Juliet M. Pullar

Living with a killer: the effects of hypochlorous acid on mammalian cells.

The production of hypochlorous acid (HOCl) by the myeloperoxidase‐H2O2‐Cl system of phagocytes plays a vital role in the ability of these cells to kill a wide range of pathogens. However, the generation of a potent oxidant is not without risk to the host, and there is evidence that HOCl contributes to the tissue injury associated with inflammation. In this review, we discuss the biological reactivity of HOCl, and detail what is known of how it interacts with mammalian cells. The outcome of exposure is dependent on the dose of oxidant, with higher doses causing necrosis, and apoptosis or growth arrest occurring with lower amounts. Glutathione (GSH) and protein thiols are easily oxidized, and are preferred targets with low, sublethal amounts of HOCl. Thiol enzymes vary in their sensitivity to HOCl, with glyceraldehyde‐3‐phosphate dehydrogenase being most susceptible. Indeed, loss of activity occurred before GSH oxidation. The products of these reactions and the ability of cells to regenerate oxidized thiols are discussed. Recent reports have indicated that HOCl can activate cell signaling pathways, and these studies may provide important information on the role of this oxidant in inflammation.

Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins

The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.

Oxidation of mitochondrial peroxiredoxin 3 during the initiation of receptor-mediated apoptosis

It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-alpha. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.

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.

Phenethyl Isothiocyanate Triggers Apoptosis in Jurkat Cells Made Resistant by the Overexpression of Bcl-2

Isothiocyanates are a class of naturally occurring chemopreventive agents known to be effective at triggering apoptosis. In this study, we show that whereas overexpression of the oncoprotein Bcl-2 renders Jurkat T-lymphoma cells resistant to a range of cytotoxic agents, phenethyl isothiocyanate is able to overcome the inhibitory action of Bcl-2 and trigger apoptosis. A 50-fold increase in Bcl-2 expression shifted the dose-response curve, with an increase in the phenethyl isothiocyanate LD(50) from 7 to 15 micromol/L, but there was still a complete loss in cell viability at doses in excess of 20 micromol/L. At these concentrations, cytotoxicity was strongly associated with caspase activation, phosphatidylserine exposure, and morphologic changes characteristic of apoptosis. Cytotoxicity was inhibited by treatment of the cells with a broad-spectrum caspase inhibitor. A structure-activity analysis showed that the phenethyl and benzyl isothiocyanates were most effective at triggering apoptosis in cells overexpressing Bcl-2 whereas phenyl isothiocyanate and benzyl thiocyanate had no proapoptotic activity. Allyl isothiocyanate also had limited efficacy despite its ability to trigger apoptosis in the parental Jurkat cell line. From this information, we propose that isothiocyanates modify a key cysteine residue in an apoptosis regulatory protein and that the aromatic side chain facilitates access to the target site. An in-depth investigation of the cellular targets of the aromatic isothiocyanates is warranted.

Bioavailability of vitamin C from kiwifruit in non-smoking males: determination of 'healthy' and 'optimal' intakes

Vitamin C is an essential nutrient in humans and must be obtained through the diet. The aim of this study was to determine vitamin C uptake in healthy volunteers after consuming kiwifruit (Actinidia chinensis var. Hort. 16A), and to determine the amount of fruit required to raise plasma vitamin C to ‘healthy’ (i.e. >50 µmol/l) and ‘optimal’ or saturating levels (i.e. >70 µmol/l). Leucocyte and urinary vitamin C levels were also determined. A total of fifteen male university students with below average levels of plasma vitamin C were selected for the study. Weekly fasting blood samples were obtained for a 4-week lead-in period and following supplementation with, sequentially, half, one, two and three Gold kiwifruit per d for 4–6 weeks each, followed by a final 4-week washout period. The results showed that addition of as little as half a kiwifruit per d resulted in a significant increase in plasma vitamin C. However, one kiwifruit per d was required to reach what is considered healthy levels. Increasing the dose of kiwifruit to two per d resulted in further increases in plasma vitamin C levels as well as increased urinary output of the vitamin, indicating that plasma levels were saturating at this dosage. Dividing the participants into high and low vitamin C groups based on their baseline plasma and leucocyte vitamin C levels demonstrated that it is critical to obtain a study population with low initial levels of the vitamin in order to ascertain a consistent effect of supplementation.

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.

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.

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.

The Effect of Hypochlorous Acid on the Expression of Adhesion Molecules and Activation of NF-κB in Cultured Human Endothelial Cells

Exposure to oxidants can up-regulate the expression of adhesion molecules in endothelial cells with a consequent increase in neutrophil attachment. Similarly, the transcription factor nuclear factor-kappaB (NF-kappaB), which controls the expression of the intercellular adhesion molecules (ICAMs), can also be activated by oxidants in some cells. We have investigated whether hypochlorous acid (HOCl), the major strong oxidant produced by neutrophils, can affect the expression of adhesion molecules on human umbilical vein endothelial cells (HUVEC) and promote neutrophil adhesion. We found that HOCl could induce an increase in neutrophil adhesion to the endothelial cells after 60 min of treatment. Activation of NF-kappaB could be detected under similar conditions. However, the dose of HOCl required for this effect resulted in considerable longer-term toxicity to the cells. Treatment of HUVEC with sublethal doses of HOCl had no effect on NF-kappaB activation, neutrophil adhesion, or the surface expression of E-selectin, ICAM-1, or P-selectin. However, pretreatment with low concentrations of HOCl prevented phorbol myristate acetate-induced von Willebrand factor expression (a marker for P-selectin). These results show that, unlike H(2)O(2), HOCl does not significantly enhance neutrophil attachment to the endothelium. Rather it may be able to inhibit the expression of adhesion molecules with important consequences for endothelial function and inflammatory vascular disease.