Redox Buffer System Research Articles

Measuring Glutathione Regeneration Capacity in Stem Cells.

Glutathione (GSH) is a chief cellular antioxidant, affecting stem cell functions. The cellular GSH level is dynamically altered by the redox buffering system and transcription factors, including NRF2. Additionally, GSH is differentially regulated in each organelle. We previously reported a protocol for monitoring the real-time GSH levels in live stem cells using the reversible GSH sensor FreSHtracer. However, GSH-based stem cell analysis needs be comprehensive and organelle-specific. Hence, in this study, we demonstrate a detailed protocol to measure the GSH regeneration capacity (GRC) in living stem cells by measuring the intensities of the FreSHtracer and the mitochondrial GSH sensor MitoFreSHtracer using a high-content screening confocal microscope. This protocol typically analyses the GRC in approximately 4 h following the seeding of the cells onto plates. This protocol is simple and quantitative. With some minor modifications, it can be employed flexibly to measure the GRC for the whole-cell area or just the mitochondria in all adherent mammalian stem cells.

Construction of Genetically Encoded Biosensors to Monitor Subcellular Compartment-Specific Glutathione Response to Chemotherapeutic Drugs in Acute Myeloid Leukemia Cells.

Glutathione (GSH), the constituent of the redox buffer system, is a scavenger of reactive oxygen species (ROS), and its ratio to oxidized glutathione (GSSG) is a key indicator of oxidative stress in the cell. Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy characterized by aberrant levels of reduced and oxidized GSH due to oxidative stress. Therefore, the real-time, dynamic, and highly sensitive detection of GSH/GSSG in AML cells is of great interest for the clinical diagnosis and treatment of leukemia. The application of genetically encoded sensors to monitor GSH/GSSG levels in AML cells is not explored, and the underlying mechanism of how the drugs affect GSH/GSSG dynamics remains unclear. In this study, we developed subcellular compartment-specific sensors to monitor GSH/GSSG combined with high-resolution fluorescence microscopy that provides insights into basal GSH/GSSG levels in the cytosol, mitochondria, nucleus, and endoplasmic reticulum of AML cells, in a decreasing order, revealing substantial heterogeneity of GSH/GSSG level dynamics in different subcellular compartments. Further, we investigated the response of GSH/GSSG ratio in AML cells caused by Prussian blue and Fe3O4 nanoparticles, separately and in combination with cytarabine, pointing to steep gradients. Moreover, cytarabine and doxorubicin downregulated the GSH/GSSG levels in different subcellular compartments. Similarly, live-cell imaging showed a compartment-specific decrease in response to various drugs, such as CB-839, parthenolide (PTL), and piperlongumine (PLM). The enzymatic activity assay revealed the mechanism underlying fluctuations in GSH/GSSG levels in different subcellular compartments mediated by these drugs in the GSH metabolic pathway, suggesting some potential therapeutic targets in AML cells.

The reversal of drug resistance by two-dimensional titanium carbide Ti2 C (2D Ti2C) in non-small-cell lung cancer via the depletion of intracellular antioxidant reserves.

BackgroundChemoresistance is a major barrier limiting the therapeutic efficacy of late stage non‐small cell lung cancer (NSCLC). In this study, we sought to use two‐dimensional titanium carbide (2D Ti2C) to reverse cisplatin resistance in NSCLC.MethodsWe first achieved favorable properties as a potential anti‐tumor agent. We then compared cell viability and cisplatin uptake in chemoresistant NSCLC cells before and after the use of 2D Ti2C. Afterwards, we explored the effects of 2D Ti2C on intracellular antioxidant reserves, followed by evaluating the subsequent changes in the expression of core drug resistance genes. Finally, we confirmed the tumor inhibitory effect and bio‐safety of 2D Ti2C in a drug‐resistant lung cancer model in nude mice.ResultsDue to the properties of thin layer, large specific surface area, and abundant reactive groups on the surface, 2D Ti2C can deplete the antioxidant reserve systems such as the glutathione redox buffer system, γ‐glutamylcysteine synthetase (γ‐GCS), glutathione peroxidase (GPx), glutathione‐S‐transferase‐Pi (GST‐π), and metallothionein (MT), thereby increasing the intracellular accumulation of cisplatin and decreasing the expression of drug resistance genes.Conclusions2D Ti2C can reverse NSCLC chemoresistance both in vitro and in vivo, suggesting that it may potentially become a novel and effective means to treat chemoresistant NSCLC in the clinic.

Multivariate Optimization of the Refolding Process of an Incorrectly Folded Fc-Fusion Protein in a Cell Culture Broth.

Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process. In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation. Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD). The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators. The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

Glutathione: A powerful but rare cofactor among Actinobacteria.

Glutathione (γ-l-glutamyl-l-cysteinylglycine, GSH) is a powerful cellular redox agent. In nature only the l,l-form is common among the tree of life. It serves as antioxidant or redox buffer system, protein regeneration and activation by interaction with thiol groups, unspecific reagent for conjugation during detoxification, marker for amino acid or peptide transport even through membranes, activation or solubilization of compounds during degradative pathways or just as redox shuttle. However, the role of GSH production and utilization in bacteria is more complex and especially little is known for the Actinobacteria. Some recent reports on GSH use in degradative pathways came across and this is described herein. GSH is used by transferases to activate and solubilize epoxides. It allows funneling epoxides as isoprene oxide or styrene oxide into central metabolism. Thus, the distribution of GSH synthesis, recycling and application among bacteria and especially Actinobacteria are highlighted including the pathways and contributing enzymes.

3,5-Bis(2-Trifluomethoxybenzylidene)-4-Piperidone Induces Apoptosis in A549 Cells Through Reactive Oxygen Species-Mediated Pathways

Medicines with the F3C-O group can increase the lipophilicity of an aromatic ring system and protect it from oxidative attacks. We have synthesized 3,5-bis(2-trifluomethoxybenzylidene)-4-piperidone (4a), an EF24 analog with ortho trifluoromethoxy group, via aldol condensation reaction and evaluated its cytotoxicity by MTT assay. Owning to the ortho-trifluoromethoxy group, compound 4a exhibited excellent cytotoxicity against A549 cells. Mechanistic investigation indicated that 4a could induce oxidative stress, including the generation of reactive oxygen species (ROS), imbalance of redox buffering system, lipid peroxidation, and collapse of mitochondrial membrane potential, and finally leading to cell apoptosis. These data indicated that the introduction of the ortho-trifluoromethoxy group into curcumin analogs may be an effective strategy to enhance the anti-cancer ROS based activity.

3,5-Bis(2-fluorobenzylidene)-4-piperidone induce reactive oxygen species-mediated apoptosis in A549 cells

The presence of the substituents in the ortho position of the aromatic ring(s) is helpful to strenghen the biological activity, highlighting a so-called ortho effect. In this paper, we synthesized six mono-carbonyl curcumin analogs with the fluorine group, which imparts a variety of properties to certain medicines. Then, the cytotoxicity against A549 and NCI-H460 cells was evaluated by the MTT assay. The results exhibited that 1d surfaced as an important lead compound displaying almost 13-fold cytotoxicity relative to curcumin against A549 cells. More importantly, 1d was more stable and more massive uptake than curcumin, which may be relationship to their cytotoxicity, apoptotic acitivity and reactive oxygen species generation. The generation of reactive oxygen species is associated with falling apart in the redox buffering system, and subsequently induces lipid peroxidation, collapse of the mitochondrial membrane potential and ultimately leads to apoptosis. These data indicated that ortho effect and leading fluorine into medicine molecular are successful strategy to improve anticancer activity of mono-carbonyl curcumin analogs.

Abstract 5210: Differential cytotoxic effects of Piperlongumine analogues and their radiotherapeutic response in breast cancer

Abstract Background: Breast cancer is the most common cancer in women in the United Kingdom. The glutathione (GSH) system is a major redox buffering system involved in regulating radioresponse of cancer cells. Overexpression has been shown to cause multidrug and radiation resistance. Its modulation may increase radiosensitivity and improve radiotherapy efficacy. Piperlongumine (PL) can reportedly inhibit GSH system function and reduce proliferation of a variety cancer cells in vitro. The current study investigates novel PL analogues to determine efficacy in triple-negative (TNBC) and luminal breast cancer cell lines, as both single agents and in combination with ionising radiation. Methods: Two novel PL analogues (LH91 and LH92) were used along with the parental drug, PL. Cell proliferation assays were conducted in MDA-MB-231 (TNBC) and T47D (luminal) lines at various drug concentrations and times. At 48hr post treatment, cells were also assessed for clonogenic survival. Clonogenic radiosensitisation was assessed by treating with PL agents or L-buthionine-S, R-sulfoximine (BSO, a well characterised GSH radiosensitiser) for 48hr then irradiation with 0-8Gy X-rays. ROS levels were evaluated using H2DCFDA flow cytometry following IC50 drug treatment alone or with subsequent exposure to 1mM H2O2 for 1hr. Results: PL, LH91 and LH92 inhibited proliferation and decreased clonogenic survival in both lines. From proliferation assays, MDA-MB-231 cells were more sensitive to PL at 48hr (P=0.047) and 72hr (P=0.01) with an IC50 dose of approx. 4μM versus approx. 12μM in T47D’s. There was a significant difference between IC50 doses of LH91 in MDA-MB-231 (5μM) and T47D (12μM) cell lines at 72hr (P=0.006), however this was not observed at 48hr treatment (P=0.178). In contrast, there was no differential sensitivity of LH92 in cell proliferation between the two cell lines at 48 and 72hr. Interestingly, in clonogenic assays, MDA-MB-231’s were more sensitive to LH92 (P=0.01) with an IC50 of 4μM versus 11μM in T47D’s. There were no significant differences in clonogenic survival between each line after treatment with PL or LH91. No radiosensitisation was observed with PL or PL analogues in MDA-MBA-231 however BSO, as a drug comparator, enhanced radiation response with a sensitizer enhancement ratio (SER) of 1.13 at 1% iso-survival. Radiation and drug combinations in T47D cells are ongoing. There were no changes in ROS levels with the drug alone in MDA-MB-231; however, with subsequent exposure of H2O2, there was a 1.7-fold increase in ROS level with LH91 at 48hr (P=0.002). Conclusion: The TNBC cell line is more sensitive to the cytotoxic effect of PL agents (PL and LH91 in cell proliferation and LH92 in clonogenic assays) in comparison to the luminal cell line; however no altered radiosensitisation was observed. The mechanism of such differential sensitivity is yet to be determined. Note: This abstract was not presented at the meeting. Citation Format: Nurul Akmaryanti Abdullah, John Moses, Li Chen Han, Sarah Storr, Aula Ammar, Stewart G. Martin. Differential cytotoxic effects of Piperlongumine analogues and their radiotherapeutic response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2017-5210

A novel quinazoline-based analog induces G2/M cell cycle arrest and apoptosis in human A549 lung cancer cells via a ROS-dependent mechanism

6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) is an excellent quinazoline-containing NF-κB inhibitor also acting as a novel anticancer agent. Considering both the medicinal significance of quinazoline scaffold and the tunable functionality of Michael acceptor-centric pharmacophores in the electrophilicity-based prooxidant strategy, we designed a novel QNZ-inspired electrophilic molecule QNZ-A by introducing a Michael acceptor unit at position-6 of quinazoline ring in QNZ. Our results identified QNZ-A as a promising selective cytotoxic agent against A549 cells. QNZ-A, by virtue of its Michael acceptor unit, induced reactive oxygen species (ROS) accumulation associated with collapse of the redox buffering system in A549 cells. This caused up-regulation of p53-inducible p21 and down-regulation of redox sensitive Cdc25C along with Cyclin B1/Cdk1, leading to a G2/M cell cycle arrest and final cell apoptosis. By contrast, QNZ-B, a reduction product of QNZ-A lacking the Michael acceptor unit failed to induce ROS generation and all these cell cycle-related events. In conclusion, this work provided a successful example of designing QNZ-directed anticancer agent by a ROS-promoting strategy and identified QNZ-A as a selective anticancer agent against A549 cells through G2/M cell cycle arrest and apoptosis via a ROS-dependent mechanism.

Hexamethoxylated Monocarbonyl Analogues of Curcumin Cause G2/M Cell Cycle Arrest in NCI-H460 Cells via Michael Acceptor-Dependent Redox Intervention.

Curcumin, derived from the dietary spice turmeric, holds promise for cancer prevention. This prompts much interest in investigating the action mechanisms of curcumin and its analogues. Two symmetrical hexamethoxy-diarylpentadienones (1 and 2) as cucumin analogues were reported to possess significantly enhanced cytotoxicity compared with the parent molecule. However, the detailed mechanisms remain unclear. In this study, compounds 1 and 2 were identified as the G2/M cell cycle arrest agents to mediate the cytotoxicity toward NCI-H460 cells via Michael acceptor-dependent redox intervention. Compared with curcumin, they could more easily induce a burst of reactive oxygen species (ROS) and collapse of the redox buffering system. One possible reason is that they could more effectively target intracellular TrxR to convert this antioxidant enzyme into a ROS promoter. Additionally, they caused up-regulation of p53 and p21 and down-regulation of redox-sensitive Cdc25C along with cyclin B1/Cdk1 in a Michael acceptor- and ROS-dependent fashion. Interestingly, in comparison with compound 2, compound 1 displayed a relatively weak ability to generate ROS but increased cell cycle arrest activity and cytotoxicity probably due to its Michael acceptor-dependent microtubule-destabilizing effect and greater GST-inhibitory activity, as well as its enhanced cellular uptake. This work provides useful information for understanding Michael acceptor-dependent and redox-mediated cytotoxic mechanisms of curcumin and its active analogues.

Insights into the importance for designing curcumin-inspired anticancer agents by a prooxidant strategy: The case of diarylpentanoids

Developing anticancer agents by a prooxidant strategy has attracted increasing attention in recent years, although it is not conventional in medicinal chemistry and is completely opposite to antioxidant therapy. In this work, a panel of diarylpentanoids as the curcumin mono-carbonyl analogs were designed and synthesized, and their cytotoxic and proapoptotic mechanisms against human lung cancer A549 cells were investigated at the frontiers of chemistry and biology. It was found that compared with curcumin, the compounds (A1, B1, and C1) bearing two ortho substituents on the aromatic rings, especially A1, exhibit significantly increased cytotoxic and proapoptotic activities through a Michael acceptor unit-dependent prooxidant-mediated mechanism. The prooxidative ability is governed not only by their electrophilicity but also by their geometry, cellular uptake and metabolic stability, and TrxR-inhibitory activity. Mechanistic investigation reveals that the compound A1 could effectively and irreversibly modify the TrxR by virtue of the above optimal biochemical parameters, and convert this antioxidant enzyme into a reactive oxygen species (ROS) promoter, resulting in a burst of the intracellular ROS including H2O2 and O2–•. The ROS generation is associated with falling apart in the redox buffering system, and subsequently induces increases in Ca2+ influx and oxidative stress, collapse of mitochondrial membrane potential, and activation of caspase-9 and caspase-3, ultimately leading to cell apoptosis. This work highlights the feasibility in designing curcumin-inspired anticancer agents by a prooxidant strategy, and gives us useful information on how to design them.

DR-06 * REACTIVE OXYGEN SPECIES MEDIATE THERAPEUTIC RESPONSE AND RESISTANCE IN GLIOBLASTOMA

Glioblastoma (GBM) resistance to therapy is the most common cause of tumor recurrence, which is ultimately fatal in 90 percent of the patients five years after initial diagnosis. A subpopulation of tumor cells with stem like properties are specifically endowed to resist or adapt to the standard therapies (e.g, radiation, chemotherapy) and lead to tumor resistance. We investigated the mechanisms underlying GBM response to treatment using the non-toxic, non-psychoactive cannabinoid derivative cannabidiol (CBD), in primary glioma stem-like cells in culture and in vivo. In culture, CBD induced an increase in reactive oxygen species (ROS) in GSC which lead to inhibition of proneural markers, cell survival, p-AKT, and self-renewal, concomitant with upregulation of anti-oxidant response gene products and mesenchymal markers. These phenotypic changes were also measured in GSC-derived xenografts from CBD-treated animals, suggesting this molecular adaptive response contributes to therapeutic response and resistance in vivo. CBD-induced mesenchymal shift was reversible by pretreatment with Tocopherol (vit E), indicating that it is ROS mediated. CBD- induced inhibition of self-renewal was mediated by activation of the AMPK-phosphor-p38-Bmi1 pathway. The anti-oxidant response cellular program was mediated by transcriptional activation of NFE2L2 and resulted in upregulation of its target genes, such as xCT. xCT is the enzymatic component of system Xc which regulates GSH levels, the main CNS redox buffering system, and plays a critical role in GBM cell survival. We hypothesized that combining CBD with xCT inhibition will exhibit an enhanced anti-tumor effect. Using genetic knockdown and the pharmacological agents Sulfasalazine and Erastin, we demonstrate that CBD and xCT inhibitors work synergistically to inhibit survival, self renewal and invasion of primary GSC. Taken together our data suggest that combination of CBD and non-toxic small molecules which regulate ROS levels constitute a promising therapeutic approach for glioblastoma.

Abstract 4406: Role of KRAS in modulating the metabolomic profile and the response of NSCLC cells to PI3K/mTOR and AMPK interfering agents

Abstract Background: Non-small-cell lung cancers (NSCLC) constitute the majority of lung cancer cases, whose risk is associated with environmental factors, such as air pollution, and smoking habits. KRAS mutations are present in about 20% of NSCLC patients, mostly at codon 12 and have been associated to tumor progression and resistance to chemotherapy in many types of cancer. Increasingly evidences seem to highlight that the definition of KRAS status is not sufficient to determine its predictive and/or prognostic role in NSCLC, and indicate that different KRAS mutations could differently impact on crucial cellular processes. An issue still to be addressed is whether different KRAS mutations may modulate cellular metabolism with possible implications on different response to anticancer treatments which specifically inhibit metabolism modulating pathways, such as the PI3K/AKT/mTOR axis. Methods: We generated from the human NSCLC cell line NCI-H1299 clones overexpressing WT KRAS, and the three most common NSCLC KRAS mutations (G12C, G12V and G12D). The clones were used to determine in vitro response to PI3K, mTOR and AMPK interfering agents. We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish the different KRAS mutated clones. Results: KRAS overexpressing clones resulted sensitive to the PI3K inhibitors BKM-120 and PIK-75 and to the PI3K/mTOR dual inhibitor BEZ-235. Metformin (acting on AMPK) did not display a cytotoxic activity on the clones at concentrations below or equal to 1mM. Metabolomics analyses revealed that clones harboring different KRAS mutations at codon 12 had different metabolic remodellings such as different redox buffering system and different glutamine-dependency. Conclusion: The preliminary findings reported herein indicate that specific KRAS mutations at codon 12 are associated to different metabolomics profiles that might have an impact to the response to anticancer treatments. Combination studies including metformin and PI3K/mTOR interfering agents and more detailed metabolomics analyses are ongoing in order to clarify whether metabolic adaptations dependent on KRAS mutants might account for the behaviour of KRAS overexpressing clones treated with different drugs. Citation Format: Elisa Caiola, Laura Brunelli, Mirko Marabese, Marina Chiara Garassino, Gabriella Farina, Massimo Broggini, Roberta Pastorelli. Role of KRAS in modulating the metabolomic profile and the response of NSCLC cells to PI3K/mTOR and AMPK interfering agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4406. doi:10.1158/1538-7445.AM2014-4406

Capturing the metabolomic diversity of KRAS mutants in non-small-cell lung cancer cells

In non-small-cell lung cancer (NSCLC), one-fifth of patients have KRAS mutations, which are considered a negative predictive factor to first-line therapy. Evidence is emerging that not all KRAS mutations have the same biological activities and possible remodeling of cell metabolism by KRAS activation might complicate the scenario. An open question is whether different KRAS mutations at codon-12 affect cellular metabolism differently with possible implications for different responses to cancer treatments. We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 (G12C, G12D, G12V) and the wild-type. The glutamine deprivation assay and real-time PCR were used to confirm the involvement of some of the metabolic pathways highlighted. Cell clones indicated distinct metabolomic profiles in KRAS wild-type and mutants. Clones harboring different KRAS mutations at codon-12 also had different metabolic remodeling, such as a different redox buffering system and different glutamine-dependency not driven by the transcriptional state of enzymes involved in glutaminolysis. These findings indicate that KRAS mutations at codon-12 are associated with different metabolomic profiles that might affect the responses to cancer treatments.

Isolation, identification and characterization of highly tellurite-resistant, tellurite-reducing bacteria from Antarctica

The tellurium oxyanion, tellurite, is extremely noxious to most living organisms. Its toxicity has been mainly related to the generation of reactive oxygen species (ROS) as well as to an unbalancing of the thiol:redox buffering system. Nevertheless, a few bacteria are capable of thriving at high tellurite concentrations. One mechanism of resistance is the enzymatic and non-enzymatic reduction of tellurite to the less toxic elemental tellurium. This reduction generates nano- to micrometric tellurium crystals that display different shapes and sizes.To date, a very limited number of highly tellurite-resistant and tellurite-reducing bacterial species are available from international culture collections. In this work, we decided to look for tellurite-reducing bacteria from an extreme environment, Antarctica. This environment exhibits a combination of several extreme factors such as high UV-radiation and desiccation and freezing conditions that impact directly on the local biodiversity. Since, as does, all these factors induce ROS formation, we hypothesized that Antarctic bacteria could also exhibit tellurite-resistance. In this context, we isolated 123 tellurite-resistant bacteria, and characterized six new tellurite-resistant and tellurite-reducing bacterial strains from samples collected in Antarctica. These strains were identified according to their 16S rRNA gene sequence as Staphylococcus hameolyticus, Staphylococcus sciuri, Acinetobacter haemolyticus, Pseudomonas lini, and two strains of Psychrobacter immobilis.The isolates display tellurite-resistance about 35- to 500-fold higher than Escherichia coli (Te-sensitive organism), and a high level of tellurite reduction which might be interesting for an application in the field of bioremediation or nanoparticle biosynthesis.

Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings

Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (EGSH) of more negative than −310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure EGSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between −280 and −290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about −238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of EGSH values that can be measured in vivo with roGFP2-based probes from about −320 mV for GRX1-roGFP2 down to about −210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an EGSH of about −260 mV in this mutant.

Disturbances in the Glutathione/Ophthalmate Redox Buffer System in the Woodchuck Model of Hepatitis Virus-Induced Hepatocellular Carcinoma

Purpose. The incidence of liver tumors is rising in USA. The purpose of this study was to evaluate liver oxido-reductive status in the presence of chronic liver disease and hepatocellular carcinoma (HCC). Methods. Glutathione species and ophthalmate (OA) concentrations were measured by LC-MS in processed plasma and red blood cells (RBC) from infected Woodchuck with hepatitis virus (WHV). Blood samples were obtained from: (i) infected animals with tumors (WHV+/HCC+), (ii) infected animals without tumors (WHV+/HCC−) and (iii) healthy animals (WHC−/HCC−). Results. The concentration of reduced glutathione (GSH) and the ratio GSH/GSG were lower in plasma from WHV+/HCC+ animals when compared to WHV+/HCC− and WHV−/HCC− (P < 0.01). In contrast, the concentration of oxidized glutathione (GSSG) was found to be higher in plasma from WHV+/HCC+ animals when compared to WHV+/HCC− and WHV−/HCC− (P < 0.01). The Glutathione species and its ratio from the RBC compartment were similar among all groups. OA concentration in both plasma and RBC was significantly higher from WHV+/HCC+ when compared to WHV+/HCC− and WHV−/HCC− (P < 0.01). Conclusions. Disturbances of the glutathione redox buffer system and higher concentrations of OA were found in the WCV+/HCC+ animal model. The role of these compounds as biomarkers of early tumor development in patients with end stage liver disease remains to be determined.

A solid-state redox buffer as interface of solid-contact ISEs

Graphite with a surface-confined redox buffer system was used as solid contact in solid-contact ion-selective electrodes (SC-ISE). Potentiostatically preconditioning of the redox buffer ensures that the ratio of oxidized and reduced groups is unity, i.e., a maximum buffer capacity. These SC-ISEs exhibited a very high reproducibility and stability of potentials. Graphite modification was achieved by generating carboxylic surface groups via oxidation with nitrosulfuric acid and subsequent modification of the acidic groups with n-(2,5-dimethoxyphenyl)ethyl-1-amine (DMPEA). Composite electrodes of modified graphite and polymethylmetharcrylate (PMMA) as binder were used as solid-contact of polymer membrane K +-and F −-sensitive SC-ISEs. The solid-state redox buffer decreased potential variation from several hundred mV to just a few mV.

Polyethylene Glycol Modified-Albumin Enhances the Cold Preservation Properties of University of Wisconsin Solution in Rat Liver and a Hepatocyte Cell Line

Liver grafts preserved in cold storage undergo changes mainly manifested by morphological modifications of the sinusoidal endothelium that result in poor graft function upon reperfusion. The present studies aimed to determine if the addition of polyethylene glycol-albumin to University of Wisconsin (Peg-AlbUW) solution ameliorates the cold preservation injuries of liver grafts. Rat livers were preserved cold with various preservation solutions and evaluated for weight changes and endothelial morphology. Solutions that preserved graft weight and endothelial morphology were tested in the isolated perfused rat liver model to assess graft function. A rat hepatocyte cell line was evaluated for both viability and glutathione concentrations emulating cold preservation and reperfusion conditions. Liver grafts preserved with Peg-AlbUW maintained their initial weight and showed a conserved endothelial morphology compared with liver grafts preserved in UW for 30 h (P<0.05). Liver grafts preserved with Peg-AlbUW had improved portal blood flow and bile secretion compared with liver grafts preserved in UW for 30 h (P<0.05). In vitro we noted comparable hepatocyte viability when cells were preserved in Peg-AlbUW versus UW under similar preservation conditions (P>0.05); glutathione concentrations (reduced and total) were significantly increased in hepatocytes preserved in 3% Peg-AlbUW compared with other preservation solutions (P<0.05). The addition of Peg-Alb to UW preservation solution ameliorated the cold preservation injuries of rat liver grafts as shown by stable liver graft weight, a better preservation of the endothelial morphology, improved portal vein blood flow, and increased bile secretion. Peg-Alb-UW solution improved the integrity of the glutathione redox buffer system of a hepatocyte cell line after cold storage and reperfusion.

Overexpression of Glucose-6-phosphate Dehydrogenase Extends the Life Span of Drosophila melanogaster

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.