reperfusion (57% recovery of RPP). Endogenous activity of glutathione peroxidase and thioredoxin reductase is dependent on an adequate supply of the micronutrient selenium. Reduced activity of these antioxidant enzymes is associated with significant reductions in myocardial function post ischemia-reperfusion.     

Diphenyl diselenide and diphenyl ditelluride differentially affect delta-aminolevulinate dehydratase from liver, kidney, and brain of mice

In the present study, the inhibitory effect of diphenyl diselenide and diphenyl ditelluride after in vitro, acute (a single dose), or chronic exposure (14 doses) was examined in mice 24 hours after the last administration. In vitro, diphenyl diselenide, and diphenyl ditelluride inhibited delta-aminolevulinate dehydratase (delta-ALA-D) from brain, liver, and kidney with a similar potency (IC50 5-10 microM), and at 120 microM, they increased the rate of dithiothreitol (DTT) and reduced glutathione (GSH) oxidation. After a single dose (sc), diphenyl diselenide (1 mmol/kg) inhibited the liver (22%, p < 0.01) and brain (27%, p < 0.01) delta-ALA-D, but it did not inhibit the kidney enzyme. After a single dose (sc), diphenyl ditelluride (0.5 mmol/kg) inhibited liver (46%, p < 0.01), kidney (21%, p < 0.05), and brain (39%, p < 0.01) delta-ALA-D. Chronic exposure to diphenyl diselenide (0.125 and 0.250 mmol/kg) caused significant (p < 0.05) increase in liver and liver-to-body weight ratio and inhibited liver (40 and 60%, respectively) and brain (21 and 40%, respectively) delta-ALA-D. Kidney delta-ALA-D was not inhibited significantly after exposure to diphenyl diselenide. Total nonprotein - SH concentration was decreased only in liver of animals exposed for 14 days to selenide. Chronic exposure to diphenyl ditelluride (0.010 and 0.025 mmol/kg) caused significant (p < 0.05) inhibition of liver (28 and 42%, respectively) and brain (23 and 54%, respectively) delta-ALA-D. Kidney delta-ALA-D was not inhibited significantly by diphenyl ditelluride. Total nonprotein--SH concentration was decreased to a different extent after acute or chronic treatment with diphenyl ditelluride depending on analyzed tissue. Hemoglobin content was decreased significantly by 17 and 22% after chronic treatment with 0.125 and 0.25 mmol/kg diphenyl diselenide, respectively. Chronic exposure to 0.010 mmol/kg diphenyl ditelluride caused a reduction of 17% in hemoglobin content that tended to be significant (p < 0.10). These results suggest that delta-ALA-D inhibition after exposure to organochalcogens may perturb heme-dependent metabolic pathway and contribute to the toxicological properties of these compounds.     

Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5 M^–1 s^–1. With 50 µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40 min, respectively, compared with 10 min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5 min with 2 µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.     

Diphenyl diselenide and diphenyl ditelluride: neurotoxic effect in brain of young rats, in vitro

This study examined whether maturity of rat brain may be relevant for the sensitivity to diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release, in vitro. Brain synaptosomes were isolated from young (14- and 30-day-old) and adult rats and incubated at different concentrations of (PhSe)₂ or (PhTe)₂. The results demonstrated that the highest concentration (100 μM) of (PhSe)₂ and (PhTe)₂ inhibited the [³H]glutamate uptake by synaptosomes of brain at all ages. In the adult brain, (PhSe)₂ did not inhibit the [³H]glutamate uptake at the lowest concentration (10 μM). The highest concentration of (PhTe)₂ inhibited the [³H]glutamate uptake more in the 14-day-old than in the 30-day-old rats or adult rats. In the 30-day-old animals, the highest concentration of (PhSe)₂, and the lowest concentration of (PhTe)₂, increased the basal [³H]glutamate release. At the highest concentration, (PhTe)₂ increased the basal and K⁺-stimulated glutamate release on all ages evaluated. The results suggest that (PhSe)₂ and (PhTe)₂ caused alterations on the homeostasis of the glutamatergic system at the pre-synaptic level. These alterations were age-, concentration-, and compound-dependent. The maturity of rat brain is relevant for the glutamatergic system sensitivity to (PhSe)₂ and (PhTe)₂ .     

Secondary structure and stability of the selenocysteine insertion sequences (SECIS) for human thioredoxin reductase and glutathione peroxidase

We have used high resolution NMR and thermodynamics to characterize the secondary structure and stability of the selenocysteine insertion sequences (SECIS) of human glutathione peroxidase (58 nt) and thioredoxin reductase (51 nt). These sequences are members of the two classes of SECIS recently identified with two distinct structures capable of directing selenocysteine incorporation into proteins in eukaryotes. UV melting experiments showed a single cooperative and reversible transition for each RNA, which indicates the presence of stable secondary structures. Despite their large size, the RNAs gave well resolved NMR spectra for the exchangeable protons. Using NOESY, the imino protons as well as the cytosine amino protons of all of the Watson-Crick base pairs were assigned. In addition, a number of non-canonical base pairs including the wobble G.U pairs were identified. The interbase-pair NOEs allowed definition of the hydrogen-bonded structure of the oligonucleotides, providing an experimental model of the secondary structure of these elements. The derived secondary structures are consistent with several features of the predicted models, but with some important differences, especially regarding the conserved sequence motifs.     

In vivo inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate

Intraperitoneal injection of rats with diethyldithiocarbamate (1.2 g/kg body wt) led to maximum diminution of superoxide dismutase activity at 1 hr by 86 and 84% in liver and red blood cell respectively with a gradual return to the normal level at 48 hr after administration of injection. Significant inhibition of selenium-dependent glutathione peroxidase was also observed, which returned to normal at 48 hr after administration of injection. However, maximum decline in its activity was at 12 hr by 52 and 73% in liver and red blood cells respectively. No significant difference in tissue level of selenium-independent glutathione peroxidase was observed during time course study after diethyldithiocarbamate administration. It is possible that inhibition of superoxide dismutase by diethyldithiocarbamate leads to accumulation of superoxide anion which in turn inactivates selenium-dependent glutathione peroxidase by its reaction with selenium at the active site of the enzyme.     

Effect of sarcolysine on glutathione peroxidase and glutathione reductase activity in sarcoma C-45

A drop of glutathione peroxidase and glutathione reductase activity was revealed in sarcoma C-45 at the period of its most intensive growth. Repeated sarcolysine injections (1.2 mg/kg, intraperitoneally) caused a sharp fall in the activity of both enzymes with a simultaneous reduction of the ratio of glutathione reductase and glutathione peroxidase activities. The important role of the glutathione enzyme redox system in the realization of antitumour action of the chemotherapeutic drugs is supposed.     

Constitutive and Aroclor 1254-induced hepatic glutathione S-transferase, peroxidase and reductase activities in genetically inbred mice

1. Constitutive and Aroclor 1254-induced hepatic glutathione (GSH) S-transferases, GSH peroxidase and GSH reductase activities were determined in 12 strains of 8-10 week-old inbred male mice. 2. The constitutive GSH S-transferase activity varied from 2.5 (SJL/JCR) to 8.9 (C57BL/6N) mumol/min/mg protein and the corresponding values for the Aroclor 1254-treated mice were in the range of 7.1-23.0 mumol/min/mg protein. Aroclor 1254 significantly induced GSH S-transferase activity in all mice, however, significant interstrain differences were found in inducibility. 3. Aroclor 1254-treatment caused a 4.2-fold induction of GSH S-transferase in NFS/NCR but only a 1.4-fold increase in AKR/NCR mice. Aroclor 1254 significantly induced GSH reductase in all strains studied while GSH peroxidase activity decreased in these mice. 4. The range of hepatic GSH levels in control and Aroclor 1254-treated mice was relatively narrow for both groups (6.59-11.25 microM/g wet tissue).     

Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measured of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050).     

Micromethods in single muscle fibers. 2. Determination of glutathione reductase and glutathione peroxidase

This paper extends the previous study for systems which control intracellular oxidative events in muscle and describes procedures suitable to assay glutathione peroxidase (GSHPx), glutathione reductase (GR), and total glutathione (GSH + GSSG) after fiber typing of individual muscle fibers. In human skeletal muscle, both GR and GSHPx activities were relatively low when compared to those of other tissue. No difference was found among fiber types (I, IIA, and IIB) with regard to GR activity, but in contrast GSHPx activity was significantly lower in type IIB fibers than in the other types. These results suggest that type IIB fibers may have a reduced ability to cope with hydroperoxides generated during oxidative stress, which, in turn, could lead to increased damage to membrane structures by lipid peroxidation or oxidation of sensitive intracellular thiol (-SH) enzymes by hydrogen peroxide. The Km of skeletal muscle GR for GSSG was 27 microM and for NADPH was 22 microM. If one assumes approximately 95% of total glutathione is present in the reduced state, then GSSG concentration would be of the order of 0.3 mmol/kg and under these conditions skeletal muscle GR would be efficient in all muscle fiber types.     

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.