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WALTHAM Centre for Pet Nutrition, Leicestershire, UK
2To whom correspondence should be addressed. E-mail: paul.heaton{at}eu.effem.com.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • DNA damage • comet assay • leukocytes • canine • feline
Cells must maintain a proper balance between the levels of free radicals, such as reactive oxygen species (ROS), and antioxidants to ensure the structural integrity of cellular components. If there is an imbalance in favor of free radicals, sensitive biological structures such as DNA, lipids and proteins could be damaged. Such damage may play a role in the etiology of several degenerative diseases such as cancer and arthritis (1,2). A variety of defense mechanisms exist to quench potentially damaging free radicals. Primary antioxidant defenses include enzymes (catalase, superoxide dismutase and glutathione peroxidase) vitamins (E and C) and other micronutrients (carotenoids and polyphenols). Secondary antioxidant defenses involve excision and repair processes that remove free-radical–induced damage (3). Despite these defense systems damage still occurs within the cell and it is thought accumulation of unrepaired DNA may contribute to a variety of disorders associated with the aging process.
Numerous epidemiological studies to date highlight the importance of consuming dietary products rich in antioxidants (4–6). Recent studies in humans have shown that supplementation with antioxidant compounds such as vitamins E and C, lycopene and ß-carotene can help reduce levels of free-radical damage (7–9). This lends support to the hypothesis that dietary products high in antioxidants potentially exert a protective effect against degenerative disorders such as cancer by a decrease in oxidative damage (10).
Studies to understand the mutual interactions of the numerous dietary antioxidants present in foods will be important to identify supplements that have a protective effect on health and help minimize DNA damage. Determining optimal antioxidant requirements will also be essential to reduce free-radical damage, but not induce formation of pro-oxidants that could enhance any deleterious effects. For example, ascorbic acid, the water-soluble form of vitamin C, plays a pivotal role in several essential metabolic processes, including help in regenerating tocopherols (vitamin E), which help quench free radicals and prevent lipid peroxidation (11). Conversely, when in excess and in the presence of transition ions such as iron and copper, ascorbic acid can become a pro-oxidant generating superoxide and hydrogen peroxide free radicals. This is particularly relevant during disease states where levels of transition ions can be higher than normal (12).
Single-cell gel electrophoresis, more commonly known as the comet assay, is a simple, sensitive and rapid method for the detection and quantitation of DNA damage by strand breaks, open repair sites, crosslinks and labile sites at the individual cell level. Following gel electrophoresis under alkaline conditions, DNA is released from the nucleus forming a comet "head" and "tail." Following fluorescent staining, the intensity of the stain is related to DNA content, with DNA damage being quantified by visual grading or computer image analysis. To date, the comet assay has been used for a variety of applications, including toxicological studies (13), exercise-induced damage (14) and measuring cell growth and DNA repair mechanisms (15). More recently, the comet assay has been used to study the effects of diet on DNA damage (8,16,17). We report herein the application of the comet assay within the canine and feline systems for future use in studying the effects that nutritional supplementation may have on protecting cells from free-radical damage.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Twelve healthy adult cats (7.2 ± 4.8 y) and 12 healthy adult dogs (4.5 ± 2.3 y) were chosen for the study. All cats had been vaccinated for feline panleukopenia virus, feline calicivirus and feline herpesvirus, and dogs vaccinated for canine distemper virus, parvovirus and adenovirus and deemed clinically healthy before commencement of the study. All animals were fed commercially available, complete diets throughout the study period and were housed at the Waltham Centre for Pet Nutrition (Leicestershire, UK), where they were housed in purpose-built, environmentally enriched facilities (18) and treated in accordance with the Centre’s research ethics and UK Home Office regulations.
Blood samples
Fasted blood samples (5 mL) were drawn from the jugular vein into lithium heparin vials and diluted 1:1 in phosphate-buffered saline (PBSa). Leukocytes were isolated over Histopaque 1083 gradients (Sigma Chemical, Poole, UK) by centrifugation at 1000 x g for 40 min. Leukocytes were washed twice in 10 mL PBSa and centrifuged at 700 x g for 10 min before counting and storing at 1 x 106 cells/mL in 90% fetal calf serum (Sigma) and 10% dimethyl sulfoxide (Sigma) at -80°C until required. Viability (assessed by trypan blue exclusion) was typically around 98%.
DNA damage evaluated by the comet assay
The comet assay was conducted according to Singh et al. (13) with slight modifications.
Hydrogen peroxide treatment and gel electrophoresis
DNA damage was induced ex vivo by exposing the leukocytes to a range of H2O2 concentrations (0–250 µmol/L diluted in PBSa) to determine the optimal level of H2O2 required to induce a significant increase in DNA damage above background endogenous DNA damage levels. Leukocytes were thawed rapidly in a 37°C water bath, washed twice in PBSa, centrifuged at 700 x g for 15 min and resuspended in PBSa at 2 x 105/mL. Cells were resuspended in 0, 10, 50, 100 and 250 µmol/L H2O2 in PBSa and incubated on ice for 5 min. Treatment on ice minimizes the possibility of cellular DNA repair.
Two layers of agarose were prepared. For the first layer, 85 µl 1% (w/v) high-melting point (HMP) agarose (Sigma) prepared at 95°C in PBSa was pipetted onto fully frosted microscope slides, covered with an 18 x 18-mm coverslip and allowed to set at 4°C for 10 min. Untreated and hydrogen peroxide–treated leukocytes were washed twice in PBSa, centrifuged at 700 x g for 15 min and resuspended at 2 x 105 in 85 µl 1% (w/v) low melting point (LMP) agarose (Sigma). The cell suspension was then pipetted over the set HMP agarose layer, covered with an 18 x 18-mm coverslip and allowed to set at 4°C for 10 min. After the coverslips were removed, the slides were immersed in prechilled lysis solution [2.5 M NaCl, 100 mM sodium EDTA, 10 mM Tris, pH adjusted to 10 using NaOH pellets, 1% Triton X-100 (v/v) (added immediately before use)] for 60 min at 4°C to remove cellular proteins.
Following lysis, slides were placed in a gel electrophoresis unit and incubated in fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 40 min at room temperature, before being electrophoresed at 25 V (300 mA) for 30 min at 4°C. All the above procedures were conducted in the dark to minimize extraneous sources of DNA damage.
Following electrophoresis, the slides were immersed in neutralization buffer (0.4 M Tris–HCl, pH 7.5) and gently washed three times for 5 min at 4°C to remove alkalis and detergents. SYBR Green (50 µL; Trevigen, Gaithersburg, MD) was added to each slide to stain the DNA, then covered with a coverslip and kept in the dark in an air-tight moist container before viewing. SYBR Green was chosen for staining damaged DNA following studies by Ward and Marples (19), demonstrating improved detection sensitivity and assay resolution of SYBR Green over alternative DNA stains.
Scoring for DNA damage
Visual and computerized image analyses of DNA damage were carried out in accordance with the protocols of Collins et al. (20,21). Slides were examined at 250x magnification on a Zeiss inverted fluorescence microscope (Zeiss, Oberkochen, Germany) at 460 nm. One hundred randomly selected nonoverlapping cells were visually assigned a score on an arbitrary scale of 0–4 (i.e., ranging from 0 = no DNA damage to 4 = extensive DNA damage) (Fig. 1) based on perceived comet tail length migration and relative proportion of DNA in the comet tail. A total damage score for each slide was derived by multiplying the number of cells assigned to each grade of damage by the numeric value of the grade and summing over all grades (giving a maximum possible score of 400, corresponding to 100 cells at grade 4). To determine whether visual scoring correlated with computerized image analysis the same cells were also scored for DNA damage using the KOMET 4.0 analysis package (Kinetic Imaging, Liverpool, UK). A variety of objective measurements including percentage DNA in tail, tail length (measured from the leading edge of the comet head) and tail moment were made. Tail moment was calculated as follows:
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A two-factor ANOVA as well as the Student–Newman–Keuls test were used to determine statistically significant differences between the different concentrations of H2O2 used to induce ex vivo DNA damage. Differences were considered significant at P < 0.05. Linear regression analysis was used to correlate visual comet scores with computerized image analysis derived scores. Values are means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As such, maintaining a balanced antioxidant status and reducing levels of oxidative stress have become the primary aims of nutritional research. One of the requirements to achieve these objectives is the ability to be able to accurately measure levels of free-radical damage and how dietary intervention may be able to reduce oxidative stress. The present study describes the application of the comet assay [modified from the original methodology described by Singh et al. (13)], for measuring markers of DNA damage by free radicals within feline and canine leukocytes for inclusion in future nutritional and oxidative stress-related studies.
Although a variety of tissues have been suggested for use in the comet assay, leukocytes are considered a good marker of actual bodily state (25). Leukocytes are more susceptible to the damaging effects of free radicals because of the high percentage of polyunsaturated fatty acids (PUFAs) in their plasma membranes and increased production of free radicals as part of their normal function. Hydrogen peroxide is believed to be one of the most potent causes of DNA damage, chromosomal alterations and gene mutations by generating hydroxyl radicals (OH·) close to the DNA molecule, by means of the Fenton reaction:
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Because hydrogen peroxide is known to damage DNA and is generated by activated leukocytes, it was considered the most appropriate candidate for inducing artificial DNA damage in the present study. As highlighted by Collins et al. (21) use of hydrogen peroxide to induce in vitro DNA damage can also reflect the antioxidant status of the blood. A study by Duthie et al. (7) demonstrated a protective effect following hydrogen peroxide treatment of lymphocytes taken from antioxidant-supplemented individuals.
To demonstrate that both feline and canine leukocytes were susceptible to DNA damage, cells were incubated with various concentrations (0–250 µmol/L) of hydrogen peroxide before analysis by the comet assay. Using visual scoring to quantify DNA damage, a statistically significant increase in DNA damage (P < 0.001) was observed when leukocytes were exposed to 10–250 µmol/L H2O2 in both feline and canine samples when compared to untreated samples. Although the data show there were no significant differences in DNA damage between 10 and 100 µmol/L H2O2 in feline samples and between 50 and 100 µmol/L H2O2 in canine samples, it was decided that 100 µmol/L would be the most suitable concentration of hydrogen peroxide to induce ex vivo DNA damage. Even though 10 and 50 µmol/L H2O2 induced significant increases in DNA damage compared to the untreated samples, the margin of difference was relatively small in comparison to the DNA damage induced at 100 µmol/L. This may present a situation where significant differences in DNA damage may not be observed if the margin of difference between endogenous DNA damage and H2O2-induced DNA damage is too narrow. Concentrations of hydrogen peroxide used in human studies to induce DNA damage have ranged from 1 µmol/L (26) up to 500 µmol/L (8). However, a high proportion of studies use concentrations between 50 and 100 µmol/L H2O2 (15,27,28), suggesting the ranges used to induce significant increases in DNA damage in feline and canine leukocytes are similar to those used in human studies.
A point of interest to note is that levels of background endogenous DNA damage vary considerably between different biological systems. The present study shows that untreated feline samples have an average comet score of 169 ± 18 (Fig. 2a) and untreated canine samples have an average comet score of 153 ± 17 (Fig. 2b). These comet scores are severalfold higher than those observed in studies using human leukocyte samples (29) and in vitro cell lines (15,30). Differing endogenous DNA damage levels may reflect the diversity between biological systems, a fact also borne out by the differences seen between endogenous DNA damage levels between human leukocytes (29) and an in vitro cell line (30). These differences highlight the need to establish comet protocols for each system to be examined. Moreover, it may be related to the feline and canine samples being frozen before comet analysis.
The second objective of this study was to determine whether a visual scoring system could be used as a valid method for categorizing damaged cells, similar to that which has been introduced with oxidative stress and nutritional studies in humans [for review see Tice et al. (31)]. The standard method employed for visual scoring involves classifying comets into five categories based on perceived length of migration and relative proportion of DNA in the tail (32,33). By assigning a numerical value to each migration class, the average extent of DNA migration among cells within a sample can be calculated. Correlations, using linear regression analysis, were made between the visual scores and the computerized image analysis parameters of percentage DNA in the tail, tail moment and tail length with feline and canine samples. In all cases visual scores and computerized image analysis parameters (Figs. 3a-c, 4a-c) demonstrated high correlations. With the highest correlation occurring between visual scoring and percentage DNA in the tail for both feline (R2 > 0.99; Fig. 3a) and canine samples (R2 > 0.97; Fig. 4a). This is in accordance with the studies of Collins et al. (20) and De Boeck et al. (34), which suggests that percentage DNA in the tail is preferentially used for measuring DNA damage by computer image analysis, given that it has important biological significance by giving a relative indication of the number of DNA strand breaks within the cell.
Care also has to be taken when interpreting tail length and tail moment data in conjunction with visual scoring of comets as a parameter for assessing DNA damage. Tail length may become saturated, particularly where high levels of DNA damage occur. This was demonstrated by the plateau effect observed with both feline and canine samples between class 2 and class 4 comets (Fig. 3c and Fig. 4c, respectively). Also, because of the DNA supercoil structure there may be a limit as to how far the DNA loops extend from the comet head independent of increasing levels of DNA damage (35); tail moment is a product of both percentage DNA in the tail and tail length and, as such, there is the possibility that induced effects may be masked. For example, if percentage DNA decreases and tail length increases, this would result in a stable tail moment. Therefore, when using tail moment as a parameter of DNA damage, data on tail length and percentage DNA in the tail should also be provided (31).
In conclusion, the comet assay could be used as a useful method for assessing levels of DNA damage as a consequence of oxidative stress in the feline and canine systems. In particular, the high correlation between visual and computerized scoring demonstrate that an experienced user could employ a visual-only scoring system, which would allow a large throughput of samples in a short time. However, it would be advisable for operators unfamiliar with the comet assay to set up individual calibration curves correlating visual and computer image analysis scores so that intra- and interobserver variation between comet interpretation is reduced to a minimum. With the capacity of the comet assay to be able to measure end products of free-radical reactions, it is a useful tool for determining the optimal effects of dietary antioxidants on a reliable biomarker of oxidative stress such as cellular DNA status in cats and dogs. Other advantages of using the comet assay include: 1) requirement of small numbers of cells per sample, and hence the need for smaller volumes of blood; 2) sensitivity of detecting low levels of DNA damage; 3) potentially high-throughput assay; 4) ease of application; and 5) flexibility with use of different cell types, and low cost.
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FOOTNOTES |
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3 Abbreviations used: H2O2, hydrogen peroxide; ROS, reactive oxygen species, PBSa, phosphate-buffered saline; HMP, high melting point; LMP, low melting point; OH·, hydroxyl radicals.
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LITERATURE CITED |
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