Thiol-containing Antioxidants Research Articles

Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III.

Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.

A cost-effective synthesis of enantiopure ovothiol A from L-histidine, its natural precursor

Ovothiol A is a naturally occurring 4-mercaptohisti dine derivative, and one of the most potent thiol-containing antioxidants. The synthesis of ovo thiol A was elaborated by introducing the thiol group onto protected 3-methyl-L-histidine via halogenation and Ullmann reaction. This enantiopure synthesis of ovothiol A, which uses L-h istidine as starting material, requires only five steps, and compared to prior synthesis, is bot h cost and time effiecent.

Plumbagin modulates leukemia cell redox status.

Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.

Eriocalyxin B-induced apoptosis in pancreatic adenocarcinoma cells through thiol-containing antioxidant systems and downstream signalling pathways.

Thiol-containing antioxidant systems play an important role in regulating cellular redox homeostasis. Several anti-cancer agents act by targeting these systems by inducing the production of reactive oxygen species (ROS). Our earlier studies have shown that Eriocalyxin B (EriB), a diterpenoid isolated from Isodon eriocalyx, possesses anti-pancreatic tumour activities in vitro and in vivo. The present study further demonstrated that only thiol-containing antioxidants, N-acetylcysteine (NAC) or dithiothreitol (DTT), inhibited EriB-induced cytotoxicity and apoptosis. EriB suppressed the glutathione and thioredoxin antioxidant systems, thus increasing the intracellular ROS levels and regulating the MAPK, NFκB pathways. Treatment with EriB depleted the intracellular thiol-containing proteins in CAPAN-2 cells. In vivo studies also showed that EriB treatment (2.5 mg/kg) reduced the pancreatic tumour weights significantly in nude mice with increased superoxide levels. Taken together, our results shed important new light on the molecular mechanisms of EriB acting as an apoptogenic agent and its therapeutic potential for pancreatic cancer.

Design, synthesis and biological evaluation of arylcinnamide hybrid derivatives as novel anticancer agents

The combination of two pharmacophores into a single molecule represents one of the methods that can be adopted for the synthesis of new anticancer molecules. A series of novel antiproliferative agents designed by a pharmacophore hybridization approach, combining the arylcinnamide skeleton and an α-bromoacryloyl moiety, was synthesized and evaluated for its antiproliferative activity against a panel of seven human cancer cell lines. In addition, the new derivatives were also active on multidrug-resistant cell lines over-expressing P-glycoprotein. The biological effects of various substituents on the N-phenyl ring of the benzamide portion were also described. In order to study the possible mechanism of action, we observed that 4p slightly increased the Reactive Oxygen Species (ROS) production in HeLa cells, but, more importantly, a remarkable decrease of intracellular reduced glutathione content was detected in treated cells compared with controls. These results were confirmed by the observation that only thiol-containing antioxidants were able to significantly protect the cells from induced cell death. Altogether our results indicate that the new derivatives are endowed with good anticancer activity in vitro, and their properties may result in the development of new cancer therapeutic strategies.

Effect of Silver Nanomaterials on the Activity of Thiol-Containing Antioxidants

The use of nanomaterials in consumer products is rapidly expanding. In most studies, nanomaterials are examined as isolated ingredients. However, consumer products such as foods, cosmetics, and dietary supplements are complex chemical matrixes. Therefore, interactions between nanomaterials and other components of the product must be investigated to ensure the product's performance and safety. Silver nanomaterials are increasingly being used in food packaging as antimicrobial agents. Thiol-containing compounds, such as reduced glutathione (GSH), cysteine, and dihydrolipoic acid, are used as antioxidants in many consumer products. In the current study, we have investigated the interaction between silver nanomaterials and thiol-containing antioxidants. The selected Ag nanomaterials were Ag coated with citrate, Ag coated with poly(vinylpyrrolidone), and Au nanorods coated with Ag in a core/shell structure. We observed direct quenching of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) by all three Ag nanomaterials to varying degrees. The Ag nanomaterials also reduced the quenching of DPPH by GSH to varying degrees. In addition, we determined that the mixture of GSH and Au@Ag nanorods held at 37 °C was less effective at quenching azo radical than at ambient temperature. Furthermore, we determined that Au@Ag nanorods significantly reduced the ability of GSH and cysteine to quench hydroxyl and superoxide radicals. The work presented here demonstrates the importance of examining the chemical interactions between nanomaterials used in products and physiologically important antioxidants.

Neuroprotection induced by N-acetylcysteine against cytosolic glutathione depletion-induced Ca2+ influx in dorsal root ganglion neurons of mice: Role of TRPV1 channels

Glutathione (GSH) and N-acetylcysteine (NAC) are thiol-containing antioxidants, and also act through a direct reaction with free radicals. Transient receptor potential vanilloid 1 (TRPV1) is the principal transduction channel serving as a polymodal detector. Despite the importance of oxidative stress in pain sensitivity, its role in TRPV1 modulation is poorly understood. NAC may also have a regulator role on TRPV1 channel activity in the dorsal root ganglion (DRG) neuron. Therefore, we tested the effects of GSH and NAC on TRPV1 channel current, Ca2+ influx, oxidative stress and caspase activity in the DRG of mice.DRG neurons were freshly isolated from mice and the neurons were incubated for 6 and 24h with buthionine sulfoximine (BSO). Pretreatment of cultured DRG neurons with NAC, results in a protection against oxidative damages. This neuroprotection is associated with the attenuation of a Ca2+ influx triggered by oxidative agents such as H2O2, 5,5′-dithiobis-(2-nitrobenzoic acid) and GSH depletion via BSO. Here, we demonstrate the contribution of cytosolic factors (related to thiol group depletion) on the activation of TRPV1 channels in this mechanism. TRPV1 channels are activated by various agents including capsaicin (CAP), the pungent component of hot chili peppers, and are blocked by capsazepine. An oxidative environment also increased CAP-evoked TRPV1 currents in the neurons. When NAC and GSH were included in the patch pipette as well as extracellularly in the chamber, TRPV1 channels were not activated by CAP and H2O2. TRPV1 inhibitors, 2-aminoethyl diphenylborinate and N-(p-amylcinnamoyl)anthranilic acid strongly reduced BSO-induced oxidative toxicity and Ca2+ influx, in a manner similar to pretreatment with NAC and GSH. Caspase-3 and -9 activities of all groups were not changed by the agonists or antagonists.In conclusion, in our experimental model, TRPV1 channels are involved in the oxidative stress-induced neuronal death, and negative modulation of this channel activity by GSH and NAC pretreatment may account for their neuroprotective activity against oxidative stress.

Metals as a cause of oxidative stress in fish: a review

This review summarizes the current knowledge on the contribution of metals to the development of oxidative stress in fish. Metals are important inducers of oxidative stress in aquatic organisms, promoting formation of reactive oxygen species through two mechanisms. Redox active metals generate reactive oxygen species through redox cycling, while metals without redox potential impair antioxidant defences, especially that of thiol-containing antioxidants and enzymes. Elevated levels of reactive oxygen species lead to oxidative damage including lipid peroxidation, protein and DNA oxidation, and enzyme inactivation. Antioxidant defences include the enzyme system and low molecular weight antioxidants. Metal-binding proteins, such as ferritin, ceruloplasmin and metallothioneins, have special functions in the detoxification of toxic metals and also play a role in the metabolism and homeostasis of essential metals. Recent studies of metallothioneins as biomarkers indicate that quantitative analysis of mRNA expression of metallothionein genes can be appropriate in cases with elevated levels of metals and no evidence of oxidative damage in fish tissue. Components of the antioxidant defence are used as biochemical markers of oxidative stress. These markers may be manifested differently in the field than in results found in laboratory studies. A complex approach should be taken in field studies of metal contamination of the aquatic environment.  

Ncf1 (p47phox) Is Essential for Direct Regulatory T Cell Mediated Suppression of CD4+ Effector T Cells

BackgroundMultiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs.Methodology/Principal FindingsTregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff.Conclusions/SignificanceWe present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.

애기장대 peroxiredoxins (Prxs)과 sulphiredoxin1 (Srx1)의 작용기작

Plants generate reactive oxygen species (ROS) as a by-product of normal aerobic metabolism or when exposed to a variety of stress conditions, which can cause widespread damage to biological macromolecules. To protect themselves from oxidative stress, plant cells are equipped with a wide range of antioxidant proteins. However, the detailed reaction mechanisms of these are still unknown. Peroxiredoxins (Prxs) are ubiquitous thiol-containing antioxidants that reduce hydrogen peroxide with an N-terminal cysteine. The active-site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid during catalysis, which leads to inactivation of peroxidase activity. This oxidation was thought to be irreversible. Recently identified small protein sulphiredoxin (Srx1), which is conserved in higher eukaryotes, reduces cysteine?sulphinic acid in yeast peroxiredoxin. Srx1 is highly induced by H₂O₂-treatment and the deletion of its gene causes decreased yeast tolerance to H₂O₂, which suggest its involvement in the metabolism of oxidants. Moreover, Srx1 is required for heat shock and oxidative stress induced functional, as well as conformational switch of yeast cytosolic peroxiredoxins. This change enhances protein stability and peroxidase activity, indicating that Srx1 plays a crucial role in peroxiredoxin stability and its regulation mechanism. Thus, the understanding of the molecular basis of Srx1 and its regulation is critical for revealing the mechanism of peroxiredoxin action. We postulate here that Srx1 is involved in dealing with oxidative stress via controlling peroxiredoxin recycling in Arabidopsis. This review article thus will be describing the functions of Prxs and Srx in Arabidopsis thaliana. There will be a special focus on the possible role of Srx1 in interacting with and reducing hyperoxidized Cys-sulphenic acid of Prxs.

System xc− and Thioredoxin Reductase 1 Cooperatively Rescue Glutathione Deficiency

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in gamma-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.

Activation of JNK and c-Jun is involved in glucose oxidase-mediated cell death of human lymphoma cells.

Mitogen-activated protein kinases (MAPK) affect the activation of activator protein-1 (AP-1), which plays an important role in regulating a range of cellular processes. However, the roles of these signaling factors on hydrogen peroxide (H(2)O(2))-induced cell death are unclear. This study examined the effects of H(2)O(2) on the activation of MAPK and AP-1 by exposing the cells to H(2)O(2) generated by either glucose oxidase or a bolus addition. Exposing BJAB or Jurkat cells to H(2)O(2) affected the activities of MAPK differently according to the method of H(2)O(2) exposure. H(2)O(2) increased the AP-1-DNA binding activity in these cells, where continuously generated H(2)O(2) led to an increase in mainly the c-Fos, FosB and c-Jun proteins. The c-Jun-NH(2)-terminal kinase (JNK)-mediated activation of c-Jun was shown to be related to the H(2)O(2)-induced cell death. However, the suppression of H(2)O(2)-induced oxidative stress by either JNK inhibitor or c-Jun specific antisense transfection was temporary in the cells exposed to glucose oxidase but not to a bolus H(2)O(2). This was associated with the disruption of death signaling according to the severe and prolonged depletion of reduced glutathione. Overall, these results suggest that H(2)O(2) may decide differently the mode of cell death by affecting the intracellular redox state of thiol-containing antioxidants, and this depends more closely on the duration exposed to H(2)O(2) than the concentration of this agent.

Sulfhydryl systems are a critical factor in the zebrafish developmental toxicity of the dithiocarbamate sodium metam (NaM)

Dithiocarbamates (DTCs) are sulfhydryls (thiol)-containing compounds, often associated with metals, and have both antioxidant and pro-oxidant abilities depending on the compound, experimental system and condition. In this study we investigated whether cell death plays a role in the manifestation of DTC-induced notochord distortions in the developing zebrafish and if thiol-containing compounds or antioxidants could modify this developmental toxicity. Sodium metam (NaM) induced notochord distortions could not be protected with the antioxidants ascorbic acid, trolox (synthetic vitamin E) or lipoic acid. However, NaM-induced distortions could be protected with co-exposure to glutathione or N-Acetyl Cysteine. Staggering the NaM and glutathione exposures in consecutive 10 h developmental windows also resulted in protection. There were no discernable changes in TUNEL positive cells, a marker of apoptotic cells, at 24 h post-fertilization (hpf) in NaM, dimethyl-dithiocarbamate, carbon disulfide, or neocuproine exposed embryos. Live NaM-exposed embryos incubated with acridine orange, a general stain for cell death, for 1 h beginning at 11, 18 and 24 hpf showed clusters of stained nuclei near the somitogenic front but not in the cells making up the notochord. Overall, induction of apoptotic pathways and widespread cell death are not involved in the manifestation of the adverse developmental outcomes following NaM exposure. However, cellular thiol status or critical sulfhydryl moieties are important considerations in the mechanisms of DTC developmental toxicity.

Melissoidesin G, a diterpenoid purified from Isodon melissoides, induces leukemic‐cell apoptosis through induction of redox imbalance and exhibits synergy with other anticancer agents

Melissoidesin G (MOG) is a new diterpenoid purified from Isodon melissoides, a plant used in Chinese traditional medicine as antitumor and anti-inflammatory agents. In our study, MOG was shown to specifically inhibit the growth of human leukemia cell lines and primary acute myeloid leukemia (AML) blasts via induction of apoptosis, with the evidence of mitochondrial DeltaPsim loss, reactive oxygen species production, caspases activation and nuclear fragmentation. Furthermore, it was shown that thiol-containing antioxidants completely blocked MOG-induced mitochondrial DeltaPsim loss and subsequent cell apoptosis, while the inhibition of apoptosis by benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone only partially attenuated mitochondrial DeltaPsim loss, indicating that MOG-induced redox imbalance is an early event upstream to mitochondrial DeltaPsim loss and caspase-3 activation. Consistently, it was found that MOG rapidly decreased the intracellular glutathione (GSH) content in a dose-dependent manner and the significance of GSH depletion in MOG-induced apoptosis was further supported by the protective effects of tert-butylhydroquinone (tBHQ) and the facilitative effects of DL-buthionine (S,R)-sulfoximine (BSO). Furthermore, it was showed that GSH depletion induced by MOG rendered some leukemia cell lines more sensitive to arsenic trioxide (As2O3), doxorubicin or cisplatin. Additionally, the synergistic apoptotic effects of MOG with As2O3 were detected in HL-60 and primary AML cells, but not in normal cells, suggesting the selective toxicity of their combination to the malignant cells. Together, we proposed that MOG alone or administered with other anticancer agents may provide a novel therapeutic strategy for leukemia.

Glutathione prevented dopamine-induced apoptosis of melanocytes and its signaling

Dopamine (DA), a monoamine neurotransmitter, is a well-known neurotoxin and plays an etiologic role in neurodegenerative disorders such as Parkinson's disease. DA exerts its toxic effect by generation of reactive oxygen species and quinone product. Vitiligo, a depigmentary disorder of the skin and hair characterized by selective destruction of melanocytes, has been reported to show increased levels of DA with onset and progression of the disease. The aim of this study is to investigate the cytotoxic effect of DA on melanocytes and to search for protective antioxidants against DA-induced toxicity. In addition, molecular mechanism of cell death was also investigated. Cells were treated with DA and cell viabilities were measured by crystal violet staining method. To investigate the cytoprotective activity of various antioxidants, vitamin C, vitamin E, Trolox, quercetin, N-acetylcysteine (NAC) and l-glutathione (GSH) were used. To study cytoprotective effects of NAC and GSH, Mel-Ab cells and cultured normal human melanocytes were pretreated with NAC or GSH, then DA solution was added. DA-induced apoptosis and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were also observed by flow cytometric analysis and Western blotting. The viability of DA-treated Mel-Ab cells significantly decreased in a dose-dependent manner while keratinocytes were much more resistant to DA-toxicity, which was a consistent finding with the selective melanocyte loss observed in vitiligo. Among various antioxidants used in this study, only thiol-containing antioxidants such as NAC or GSH inhibited both JNK and p38 MAPK activation and apoptosis, indicating the unique protective capacity of thiol compounds. Cultured normal human melanocytes were also susceptible to DA and thiol compounds were very efficiently protective against DA-induced cytotoxicity. DA-induced apoptosis and cytoprotective effect of thiol compounds shown in this study could be a clue to understand pathogenesis of viltigo and provide a new therapeutic strategy.

Effects of antioxidants on glutathione levels and clinical recovery from the malnutrition syndrome kwashiorkor – a pilot study

Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many African countries, and repeatedly has been reported to be associated with oxidative stress. The therapy of kwashiorkor is still ineffective. In this pilot study, we tested the hypothesis that oral application of thiol-containing antioxidants increases glutathione status and is beneficial for the clinical recovery of kwashiorkor patients. The longitudinal clinical intervention study was carried out at St Joseph's Hospital, Jirapa, Ghana. Children with severe kwashiorkor were randomly assigned to either a standard treatment (ST) receiving a therapeutic protocol based on the recommendations of the WHO or to one of three study groups receiving in addition 2 × 600 mg reduced glutathione or 2 × 50 mg α-lipoic acid or 2 × 100 mg N-acetylcysteine per day. Patients were followed up clinically and biochemically for 20 days and compared with 37 healthy controls. Both glutathione and α-lipoic acid supplementation had positive effects on survival. Also, the blood glutathione concentrations correlated positively with survival rates. Furthermore, the initial skin lesions, glutathione and total protein concentrations were found to be strong predictors of survival. The data strongly suggest that a therapy restoring the antioxidative capacity by applying cysteine equivalents in the form of glutathione and/or α-lipoic acid is beneficial for biochemical and clinical recovery of kwashiorkor patients.

Effect of methylglyoxal on intracellular calcium levels and viability in renal tubular cells

Methylglyoxal (2-oxopropanal), a physiological glucose metabolite, is a highly reactive dicarbonyl compound that can induce stress in cells and cause apoptotic cell death. This study examines the early signaling effects of methylglyxal on renal cells. It was found that methylglyoxal caused a slow and sustained rise of intracellular Ca 2+ concentration ([Ca 2+] i) in a concentration-dependent manner (EC 50=1.8 mM). Methylglyoxal also induced a [Ca 2+] i rise when extracellular Ca 2+ was removed, but the magnitude was reduced by 80%. Depletion of intracellular Ca 2+ stores with thapsigargin (TG), an endoplasmic reticulum (ER) Ca 2+ pump inhibitor, did not affect methylglyoxal's effect. In Ca 2+-free medium, the methylglyoxal-induced [Ca 2+] i rise was abolished by depleting stored Ca 2+ with carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Methylglyoxal-caused [Ca 2+] i rise in the Ca 2+-containing medium was not affected by modulation of protein kinase C activity, presence of voltage-gated Ca 2+ channel blockers, or preincubation with thiol-containing antioxidants. U73122, an inhibitor of phospholipase C, abolished ATP (but not methylglyoxal)-induced [Ca 2+] i rise. Furthermore, the [Ca 2+] i-elevating effect of methylglyoxal was cell type-dependent, because methylglyoxal failed to cause [Ca 2+] i rises in CHO-K1, neutrophils, or platelets. Pretreatment with methylglyoxal for 0–24 h decreased cell viability in a concentration- and time-dependent manner. Meanwhile, methylglyoxal-induced cell death involved apoptotic and necrotic events, the former being the dominant. These findings suggest that methylglyoxal induced a significant rise in [Ca 2+] i in Madin–Darby canine kidney (MDCK) renal tubular cells by stimulating both extracellular Ca 2+ influx and CCCP-sensitive intracellular Ca 2+ release via as yet unidentified mechanisms. The cell type-specific Ca 2+ signaling may play an important role in the early process of cytotoxic action of methylglyoxal.

ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as 'damage' or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid. Cysteine-sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine-sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass M(r) = 13,000, which we have named sulphiredoxin (identified by the US spelling 'sulfiredoxin', in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine-sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides and control hydroperoxide-mediated signalling in mammals. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine-sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine-sulphinic acid modifications, and in signalling pathways involving protein oxidation.

Oxidative stress and aging.

Free radical-derived reactive oxygen species (ROS) are constantly generated in most living tissue and can potentially damage DNA, proteins and lipids. "Oxidative stress" occurs if ROS reach abnormally high concentrations. Harman was the first to propose that the damaging effects of ROS may play a key role in the mechanism of aging. Genetic studies of such distantly related species as C. elegans, Drosophila melanogaster, and mice support this hypothesis. However, ROS are not only a cause of structural damage, but also physiologically important mediators in biological signaling processes. Abnormally high levels of ROS may therefore lead to dysregulation of redox-sensitive signaling pathways. The redox-sensitive targets in these pathways are often signaling proteins with redox-sensitive cysteine residues which are oxidized to sulfenic acid moieties and mixed disulfides, thereby altering the signaling function of the protein. Because the formation of these mixed disulfides can also occur through a prooxidative shift in the intracellular thiol/disulfide redox status (REDST), the respective signaling pathways respond not only to ROS but also to changes in REDST. Information about the concentration of ROS in living tissue is scarce, but aging-related changes in REDST are well documented. Several studies with cell cultures or experimental animals have shown that the oxidative shift in the intracellular glutathione REDST is typically associated with cellular dysfunction. Complementary studies in humans have shown that oxidative changes in the plasma (i.e., extracellular) REDST are correlated with aging-related pathophysiological processes. The available evidence suggests that these changes play a key role in various conditions which limit the human life span. Several attempts have been made to ameliorate the consequences of aging by thiol-containing antioxidants, but this approach requires a detailed knowledge of the effects of thiol-containing antioxidants on cysteine homeostasis, REDST, and redox-sensitive signaling pathways of the host.

Tumor cell growth inhibition and extracellular signal-regulated kinase (ERK) phosphorylation by novel K vitamins

2-(2-Hydroxy-ethylsulfanyl)-3-methyl-1,4-naphthoquinone or CPD-5, a K vitamin analog, was previously indicated to be a potent growth inhibitor for Hep 3B hepatoma cells in vitro. Here, we show that CPD-5 and two newly synthesized analogs, 2-(2-hydroxy-ethylsulfanyl)-3-methyl-5-nitro-1,4-naphthoquinone (PD-37) and 2-(2-hydroxy-ethylsulfanyl)-3-methyl-5-acetylamino-1,4-naphthoquinone (PD-42), are potent growth inhibitors of 13 different human cancer cell lines, with IC50 values in the range of 3-54 μM. Phospho-ERK was induced by each of three K vitamin analogs in every cell line in a dose-dependent manner, at growth inhibitory doses. ERK phosphorylation and growth inhibitory effects were strongly correlated, with p = 0.0080 for CPD-5, p = 0.0076 for PD-37 and p = 0.0251 for PD-42. The induction of phospho-ERK and growth inhibition were antagonized by thiol-containing anti-oxidants, but not by catalase, consistent with a possible arylating mechanism. The data show a novel class of growth inhibitors with a wide spectrum of action that induces ERK hyper-phosphorylation, as a possible new growth inhibitory feature.