Christina Dunster

Changes in Circulatory Antioxidant Status in Horses during Prolonged Exercise

Prolonged low-medium intensity exercise is associated with increased oxidative stress in humans. We hypothesized that competitive equine endurance racing would induce changes in circulatory antioxidants and produce systemic oxidative stress. Forty horses competing in a 140-km endurance race in warm conditions [shade temperature 15-19 degrees C; 62-88% relative humidity (%RH)] were sampled before (Pre), immediately after exercise (End) and at approximately 16 h into recovery (+16 h). Plasma ascorbic acid concentration was not different between Pre [11.1 (median); 4.6-20.3 micromol/L (range)] and End [9.7; 3.0-38.9 (range) micromol/L] but was significantly decreased at +16 h (5.5; 2.8-15.5 micromol/L; P < 0.05). Total red cell hemolysate glutathione (TGSH) concentration was significantly reduced by exercise (Pre 1261; 883-1532 micromol/L; End 1065; 757-1334 micromol/L; P < 0.05) and at +16 h recovery (1032; 752-1362 micromol/L; P < 0.05). Glutathione redox ratio was unchanged by exercise but was significantly decreased at +16 h compared with that at both Pre and End (P < 0.05). The concentration of total barbituric acid reactive substances (TBARS) in plasma was increased compared with that at Pre (309; 66-1048 nmol/L), both at End (408; 170-1196 nmol/L; P < 0.05) and +16 h (380; 99-1161 nmol/L; P < 0.05). alpha-Tocopherol was unchanged by exercise or recovery. Mean race speed was 16.5 +/- 1.6 km/h and ranged from 13.9 to 19.7 km/h. Mean speed during competition in horses that completed the full 140 km (n = 28) was significantly correlated with end of exercise ascorbic acid (r = 0.622; P = 0.0004). Although there were increases in creatine phosphokinase (CK), aspartate aminotransferase (AST) and TBARS and a loss of TGSH, this study failed to demonstrate evidence of classical oxidative stress.

Air pollution and the elderly: oxidant/antioxidant issues worth consideration

The elderly are frequently classified as being particularly susceptible to air pollution. The basis of this increased sensitivity is not known but it is likely that it is linked to age-related impaired function of the lung. However, given this uncertainty and increased impact of air pollution of this section of the community there is a need to explore the mechanisms involved. Gaseous air pollutants such as ozone and nitrogen dioxide, or many of the components adsorbed onto the surface of respirable particles, are either powerful oxidants or capable of generating free radicals. If unabated, oxidants can cause injury to the delicate cells that line the respiratory tract. Small molecular weight antioxidant defences present in respiratory tract lining fluid (RTLF) represent the first line of defence against a range of oxidants that enter the lung. The quantity and quality of the RTLF airways antioxidant network are, therefore, likely to be important determinants of the impact of the oxidant challenge on the underlying respiratory epithelium. As yet, comprehensive information on the distribution and variability of respiratory tract lining fluid antioxidant defences is only available in young, mainly healthy volunteers. Studies undertaken in patients with a range of respiratory diseases suggest that marked changes in the distribution of respiratory tract lining fluid antioxidants can occur. Information is not currently available on the impact of ageing on the respiratory tract lining fluid antioxidant defence network. As several respiratory tract lining fluid antioxidants are of dietary origin, the elderly, who often have different dietary patterns to younger individuals, may have decreased availability of important antioxidants. Given these possibilities, a better understanding of respiratory tract lining fluid antioxidant defences in the aged lung is warranted.

Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of α- and γ-tocopherol in human plasma

Alpha- and gamma-tocopherol (alpha- and gamma-T, respectively) metabolite analysis is of key relevance in the study of vitamin E metabolism. Whilst there is information on urinary excretion of the two major metabolites of these vitamin E homologues, namely the 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), their concentration and response to supplements in plasma remains poorly investigated. In this study we describe a gas chromatography-mass spectrometry (GC/MS)-based assay to measure both alpha- and gamma-T and their corresponding CEHC metabolites in human plasma. As an example of the application of this method we report data obtained following the supplemention of two healthy volunteers with 100 mg of deuterium-labeled gamma-T acetate (d(2)-gamma-TAC). Under routine analytical conditions a good linearity in the range 0.0025--1 microM was observed for both the alpha- and gamma-CEHC deuterated standards. In plasma samples, the detection limit for alpha- and gamma-CEHC was 2.5 and 5 nmol/l, respectively. The minimum amount of plasma required for the assay was 500 microl. The plasma concentrations of alpha-CEHC and gamma-CEHC in unsupplemented healthy subjects were 12.6 +/-7.5 and 160.7 +/- 44.9 nmol/l, respectively. In the two volunteers supplemented with 100 mg of d(2)-gamma-TAC, plasma d(2)-gamma-T concentrations increased 250 to 450-fold 6 h postsupplementation. Plasma and urinary d(2)-gamma-CEHC concentrations increased 20 to 40-fold 9--12 h postsupplementation. Interestingly, the acute increase in d(2) gamma-T did not significantly affect the baseline plasma concentrations of d(0)-gamma-T and only slight lowered alpha-T concentrations. Likewise, plasma alpha-CEHC levels were not influenced and urinary excretion of alpha-CEHC were unaltered. This GC/MS method provides a versatile and accurate mean for assessing carboxyethyl-hydroxychroman metabolites of vitamin E in plasma.

Increased Oxidative Burden Associated with Traffic Component of Ambient Particulate Matter at Roadside and Urban Background Schools Sites in London

As the incidence of respiratory and allergic symptoms has been reported to be increased in children attending schools in close proximity to busy roads, it was hypothesised that PM from roadside schools would display enhanced oxidative potential (OP). Two consecutive one-week air quality monitoring campaigns were conducted at seven school sampling sites, reflecting roadside and urban background in London. Chemical characteristics of size fractionated particulate matter (PM) samples were related to the capacity to drive biological oxidation reactions in a synthetic respiratory tract lining fluid. Contrary to hypothesised contrasts in particulate OP between school site types, no robust size-fractionated differences in OP were identified due high temporal variability in concentrations of PM components over the one-week sampling campaigns. For OP assessed both by ascorbate (OPAA m−3) and glutathione (OPGSH m−3) depletion, the highest OP per cubic metre of air was in the largest size fraction, PM1.9–10.2. However, when expressed per unit mass of particles OPAA µg−1 showed no significant dependence upon particle size, while OPGSH µg−1 had a tendency to increase with increasing particle size, paralleling increased concentrations of Fe, Ba and Cu. The two OP metrics were not significantly correlated with one another, suggesting that the glutathione and ascorbate depletion assays respond to different components of the particles. Ascorbate depletion per unit mass did not show the same dependence as for GSH and it is possible that other trace metals (Zn, Ni, V) or organic components which are enriched in the finer particle fractions, or the greater surface area of smaller particles, counter-balance the redox activity of Fe, Ba and Cu in the coarse particles. Further work with longer-term sampling and a larger suite of analytes is advised in order to better elucidate the determinants of oxidative potential, and to fuller explore the contrasts between site types.

Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats.

•  High‐altitude pregnancy is associated with reduced oxygenation and placental complications, which can affect maternal and fetal outcome. However, most high‐altitude populations are also impoverished and because maternal undernutrition itself is known to promote placental problems, the extent to which complications during high‐altitude pregnancy could be due to maternal oxygen and/or nutrient restriction remains unclear. •  The aim of the study was to investigate whether reduced placental oxygenation, independent of maternal undernutrition, increases maternal and placental oxidative stress and whether maternal treatment with vitamin C is protective. •  The study shows that hypoxic pregnancy increased maternal circulating and placental molecular indices of oxidative stress. •  Maternal vitamin C treatment was protective and increased birth weight. •  The study offers insight to mechanism and intervention against the effects of high altitude on pregnancy.

Vitamin C Protects Against Hypochlorous Acid–Induced Glutathione Depletion and DNA Base and Protein Damage in Human Vascular Smooth Muscle Cells

Hypochlorous acid (HOCl), generated by myeloperoxidase released from activated macrophages, is thought to contribute to vascular dysfunction and oxidation of low density lipoproteins (LDLs) in atherogenesis. We have previously shown that HOCl exposure can cause chlorination and oxidation of isolated DNA and that vitamin C protects human arterial smooth muscle cells against oxidized LDL–mediated damage. We report in the present study that vitamin C attenuates HOCl-induced DNA base and protein damage and depletion of intracellular glutathione (GSH) and ATP in human arterial smooth muscle cells. Cells were pretreated in the absence or presence of 100 &mgr;mol/L vitamin C (24 hours) and then exposed to HOCl (0 to 500 &mgr;mol/L, 0 to 60 minutes) in the absence of vitamin C. Intracellular GSH and ATP levels were depleted by HOCl treatment, and gas chromatography–mass spectroscopy revealed a concentration- and time-dependent increase in DNA base oxidation and protein damage (measured as 3-chlorotyrosine). Pretreatment of smooth muscle cells with vitamin C significantly reduced the extent of HOCl-induced DNA and protein damage and attenuated decreases in intracellular ATP and GSH. Our findings suggest that physiological levels of vitamin C provide an important antioxidant defense against HOCl-mediated injury in atherosclerosis.

In vivo prooxidant state in werner syndrome (WS) : Results from three WS patients and two WS heterozygotes

The hypothesis was tested that Werner syndrome (WS) phenotype might be associated with an in vivo prooxidant state. A set of redox-related endpoints were measured in three WS patients, two of their parents, and 99 controls within a study of some cancer-prone and/or ageing-related genetic disorders. The following analytes were measured: (a) leukocyte 8-hydroxy-2′-deoxyguanosine; (b) glutathione from whole blood, and (c) plasma levels of glyoxal, methylglyoxal, 8-isoprostane, and some plasma antioxidants (uric acid, ascorbic acid, α- and γ-tocopherol). Leukocyte 8-hydroxy-2′-deoxyguanosine levels showed a significant increase in the 3 WS patients vs. 85 controls (p<10−7). The disulfide glutathione:glutahione ratio was significantly altered in WS patients (p=0.005). Glyoxal and methylglyoxal levels were significantly increased (p=0.018 and p=0.007, respectively). The plasma levels of uric acid (p=0.002) and ascorbic acid (p=0.003) were also increased significantly in WS patients and in their parents. No significant alterations were found in the plasma levels of α- and γ-tocopherol, nor of 8-isoprostane. This is the first report of in vivo alterations of oxidative stress parameters in WS patients. Further investigations on more extensive study populations are warranted to verify the relevance of an in vivo prooxidant state in WS patients.

Physicochemical and toxicological profiling of ash from the 2010 and 2011 eruptions of Eyjafjallajökull and Grímsvötn volcanoes, Iceland using a rapid respiratory hazard assessment protocol.

The six week eruption of Eyjafjallajökull volcano in 2010 produced heavy ash fall in a sparsely populated area of southern and south eastern Iceland and disrupted European commercial flights for at least 6 days. We adopted a protocol for the rapid analysis of volcanic ash particles, for the purpose of informing respiratory health risk assessments. Ash collected from deposits underwent a multi-laboratory physicochemical and toxicological investigation of their mineralogical parameters associated with bio-reactivity, and selected in vitro toxicology assays related to pulmonary inflammatory responses. Ash from the eruption of Grímsvötn, Iceland, in 2011 was also studied. The results were benchmarked against ash from Soufrière Hills volcano, Montserrat, which has been extensively studied since the onset of eruptive activity in 1995. For Eyjafjallajökull, the grain size distributions were variable: 2-13 vol% of the bulk samples were <4 µm, with the most explosive phases of the eruption generating abundant respirable particulate matter. In contrast, the Grímsvötn ash was almost uniformly coarse (<3.5 vol%<4 µm material). Surface area ranged from 0.3 to 7.7 m2 g(-1) for Eyjafjallajökull but was very low for Grímsvötn (<0.6 m2 g(-1)). There were few fibre-like particles (which were unrelated to asbestos) and the crystalline silica content was negligible in both eruptions, whereas Soufrière Hills ash was cristobalite-rich with a known potential to cause silicosis. All samples displayed a low ability to deplete lung antioxidant defences, showed little haemolysis and low acute cytotoxicity in human alveolar type-1 like epithelial cells (TT1). However, cell-free tests showed substantial hydroxyl radical generation in the presence of hydrogen peroxide for Grímsvötn samples, as expected for basaltic, Fe-rich ash. Cellular mediators MCP-1, IL-6, and IL-8 showed chronic pro-inflammatory responses in Eyjafjallajökull, Grímsvötn and Soufrière Hills samples, despite substantial differences in the sample mineralogy and eruptive styles. The value of the pro-inflammatory profiles in differentiating the potential respiratory health hazard of volcanic ashes remains uncertain in a protocol designed to inform public health risk assessment, and further research on their role in volcanic crises is warranted.

Redox modulation of the fetal cardiovascular defence to hypoxaemia

Episodes of hypoxia in utero present a potentially serious challenge to the fetus, but are counteracted by defence responses including marked redistribution of blood flow from peripheral circulations to the brain. Here, we report the novel observation that the oxidant tone is an important modulator of this cardiovascular defence. Using pregnant Welsh Mountain sheep surgically prepared for long‐term recording, we investigated in vivo the effects on the fetal cardiovascular defence to acute hypoxaemia of fetal treatment with the antioxidant vitamin C. The mechanisms via which vitamin C may affect the vascular oxidant tone were investigated by monitoring fetal plasma concentrations of nitrates and nitrites, by determining changes in the activity of superoxide dismutase (SOD) in fetal plasma, and by investigating the effect of vitamin C treatment on the fetal cardiovascular defence to hypoxaemia following nitric oxide (NO) synthase blockade. Fetal treatment with vitamin C markedly depressed the normal femoral constrictor response to acute hypoxaemia in the fetus (5.2 ± 1.0 vs. 1.1 ± 0.3 mmHg (ml min−1)−1, mean ±s.e.m.; P < 0.05) an effect which was completely restored following NO synthase blockade (6.2 ± 1.3 mmHg (ml min−1)−1). Compared to saline infusion, fetal treatment with vitamin C during acute hypoxaemia also significantly increased fetal plasma SOD activity from normoxic baseline (−8.9 ± 6.5 vs. 15.0 ± 6.6% inhibition, P < 0.05) and decreased the plasma concentration ratio of nitrate:nitrite from normoxic baseline (ΔNO3−:NO2−; 0.15 ± 0.30 vs. −0.29 ± 0.11, P < 0.05). The data provide in vivo evidence of redox modulation of redistribution of blood flow in the fetus, part of the fetal brain sparing during acute hypoxaemic stress.

Oxidative potential of smoke from burning wood and mixed biomass fuels

Abstract More than half the worlds population still rely on burning biomass fuels to heat and light their homes and cook food. Household air pollution, a common component of which is inhalable particulate matter (PM), emitted from biomass burning is associated with increased vulnerability to respiratory infection and an enhanced risk of developing chronic obstructive pulmonary disease. In the light of an emerging hypothesis linking chronic PM exposure during childhood and increased vulnerability to respiratory diseases in adulthood, in a chain of events involving oxidative stress, reduced immunity and subsequent infection, the aim of this study was to characterise the oxidative potential (OP) of PM collected during the burning of wood and mixed biomass, whilst cooking food in the Kathmandu Valley, Nepal. Our assessments were based on the capacity of the particles to deplete the physiologically relevant antioxidants from a validated, synthetic respiratory tract lining fluid (RTLF). Incubation of mixed biomass and wood smoke particles suspensions with the synthetic RTLF for 4 h resulted in a mean loss of ascorbate of 64.76 ± 16.83% and 83.37 ± 14.12% at 50 μg/ml, respectively. Reduced glutathione was depleted by 49.29 ± 15.22% in mixed biomass and 65.33 ± 13.01% in wood smoke particles under the same conditions. Co-incubation with the transition metal chelator diethylenetriaminepentaacetate did not inhibit the rate of ascorbate oxidation, indicating a negligible contribution by redox-active metals in these samples. The capacity of biomass smoke particles to elicit oxidative stress certainly has the potential to contribute towards negative health impacts associated with traditional domestic fuels in the developing world.