Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone 'Compound 1' caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1's MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols. © 2004 Elsevier B.V. All rights reserved.

The antioxidant enzyme peroxiredoxin-2 is depleted in lymphocytes seven days after ultra-endurance exercise

Purpose: Peroxiredoxin-2 (PRDX-2) is an antioxidant and chaperone-like protein critical for cell function. This study examined whether the levels of lymphocyte PRDX-2 are altered over one month following ultra-endurance exercise. Methods: Nine middle-aged men undertook a single-stage, multi-day 233 km (145 mile) ultra-endurance running race. Blood was collected immediately before (PRE), upon completion/retirement (POST), and following the race at DAY 1, DAY 7 and DAY 28. Lymphocyte lysates were examined for PRDX-2 by reducing SDS-PAGE and western blotting. In a sub-group of men who completed the race (n = 4) PRDX-2 oligomeric state (indicative of redox status) was investigated. Results: Ultra-endurance exercise caused significant changes in lymphocyte PRDX-2 (F (4,32) 3.409, p=0.020, ?(2) =0.299): seven-days after the race, PRDX-2 levels in lymphocytes had fallen to 30% of pre-race values (p=0.013) and returned to near-normal levels at DAY 28. Non-reducing gels demonstrated that dimeric PRDX-2 (intracellular reduced PRDX-2 monomers) was increased in 3 of 4 race completers immediately post-race, indicative of an "antioxidant response". Moreover, monomeric PRDX-2 was also increased immediately post-race in 2 of 4 race-completing subjects, indicative of oxidative damage, which was not detectable by DAY 7. Conclusions: Lymphocyte PRDX-2 was decreased below normal levels 7 days after ultra-endurance exercise. Excessive accumulation of reactive oxygen species induced by ultra-endurance exercise may underlie depletion of lymphocyte PRDX-2 by triggering its turnover after oxidation. Low levels of lymphocyte PRDX-2 could influence cell function and might, in part, explain reports of dysregulated immunity following ultra-endurance exercise.

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes.

Effects of dihydrolipoic acid (DHLA), α-lipoic acid. N-acetyl cysteine and ascorbate on xenobiotic-mediated methaemoglobin formation in human erythrocytes in vitro

α-Lipoic acid, dihydrolipoic acid (DHLA), N-acetyl cysteine and ascorbate were compared with methylene blue for their ability to attenuate and/or reduce methaemoglobin formation induced by sodium nitrite, 4-aminophenol and dapsone hydroxylamine in human erythrocytes. Neither α-lipoic acid, DHLA, N-acetyl cysteine nor ascorbate had any significant effects on methaemoglobin formed by nitrite, either from pre-treatment, simultaneous addition or post 30 min addition of the agents up to the 60 min time point, although N-acetyl cysteine did reduce methaemoglobin formation at 120 min (P<0.05). In all three treatment groups at 30, 60 and 120 min, there were no significant effects mediated by DHLA or N-acetyl cysteine on 4-aminophenol (1 mM)-mediated haemoglobin oxidation. Ascorbate caused marked significant reductions in 4-aminophenol methaemoglobin in all treatment groups at 30-120 min except at 30 min in the simultaneous addition group (P<0.0001). Neither α-lipoic acid, nor N-acetyl cysteine showed any effects on hydroxylamine-mediated methaemoglobin formation at 30 and 60 in all treatment groups. In contrast, DHLA significantly reduced hydroxylamine-mediated methaemoglobin formation at all three time points after pre-incubation and simultaneous addition (P<0.001), while ascorbate was ineffective. Compared with methylene blue, which was effective in reducing methaemoglobin formation by all three toxins (P<0.01), ascorbate was only highly effective against 4-aminophenol mediated methaemoglobin, whilst the DHLA-mediated attenuation of dapsone hydroxylamine-mediated methaemoglobin formation indicates a possible clinical application in high-dose dapsone therapy. © 2003 Elsevier B.V. All rights reserved.

Monitoring changes in thioredoxin and over-oxidised peroxiredoxin in response to exercise in humans

Introduction. Peroxiredoxin (PRDX) and thioredoxin (TRX) are antioxidant proteins that control cellular signalling and redox balance, although their response to exercise is unknown. This study aimed to assess key aspects of the PRDX-TRX redox cycle in response to three different modes of exercise. Methods. Healthy males (n = 10, mean ± SD: 22 ± 3 yrs) undertook three exercise trials on separate days: two steady-state cycling trials at moderate (60% VO2MAX; 27 min, MOD) and high (80% VO2MAX; 20 min, HIGH) intensities, and a low-volume high-intensity interval training trial (10 × 1 min 90% VO2MAX, LV-HIIT). Peripheral blood mononuclear cells were assessed for TRX-1 and over-oxidised PRDX (isoforms I-IV) protein expression before, during, and 30 min following exercise (post + 30). The activities of TRX reductase (TRX-R) and the nuclear factor kappa B (NF-κB) p65 subunit were also assessed. Results. TRX-1 increased during exercise in all trials (MOD, + 84.5%; HIGH, + 64.1%; LV-HIIT, + 205.7%; p < 05), whereas over-oxidised PRDX increased during HIGH only (MOD, - 28.7%; HIGH, + 202.9%; LV-HIIT, - 22.7%; p < .05). TRX-R and NF-κB p65 activity increased during exercise in all trials, with the greatest response in TRX-R activity seen in HIGH (p < 0.05). Discussion. All trials stimulated a transient increase in TRX-1 protein expression during exercise. Only HIGH induced a transient over-oxidation of PRDX, alongside the greatest change in TRX-R activity. Future studies are needed to clarify the significance of heightened peroxide exposure during continuous high-intensity exercise and the mechanisms of PRDX-regulatory control.

Lymphocyte peroxiredoxin-2 levels are depleted one week after ultra-endurance exercise

Peroxiredoxin-2 (PRDX-2) belongs to a family of thiol containing proteins and is important for antioxidant defense, redox signaling and cell function. This study examined whether lymphocyte PRDX-2 levels are altered over one month following ultra-endurance exercise. Nine middle-aged men participated in a 145 mile ultra-endurance running race event. Blood drawing was undertaken immediately before, upon completion/retirement, and at one, seven and twenty eight-days following the race. PRDX-2 levels were examined at each time-point, for all participants (n=9) by reducing SDS-PAGE and western blotting. Further analysis using non-reducing SDS-PAGE and western blotting was undertaken in a sub-group of men who completed the race (n = 4) to investigate PRDX-2 oligomeric state (indicative of oxidation state). Ultra-endurance exercise caused a significant alteration in lymphocyte PRDX-2 levels (F(4,32) 3.409, p=0.020, η2 =0.299): seven-days after the race PRDX-2 levels fell by 70% (p=0.013) and at twenty eight-days after the race returned to near-normal levels. PRDX-2 dimers (intracellular reduced PRDX-2 monomers) in three of the four participants, who finished the race, were increased upon race completion. Furthermore, PRDX-2 monomers (intracellular over-oxidized PRDX-2 monomers) in two of these four participants were present upon race completion, but absent seven-days after the race. This study found that PRDX-2 levels in lymphocytes were reduced below normal levels seven-days after an ultra-endurance running race. We suggest that excessive reactive oxygen species production, induced by ultra-endurance exercise may, in part, explain the depletion of lymphocyte PRDX-2 by triggering its turnover after oxidation.

Synthesis and application of novel probes for the detection of cysteine sulphenic acids

Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 serine mutant is resistant to 2-AAPA–induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles

Glutaredoxin-1 Is up-regulated in preeclampsia and increases soluble Flt-1 expression

INTRODUCTION: Preeclampsia is a vascular disorder in pregnancyand is biochemical characterization by high soluble Flt-1 and lowplacenta growth factor as well as an imbalance in redox homeostasis.During conditions of high oxidative stress, cysteine residues on keyproteins are reversibly altered by S-glutathionylation, modifying theirfunction. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics toproteins with redox-sensitive cysteines. The role of Glrx in preeclampsiais unknown.METHODS: Immunohistochemistry and Western blot analysis for Glrx orglutathione were conducted on human placenta samples collected pre-termfrom early onset preeclamptic patients (n=10) or non-preeclamptic induceddeliveries (n=9). Human endothelial cells were infected with adenovirusencoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1%O2, 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCRand ELISA were used to measure sFlt-1 at mRNA and protein level.RESULTS: Immunohistochemical staining for GSH revealed lowerS-glutathionylation adducts in preeclampsia placenta in comparison tocontrols. Glrx expression, which catalyses de-glutathionylation wasenhanced in early onset preeclampsia compared to pre-term controlsamples. In contrast, no change was observed in preeclamptic and IUGRplacentas at full term. In endothelial cells overexpressing Glrx, sFlt-1expression was dramatically enhanced at mRNA (3-fold P<0.05) andprotein level (5 fold P>0.01, n=4) after hypoxia andoverexpressing Glrxin mice enhanced levels of circulating sFlt-1 during in vivo ischemia.CONCLUSIONS: Enhanced Glrx expression in preeclamptic placentain line with an apparent decrease in S-glutathionylation may leavekey proteins susceptible to irreversible oxidation in conditions of highoxidative stress.