Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

Does Risk of Hyperhomocysteinemia Depend on Thiol-Disulfide Exchange Reactions of Albumin and Homocysteine?

Significance: Increased plasma concentrations of total homocysteine (tHcy; mild-moderate hyperhomocysteinemia: 15-50 µM tHcy) are considered an independent risk factor for the onset/progression of various diseases, but it is not known about how the increase in tHcy causes pathological conditions. Recent Advances: Reduced homocysteine (HSH ∼1% of tHcy) is presumed to be toxic, unlike homocystine (∼9%) and mixed disulfide between homocysteine and albumin (HSS-ALB; homocysteine [Hcy]-albumin mixed disulfide, ∼90%). This and other notions make it difficult to explain the pathogenicity of Hcy because: (i) lowering tHcy does not improve pathological outcomes; (ii) damage due to HSH usually emerges at supraphysiological doses; and (iii) it is not known why tiny increments in plasma concentrations of HSH can be pathological. Critical Issues: Albumin may have a role in Hcy toxicity, because HSS-ALB could release toxic HSH via thiol-disulfide (SH/SS) exchange reactions in cells. Similarly, thiol-disulfide exchange processes of reduced albumin (albumin with free SH group of Cys34 [HS-ALB]) or N-homocysteinylated albumin are plausible alternatives for initiating Hcy pathological events. Adverse effects of albumin and other data reviewed here suggest the hypothesis of a role of albumin in Hcy toxicity. Future Directions: HSS-ALB might be involved in disruption of the antioxidant/oxidant balance in critical tissues (brain, liver, kidney). Since homocysteine-albumin mixed disulfide is a possible intermediate of thiol-disulfide exchange reactions, we suggest that homocysteinylated albumin could be a new pathological factor, and that studies on the redox role of albumin and mixed disulfide production via thiol-disulfide exchange reactions could offer new therapeutic insights for reducing Hcy toxicity.

N-Acetylcysteine or Sodium Selenite Prevent the p38-Mediated Production of Proinflammatory Cytokines by Microglia during Exposure to Mercury (II).

Mercury (Hg) is known for its neurotoxicity and is reported to activate microglia cells at low exposure levels. Since mercury decreases the activity of the glutathione and thioredoxin systems, we hypothesize that Hg would, in turn, disrupt microglia homeostasis by interfering with redox regulation of signaling pathways. Thus, in this work, we analyzed the effect of exposure to Hg2+ on nuclear translocation and activation of NF-kB (p50) and p38 and pro-inflammatory gene transcription (IL-1ß; iNOS, TNF-alpha) considering the interaction of Hg with the glutathione system and thioredoxin systems in microglial cells. N9 (mouse) microglia cells were exposed to different concentrations of Hg2+ and the 24 h EC50 for a reduction in viability was 42.1 ± 3.7 µM. Subsequent experiments showed that at sub-cytotoxic levels of Hg2+, there was a general increase in ROS (≈40%) accompanied by a significant depletion (60-90%) of glutathione (GSH) and thioredoxin reductase (TrxR) activity. Upon 6 h of exposure to Hg2+, p38 (but not p50) accumulated in the nucleus (50% higher than in control), which was accompanied by an increase in its phosphorylation. Transcript levels of both IL1-ß and iNOS were increased over two-fold relative to the control. Furthermore, pre-exposure of cells to the p38 inhibitor SB 239063 hindered the activation of cytokine transcription by Hg2+. These results show that disruption of redox systems by Hg2+ prompts the activation of p38 leading to transcription of pro-inflammatory genes in microglia cells. Treatment of N9 cells with NAC or sodium selenite-which caused an increase in basal GSH and TrxR levels, respectively, prevented the activation of p38 and the transcription of pro-inflammatory cytokines. This result demonstrates the importance of an adequate nutritional status to minimize the toxicity resulting from Hg exposure in human populations at risk.

The optimized quantum dot mediated thermometry reveals isoform specific differences in efficiency of myosin extracted from muscle mini bundles.

The biophysical function of myosin in vitro has been extensively investigated in different motility assays, but the study of myosin ATPase properties at the fiber level is insufficiently investigated. In this study, quantum dot (QD) mediated thermometry measurements were optimized to measure the efficiency of myosin extracted from muscle mini bundles. A reduction in fluorescent intensity of QD reflects an increase in temperature caused by the heat released during ATP hydrolysis and denotes the efficiency of the motor protein myosin. The procedure for extracting myosin was similar to the single fiber in vitro motility assay with some small modifications, and the concentration of myosin was represented by the extracted total protein since the ratio of extracted myosin to total protein was constant. Moreover, the efficiencies of myosin extracted from preparations containing different myosin heavy chain isoforms reveal lower efficiency of slow compared to fast myosin isoforms. Specifically, more heat was released in slow myosin enzymatic reaction, resulting in faster decay of QD fluorescence intensity. Hence, the optimized QD mediated thermometry provides a novel and sensitive approach to evaluate efficiency of myosin ATPase obtained from small muscle samples, representing a significant advantage in the clinical evaluation of neuromuscular disorders.

Mitochondrial depletion of glutaredoxin 2 induces metabolic dysfunction-associated fatty liver disease in mice.

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that facilitates glutathionylation/de-glutathionylation of target proteins. The main variants of Grx2 are the mitochondrial Grx2a and the cytosolic Grx2c. The aim of this study was to investigate the specific role of mitochondrial Grx2 in vivo using a mitochondrial Grx2 depleted (mGD) mouse model. mGD mice displayed an altered mitochondrial morphology and functioning. Furthermore, the lack of Grx2 in the mitochondrial compartment is responsible for increased blood lipid levels under a normal diet, a metabolic dysfunction-associated fatty liver disease (MAFLD) phenotype and a decreased glycogen storage capacity. In addition, depleting Grx2a leads to an alteration in abundance and in glutathionylation pattern of different mitochondrial enzymes, highlighting the selective role of Grx2 in the regulation of metabolic pathways. Overall, our findings identify the involvement of mitochondrial Grx2a in the regulation of cell metabolism and highlight a previously unknown association between Grx2 and MAFLD.

A substitution in the glutathione reductase lowers electron leakage and inflammation in modern humans.

Glutathione reductase is a critical enzyme for preventing oxidative stress and maintaining a reduced intracellular environment. Almost all present-day humans carry an amino acid substitution (S232G) in this enzyme relative to apes and Neanderthals. We express the modern human and the ancestral enzymes and show that whereas the activity and stability are unaffected by the amino acid substitution, the ancestral enzyme produces more reactive oxygen species and increases cellular levels of transcripts encoding cytokines. We furthermore show that the ancestral enzyme has been reintroduced into the modern human gene pool by gene flow from Neanderthals and is associated with multiple traits in present-day people, including increased susceptibility for inflammatory-associated disorders and vascular disease.

Thiol-Based Antioxidants and the Epithelial/Mesenchymal Transition in Cancer.

Significance: The epithelial/mesenchymal transition (EMT) is commonly associated with tumor metastasis. Oxidative and nitrosative stress is maintained in cancer cells and is involved in the EMT. Cancer cells are endowed with high levels of enzymatic and nonenzymatic antioxidants, which counteract the effects of oxidative and nitrosative stress. Thiol-based antioxidant systems such as the thioredoxin/thioredoxin reductase (Trx/TrxR) and glutathione/glutaredoxin (GSH/Grx) are continually active in cancer cells, while the thioredoxin-interacting protein (Txnip), the negative regulator of the Trx/TrxR system, is downregulated. Recent Advances: Trx/TrxR and GSH/Grx systems play a major role in maintaining EMT signaling and cancer cell progression. Critical Issues: Enhanced stress conditions stimulated in cancer cells inhibit EMT signaling. The elevated expression levels of the Trx/TrxR and GSH/Grx systems in these cells provide the antioxidant protection necessary to guarantee the occurrence of the EMT. Future Directions: Elevation of the intracellular reactive oxygen species and nitric oxide concentrations in cancer cells has been viewed as a promising strategy for elimination of these cells. The development of inhibitors of GSH synthesis and of the Trx/TrxR system together with genetic-based strategies to enhance Txnip levels may provide the necessary means to achieve this goal. Antioxid. Redox Signal. 36, 1037-1050.

Glutaredoxin: Discovery, redox defense and much more.

Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.

Supplemental Ascorbate Diminishes DNA Damage Yet Depletes Glutathione and Increases Acute Liver Failure in a Mouse Model of Hepatic Antioxidant System Disruption.

Cellular oxidants are primarily managed by the thioredoxin reductase-1 (TrxR1)- and glutathione reductase (Gsr)-driven antioxidant systems. In mice having hepatocyte-specific co-disruption of TrxR1 and Gsr (TrxR1/Gsr-null livers), methionine catabolism sustains hepatic levels of reduced glutathione (GSH). Although most mice with TrxR1/Gsr-null livers exhibit long-term survival, ~25% die from spontaneous liver failure between 4- and 7-weeks of age. Here we tested whether liver failure was ameliorated by ascorbate supplementation. Following ascorbate, dehydroascorbate, or mock treatment, we assessed survival, liver histology, or hepatic redox markers including GSH and GSSG, redox enzyme activities, and oxidative damage markers. Unexpectedly, rather than providing protection, ascorbate (5 mg/mL, drinking water) increased the death-rate to 43%. In adults, ascorbate (4 mg/g × 3 days i.p.) caused hepatocyte necrosis and loss of hepatic GSH in TrxR1/Gsr-null livers but not in wildtype controls. Dehydroascorbate (0.3 mg/g i.p.) also depleted hepatic GSH in TrxR1/Gsr-null livers, whereas GSH levels were not significantly affected by either treatment in wildtype livers. Curiously, however, despite depleting GSH, ascorbate treatment diminished basal DNA damage and oxidative stress markers in TrxR1/Gsr-null livers. This suggests that, although ascorbate supplementation can prevent oxidative damage, it also can deplete GSH and compromise already stressed livers.

Inhibition of Thioredoxin Reductase by Triosmium Carbonyl Clusters.

Tumor cells are characterized by increased reactive oxygen species production in parallel with an enhanced antioxidant system to avoid oxidative damage. The inhibition of antioxidant systems is an effective way to kill cancer cells, and the thioredoxin system or, more specifically, the cytosolic selenocysteine-containing enzyme thioredoxin reductase (TrxR) has become an interesting target for cancer therapy. We show here that the known cytotoxic and apoptosis-inducing osmium carbonyl cluster Os3(CO)10(NCCH3)2 (1) is a nonsubstrate inhibitor of mammalian TrxR, with an IC50 of 5.3 ± 0.9 µM. It inhibits TrxR selectively over the closely related glutathione reductase (GR) and in the presence of excess reduced glutathione (GSH). This inhibition has also been demonstrated in cell lysates, suggesting that TrxR inhibition is a potential apoptotic pathway for 1.

Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2-R1-dependent ribonucleotide reductase.

Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport. After each catalytic cycle the active site contains a disulfide, which has to be reduced for turnover. Thioredoxin (Trx) and glutaredoxin (Grx) systems have been implicated as electron donors for the RNR disulfide reduction via the swinging arm. Using mouse R1-R2 and R1-p53R2 complexes, we found here that the catalytic efficiency of the GSH-Grx system is 4-6 times higher than that of the Trx1 system. For both complexes, the Vmax values for Grx are strongly depended on GSH concentrations. The GSH disulfide resulting from the Grx reaction was reduced by NADPH and GSH reductase and this enzyme was essential because reaction with GSH alone yielded only little activity. These results indicate that C-terminal shuttle dithiols of mammalian R1 have a crucial catalytic role and that the GSH-Grx system favors the R1-p53R2 enzyme for DNA replication in hypoxic conditions, mitochondrial DNA synthesis, and in DNA repair outside the S-phase.

The combination of ascorbate and menadione causes cancer cell death by oxidative stress and replicative stress.

The combination of ascorbate and menadione (VC:VK3 = 100:1) is an investigational treatment for cancer under clinical trials. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, can be taken up by cells via glucose transporters, over-expressed in many cancer cells. It has been known that the combination of VC/VK3 kills cancer cells by inducing hydrogen peroxide (H2O2) via a redox cycling reaction. However, the mechanism has not been fully understood yet. Intracellularly, DHA is reduced to ascorbate by NADPH via GSH and glutaredoxin as well as by thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR). These two systems are also critical as electron donors for ribonucleotide reductase (RNR), which produces deoxyribonucleotides de novo for DNA replication and DNA repair and is highly expressed in tumor cells. We found that RNR was highly sensitive to VC/VK3 in vitro with similar effects as observed with H2O2. In cancer cells, VC/VK3 inhibited RNR mainly by targeting its R2 subunit. More importantly, both the Trx and GSH systems were oxidized by the combination, which resulted in the loss of GSH, increased protein glutathionylation, and highly oxidized Trx1. The mechanism of cell death induced by VC/VK3 was also elucidated. We found that VC/VK3 inhibited glutathione peroxidase activity and led to an elevated level of lipid peroxidation, which triggered apoptosis-inducing factor (AIF) mediated cell death pathway. Therefore, the combination not only induced replicative stress by inhibiting RNR, but also oxidative stress by targeting anti-oxidant systems and triggered AIF-mediated cancer cell death.

Enzymatic glutaredoxin-dependent method to determine glutathione and protein S-glutathionylation using fluorescent eosin-glutathione.

Glutathione is an abundant low-molecular-weight thiol, up to 10 mM in mammalian cells, and exists in three major forms: reduced sulphydryl (GSH), glutathione disulfide (GSSG) or bound to Cys residues in proteins (PSSG). The ratio GSH/GSSG has been used as an indicator of the cells redox level but this parameter can also be estimated by the quantification of PSSG. In fact, PSSGs have the advantage of being more stable than GSSG. Here we present a highly sensitive fluorescent-based method for detection of low concentrations of glutathione in complex samples such as cell lysates, tissues and plasma. The method is based on our previously described protocol to study Glutaredoxin (Grx) activity. The whole procedure was optimized to measure the fluorescence increase of the di-eosin-glutathione disulfide (Di-E-GSSG) reduced by Grx in the presence of Glutathione Reductase and NADPH, keeping GSH as the limiting factor to drive the reaction. The methods to selectively measure PSSG are expensive and not widely accessible, therefore we optimized our glutaredoxin protocol to quantify this post-translational modification using common laboratory equipments. Overall, our method has simplicity and rapidity combined with high sensitivity as its main advantages; therefore, it may be particularly suitable for large-scale clinical studies.

Inhibition of the glutaredoxin and thioredoxin systems and ribonucleotide reductase by mutant p53-targeting compound APR-246.

The tumor suppressor p53 is commonly inactivated in human tumors, allowing evasion of p53-dependent apoptosis and tumor progression. The small molecule APR-246 (PRIMA-1Met) can reactive mutant p53 in tumor cells and trigger cell death by apoptosis. The thioredoxin (Trx) and glutaredoxin (Grx) systems are important as antioxidants for maintaining cellular redox balance and providing electrons for thiol-dependent reactions like those catalyzed by ribonucleotide reductase and peroxiredoxins (Prxs). We show here that the Michael acceptor methylene quinuclidinone (MQ), the active form of APR-246, is a potent direct inhibitor of Trx1 and Grx1 by reacting with sulfhydryl groups in the enzymes. The inhibition of Trx1 and Grx1 by APR-246/MQ is reversible and the inhibitory efficiency is dependent on the presence of glutathione. APR-246/MQ also inhibits Trxs in mutant p53-expressing Saos-2 His-273 cells, showing modification of Trx1 and mitochondrial Trx2. Inhibition of the Trx and Grx systems leads to insufficient reducing power to deoxyribonucleotide production for DNA replication and repair and peroxiredoxin for removal of ROS. We also demonstrate that APR-246 and MQ inhibit ribonucleotide reductase (RNR) in vitro and in living cells. Our results suggest that APR-246 induces tumor cell death through both reactivations of mutant p53 and inhibition of cellular thiol-dependent redox systems, providing a novel strategy for cancer therapy.

Glutathione Fine-Tunes the Innate Immune Response toward Antiviral Pathways in a Macrophage Cell Line Independently of Its Antioxidant Properties.

Glutathione (GSH), a major cellular antioxidant, is considered an inhibitor of the inflammatory response involving reactive oxygen species (ROS). However, evidence is largely based on experiments with exogenously added antioxidants/reducing agents or pro-oxidants. We show that depleting macrophages of 99% of GSH does not exacerbate the inflammatory gene expression profile in the RAW264 macrophage cell line or increase expression of inflammatory cytokines in response to the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); only two small patterns of LPS-induced genes were sensitive to GSH depletion. One group, mapping to innate immunity and antiviral responses (Oas2, Oas3, Mx2, Irf7, Irf9, STAT1, il1b), required GSH for optimal induction. Consequently, GSH depletion prevented the LPS-induced activation of antiviral response and its inhibition of influenza virus infection. LPS induction of a second group of genes (Prdx1, Srxn1, Hmox1, GSH synthase, cysteine transporters), mapping to nrf2 and the oxidative stress response, was increased by GSH depletion. We conclude that the main function of endogenous GSH is not to limit inflammation but to fine-tune the innate immune response to infection.

Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.

Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5µM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells.

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase.

Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells.

Accumulating evidence suggests that natural bioactive compounds, alone or in combination with traditional chemotherapeutic agents, could be used as potential therapies to fight cancer. In this study, we employed four natural bioactive compounds (curcumin, resveratrol, melatonin, and silibinin) and studied their role in redox control and ability to promote apoptosis in androgen sensitive and insensitive prostate cancer cells. Here is shown that curcumin and resveratrol promote ROS production and induce apoptosis in LNCaP and PC-3. An increase in reactive species is a trigger event in curcumin-induced apoptosis and a consequence of resveratrol effects on other pathways within these cells. Moreover, here we demonstrated that these four compounds affect differently one of the main intracellular redox regulator, the thioredoxin system. Exposure to curcumin and resveratrol promoted TRX1 oxidation and altered its subcellular location. Furthermore, resveratrol diminished TRX1 levels in PC-3 cells and increased the expression of its inhibitor TXNIP. Conversly, melatonin and silibinin only worked as cytostatic agents, reducing ROS levels and showing preventive effects against TRX oxidation. All together, this work explores the effect of compounds currently tested as chemo-preventive agents in prostate cancer therapy, on the TRX1 redox state and function. Our work shows the importance that the TRX system might have within the differences found in their mechanisms of action. These bioactive compounds trigger different responses and affect ROS production and redox systems in prostate cancer cells, suggesting the key role that redox-related pathways might play in processes like differentiation or survival in prostate cancer.

Homocysteinemia control by cysteine in cerebral vascular patients after methionine loading test: evidences in physiological and pathological conditions in cerebro-vascular and multiple sclerosis patients.

The toxicity risk of hyperhomocysteinemia is prevented through thiol drug administration which reduces plasma total homocysteine (tHcy) concentrations by activating thiol exchange reactions. Assuming that cysteine (Cys) is a homocysteinemia regulator, the hypothesis was verified in healthy and pathological individuals after the methionine loading test (MLT). The plasma variations of redox species of Cys, Hcy, cysteinylglycine, glutathione and albumin (reduced, HS-ALB, and at mixed disulfide, XSS-ALB) were compared in patients with cerebral small vessels disease (CSVD) (n = 11), multiple sclerosis (MS) (n = 12) and healthy controls (n = 11) at 2-4-6 h after MLT. In MLT-treated subjects, the activation of thiol exchange reactions provoked significant changes over time in redox species concentrations of Cys, Hcy, and albumin. Significant differences between controls and pathological groups were also observed. In non-methionine-treated subjects, total Cys concentrations, tHcy and thiol-protein mixed disulfides (CSS-ALB, HSS-ALB) of CSVD patients were higher than controls. After MLT, all groups displayed significant cystine (CSSC) increases and CSS-ALB decreases, that in pathological groups were significantly higher than controls. These data would confirm the Cys regulatory role on the homocysteinemia; they also explain that the Cys-Hcy mixed disulfide excretion is an important point of hyperhomocysteinemia control. Moreover, in all groups after MLT, significant increases in albumin concentrations, named total albumin (tALB) and measured as sum of HS-ALB (spectrophometric), and XSS-ALB (assayed at HPLC) were observed. tALB increases, more pronounced in healthy than in the pathological subjects, could indicate alterations of albumin equilibria between plasma and other extracellular spaces, whose toxicological consequences deserve further studies.

Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier.

Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos(-/-) and wild-type mice. Inos(-/-) mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos(-/-) mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.