Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Amyloid formation and depolymerization of tumor suppressor p16INK4a are regulated by a thiol-dependent redox mechanism.

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

Ascorbate Uptake and Retention by Breast Cancer Cell Lines and the Intracellular Distribution of Sodium-Dependent Vitamin C Transporter 2.

Ascorbate plays a vital role as a co-factor for a superfamily of enzymes, the 2-oxoglutarate dependent dioxygenases (2-OGDDs), which govern numerous pathways in cancer progression, including the hypoxic response and the epigenetic regulation of gene transcription. Ascorbate uptake into most cells is through active transport by the sodium-dependent vitamin C transporter 2 (SVCT2). The aims of this study were to determine the kinetics of ascorbate uptake and retention by breast cancer cell lines under various oxygen conditions, and to investigate the role of SVCT2 in mediating ascorbate uptake and intracellular trafficking. Human MDA-MB231 cells accumulated up to 5.1 nmol ascorbate/106 cells, human MCF7 cells 4.5 nmol/106 cells, and murine EO771 cells 26.7 nmol/106 cells. Intracellular ascorbate concentrations decreased rapidly after reaching maximum levels unless further ascorbate was supplied to the medium, and there was no difference in the rate of ascorbate loss under normoxia or hypoxia. SVCT2 was localised mainly to subcellular compartments, with the nucleus apparently containing the most SVCT2 protein, followed by the mitochondria. Much less SVCT2 staining was observed on the plasma membrane. Our data showed that careful management of the doses and incubation times with ascorbate in vitro allows for an approximation of in vivo conditions. The localisation of SVCT2 suggests that the distribution of ascorbate to intracellular compartments is closely aligned to the known function of ascorbate in supporting 2-OGDD enzymatic functions in the organelles and with supporting antioxidant protection in the mitochondria.

The Role of Physiological Vitamin C Concentrations on Key Functions of Neutrophils Isolated from Healthy Individuals.

Vitamin C (ascorbate) is important for neutrophil function and immune health. Studies showing improved immune function have primarily used cells from scorbutic animals or from individuals with infectious conditions or immune cell disorders. Few studies have focused on the requirements of neutrophils from healthy adults. Therefore, we have investigated the role of vitamin C, at concentrations equivalent to those obtained in plasma from oral intakes (i.e., 50-200 µmol/L), on key functions of neutrophils isolated from healthy individuals. Cells were either pre-loaded with dehydroascorbic acid, which is rapidly reduced intracellularly to ascorbate, or the cells were activated in the presence of extracellular ascorbate. We measured the effects of enhanced ascorbate uptake on the essential functions of chemotaxis, oxidant production, programmed cell death and neutrophil extracellular trap (NET) formation. We found that neutrophils isolated from healthy individuals already had replete ascorbate status (0.35 nmol/106 cells), therefore they did not uptake additional ascorbate. However, they readily took up dehydroascorbic acid, thus significantly increasing their intracellular ascorbate concentrations, although this was found to have no additional effect on superoxide production or chemotaxis. Interestingly, extracellular ascorbate appeared to enhance directional mobilityin the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Stimulation of the cells in the presence of ascorbate significantly increased intracellular ascorbate concentrations and, although this exhibited a non-significant increase in phosphatidylserine exposure, NET formation was significantly attenuated. Our findings demonstrate the ability of neutrophils to regulate their uptake of ascorbate from the plasma of healthy humans to maintain an optimal level within the cell for proper functioning. Higher oral intakes, however, may help reduce tissue damage and inflammatory pathologies associated with NET formation.

Prolonged exposure to hypoxia induces an autophagy-like cell survival program in human neutrophils.

Neutrophils contribute to low oxygen availability at inflammatory sites through the generation of reactive oxidants. They are also functionally affected by hypoxia, which delays neutrophil apoptosis. However, the eventual fate of neutrophils in hypoxic conditions is unknown and this is important for their effective clearance and the resolution of inflammation. We have monitored the survival and function of normal human neutrophils exposed to hypoxia over a 48 h period. Apoptosis was delayed, and the cells remained intact even at 48 h. However, hypoxia promoted significant changes in neutrophil morphology with the appearance of many new cytoplasmic vesicles, often containing cell material, within 5 hours of exposure to low O2 . This coincided with an increase in LC3B-II expression, indicative of autophagosome formation and an autophagy-like process. In hypoxic conditions, neutrophils preferentially lost myeloperoxidase, a marker of azurophil granules. Short-term (2 h) hypoxic exposure resulted in sustained potential to generate superoxide when O2 was restored, but the capacity for oxidant production was lost with longer periods of hypoxia. Phagocytic ability was unchanged by hypoxia, and bacterial killing by neutrophils in both normoxic and hypoxic conditions was substantially diminished after 24 hours. However, pre-exposure to hypoxia resulted in an enhanced ability to kill bacteria by oxidant-independent mechanisms. Our data provide the first evidence for hypoxia as a driver of neutrophil autophagy that can influence the function and ultimate fate of these cells, including their eventual clearance and the resolution of inflammation.

Physiological Concentrations of Blueberry-Derived Phenolic Acids Reduce Monocyte Adhesion to Human Endothelial Cells.

SCOPE: Blueberry polyphenols are thought to confer cardiovascular health benefits, but have limited bioavailability. They undergo extensive metabolism and their phenolic acid metabolites are likely to be the mediators of bioactivity. The effect of blueberry-derived phenolic acids on one aspect of inflammation, monocyte adhesion to vascular endothelial cells, is investigated. METHODS AND RESULTS: The major blueberry-derived phenolic acids in human plasma are identified and quantified. Three test mixtures representing compounds present at 0-4 h (Early), 4-24 h (Late), or 0-24 h (Whole) are used to investigate the effect on adhesion of monocytes to tumor necrosis factor alpha (TNFα)-activated endothelial cells. The Late mixture reduces monocyte adhesion, but there is no effect of the Early or Whole mixtures. Exclusion of syringic acid from each mixture results in inhibition of monocyte adhesion. Exposure to the phenolic acid mixtures has no effect on the endothelial surface expression of adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or E-selectin, suggesting that other molecular mechanisms are responsible for the observed effect. CONCLUSION: This study shows that physiological concentrations of blueberry polyphenol metabolites can help maintain cardiovascular health by regulating monocyte adhesion to the vascular endothelium.

High Vitamin C Status Is Associated with Elevated Mood in Male Tertiary Students.

Micronutrient status is thought to impact on psychological mood due to the role of nutrients in brain structure and function. The aim of the current study was to investigate the association of vitamin C status with mood state in a sample of male tertiary students. We measured fasting plasma vitamin C levels as an indicator of vitamin C status, and subjective mood was determined using the Profile of Mood States (POMS) questionnaire. One hundred and thirty-nine male students aged 18 to 35 years were recruited from local tertiary institutes in Christchurch, New Zealand. The average plasma vitamin C concentration was 58.2 ± 18.6 (SD) µmol/L and the average total mood disturbance score was 25.5 ± 26.6 (possible score -32 to 200 measuring low to high mood disturbance, respectively). Plasma vitamin C concentration was inversely correlated with total mood disturbance as assessed by POMS (r = -0.181, p < 0.05). Examination of the individual POMS subscales also showed inverse associations of vitamin C status with depression, confusion, and anger. These findings suggest that high vitamin C status may be associated with improved overall mood in young adult males.

Bioavailable Blueberry-Derived Phenolic Acids at Physiological Concentrations Enhance Nrf2-Regulated Antioxidant Responses in Human Vascular Endothelial Cells.

SCOPE: Blueberry consumption is believed to confer a cardiovascular health advantage, but the active compounds and effects require characterization. This study aims to identify the polyphenol metabolites in plasma after blueberry juice intake and determine their bioactivity on endothelial cells. METHODS AND RESULTS: Three healthy individuals are recruited to obtain profiles of bioavailable plasma polyphenol metabolites following intake of blueberry juice. Of 33 phenolic compounds screened, 12 aglycone phenolic acids are detected and their maximum plasma concentrations and circulation time determined. Using this information, the effect of three physiologically relevant mixtures of blueberry-derived phenolic acids is investigated for their ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2)-nuclear translocation and downstream gene expression in human endothelial cells. Pretreatment with the phenolic acids for 18 h results in a significant upregulation of the Nrf2-regulated antioxidant response proteins heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), following 6 h exposure to 2.5 µm H2 O2 . CONCLUSION: Physiologically relevant concentrations of blueberry-derived aglycone phenolic acids can induce Nrf2-regulated antioxidant response proteins in vascular endothelial cells in response to low µm concentrations of H2 O2 . Our results represent an advance over previous studies that have used single compounds or high concentrations in cell-based investigations.

Marginal Ascorbate Status (Hypovitaminosis C) Results in an Attenuated Response to Vitamin C Supplementation.

Inadequate dietary intake of vitamin C results in hypovitaminosis C, defined as a plasma ascorbate concentration ≤23 µmol/L. Our objective was to carry out a retrospective analysis of two vitamin C supplementation studies to determine whether supplementation with 50 mg/day vitamin C is sufficient to restore adequate ascorbate status (≥50 µmol/L) in individuals with hypovitaminosis C. Plasma ascorbate data from 70 young adult males, supplemented with 50 or 200 mg/day vitamin C for up to six weeks, was analyzed. Hypovitaminosis C status was identified based on plasma ascorbate being ≤23 µmol/L and the response of these individuals to vitamin C supplementation was examined. Of the participants consuming 50 mg/day vitamin C for up to six weeks, those with hypovitaminosis C at baseline achieved plasma concentrations of only ~30 µmol/L, whereas the remainder reached ~50 µmol/L. Participants who consumed 200 mg/day vitamin C typically reached saturating concentrations (>65 µmol/L) within one week, while those with hypovitaminosis C required two weeks to reach saturation. Regression modelling indicated that the participants' initial ascorbate status and body weight explained ~30% of the variability in the final ascorbate concentration. Overall, our analysis revealed that supplementation with 50 mg/day vitamin C, which resulted in a total dietary vitamin C intake of 75 mg/day, was insufficient to achieve adequate plasma ascorbate concentrations in individuals with hypovitaminosis C. Furthermore, increased body weight had a negative impact on ascorbate status.

Pharmacokinetic and anti-cancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice.

Despite recent evidence for an anti-tumour role for high-dose ascorbate, potential mechanisms of action are still unclear. At mM concentrations that are achieved with high-dose intravenous administration, autoxidation of ascorbate can generate cytotoxic levels of H2O2. Ascorbate is also a required co-factor for the hydroxylases that suppress the transcription factor hypoxia-inducible factor (HIF-1). HIF-1 supports an aggressive tumour phenotype and is associated with poor prognosis, and previous studies have shown that optimizing intracellular ascorbate levels down-regulates HIF-1 activation. In this study we have simultaneously measured ascorbate concentrations and the HIF-1 pathway activity in tumour tissue following high dose ascorbate administration, and have studied tumour growth and physiology. Gulo-/- mice, a model of the human ascorbate dependency condition, were implanted with syngeneic Lewis lung tumours, 1g/kg ascorbate was administered into the peritoneum, and ascorbate concentrations were monitored in plasma, liver and tumours. Ascorbate levels peaked within 30min, and although plasma and liver ascorbate returned to baseline within 16h, tumour levels remained elevated for 48h, possibly reflecting increased stability in the hypoxic tumour environment. The expression of HIF-1 and its target proteins was down-regulated with tumour ascorbate uptake. Elevated tumour ascorbate levels could be maintained with daily administration, and HIF-1 and vascular endothelial growth factor protein levels were reduced in these conditions. Increased tumour ascorbate was associated with slowed tumour growth, reduced tumour microvessel density and decreased hypoxia. Alternate day administration of ascorbate resulted in lower tumour levels and did not consistently decrease HIF-1 pathway activity. Levels of sodium-dependent vitamin C transporters 1 and 2 were not clearly associated with ascorbate accumulation by murine tumour cells in vitro or in vivo. Our results support the suppression of the hypoxic response by ascorbate as a plausible mechanism of action of its anti-tumour activity, and this may be useful in a clinical setting.

Enhanced human neutrophil vitamin C status, chemotaxis and oxidant generation following dietary supplementation with vitamin C-rich SunGold kiwifruit.

Neutrophils are the body's primary defenders against invading pathogens. These cells migrate to loci of infection where they engulf micro-organisms and subject them to an array of reactive oxygen species and antimicrobial proteins to effect killing. Spent neutrophils subsequently undergo apoptosis and are cleared by macrophages, thereby resolving the inflammatory episode. Neutrophils contain high concentrations of vitamin C (ascorbate) and this is thought to be essential for their function. This may be one mechanism whereby vitamin C enhances immune function. The aim of our study was to assess the effect of dietary supplementation with vitamin C-rich SunGold kiwifruit on four important functions of neutrophils: chemotaxis, oxidant generation, extracellular trap formation, and apoptosis. Fourteen young men (aged 18-30 years) with suboptimal plasma vitamin C status (<50 µmol/L) were supplemented for four weeks with two SunGold kiwifruit/day. Plasma vitamin C status was monitored weekly and neutrophil vitamin C levels were assessed at baseline and post-intervention. Neutrophil function assays were carried out on cells isolated at baseline and post-intervention. Plasma vitamin C levels increased to >70 µmol/L (p < 0.001) within one week of supplementation and there was a significant increase in neutrophil vitamin C status following four weeks' intervention (p = 0.016). We observed a significant 20% increase in neutrophil chemotaxis post-intervention (p = 0.041) and also a comparable increase in oxidant generation (p = 0.031). Supplementation did not affect neutrophil extracellular trap formation or spontaneous apoptosis. Our data indicate that supplementation with vitamin C-rich kiwifruit is associated with improvement of important neutrophil functions, which would be expected to translate into enhanced immunity.

A randomised cross-over pharmacokinetic bioavailability study of synthetic versus kiwifruit-derived vitamin C.

Kiwifruit are a rich source of vitamin C and also contain numerous phytochemicals, such as flavonoids, which may influence the bioavailability of kiwifruit-derived vitamin C. The aim of this study was to compare the relative bioavailability of synthetic versus kiwifruit-derived vitamin C using a randomised cross-over pharmacokinetic study design. Nine non-smoking males (aged 18-35 years) received either a chewable tablet (200 mg vitamin C) or the equivalent dose from gold kiwifruit (Actinidia chinensis var. Sungold). Fasting blood and urine were collected half hourly to hourly over the eight hours following intervention. The ascorbate content of the plasma and urine was determined using HPLC with electrochemical detection. Plasma ascorbate levels increased from 0.5 h after the intervention (P = 0.008). No significant differences in the plasma time-concentration curves were observed between the two interventions (P = 0.645). An estimate of the total increase in plasma ascorbate indicated complete uptake of the ingested vitamin C tablet and kiwifruit-derived vitamin C. There was an increase in urinary ascorbate excretion, relative to urinary creatinine, from two hours post intervention (P < 0.001). There was also a significant difference between the two interventions, with enhanced ascorbate excretion observed in the kiwifruit group (P = 0.016). Urinary excretion was calculated as ~40% and ~50% of the ingested dose from the vitamin C tablet and kiwifruit arms, respectively. Overall, our pharmacokinetic study has shown comparable relative bioavailability of kiwifruit-derived vitamin C and synthetic vitamin C.

The bioavailability of vitamin C from kiwifruit.

Vitamin C is an essential component of the diet for humans, and an adequate intake is important not only for the prevention of scurvy but also to limit the risk of developing chronic diseases such as heart disease and cancer. To achieve a regular and adequate intake, daily consumption of fresh fruit and vegetables is recommended. The vitamin C content of food varies widely, however, and plasma levels generally reflect the amount consumed, regardless of its origin. Kiwifruit are one of the premier dietary sources of vitamin C, with all commercially important varieties having high content, and with one serving delivering the bulk of the recommended dietary intake. Recent studies have shown that the addition of kiwifruit to a marginal vitamin C diet markedly improves plasma vitamin C levels and can increase them to both healthy and optimal levels.

Human skeletal muscle ascorbate is highly responsive to changes in vitamin C intake and plasma concentrations.

BACKGROUND: Vitamin C (ascorbate) is likely to be essential for skeletal muscle structure and function via its role as an enzyme cofactor for collagen and carnitine biosynthesis. Vitamin C may also protect these metabolically active cells from oxidative stress. OBJECTIVE: We investigated the bioavailability of vitamin C to human skeletal muscle in relation to dietary intake and plasma concentrations and compared this relation with ascorbate uptake by leukocytes. DESIGN: Thirty-six nonsmoking men were randomly assigned to receive 6 wk of 0.5 or 2 kiwifruit/d, an outstanding dietary source of vitamin C. Fasting blood samples were drawn weekly, and 24-h urine and leukocyte samples were collected before intervention, after intervention, and after washout. Needle biopsies of skeletal muscle (vastus lateralis) were carried out before and after intervention. RESULTS: Baseline vastus lateralis ascorbate concentrations were ~16 nmol/g tissue. After intervention with 0.5 or 2 kiwifruit/d, these concentrations increased ~3.5-fold to 53 and 61 nmol/g, respectively. There was no significant difference between the responses of the 2 groups. Mononuclear cell and neutrophil ascorbate concentrations increased only ~1.5- and ~2-fold, respectively. Muscle ascorbate concentrations were highly correlated (P < 0.001) with dietary intake (R = 0.61) and plasma concentrations (R = 0.75) in the range from 5 to 80 µmol/L. CONCLUSIONS: Human skeletal muscle is highly responsive to vitamin C intake and plasma concentrations and exhibits a greater relative uptake of ascorbate than leukocytes. Thus, muscle appears to comprise a relatively labile pool of ascorbate and is likely to be prone to ascorbate depletion with inadequate dietary intake. This trial was registered at the Australian New Zealand Clinical Trials Registry (www.anzctr.org.au) as ACTRN12611000162910.

Mood improvement in young adult males following supplementation with gold kiwifruit, a high-vitamin C food.

Enhanced intakes of fruit and vegetables have been associated with improved psychological well-being. We investigated the potential mood-enhancing effects of kiwifruit, a fruit rich in vitamin C and a number of other important micronutrients. Young adult males (n 35) were supplemented with either half or two kiwifruit/d for 6 weeks. Profile of Mood States questionnaires were completed at baseline and following the intervention. No effect on overall mood was observed in the half a kiwifruit/d group; however, a 35 % (P = 0·06) trend towards a decrease in total mood disturbance and a 32 % (P = 0·063) trend towards a decrease in depression were observed in the two kiwifruit/d group. Subgroup analysis indicated that participants with higher baseline mood disturbance exhibited a significant 38 % (P = 0·029) decrease in total mood disturbance, as well as a 38 % (P = 0·048) decrease in fatigue, 31 % (P = 0·024) increase in vigour and a 34 % (P = 0·075) trend towards a decrease in depression, following supplementation with two kiwifruit/d. There was no effect of two kiwifruit/d on the mood scores of participants with lower baseline mood disturbance. Dietary intakes and body status of specific micronutrients indicated a significant increase in the participants' vitamin C intakes and corresponding plasma levels of the vitamin. The results indicate that enhanced intake of kiwifruit by individuals with moderate mood disturbance can improve overall mood.

A randomized steady-state bioavailability study of synthetic versus natural (kiwifruit-derived) vitamin C.

Whether vitamin C from wholefoods has equivalent bioavailability to a purified supplement remains unclear. We have previously showed that kiwifruit provided significantly higher serum and tissue ascorbate levels than synthetic vitamin C in a genetically vitamin C-deficient mouse model, suggesting a synergistic activity of the whole fruit. To determine if these results are translatable to humans, we carried out a randomized human study comparing the bioavailability of vitamin C from kiwifruit with that of a vitamin C tablet of equivalent dosage. Thirty-six young non-smoking adult males were randomized to receive either half a gold kiwifruit (Actinidia Chinensis var. Hort 16A) per day or a comparable vitamin C dose (50 mg) in a chewable tablet for six weeks. Ascorbate was monitored weekly in fasting venous blood and in urine, semen, leukocytes, and skeletal muscle (vastus lateralis) pre- and post-intervention. Dietary intake of vitamin C was monitored using seven day food and beverage records. Participant ascorbate levels increased in plasma (P < 0.001), urine (P < 0.05), mononuclear cells (P < 0.01), neutrophils (P < 0.01) and muscle tissue (P < 0.001) post intervention. There were no significant differences in vitamin C bioavailability between the two intervention groups in any of the fluid, cell or tissue samples tested. Overall, our study showed comparable bioavailability of synthetic and kiwifruit-derived vitamin C.

Dietary ascorbate intake affects steady state tissue concentrations in vitamin C-deficient mice: tissue deficiency after suboptimal intake and superior bioavailability from a food source (kiwifruit).

BACKGROUND: Humans acquire vitamin C (ascorbate) from their diet, and optimal tissue concentrations are required to maintain its enzyme cofactor and antioxidant activities. How dietary intake affects tissue concentrations is difficult to monitor and has generally been based on the measurement of plasma concentrations. OBJECTIVE: We aimed to determine the effect of various ascorbate intakes on tissue concentrations in the Gulo mouse model of vitamin C deficiency and to compare the effectiveness of delivery when ascorbate was added to the drinking water or obtained through a fruit source (kiwifruit). DESIGN: Gulo(-/-) mice were fed various amounts of ascorbate for 1 mo, either in their drinking water or as a kiwifruit gel. Tissue vitamin C content was measured and compared with concentrations in wild-type mice. RESULTS: Ascorbate concentrations in serum, liver, kidney, heart, and white blood cells were extremely labile and were well below concentrations observed in the wild-type mice when serum concentrations were below saturation. All tissues except for brain were rapidly depleted when intake was stopped. Consumption of a preparation of fresh kiwifruit (either green or gold varieties) resulted in up to 5 times more effective delivery to tissues than when ascorbate was administered via the drinking water. CONCLUSIONS: Subsaturation concentrations of plasma ascorbate resulted in severe deficiency in many tissues, and saturating amounts were required to achieve tissue concentrations similar to those found in wild-type animals. It is possible that the bioavailability of ascorbate is superior from some foods, such as kiwifruit. These results have important implications for human nutrition.

Hypothiocyanous acid is a potent inhibitor of apoptosis and caspase 3 activation in endothelial cells.

Hypothiocyanous acid (HOSCN) is a common, thiol-specific oxidant with strong antibacterial activity. It is thought to be nontoxic to mammalian cells, although its ability to specifically target intracellular thiols may potentially cause cellular dysfunction. In this study we demonstrate specific effects of HOSCN on human endothelial cells, with exposure to high concentrations resulting in morphology changes unlike those seen with other oxidants. Effects were time- and dose-dependent and were accompanied by loss of total cell thiols and GSH and by inactivation of glyceraldehyde-3-phosphate dehydrogenase. High-dose exposure was cytotoxic, but lesser doses did not cause cell death, and apoptosis was not initiated by any concentration of HOSCN. In fact, initiation of apoptosis was blocked by minimal HOSCN exposure, with activation of caspase 3 and cleavage of the proenzyme being prevented. This was unlikely to be due to direct oxidation of the caspase 3 active-site cysteine and suggests alternative targeting of the caspase pathway. The survival of endothelial cells when HOSCN is present together with an inducer of apoptosis suggests that HOSCN differs from most other oxidants and could affect endothelial cell survival pathways in a way that may have an impact on vascular function.

Oxidation of a eukaryotic 2-Cys peroxiredoxin is a molecular switch controlling the transcriptional response to increasing levels of hydrogen peroxide.

Although activation of the AP-1-like transcription factor Pap1 in Schizosaccharomyces pombe is important for oxidative stress-induced gene expression, this activation is delayed at higher concentrations of peroxide. Here, we reveal that the 2-Cys peroxiredoxin (2-Cys Prx) Tpx1 is required for the peroxide-induced activation of Pap1. Tpx1, like other eukaryotic 2-Cys Prxs, is highly sensitive to oxidation, which inactivates its thioredoxin peroxidase activity. Our data suggest that the reduced thioredoxin peroxidase-active form of Tpx1 is required for the peroxide-induced oxidation and nuclear accumulation of Pap1. Indeed, in contrast to the previously described role for Tpx1 in the activation of the Sty1 stress-activated protein kinase by peroxide, we find that both catalytic cysteines of Tpx1 are required for Pap1 activation. Moreover, overexpression of the conserved sulfiredoxin Srx1, which interacts with and reduces Tpx1, allows rapid activation of Pap1 at higher concentrations of H(2)O(2). Conversely, loss of Srx1 prevents the reduction of oxidized Tpx1 and prolongs the inhibition of Pap1 activation. Collectively, these data suggest that redox regulation of the thioredoxin peroxidase activity of Tpx1 acts as a molecular switch controlling the transcriptional response to H(2)O(2). Furthermore, they reveal that a single eukaryotic 2-Cys Prx regulates peroxide signaling by multiple independent mechanisms.

A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase.

Oxidative stress-induced cell damage is an important component of many diseases and ageing. In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress. 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress. Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1. Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1. We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation. Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress.