Nicola C. Smith

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.

Effects of exercise intensity and environmental stress on indices of oxidative stress and iron homeostasis during exercise in the horse.

The effects of prolonged variable-intensity and short-term high-intensity exercise on indices of oxidative stress and iron homeostasis were compared in six fit horses under cool [20°C, 40% relative humidity (RH)] or hot/humid (30°C, 80% RH) environmental conditions. The exercise protocols were designed to simulate equine competition, including racing (intense exercise) or the speed and endurance phase of a 3-day event (prolonged exercise). Increased plasma concentrations of lipid hydroperoxides and haemolysate concentrations of oxidised glutathione (GSSG) were measured within 30 min of the completion of exercise, indicating production of reactive oxygen species (ROS) and lipid membrane peroxidation. The horses were unable to complete the prolonged exercise protocol at high temperature and humidity. This coincided with higher maximal values of lipid hydroperoxides [138.2 (17.7) μM and GSSG [110.6 (18.2) μM], compared to high-intensity [105.2 (14.9) μM and 63.6 (8.6) μM, respectively] or prolonged [100.7 (18.7) μM and 86.2 (9.1) μM, respectively] exercise performed under cooler environmental conditions. Significant correlations were found between the duration of the final stage of exercise during hot/humid environmental conditions and increased levels of lipid hydroperoxides (r = 0.85), GSSG (r = 0.94), xanthine (r = 0.92) and uric acid (r = 0.96). Excerise also decreased the iron (Fe)-binding antioxidant activity of the plasma and increased the total plasma Fe levels, although this was only significant for prolonged exercise in ambient conditions. There was no detectable free Fe in the plasma at any stage of exercise. Other changes in biochemical parameters had returned to pre-exercise levels within 24 h after exercise. The results show that exercise can induce changes in biochemical parameters that are indicative of oxidative stress in the fit horse and that this was, exacerbated during exercise at high temperature and humidity.

Effect of nutritional antioxidant supplementation on systemic and pulmonary antioxidant status, airway inflammation and lung function in heaves-affected horses

An oxidant/antioxidant imbalance in favour of oxidants has been identified as playing a decisive role in the pathogenesis of chronic inflammatory airway diseases. Nutritional antioxidant supplementation might reduce oxidative damage by enhancement of the antioxidant defence, thereby modulating inflammatory processes. In a placebo-controlled, blind study, it was tested whether a dietary antioxidant supplement administered for 4 weeks would improve lung function and reduce airway inflammation in heaves-affected horses. Eight horses in clinical remission of heaves were investigated at rest and after a standardised exercise test before and after treatment with an antioxidant supplement (consisting of a mixture of natural antioxidants including vitamins E and C and selenium from a variety of sources) or placebo (oatfeed pellets without additive). Pulmonary function and exercise tolerance were monitored; systemic and pulmonary lining fluid uric acid, glutathione and 8-epi-PGF(2alpha) were analysed, and bronchoalveolar lavage (BAL) cytology and inflammatory scoring of the airways were performed. The antioxidant treatment significantly improved exercise tolerance and significantly reduced endoscopic inflammatory score. Plasma uric acid concentrations were significantly reduced, suggesting downregulation of the xanthine-dehydrogenase and xanthine-oxydase pathway. Haemolysate glutathione showed a nonsignificant trend to increase, while plasma 8-epi-PGF(2alpha) remained unchanged. Pulmonary markers and BAL cytology were not significantly affected by antioxidant supplementation. The present study suggests that the antioxidant supplement tested modulated oxidant/antioxidant balance and airway inflammation of heaves-affected horses.

Effect of chronic airway inflammation and exercise on pulmonary and systemic antioxidant status of healthy and heaves-affected horses.

In heaves-affected horses the relation between oxidant status, airway inflammation (AI) and pulmonary function (PF) is unknown. The oxidant status of blood and pulmonary epithelial lining fluid (PELF) of healthy (H, n = 6) and heaves-affected horses in clinical remission (REM, n = 6) and in crisis (CR, n = 7) was assessed at rest, during and after standardised exercise test by measurement of reduced and oxidised glutathione, glutathione redox ratio [GRR%]; uric acid and 8-epi-PGF2alpha. Oxidant status was related to PF parameters (mechanics of breathing and arterial blood gas tension) and Al parameters (bronchoalveolar lavage [BAL] neutrophil % and AI score). Haemolysate glutathione was significantly different between groups and was correlated with PF and AI parameters; GRR in PELF was increased during CR and was correlated with PF and AI parameters. Exercise induced an increase of plasma uric acid that was significantly higher both in REM and CR. PELF 8-epi-PGF2alpha was significantly increased in CR and correlated with PF and AI parameters. These results suggest that oxidative stress occurring in heaves is correlated with PF and AI and may be locally assessed by PELF glutathione status, uric acid and 8-epi-PGF2alpha. Systemic repercussions are reflected by assay of GSH in resting horses and by uric acid in exercising horses.

Airway inflammation in cadmium-exposed rats is associated with pulmonary oxidative stress and emphysema

The aim of this study was to test the hypothesis that pulmonary inflammation and emphysema induced by cadmium (Cd) inhalation are associated with pulmonary oxidative stress. Two groups of Sprague Dawley rats were used: one vehicle-exposed group undergoing inhalation of NaCl (0.9%, n = 24) and one Cd-exposed group undergoing inhalation of CdCl2 (0.1%, n = 24). The animals in the vehicle-and Cd-exposed groups were divided into 4 subgroups (n = 6 per group), which underwent either a single exposure (D2) of 1H or repeated exposures 3 times/week for 1H for a period of 3 weeks (3W), 5 weeks (5W) or 5 weeks followed by 2 weeks without exposure (5W+2). At sacrifice, the left lung was fixed for histomorphometric analysis (median inter-wall distance, MIWD), whilst bronchoalveolar lavage fluid (BALF) was collected from the right lung. Cytological analysis of BALF was performed and BALF was analysed for oxidant markers 8-iso-PGF2α, uric acid (UA), reduced (AA) and oxidised ascorbic acid (DHA) and reduced (GSH) and oxidised glutathione (GSSG). Cd-exposure induced a significant increase of BALF macrophages and neutrophils. 8-iso-PGF2α, UA, GSH and GSSG were significantly increased at D2. At 5W and 5W+2, AA and GSH were significantly lower in Cd-exposed rats, indicating antioxidant depletion. MIWD significantly increased in all repeatedly Cd-exposed groups, suggesting development of pulmonary emphysema. 8-iso-PGF2α and UA were positively correlated with macrophage and neutrophil counts. GSH, GSSG and 8-iso-PGF2α were negatively correlated with MIWD, indicating that Cd-induced emphysema could be associated with pulmonary oxidative stress.

Breath Condensate Hydrogen Peroxide Correlates with Both Airway Cytology and Epithelial Lining Fluid Ascorbic Acid Concentration in the Horse

The relationship between hydrogen peroxide (H2O2) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H2O2 in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and α-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H2O2 than control horses and RAO-affected horses in the absence of inflammation (2.0±0.5 μmol/l, 0.4±0.2 μmol/l and 0.9±0.2 μmol/l H2O2, respectively; p<0.0001). The concentration of breath condensate H2O2 was related inversely to the concentration of ascorbic acid in ELF (r=-0.80; p<0.0001) and correlated positively with TW inflammation score (r=0.76, p<0.0001) and BAL neutrophil count (r=0.80, p<0.0001). We conclude that the concentration of H2O2 in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.

Effect of allopurinol on the formation of reactive oxygen species during intense exercise in the horse

Allopurinol was administered to six horses in a cross-over study to determine the relative contribution of xanthine oxidase (XO) activity to the formation of reactive oxygen species (ROS) in the horse during intense exercise. Exercise increased the mean (SEM) plasma lipid hydroperioxide concentration to a maximum of 492.7 (33.4) microM within one minute of exercise completion and maximum levels of both oxidised glutathione (GSSG) in haemolysates of red blood cells and the glutathione redox ratio (GRR) occurred 20 minutes after exercise (87.2 [12.2] microM and 8.9 [0.9] per cent, respectively). Allopurinol significantly reduced lipid hydroperoxides, GSSG and the GRR at the corresponding maximal times after exercise measured during control exercise (217.5 [32.1] microM. 63.8 [8.6] microM and 6.8 [0.7] per cent, respectively). Significantly higher levels of hypoxanthine and xanthine were measured after exercise in the plasma of horses that received allopurinol than in control horses, although uric acid levels remained constant. In control horses, plasma uric acid concentrations increased after exercise to a maximum 20 minutes after exercise of 28.1 (2.6) microM, significantly higher than in horses given allopurinol (9.6 [1.3] microM). The results show that the inhibition of XO by allopurinol leads to a decrease in the formation of ROS during exercise, and thus a reduction in oxidative stress.

Nitric oxide and exercise in the horse.

1. The effects of exercise on the production rate of nitric oxide (NO) in exhaled air (VNO) and the effects of inhaled NO (80 p.p.m.) on cardiovascular and respiratory parameters were investigated in five Throughbred horses. 2. The concentration of NO ([NO]) in exhaled air collected from within the nasal opening was lower when collected at a high flow rate of 80 l min‐1 than at a low flow rate of 20 l min‐1: when trotting at 3.7 m s‐1 the values were 0.78 +/‐ 0.15 and 1.23 +/‐ 9.14 p.p.b., respectively, and when cantering at 9 m s‐1 the values were 1.69 +/‐ 0.31 and 2.25 +/‐ 0.32 p.p.b., respectively. 3. Nebulized methoxamine (40 mg ml‐1 for 60 s), an alpha 1‐adrenergic agonist, further reduced [NO] during the 9 m s‐1 canter to 1.05 +/‐ 0.14 and 1.99 +/‐ 0.41 p.p.b. when collected at 80 and 20 l min‐1, respectively, and induced cyclical changes in the breathing pattern. 4. Exercise induced a linear increase in VNO with work intensity to a maximum (428.1 +/‐ 31.6 pmol min‐1 kg‐1) which coincided with the maximal oxygen uptake for the horses (138.3 +/‐ 11.7 ml min‐1 kg‐1), although a further increase in VNO (779.3 +/‐ 38.4 pmol min‐1 kg‐1) occurred immediately after exercise. The changes in VNO correlated well with the tidal volume (r = 0.968; P < 0.01) and the haematocrit (r = 0.855; P < 0.01). 5. In the first 2 min of high intensity exercise, inhaled NO (80 p.p.m.) significantly (P < 0.05) reduced the pulmonary artery pressure: during the first minute, pulmonary artery pressure was 83.1 +/‐ 7.6 mmHg compared with a control value of 94.4 +/‐ 6.3 mmHg, and during the second minute, 84.2 +/‐ 7.1 mmHg compared with a control value of 98.4 +/‐ 4.7 mmHg. There were no other significant changes in cardiovascular or respiratory indices, including cardiac output, measured during exercise between control and inhaled NO tests. 6. The results show that exhaled NO is released from the airways of the horse and may contribute to the regulation of pulmonary vascular tone during exercise.

Effect of acute airway inflammation on the pulmonary antioxidant status.

Effects of acute airway inflammation induced by organic dust inhalation on pulmonary antioxidant status were investigated in healthy horses and horses affected by recurrent airway obstruction. Exposure to organic dust induced acute airway neutrophilia, which was associated with increases in elastase and decreases in ascorbic acid concentrations in bronchoalveolar lavage fluid. However, markers of oxidative stress were unaffected, as was hydrogen peroxide in breath condensate. Decreases in ascorbic acid correlated with increased respiratory resistance (P = .001) when both groups were combined. In conclusion, acute neutrophilic airway inflammation does not result in significant evidence of oxidative stress in horses affected by recurrent airway obstruction.

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone.

REASONS FOR PERFORMING STUDY Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.