Major Redox Systems Research Articles

Differences in Redox Regulatory Systems in Human Lung and Liver Tumors Suggest Different Avenues for Therapy.

A common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated dependence on these systems in cancers suggests that targeting these systems may provide an avenue for retarding the malignancy process. Herein, we examined the redox regulatory systems in human liver and lung cancers by comparing human lung adenocarcinoma and liver carcinoma to their respective surrounding normal tissues. Significant differences were found in the two major redox systems, the thioredoxin and glutathione systems. Thioredoxin reductase 1 levels were elevated in both malignancies, but thioredoxin was highly upregulated in lung tumor and only slightly upregulated in liver tumor, while peroxiredoxin 1 was highly elevated in lung tumor, but downregulated in liver tumor. There were also major differences within the glutathione system between the malignancies and their normal tissues. The data suggest a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system.

Thioredoxin-dependent regulation of AIF-mediated DNA damage

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.

Large-Scale Bleaching of Red Beds Related To Upward Migration of Hydrocarbons: Los Chihuidos High, Neuquen Basin, Argentina

Abstract The striking arrangement of colorful facies in the sandstones outcrops of the Los Chihuidos high, Argentina, has been attributed to the surface expression of hydrocarbon-induced alteration. The Huincul Formation (late Cenomanian–early Turonian) is part of the thick continental red-bed-dominated Neuquen Group, which consists of meandering fluvial deposits developed in a foreland basin. Visual distinctions reveal four diagenetic facies: red, white, gray, and minor brown sandstones. Differences in the diagenetic mineralogy among sandstones facies are interpreted to depend on the degree of interaction with hydrocarbon-bearing solutions and the relative position of the redox roll front developed during this process. The red facies represents the original oxidized sandstone, with hematite, kaolinite, quartz and albite overgrowths, and calcite cement precipitated under oxidizing conditions during the regional burial diagenesis. The white facies formed during the interaction of red beds with reduced fluids (in presence of hydrocarbons or organic acids), where clasts and cement were partially dissolved, and iron was reduced to Fe2+ due to redox reactions and removed, resulting in the sandstone decoloration and in a marked development of the secondary porosity. Minor montmorillonite replaced kaolinite and detrital feldspars, and a poikilotopic calcite precipitated, filling the pores. The gray and the brown sandstone bound the redox front, which is characterized by the presence of abundant clay minerals and oxide minerals enriched in vanadium and copper. Montmorillonite and secondary hematite are dominant in the more oxidized gray sandstones whereas interstratified chlorite–montmorillonite is more abundant in the more reduced brown sandstones. The coexistence of these minerals indicates metastable reducing–oxidizing conditions at the redox front. Based on diagenetic characteristics, tectonic framework, maturation timing of source rocks, and petroleum-emplacement history, hydrocarbons could have migrated into the Huincul Formation during the Tertiary uplift, but later erosion could have released the hydrocarbons and exhumed the reservoir. This multi-scale investigation of diagenetic features related to burial history and to structure-controlled upflow of hydrocarbons in a succession of siliciclastic rocks enhances modeling of a regional-scale redox system worldwide. Our study offers new insight into the application of authigenic minerals to trace hydrocarbon pathways along red beds and to evaluate the reservoir quality, in the understanding the importance of fluid compositions, mixing, and fluid–rock interaction along a major redox system.

S-D-Lactoylglutathione can be an alternative supply of mitochondrial glutathione

The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, in defending mitochondrial DNA integrity and protecting mitochondrial-derived ROS, and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. To demonstrate SLG transport from cytosol to mitochondria we used radiolabeled compounds and the results showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. Also, the incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria, demonstrating transport not linked to ATP presence. As well mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data were confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II in inter-membrane space and mitochondrial matrix and we investigated the role of SLG in whole cells. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.

Measurement of ascorbic acid concentration and glutathione peroxidase activity in biological samples collected from horses with recurrent airway obstruction

To measure the ascorbic acid (AA) concentration in bronchoalveolar lavage fluid (BALF) and cellular glutathione peroxidase (cGPx) activity in RBCs and WBCs from peripherally obtained blood and in cells from BALF to determine whether differences existed between the 2 major redox systems in recurrent airway obstruction (RAO)-affected and -nonaffected (control) horses and between systemic and local pulmonary responses in the glutathione redox system. 16 adult horses in pairs: 8 healthy (control) and 8 RAO-affected horses. Physical examination data and biological samples were collected from horses before (remission), during, and after (recovery) environmental challenge with dusty straw and hay. At each stage, BALF cell AA concentration and RBC, WBC, and BALF cell cGPx activity were measured. Compared with control horses, RAO-affected horses had significantly higher cGPx activity in RBCs at all points and in WBCs during remission and challenge. The BALF cell cGPx activity was higher in RAO-affected horses during recovery than during remission The BALF cell AA concentration did not differ significantly in control horses at any point, but total and free AA concentrations were significantly lower in RAO-affected horses during the challenge period than during remission and recovery periods. High cGPx activity suggested this redox system was upregulated during exposure to dusty straw and hay to combat oxidative stress, as AA was depleted in RAO-affected horses. The relative delay and lack of comparative increase in cGPx activity within the local environment (represented by BALF cells), compared with that in RBCs and WBCs, might contribute to disease in RAO-affected horses.

Dietary Sulfur Amino Acid Supplementation Reduces Small Bowel Thiol/Disulfide Redox State and Stimulates Ileal Mucosal Growth after Massive Small Bowel Resection in Rats

Following massive small bowel resection in animal models, the remnant intestine undergoes a dynamic growth response termed intestinal adaptation. Cell growth and proliferation are intimately linked to cellular and extracellular thiol/disulfide redox states, as determined by glutathione (GSH) and GSH disulfide (GSSG) (the major cellular redox system in tissues), and cysteine (Cys) and its disulfide cystine (CySS) (the major redox system in plasma), respectively. The study was designed to determine whether dietary supplementation with sulfur amino acids (SAA) leads to a greater reduction in thiol/disulfide redox state in plasma and small bowel and colonic mucosa and alters gut mucosal growth in an established rat model of short bowel syndrome (SBS). Adult rats underwent 80% jejunal-ileal resection (RX) or small bowel transection (surgical control) and were pair-fed either isonitrogenous, isocaloric SAA-adequate (control) or SAA-supplemented diets (218% increase vs. control diet). Plasma and gut mucosal samples were obtained after 7 d and analyzed for Cys, CySS, GSH, and GSSG concentrations by HPLC. Redox status (Eh) of the Cys/CySS and GSH/GSSG couples were calculated using the Nernst equation. SAA supplementation led to a greater reduction in Eh GSH/GSSG in jejunal and ileal mucosa of resected rats compared with controls. Resected SAA-supplemented rats showed increased ileal adaptation (increased full-thickness wet weight, DNA, and protein content compared with RX control-fed rats; increased mucosal crypt depth and villus height compared with all other study groups). These data suggest that SAA supplementation has a trophic effect on ileal adaptation after massive small bowel resection in rats. This finding may have translational relevance as a therapeutic strategy in human SBS.

Simultaneous expression of glutathione, thioredoxin-1, and their reductases in nerve transected hypoglossal motor neurons of rat

Anti-oxidative stress responses are crucial for the survival of nerve-injured motor neurons. Herein, we examined changes in expression of glutathione reductase (GSHr), thioredoxins (TRX1 and TRX2), and thioredoxin reductases (TRXr1 and TRXr2), important constituents of anti-oxidative pathways, following rat hypoglossal nerve transection. RT-PCR and in situ hybridization demonstrated that GSHr, TRX1, and TRXr1 mRNAs were significantly up-regulated during the first few weeks in nerve-injured motor neurons, while TRX2 and TRXr2 mRNAs were unchanged throughout 8 weeks after nerve transection. The up-regulation of GSH, GSHr, TRX1, and TRXr1 proteins in injured neurons was confirmed by immunohistochemical analysis. Western blotting also demonstrated up-regulation of GSHr, TRX1, and TRXr1 in injured neurons. These data suggest that the two major redox systems, GSH/GSHr and TRX1/TRXr1, are simultaneously activated in injured neurons, and likely provide protection of injured neurons against oxidative stress.

Alternative splicing involving the thioredoxin reductase module in mammals: a glutaredoxin-containing thioredoxin reductase 1.

Thioredoxin reductase 1 (TR1) is a key component in the thioredoxin system, one of major redox systems in mammals that links NADPH and thiol-dependent processes. Mammalian TR1 genes are known to be regulated by alternative splicing. In this report, comparative genomic analyses were used to identify and characterize species-specific and common alternative forms of mammalian TR1 genes. Six human TR1 isoforms were identified that were derived from a large number of transcripts and differed in their N-terminal sequences. One isoform resulted from exons located 30-70 kb upstream of the previously identified core TR1 promoter and was composed of a basic TR1 module fused to a glutaredoxin (Grx) domain that contained an unusual active site CTRC sequence. This TR1 form occurred in humans, dogs, and chimpanzees but was inactivated in mice and rats. The CTRC motif in the human enzyme made the N-terminal domain inactive in the Grx assays tested. However, when mutated to CPYC, an active site present in most Grxs, the Grx domain was active. In addition, the presence of the Grx domain interfered with the TR1 activity, distinguishing this enzyme from other proteins with Grx and TR fusions. The data suggest that the fusion of the basic TR1 module and variable N-terminal sequences links the pyridine nucleotide thiol/disulfide oxidoreductase pathway to specific cellular redox functions and may control spatial and temporal expression of TR1 transcripts. Our data also suggest that various N-terminal extensions in mammalian TRs are often expressed in testes.

Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems.

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.