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Centers for Equine Studies and * Preventive Medicine, Animal Health Trust, Kentford, Newmarket, Suffolk, CB8 7UU, UK and Equine Studies Group, WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, Leicestershire LE14 4RT, UK
3 To whom correspondence should be addressed. E-mail: david.marlin{at}aht.org.uk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • single-cell gel electrophoresis • horse • recurrent airway obstruction • aging • foals
Introduction
Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals. This imbalance can be caused by either increased free radical formation or decreased antioxidant capacity (1). In recent years, it has become clear that high levels of oxidative stress may damage sensitive biological structures such as DNA, lipids, and proteins, and such damage may play a role in the etiology of several degenerative diseases such as cancer and arthritis (2,3). In addition, oxidant-antioxidant imbalances are suggested to be the basis of tissue damage in many conditions that affect human lungs (4,5). As well as disease, oxidative stress is implicated in the mechanism of aging (6,7).
Recent work shows the importance of oxidative stress in both equine lungs and peripheral blood and indicates that animals that suffer from recurrent airway obstruction (RAO),4 a condition with many similarities to human asthma, may have a disturbed oxidant-antioxidant equilibrium (8–10). Equine chronic obstructive pulmonary disease (COPD) (11), which is also referred to as "heaves" and, more recently, as RAO (12), is the most common equine lung disease. A recent study (13) suggests that a dietary antioxidant cocktail improves lung function of RAO-affected horses by modulating the oxidant-antioxidant balance and airway inflammation.
Many different techniques have been used to assess in vivo, ex vivo, and in vitro oxidative DNA damage including alkaline filter elution (14), chromosomal aberrations (15), measurement of micronuclei in cells (16), measurement of 8-hydroxydeoxyguanosine (8-OHDG), and the comet assay (17). Presently, the most-used approaches to quantify DNA damage are 8-OHDG measurement and the comet assay.
The comet assay, which is also called the single-cell gel electrophoresis technique, is a simple, sensitive, and rapid method that can be used to estimate DNA damage at the individual cell level through strand breaks, open repair sites, cross-links, and labile sites (17–19). The assay works on the principle that free radicals such as reactive oxygen species (ROS) cause breaks in the DNA and/or base oxidation.
To date, the comet assay has been used for a variety of applications including studies on toxicology [review (20)], pollution (21,22), aging (23,24), exercise (25,26), training (27), and measurement of cell-growth and DNA-repair mechanisms (28,29). The comet assay has also been used to study the effects of diet and antioxidant supplementation on oxidative DNA damage [see review (30)].
The majority of studies using the comet technique have been performed on humans or laboratory animals with few reports on companion animals (i.e., cats, dogs, and horses). Heaton and colleagues (31) recently described application of the comet assay to cat and dog lymphocytes. They subsequently used this approach to evaluate the effects of dietary antioxidant supplementation in dogs and demonstrated that supplementation reduced both endogenous and exogenous oxidative DNA damage (32).
The peripheral blood mononuclear cell (PBMC) is the most frequently studied cell type using the comet technique, although any nucleated cell type may be studied including neurons (33), hepatocytes (26), pulmonary cells (34), and spermatozoa (35). To the best of our knowledge, there is only one previous publication on the use of the comet assay in horses (35), which was to determine DNA damage induced by freezing and storing spermatozoa.
The aim of this study was to apply the comet assay to lymphocyte-enriched equine PBMC samples and to subsequently use this technique to investigate 1) the effects of age on PBMC endogenous DNA damage and susceptibility to ROS-induced damage in vitro; 2) the relationship between systemic antioxidant status, PBMC endogenous DNA damage, and susceptibility to ROS-induced damage in vitro; and 3) the effects of RAO on PBMC endogenous DNA damage and susceptibility to ROS-induced damage in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We studied 22 clinically healthy animals (4 horses and 18 ponies) that were identified based on the basis of history and clinical examination, and 8 animals that were diagnosed with RAO (4 horses and 4 ponies). The 18 clinically healthy ponies were allocated to three groups, each of which contained 6 Welsh Mountain ponies: young (6 foals: 3 colts and 3 fillies; mean age, 3.8 mo; range, 3–4 mo), mature (6 geldings; mean age, 3.8 y; range, 3–4 y), and aged (6 geldings; all >24 y of age).
The 8 animals with RAO (4 horses, 1 mare, and 3 geldings: mean age, 17 y; range, 11–22 y; and 4 ponies, 1 mare, and 3 geldings: mean age, 16 y; range, 11–23 y) were diagnosed by demonstration of marked increases in airway resistance and >50% neutrophils in bronchoalveolar lavage fluid after environmental challenge (12) but were free of airway inflammation at the time of study. The RAO-affected animal group was breed-matched with a group of 8 healthy non-RAO–affected control animals (4 horses, all mares: mean age, 7 y; range, 4–9 y; and 4 ponies, all geldings: mean age, 4.8 y; range, 4–6 y). All animals were kept on bare pasture and were fed supplemental haylage or hay.
The experimental protocol was approved by the Ethics Committee of the Animal Health Trust and conformed to the Home Office Animals (Scientific Procedures) Act 1986.
Experimental details
Intraassay variation was determined by analyzing 20 gels (10 each for endogenous and exogenous DNA damage) in a single assay from a single sample. Interassay variation was assessed by using a single, large, blood sample to produce five cell aliquots. A fresh (i.e., previously unthawed) aliquot was analyzed in each of five different assays for both endogenous and exogenous DNA damage. Cell isolation, comet assay, and identification of the optimal H2O2 dose for determining susceptibility to exogenous DNA damage were investigated by analyzing four gels for each of six horses that were treated with 0, 25, 50, and 100 µM H2O2. Blood samples were taken from a group of six Welsh Mountain ponies (aged 4 y) on three consecutive days prefeeding at 0900 h to determine day-to-day variability in endogenous and exogenous oxidative DNA damage and repeatability of the comet assay for horses. To determine whether RAO-affected horses had higher systemic oxidative stress and DNA damage and/or increased sensitivity to exogenous DNA damage, samples were analyzed from eight non-RAO–affected and eight RAO-affected horses that were in clinical remission and kept at pasture for at least 3 mo. To determine whether there was any relationship between endogenous or exogenous DNA damage, blood or plasma antioxidant status, or markers of oxidative damage, paired blood samples were collected from the 22 clinically healthy animals (4 horses and 18 ponies) and analyzed for plasma reduced and oxidized ascorbic acid (AA), 
-tocopherol, uric acid, and malondialdehyde (MDA), and red blood cell (RBC) hemolysate reduced and oxidized glutathione. DNA damage was assessed by comet assay on PBMC-enriched fractions from the paired samples.
Blood-sample collection
Blood samples (30 mL) were collected with needle and syringe from the jugular vein before feeding at 0900 h on each occasion. Blood (15 mL) was placed into plain glass tubes that contained 0.5 mL of preservative-free heparin (15 IU/mL of PBS). An additional 15 mL of blood was collected into tubes with EDTA (5 mL) and lithium-heparin (2 x 5 mL) for determination of antioxidant status. All blood samples were immediately placed on ice.
Isolation of lymphocyte-enriched mononuclear cell fraction from peripheral blood
Within 10 min of collection, 5 mL of heparinized blood was layered onto an equal volume of a density-gradient–centrifugation medium (Ficoll-Paque Plus, 1.077 g/mL density, Amersham Biosciences, Buckinghamshire, UK) in 15-mL plastic, V-bottomed centrifuge tubes at room temperature. The tubes were then centrifuged at room temperature for 40 min at 1000 x g. The top layer of serum was discarded, and the lymphocyte layer was harvested. Cold PBS (4°C) was added to the harvested cells to yield a total volume of 10 mL. The harvested lymphocytes in PBS were then centrifuged for 10 min at 4°C and 700 x g. The supernatant was removed, the cell pellet was resuspended in 1 mL of cold PBS, and additional cold PBS was added to make a total volume of 10 mL. The resuspended pellet was centrifuged for 10 min at 4°C and 700 x g, and the supernatant was discarded. The cell pellet was resuspended in 1 mL of cold PBS, and additional cold PBS was added to make a 5-mL total volume. The resuspended cell suspension was returned to ice. A manual cell count and an estimate of cell viability were performed using Trypan blue stain. The cell suspension was centrifuged for an additional 10 min at 4°C and 700 x g. The supernatant was decanted, and the lymphocyte pellet was resuspended in freezing medium (10% dimethylsulfoxide and 90% fetal calf serum) to yield 3 x 106 cells/mL. Aliquots of lymphocytes were placed in cryotubes, frozen slowly, and stored in a thick-walled polystyrene box at –80°C.
To determine the degree of enrichment of the PBMC fraction, samples were collected from seven horses, and a differential cell count was performed before and after density-gradient enrichment (Table 1).
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Within 5 min of collection, blood was centrifuged at 500 x g for 10 min at 4°C. After centrifugation, samples were returned to ice. For AA analysis, 500 µL of EDTA-plasma was transferred with a pipette into an amber Eppendorf tube with 500 µL of 10% metaphosphoric acid/1 mM Na2-EDTA for deproteinization. The sample was mixed on a vortex, snap-frozen in liquid nitrogen, and stored at –80°C. For dehydroascorbate, 300 µL of plasma was transferred with a pipette into an amber Eppendorf tube with 200 µL of 10 mM dithiothreitol, mixed on a vortex, and allowed to stand at room temperature for 10 min. We added 500 µL of 10% metaphosphoric acid with 1 mM Na2-EDTA, and the sample was mixed on a vortex, snap-frozen, and stored at –80°C. For plasma vitamin E analysis, 1-mL aliquots of lithium-heparin–plasma were placed in 1.8-mL cryotubes, snap-frozen, and stored in liquid nitrogen. For plasma MDA analysis, 1 mL of lithium-heparin–plasma was transferred with a pipette into an Eppendorf tube with 100 µL of BHT (100 mg/mL of ethanol) and 100 µL of desferal (100 mg/mL of water) and stored at –80°C. For uric acid and iron analyses, 0.5 mL of lithium-heparin–plasma was stored in Eppendorf tubes at –80°C. For determination of glutathione in RBC hemolysate, 0.5 mL of RBCs from a centrifuged lithium-heparin sample were transferred with a pipette into a 1.8-mL cryotube with 0.5 mL of 0.9% saline and 2 mM EDTA, mixed gently, snap-frozen, and stored in liquid nitrogen. Glutathione, AA, and -tocopherol were chosen as indicators of systemic antioxidant capacity based on previous in vitro work on humans (36) and in vivo work on horses (9).
Comet assay
The comet assay procedure was performed essentially as described by Hartmann et al. (19). Cryopreserved PBMCs were thawed and washed in PBS as described by Heaton et al. (31). The volume of suspension that contained 40,000 cells was calculated, and this volume was added to individual Eppendorf tubes. Two tubes were prepared per sample: one to which PBS was added to the PBMCs (to assess endogenous DNA damage) and one to which 50 µL of 1 mM H2O2 was added (to induce exogenous DNA damage). Cold PBS was added to the cells to yield a total volume of 1 mL. After the addition of H2O2 and PBS, the cells were incubated on ice for 5 min and then immediately centrifuged for 4 min at 4°C and 200 x g. Slide preparation, lysis, alkaline incubation, electrophoresis, and neutralization were carried out according to Heaton et al. (31) with the exception that incubation was carried out at 4°C to prevent enzymatic repair. Samples were analyzed in duplicate (i.e., four slides per sample: two for endogenous damage, and two for exogenous damage). Slides were then stored in a small volume of neutralizing buffer at 4°C in the dark until staining and scoring was performed (within 24 h).
The gels on each slide were stained with 50 µL of SYBRgreen (Trevigen, Gaithersburg, MD) and diluted 1:10,000 in Tris-EDTA buffer. A 22 x 22-mm coverslip was placed over the gel, and the slides were examined using a 20x objective on an epifluorescence microscope (Leica Microsystems, Milton Keynes, UK) with a 460- and 500-nm excitation and emission spectra filter cube (fluorescein isothiocyanate filter). Images were captured using a digital video camera with live imaging facility (Leica Microsystems) and saved to compact disk. Captured images (stored as TIFF files) were scored using Komet 6.0 software (Kinetic Imaging, Liverpool, UK) to measure the percentage of DNA in the comet tail. We scored 100 randomly selected cells for each slide. Only cells that did not overlap and had a clear margin surrounding them were scored. In addition, to allow comparison with other publications, we scored some images visually for direct comparison to Komet 6.0 computer-generated scores. For visual scoring, the comets scored during computer analysis were subsequently assigned a classification from 0 to 4 (0 corresponding to no DNA damage, and 4 indicating maximum DNA damage; Fig. 1) with the scorer unaware of the computer score. An overall score was obtained for each gel (slide) by multiplying the number of cells assigned to each class by the numeric value of the class to yield an overall score ranging from 0 (corresponding to no DNA damage) to 400 (corresponding to maximum DNA damage) arbitrary units.
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Antioxidant analysis
Chemicals were purchased from Sigma Chemicals, Rathburn Chemicals, and ICN Biomedicals. HPLC with electrochemical detection was used to measure reduced and oxidized glutathione (GSH and GSSG, respectively) in RBC hemolysates (37). The glutathione redox ratio was calculated by dividing GSSG by the total concentration of glutathione (GSH and GSSG). Plasma uric acid levels were determined using a commercial kit (no. 685-10, Sigma). Plasma total iron concentration was analyzed according to the method of Pepper et al. (38). Plasma MDA concentration was determined by HPLC with fluorometric detection based on the method of Young and Trimble (39). Plasma AA concentration was analyzed by HPLC with UV detection as described previously (40). The AA redox ratio was calculated by dividing the dehydroascorbate concentration by the total AA concentration. The concentration of -tocopherol in plasma was measured by HPLC according to the method described by Kelly et al. (41).
Statistical analysis
Data are presented as means ± SD unless otherwise stated. Linear regression analysis and correlation (Spearman's R) were used to compare visual and computer scoring results. A one-way ANOVA was applied to data obtained for samples taken on three consecutive days to determine day-to-day variability and repeatability of the comet assay in equine blood samples. Comparisons between healthy and RAO-affected horses were made using an unpaired t test. The effects of age on endogenous oxidative DNA damage and susceptibility to exogenous oxidative DNA damage were investigated by correlation and linear regression and by one-way ANOVA after animals were grouped into young, mature, and aged categories. Where significance was reached (P < 0.05), post-hoc comparisons were carried out using the Student-Newman-Keuls test. The relationships between systemic antioxidants, markers of oxidative damage, and iron were investigated using Pearson's correlation. Significance was taken as P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The differential cell counts for seven whole blood samples from three horses (one sample from two animals and two samples from the third animal) and two ponies (one sample from one animal and two samples from the other animal) before and after PBMC enrichment are shown in Table 1. The PBMC fraction (sum of lymphocytes and monocytes) was increased from 40 ± 10 to 96 ± 2% (P < 0.0001). The lymphocyte fraction was significantly increased from 38 ± 10% in whole blood to 78 ± 6% after enrichment (P < 0.0001).
Examples of different comet classes
Example images of each of the different classes of DNA damage in equine PBMCs are shown in Figure 1.
Comparison of visual scoring with Komet 6.0
There was excellent correlation (r = 1.000; P < 0.0001) between visual scores of DNA damage and percentage of tail DNA calculated using Komet 6.0 software (Fig. 2).
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The mean intraassay coefficient of variation for the percentage of tail DNA for a single sample was 4.7% for endogenous DNA damage and 9.7% for exogenous DNA damage. Interassay coefficients of variation for a single sample in each of five different assays were 7.3 and 8.3% for endogenous and exogenous DNA damage, respectively.
Day-to-day variation
The scores for percentage of tail DNA for endogenous and exogenous DNA damage in the group of six mature Welsh Mountain ponies are shown in Figure 3. There was no significant effect of day on either endogenous, exogenous, or exogenous (corrected) DNA damage (P > 0.05).
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DNA damage (i.e., increasing percentage of tail DNA) was linearly related to increasing H2O2 concentration (Fig. 4). On the basis of these results, 50 µM H2O2 was used in all other assays to estimate exogenous DNA damage.
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There was no significant difference in endogenous, exogenous, or exogenous corrected DNA damage between mature and aged ponies (Fig. 5). However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). There were no differences between any of the age groups in sensitivity to exogenous DNA damage before or after correction for endogenous damage (ANOVA; P > 0.05).
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Both endogenous and exogenous DNA damage were significantly greater in the RAO-affected horses in remission compared with the non-RAO–affected control animals (P = 0.009 and P = 0.003, respectively; Fig. 6). However, after correction for endogenous damage, there was no significant difference in sensitivity to H2O2 treatment between the two groups (exogenous damage, corrected; Fig. 6).
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There was a significant negative correlation between exogenous (corrected for endogenous) and endogenous percentage of tail DNA (r =–0.574; P = 0.005).
Relationship between DNA damage and antioxidant status
There was a significant nonlinear correlation between RBC GSH concentration and endogenous percentage of tail DNA (r = 0.720; P < 0.001; Fig. 7). There were no significant correlations between endogenous, exogenous, or exogenous (corrected) percentages of tail DNA and any of the other markers of antioxidant status, oxidative stress, or lipid peroxidation.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We include three refinements in our application of the comet-assay technique to equine PBMCs that do not appear to have been used previously. First, we took differential cell counts on blood samples before and after PBMC isolation and lymphocyte enrichment and demonstrated the extent of the lymphocyte enrichment with our technique. Although there was a marked increase in the lymphocyte proportion after gradient centrifugation (38 to 78%), the monocyte proportion also increased significantly (from a mean of 2 to 17%; range, 11–27%). We have therefore chosen to call the sample on which comet was performed a lymphocyte-enriched PBMC fraction. Second, as far as we know, our study is the first to report day-to-day variation in the comet assay for animals maintained on the same management and sampled at the same time of day on three consecutive days. Finally, in studies where susceptibility to exogenous damage was estimated after H2O2 treatment, it does not appear that any account was made of the endogenous damage, although the pool of cells used for each of these assays was the same. For example, in comparing RAO- and non-RAO–affected animals in this study, RAO-affected animals appear to have increased exogenous damage. However, they also have increased endogenous damage, and if the exogenous damages for both RAO- and non-RAO–affected animals are adjusted by subtracting the existing endogenous damage, the apparent increased susceptibility of RAO-affected horses is removed. We therefore believe it is more appropriate to express exogenous damage after correction for endogenous damage.
Heaton et al. (31) used almost identical methodology for the comet assay in cats and dogs. The main differences are that in their study, blood was diluted 1:1 in PBS before centrifugation, and results were presented as arbitrary DNA damage using visual scoring, although a comparison was undertaken against Komet 6.0 software, and a strong correlation was obtained with percentage of tail DNA. In this study in healthy, mature horses, endogenous percentage of tail DNA averaged 19% (range, 9–31%), which is slightly lower than the mean for dogs (
25% tail DNA) and cats (30% tail DNA) (31). These differences cannot be due to the scoring, because Komet 6.0 was used in both our study and that of Heaton et al. (31); however, differences could be due to the dilution step with PBS, other interlaboratory differences, or true species differences. After incubation of PBMCs from mature horses with 50 µM H2O2, the increase in percentage of tail DNA (i.e., exogenous damage, corrected) was 13% and was similar to that estimated by Heaton et al. (31) (17% for percentage of tail DNA corrected for endogenous damage). In this study, the slope of the relationship between percentage of tail DNA (as assessed by Komet 6.0) and visually scored cells is similar to that reported for both feline and canine leukocytes (31).
Relatively few authors report coefficients of variation for the comet assay. In this study, the intra- and interassay coefficients of variation were 4.7 and 7.3% for endogenous DNA damage and 9.7 and 8.3% for exogenous DNA damage, respectively. These intraassay coefficients of variation are comparable to those reported for human sperm: 4% for endogenous damage and 9% for irradiated sperm (an alternative approach to H2O2 incubation to assess susceptibility to exogenous damage) (42).
Endogenous PBMC DNA damage was not different between mature (4 y of age) and aged (>24 y of age) ponies in this study. Two studies by the same group have demonstrated no difference in endogenous DNA damage in human peripheral blood lymphocytes in subjects 25–91 y of age (23) or between subjects younger or older than 60 y of age (24). In contrast, Piperakis et al. (43) found lower endogenous DNA damage in the lymphocytes of human subjects 20–25 y of age than in those 55–60 y of age. As far as we know, this is the first study to compare endogenous DNA damage in very young, mature, and aged groups of any species. The lower level of endogenous damage in young pony foals could result from less exposure to free radicals and/or ROS or to better in vivo DNA-repair mechanisms (23,44).
PBMCs from RAO-affected animals in clinical remission had greater endogenous DNA damage than control animals but no difference in susceptibility to H2O2-induced damage. The greater damage may be due either to more ROS or free radical production, a lower capacity for repair, or a lower antioxidant capacity. Horses with RAO were shown to have lower systemic and pulmonary antioxidant capacity (8,9,45), and low antioxidant status was correlated with increased DNA damage in human blood lymphocytes using the comet technique (46). There is increasing evidence that asthma in humans (which shows many similarities to equine RAO) has both pulmonary and systemic effects (47). For example, polymorphonuclear neutrophils from asthmatic patients were shown to produce more superoxide than in nonasthmatic control subjects (48). However, in equine neutrophils, superoxide production was not different between RAO-affected horses in remission and non-RAO–affected control animals (49). Similarly, oxygen radical production of phagocytes isolated from peripheral blood measured using a modified nitroblue tetrazolium test was not different between RAO-affected and non-RAO–affected animals (50).
There are several possible explanations for the apparent discrepancy between human and equine studies in RAO and asthma and between previous work on equine polymorphonuclear neutrophils and our findings in this study. First, our RAO-affected horses may not have been in full remission, although this is unlikely; all RAO-affected horses were at pasture 24 h/d for at least 3 mo, and all underwent regular airway endoscopy. Second, the DNA damage in RAO-affected horses in remission may be caused by production of ROS or other free radicals other than superoxide and/or by cells other than neutrophils. Third, there was an age difference between the RAO-affected horses (mean age, 16 y) and the non-RAO–affected control animals (mean age, 6 y), but in healthy ponies, there was no significant difference in DNA damage between mature and aged horses.
When the relationships between antioxidant status and markers of oxidative stress and damage in plasma and RBCs and DNA damage in PBMCs were examined (excluding the RAO-affected animals), the only significant correlation was between endogenous DNA damage and RBC GSH, and animals with the most DNA damage had the highest GSH concentrations. This is consistent with observations of human smokers, where increased exposure to oxidants from chronic cigarette smoke results in increased concentrations of glutathione in epithelial lining fluid (51). Glutathione appears to be a sensitive marker of degree of inflammation, and many inflammatory mediators appear to regulate glutathione synthesis (52). For example, the proinflammatory cytokine TNF- increases glutathione production in human alveolar epithelial cells, whereas anti-inflammatory treatment with dexamethasone decreases glutathione concentration in these cells (53). Asthmatic patients show increased GSH concentrations in RBC hemolysate (54) and bronchoalveolar lavage fluid (55). Similarly, horses with RAO in remission have markedly increased glutathione concentrations in the epithelial lining fluid (9).
In conclusion, the comet assay can be performed with high precision on equine lymphocyte–enriched PBMCs, and in healthy animals, the assay shows low day-to-day variation. Both visual scoring and analysis of the percentage of tail DNA using Komet 6.0 appear to be acceptable for quantifying the degree of DNA damage. Preliminary data from application of the technique described has shown that endogenous but not exogenous DNA damage is greater in mature and aged ponies compared with young foals; endogenous DNA damage is greater in RAO-affected horses in clinical remission; and greater endogenous DNA damage is associated with increased RBC glutathione concentrations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This study was funded by a grant from WALTHAM.
4 Abbreviations used: AA, ascorbic acid; COPD, chronic obstructive pulmonary disease; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, malondialdehyde; 8-OHDG, 8-hydroxydeoxyguanosine; PBMCs, polymorphonuclear cells; RAO, recurrent airway obstruction; ROS, reactive oxygen species.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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