Chemical and enzymatic reductive activation of acylfulvene to isomeric cytotoxic reactive intermediates

Acylfulvenes (AFs), a class of semisynthetic analogues of the sesquiterpene natural product illudin S, are cytotoxic toward cancer cells. The minor structural changes between illudin S and AFs translate to an improved therapeutic window in preclinical cell-based assays and xenograft models. AFs are, therefore, unique tools for addressing the chemical and biochemical basis of cytotoxic selectivity. AFs elicit cytotoxic responses by alkylation of biological targets, including DNA. While AFs are capable of direct alkylation, cytosolic reductive bioactivation to an electrophilic intermediate is correlated with enhanced cytotoxicity. Data obtained in this study illustrate chemical aspects of the process of AF activation. By tracking reaction mechanisms with stable isotope-labeled reagents, enzymatic versus chemical activation pathways for AF were compared for reactions involving the NADPH-dependent enzyme prostaglandin reductase 1 (PTGR1) or sodium borohydride, respectively. These two processes resulted in isomeric products that appear to give rise to similar patterns of DNA modification. The chemically activated isomer has been newly isolated and chemically characterized in this study, including an assessment of its relative stereochemistry and stability at varying pH and under bioassay conditions. In mammalian cancer cells, this chemically activated analogue was shown to not rely on further cellular activation to significantly enhance cytotoxic potency, in contrast to the requirements of AF. On the basis of this study, we anticipate that the chemically activated form of AF will serve as a useful chemical probe for evaluating biomolecular interactions independent of enzyme-mediated activation.     

Recent advances in the development of thioredoxin reductase inhibitors as anticancer agents

Redox homeostasis is crucial for the cellular viability and normal function which balance is maintained by two major cellular antioxidant systems, including glutathione system and thioredoxin system. Thioredoxin system, including thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, exhibits a wide range of functions such as regulation of redox state and cell apoptosis. Particularly, Trx functions as a protein disulfide reductase which is essential for the function of Trx system. However, the bioactivity of Trx is closely dependent on its reducing form. According to the information, TrxR is the only cellular enzyme to catalyze the NADPH-dependent reduction of Trx. Besides the reduction of some protein disulfide like Trx, TrxR still has a broad substrate specificity to reduce some small molecules like 5, 5 '-dithiobis-2- nitrobenzoic acid (DTNB). The reduction of Trx or its own direct action towards its various substrates endows TrxR with a wide range of cellular functions. Recent studies have elucidated that TrxR was upregulated in many malignant tumors and inhibition of TrxR could prevent the tumor initiation and progression, suggesting TrxR to be a promising target for cancer therapy and the high nucleophilic and accessible selenocysteine (Sec) active site might be the prime target for drug design. Various kinds of TrxR inhibitors have been developed as anticancer agents for years. In this review, TrxR inhibitors are divided into three classes, including metal-containing inhibitors, naturally occurring products and their derivatives and other newly emerged inhibitors. The last five years reports about TrxR inhibitors of each class will be introduced and their novel inhibiting mechanisms will be discussed.     

Short-interfering RNA-mediated silencing of thioredoxin reductase 1 alters the sensitivity of HeLa cells toward cadmium

The mammalian thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that regulates the thioredoxin system, one of the major systems that maintain the intracellular redox balance. We previously reported that cytosolic TrxR (TrxR1), one of three mammalian TrxR isozymes, was induced by treatment with cadmium. In the present study, to study the role of cadmium-induced TrxR1 in cellular defense, we silenced the expression of TrxR1 in HeLa cells by using small interfering RNA and examined the effect of TrxR1 silencing on the sensitivity of the cells toward cadmium. We found that the gene silencing of TrxR1 had a dual effect on cadmium-induced cell death, depending on the concentration of cadmium. The TrxR1 silencing increased the sensitivity toward a low dose (less than 10 microM) of cadmium but decreased the sensitivity toward a high dose of cadmium. These results suggested that TrxR1 might play an important role in the cellular defense system against cadmium in two ways. TrxR1 might rescue the cells from a low dose of cadmium-induced moderate injury, while it might promote the death of cells severely injured by a high dose of cadmium.     

The potent and novel thiosemicarbazone chelators di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone and 2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone affect crucial thiol systems required for ribonucleotide reductase activity

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone possesses potent and selective antitumor activity. Its cytotoxicity has been attributed to iron chelation leading to inhibition of the iron-containing enzyme ribonucleotide reductase (RR). Thiosemicarbazone iron complexes have been shown to be redox-active, although their effect on cellular antioxidant systems is unclear. Using a variety of antioxidants, we found that only N-acetylcysteine significantly inhibited thiosemicarbazone-induced antiproliferative activity. Thus, we examined the effects of thiosemicarbazones on major thiol-containing systems considering their key involvement in providing reducing equivalents for RR. Thiosemicarbazones significantly (p < 0.001) elevated oxidized trimeric thioredoxin levels to 213 ± 5% (n = 3) of the control. This was most likely due to a significant (p < 0.01) decrease in thioredoxin reductase activity to 65 ± 6% (n = 4) of the control. We were surprised to find that the non-redox-active chelator desferrioxamine increased thioredoxin oxidation to a lower extent (152 ± 9%; n = 3) and inhibited thioredoxin reductase activity (62 ± 5%; n = 4), but at a 10-fold higher concentration than thiosemicarbazones. In contrast, only the thiosemicarbazones significantly (p < 0.05) reduced the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant. All chelators significantly decreased RR activity, whereas the NADPH/NADP(total) ratio was not reduced. This was important to consider because NADPH is required for thiol reduction. Thus, thiosemicarbazones could have an additional mechanism of RR inhibition via their effects on major thiol-containing systems.     

Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties

Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme thioredoxin reductase (TrxR) is considered as the most relevant molecular target for these species. The here investigated gold(I) complexes with benzimidazole derived N-heterocyclic carbene (NHC) ligands represent a promising class of gold coordination compounds with a good stability against the thiol glutathione. TrxR was selectively inhibited by in comparison to the closely related enzyme glutathione reductase, and all complexes triggered significant antiproliferative effects in cultured tumor cells. More detailed studies on a selected complex revealed a distinct pharmacodynamic profile including the high increase of reactive oxygen species formation, apoptosis induction, strong effects on cellular metabolism (related to cell surface properties, respiration, and glycolysis), inhibition of mitochondrial respiration and activity against resistant cell lines.     

Thioredoxin reductase inhibition reduces relaxation by increasing oxidative stress and s-nitrosylation in mouse aorta

Oxidative stress is well known to lead to vascular dysfunction. Thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin. Reduced thioredoxin plays a role in cellular antioxidative defense and in decreasing S-nitrosylation. It is not known whether TrxR affects vascular reactivity. We hypothesized that TrxR inhibition decreases vascular relaxation via increased oxidative stress and S-nitrosylation. Aortic rings from C57BL/6 mice were treated with the TrxR inhibitor, 1-chloro-2,4-dinitrobenzene (DNCB), or auranofin for 30 minutes. Vascular relaxation to acetylcholine was measured in the rings contracted with phenylephrine. DNCB and auranofin reduced relaxation compared with vehicle (vehicle Emax = 71 ± 3%, DNCB Emax = 53 ± 3%; P < 0.05). The antioxidants, apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), and tempol (superoxide dismutase mimetic) normalized impaired relaxation by DNCB in aorta (DNCB Emax = 53 ± 3%, DNCB + tempol Emax = 66 ± 3%; P < 0.05). In addition, DNCB reduced sodium nitroprusside-induced relaxation. DNCB increased soluble guanylyl cyclase (sGC) S-nitrosylation and decreased sGC activity. These data suggest that TrxR regulates vascular relaxation via antioxidant defense and sGC S-nitrosylation. TrxR may be an enzyme to approach for treatment of vascular dysfunction and arterial hypertension.     

Cyclophosphamide as a potent inhibitor of tumor thioredoxin reductase in vivo

Cyclophosphamide (CTX) is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents. It is one of the most frequently used antitumor agents for the treatment of a broad spectrum of human cancers. Thioredoxin reductase (TrxR) catalyze the NADPH-dependent reduction of thioredoxin and play an important role in multiple cellular events related to carcinogenesis including cell proliferation, apoptosis, and cell signaling. This enzyme represents a promising target for the development of cytostatic agents. The purpose of this study is to determine whether CTX could target TrxR in vivo. Lewis lung carcinoma and solid H22 hepatoma treated with 50-250 mg/kg CTX for 3 h lost TrxR activity in a dose-dependent fashion. Over 75% and 95% of TrxR activity was lost at the dose of 250 mg/kg. There was, however, a recovery of TrxR activity such that it attained normal levels by 120 h after a dose of 250 mg/kg. In addition, we found that CTX caused a preferential TrxR inhibition over other antioxidant enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase. We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo since CTX is needed to be activated by liver cytochrome P450 enzymes. The time course and dose-dependent changes of cellular TrxR activity were similar with those in tumor tissue. CTX caused a dose-dependent cellular proliferation inhibition which was positively correlated with TrxR inhibition at 3 h. Furthermore, when 3 h CTX-treated cells with various TrxR backgrounds, harvested from ascites-bearing mice, were implanted into mice, the proliferations of these cells were again proportionally dependent on TrxR activity. The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX.     

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, ‘auranofin-like’ gold(I) complexes all containing the [Au(PEt₃)]⁺ synthon and the ligands: Cl⁻, Br⁻, cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.     

The role of thioredoxin reductase activity in selenium-induced cytotoxicity

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.     

Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo.     

Pyrroloquinoline quinone modulates the kinetic parameters of the mammalian selenoprotein thioredoxin reductase 1 and is an inhibitor of glutathione reductase

Pyrroloquinoline quinone (PQQ) is a redox active cofactor for bacterial quinoproteins. Dietary PQQ also has prominent physiological effects in mammals although no mammalian quinoprotein has yet been conclusively identified. Here we found that PQQ has substantial effects on the redox active mammalian selenoprotein thioredoxin reductase 1 (TrxR1). PQQ efficiently inhibited the activity of TrxR1 with its main native substrate thioredoxin and acted as a low efficiency substrate in a Sec-dependent TrxR1-catalyzed reduction. Interestingly, PQQ also stimulated redox cycling of TrxR1 with another quinone substrate, juglone, as much as 13-fold (k(cat)/K(m) increased from 105 min(-1) μM(-1) to 1331 min(-1) μM(-1) for juglone in the presence of 50 μM PQQ, mainly through a lowered apparent K(m) for juglone). Glutathione reductase was also inhibited by PQQ but in contrast to the effects of PQQ on TrxR1, its quinone reduction was not further stimulated. These results reveal that glutathione reductase and the mammalian selenoprotein TrxR1 are direct PQQ protein targets, although not being genuine quinoproteins. These findings may help explain several of the effects of PQQ seen in mammals.     

Effects of selenium deficiency on expression of selenoproteins in bovine arterial endothelial cells.

Damage to the vascular endothelium by reactive oxygen species causes many cardiovascular diseases including atherosclerosis. Such damage can be prevented by selenium (Se), which is thought to exert its actions mainly through the expression of selenoproteins. Se deficiency increased the susceptibility to tert-butylhydroperoxide (t-BuOOH) and enhanced lipid peroxidation in bovine arterial endothelial cells (BAEC). We investigated the effects of Se deficiency on the expression of the selenoproteins in BAEC. 75Se metabolic labeling analysis and RT-PCR analysis revealed that BAEC expressed two glutathione peroxidase (GPx) isozymes, cytosolic GPx (cGPx) and phospholipid hydroperoxide GPx (PHGPx), three thioredoxin reductase (TrxR) isozymes, TrxR1, TrxR2 and TrxR3, and selenoprotein P (SelP). Se deficiency reduced both enzyme activity and mRNA level of cGPx, but did not affect those of PHGPx. SelP mRNA level was also reduced by Se deficiency, although the extent of reduction was much smaller than that of cGPx mRNA. We further found that TrxR activity was also decreased by Se deficiency but none of the mRNA levels of TrxR isozymes were reduced. These results indicate that vascular endothelial cells express several selenoproteins including cGPx, PHGPx, TrxR isozymes and SelP which might play important roles in the defense system against oxidative stresses and that the expressions of these selenoproteins are differently regulated by Se status.     

Review: Exploring anticarcinogenic agents in a rat hepatocarcinogenesis model--focus on selenium and statins

In this review, we describe a rat model for chemically induced hepatocarcinogenesis that can be used for studying the anticarcinogenic effects of different agents. In this model the process of carcinogenesis can be followed through the different stages of initiation, promotion and progression. Mechanistic studies of anticarcinogenic agents can be carried out and two examples are given by studies on selenium and statins as anticarcinogenic agents. These compounds suppress cancer via different mechanisms. In the case of selenium the induction of glutathione peroxidase 4 and inhibition of lipid peroxidation might be a part of the anticarcinogenic effect. In the case of statins, the inhibition of ubiquinone synthesis, as well as of the selenium-containing enzyme thioredoxin reductase 1 (TrxR1) might explain their anticarcinogenic properties. Interestingly, also in the case of selenium the inhibited carcinogenesis was associated with reduced TrxR activity, indicating an important role for this enzyme in carcinogenesis.     

Inhibition of thioredoxin reductase by auranofin induces apoptosis in adriamycin-resistant human K562 chronic myeloid leukemia cells

Mammalian thioredoxin reductase (TrxR) catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx) and plays a central role in regulating cellular redox homeostasis, cell growth and apoptosis. Increasing evidence shows that TrxR is over-expressed or constitutively active in many tumor cells. Moreover, TrxR appears to contribute to increased tumor cell growth and a resistance to chemotherapy. In this study, we evaluated the activity of TrxR in adriamycin-resistant leukemic cells (K562/ADM) and adriamycin-sensitive parental lines (K562), and found that TrxR activity was higher in the drug resistant cell sublines K562/ADM than in K562 drug sensitive parental cells. Auranofin, a gold(I) compound clinically used as an antirheumatic agent, reduced TrxR activity and was more effective than adriamycin in decreasing cell viability in K562/ADM cells. In addition, auranofin induced apoptosis in dose-dependent manners, accompanied by caspase-3 activation in K562/ADM cells. Our results demonstrate that inhibition of TrxR and induction of apoptosis by auranofin provides its ability in overcoming adriamycin resistance in K562/ADM cells.     

Gold(I) carbene complexes causing thioredoxin 1 and thioredoxin 2 oxidation as potential anticancer agents

Gold(I) complexes with 1,3-substituted imidazole-2-ylidene and benzimidazole-2-ylidene ligands of the type NHC-Au-L (NHC = N-heterocyclic carbene L = Cl or 2-mercapto-pyrimidine) have been synthesized and structurally characterized. The compounds were evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), as well in the nontumorigenic human embryonic kidney cell line (HEK-293T), showing in some cases important cytotoxic effects. Some of the complexes were comparatively tested as thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibitors, directly against the purified proteins or in cell extracts. The compounds showed potent and selective TrxR inhibition properties in particular in cancer cell lines. Remarkably, the most effective TrxR inhibitors induced extensive oxidation of thioredoxins (Trxs), which was more relevant in the cancerous cells than in HEK-293T cells. Additional biochemical assays on glutathione systems and reactive oxygen species formation evidenced important differences with respect to the classical cytotoxic Au(I)-phosphine compound auranofin.     

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.     

Evidences for a role of glutathione peroxidase 4 (GPx4) in methylmercury induced neurotoxicity in vivo

We evaluated the activity and expression of antioxidant enzymes in the cerebellum and cortex of Swiss adult male mice exposed to methylmercury (MeHg) in drinking water (40mg/L) during 21 days. The activity of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were determined spectrophotometrically. The expression (protein levels) of GPx1 and GPx4 isoforms, TrxR1 as well as heat shock protein 70 (HSP70) were evaluated using specific antibodies and normalized by actin levels. The exposure of mice to MeHg caused a significant impairment in locomotors performance in the open field test (crossings and rearing). This result was followed by a significant reduction of GPx and TrxR activities in the cerebellum and cortex when compared to untreated animals. We also observed a substantial decrease in GPx1, GPx4 and TrxR1 protein levels in the cerebellum, while in the cerebral cortex, only GPx4 and TrxR1 were decreased after MeHg treatment. The activities of the antioxidant enzymes GR, GST, CAT and SOD were increased in the cerebellum after MeHg administration to mice. In contrast, only CAT was increased in the cerebral cortex of MeHg-treated animals. The expression of HSP70 was up-regulated only in the cerebellum where MeHg-exposed mice showed a significant increase in the immunocontent of HSP70 when compared to controls. This is the first report showing a role for GPx4 in the neurotoxicity induced by MeHg in vivo. In addition, our data indicates that the selenoproteins GPx and TrxR as main targets during MeHg exposure, which may be considered in biomarker studies.     

Paraquat-induced oxidative stress and dysfunction of cellular redox systems including antioxidative defense enzymes glutathione peroxidase and thioredoxin reductase.

We examined if paraquat-induced oxidative stress and cytotoxicity in pulmonary microvascular endothelial cells are associated with cellular redox systems such as the glutathione system and the thioredoxin system. Loss of viability, accompanied by marked decreases in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thioredoxin reductase activities, occurred 48 h after exposure to 1mM paraquat. These changes were preceded by an increased production of hydrogen peroxide after the decrease in glutathione peroxidase activity. Glutaredoxin activity was not decreased even after exposure to paraquat for 48 h, whereas thioredoxin activity was slightly decreased at 48 h. Unexpectedly, the activity of peroxiredoxin, a non-selenoenzyme, was almost completely lost at 24h. Loss of GAPDH activity and viability was notably aggravated by mercaptosuccinate. Selenium supplementation suppressed the loss of activities of glutathione peroxidase and thioredoxin reductase and alleviated paraquat-induced cytotoxicity. An in vitro experiment demonstrated that GAPDH was highly susceptible to reactive oxygen species generated in the xanthine-xanthine oxidase system, whereas thioredoxin reductase was considerably resistant. Taken together, the results suggest that the reduced regenerative ability of oxidatively damaged proteins including GAPDH due to the inactivation of thioredoxin reductase and glutathione peroxidase by paraquat may contribute to increasing oxidative stress, leading to cell death.     

Further insight into the impact of sodium selenite on selenoenzymes: high-dose selenite enhances hepatic thioredoxin reductase 1 activity as a consequence of liver injury

Selenium (Se) at supranutritional levels can enhance the activity of glutathione S-transferase (GST), whose gene is a target of nuclear factor erythroid-2 related factor 2 (Nrf2). Recent studies indicated that the thioredoxin reductase 1 (TrxR1) gene could also be targeted by Nrf2. Thus, high-dose Se may stimulate TrxR1 provided it enhances GST activity. Indeed, one study found that Se at supranutritional levels transiently increased hepatic TrxR1 activity. However, another study reported that supranutritional Se had no such effect on hepatic TrxR1 activity. In view of this discrepancy, the present research investigated whether high-dose Se has any impact on hepatic TrxR1. Moreover, we investigated whether Se preferentially activates GST over TrxR1. We observed that when sodium selenite (SS) caused liver injury, both hepatic TrxR1 activity and hepatic GST activity increased. Further experiments indicated that SS increased hepatic GST activity at either toxic or high but non-toxic dose levels; however, increase in hepatic TrxR1 activity occurred only at toxic levels, suggesting that enhanced TrxR1 activity correlates with liver injury. To corroborate this, we showed that hepatotoxic agents, thioacetamide or carbon tetrachloride, caused marked increases in hepatic TrxR1 activity. In conclusion, high-dose SS indeed can enhance hepatic TrxR1 activity, but only on the condition that it causes liver injury. High-dose SS affects hepatic GST more readily than hepatic TrxR1. Thus, the cancer-preventive mechanism of Se at non-toxic supranutritional levels relies more on its modulation of GST rather than TrxR1, at least in liver tissue.     

Inhibition of the thioredoxin system in the brain and liver of zebra-seabreams exposed to waterborne methylmercury

Mercury compounds were recently found to interact in vitro with the thioredoxin system, inhibiting both Thioredoxin (Trx) and Thioredoxin reductase (TrxR). In order to evaluate if Trx and TrxR are affected in vivo by methylmercury (MeHg), we exposed juvenile zebra-seabreams to different concentrations of this toxicant in water for 28 days followed by a 14-day depuration period. Methylmercury accumulated to a larger extent in the kidney and liver of fishes, but decreased significantly during the depuration. During the exposure, MeHg percentage in the liver reached levels above 90% of total mercury (HgT) decreasing to 60% of HgT by the end of the depuration period. In the kidney, MeHg accounted for 50-70% of HgT. In the brain and muscle, mercury accumulated throughout the exposure with all mercury being MeHg. The total mercury kept increasing in these organs during the depuration period. However, in the brain, this increase in HgT was accompanied by a decrease in the MeHg percentage (~ 10%). In the liver, both Trx and TrxR activities were significantly reduced (TrxR - 40%; Trx - 70%) by the end of the exposure, but recovered to control levels (100%) during the depuration. In the brain, both enzymes where inhibited during the depuration period (TrxR - 75%; Trx - 70%) when some production of inorganic mercury was detected. Activity of glutathione reductase showed increased levels when TrxR activity was low, suggesting complementarity between both systems. These results indicate that in vivo the thioredoxin system is a toxicological target for MeHg with TrxR being particularly affected.