Changes to Levels of DNA Damage and Apoptotic Resistance

EXPANDED ABSTRACT

KEY WORDS: * canine * aging * oxidative stress * DNA damage * apoptosis

It was previously reported that the immune system in humans and other mammals goes through a process of age-related remodeling, which necessarily involves a decline in some aspects of immunity accompanied by an enhancement of others (1,2). However, the net result is often an overall drop in the host’s capability to deal with immune challenges, which may in turn contribute to the observed increase in morbidity and mortality in the elderly (3).

Several hypotheses have been put forward to explain the reasoning behind this “immunoaging” phenomenon. The freeradical theory of aging proposes that the process of age-related deterioration in mammals is affected to a great extent by the production of highly reactive free radicals (4). Free radicals, such as reactive oxygen species (ROS)4, are atoms, molecules, or compounds possessing one or more unpaired electrons, which cause them to be attracted to sites of high electron density such as compounds with nitrogen atoms (e.g., proteins, DNA, and RNA) or carbon-carbon double bonds (e.g., lipids). If they are not neutralized by antioxidant enzymes (e.g., catalase, superoxide dismutase), metal-binding proteins (e.g., ceruloplasmin, ferritin), or antioxidants (e.g., vitamin E, vitamin C), ROS can cause structural damage to these important biomolecules (5).

Unrepaired DNA may force the cell into premature apoptosis (6,7), which in turn may contribute to the depletion of cellular populations (e.g., lymphocytes) previously reported in aging Labrador Retriever dogs (8,9). Alternatively, unrepaired DNA damage may result in permanent cell-cycle arrest, termed replicative senescence (10), which renders cells of the immune system unable to divide when stimulated by an antigen. Because previous studies demonstrated that poor T-cell function positively correlates with impending mortality in aging humans (11,12), it is important to understand how DNA damage and an individual’s ability to counteract the effects may contribute to the process of immunoaging.

The effect of free radicals is of particular relevance to components of the immune system when it is considered that activated macrophages and neutrophils secrete ROS and reactive nitrogen intermediates at the site of an immune response (13), and that T cells, when activated, were shown to contain elevated levels of ROS (14).

The aims of this study were to determine the total antioxidant potential of whole blood plasma together with spontaneous and induced levels of both DNA damage and apoptosis in peripheral blood mononuclear cells (PBMC) in the absence or presence of oxidative challenge, respectively. These parameters enable the identification of any potential age-related changes in resistance to DNA damage in Labrador Retriever dogs and hence speculate as to any effects on immunoaging.

MATERIALS AND METHODS

Animals

A study previously published by this research group consisted of 9 79 Labrador Retriever dogs split into four age groups 1.1-3.1 y, 3.9-5.4 y, 6.2-7.7 y, and 8.5-12.8 y (8). We found that only the extreme groups were significantly different from one another in immunological parameters such as white blood cell, T-cell, CD4, and CD8 counts, and hence those age groups were chosen for this study.

Accordingly, 34 Labrador Retriever dogs were equally split into two age groups: “young” (1.1-3.1 y; 7 male, 10 female) and “senior” (8.512.8 y; 3 male, 14 female). All dogs had received routine vaccinations (canine distemper virus, parvovirus, and adenovirus) and were deemed to be clinically healthy. All dogs were fed commercially available complete diets throughout the study, at an energy level that maintained constant body weight, and were housed in purpose-built, environmentally enriched facilities (15) at the WALTHAM Centre for Pet Nutrition (Leicestershire, UK). The dogs were treated in accordance with WALTHAM’s research ethics and UK home office regulations.

Blood samples and preparation of plasma

Food-deprived blood samples were drawn in the morning between 0800 and 1000 h by jugular venopuncture into lithium heparin vials. Samples reached the laboratory within 30 min and were prepared for analysis immediately.

Plasma was separated from fresh heparinized venous blood by centrifugation at 2300 g for 10 min at 4C. Plasma was removed and assayed immediately.

Preparation of PBMC

PBMC were isolated from fresh heparinized venous blood samples by centrifugation at 1200 g for 40 minover Histopaque-1077 density gradient (density 1.077 g/mL; Sigma-Aldrich, Poole, UK). Harvested cells were washed twice in phosphate-buffered saline (PBS) before counting. A volume containing 2 10^sup 6^ cells was removed for use in the apoptosis assessment assay. The remainder were frozen slowly at a concentration of 1 10^sup 6^ cells/ml in 90% fetal calf serum (Invitrogen, Paisley, UK) and 10% dimethyl sulfoxide (Sigma- Aldrich) to <80C until required.

Total antioxidant potential of plasma

The ferric reducing antioxidant power (FRAP) assay was carried out according to Benzie and Strain (16). Briefly, FRAP reagent was prepared by mixing 300 mM acetate buffer (pH3.6), 10mM 2,4,6-tri (2- pyridyl)-s-triazine (Fluka Chemicals, Buchs, Switzerland) in 40 mM hydrochloric acid (BDH, Poole, UK); and 20 mM iron (III) chloride (BDH) in a ratio of 10:1:1. Plasma was separated from fresh heparinized venous blood and 100 l was diluted with 40 l distilled water and mixed with 3 ml FRAP reagent. Treated samples were analyzed in duplicate using a Cobas Fara II (Roche Diagnostic Systems, Basel, Switzerland) according to manufacturer’s instructions. Ascorbic acid standards and commercial quality controls were run simultaneously to ensure experimental accuracy. Results were calculated as FRAP values (M).

DNA damage assessment in PBMC

DNA damage was assessed by the comet assay using methodology described by Heaton et al. (17), with further modifications. Briefly, frozen PBMC aliquots were gently thawed and washed in PBS at 4 7deg;C before being incubated on ice for 5 min in the presence of 0 or 50 M hydrogen peroxide in PBS. The cells were then centrifuged at 200 g for 4 min at 4C and the pellet gently resuspended in 85 l of 1% (w/v) low melting point agarose (Type VII: low gelling temperature; Sigma-Aldrich) at 37C. This mixture was pipetted onto a frosted slide onto 85 l preset 1% (w/v) high melting point agarose (Type I: low EEO; Sigma-Aldrich) and immediately covered with a coverslip to form a microgel. Slides were left at 4C for 10 min to allow the agarose to set. The coverslip was removed and slides were immersed in prechilled lysis solution [2.5 M sodium chloride, 0.1 M EDTA, 0.001 M Tris, 1% (v/v) Triton X-100, pH 10.0] to remove cellular proteins. After lysis for at least 65 min at 4C, the slides were placed in electrophoresis buffer [0.3 M sodium hydroxide, 1 mM EDTA, pH 13.0] for 40 min to allow unwinding of the DNA. Slides were then electrophoresed at 20 V, 300 mA for 30 min, before being washed three times in neutralizing buffer [0.4 M Tris, pH 7.5] to remove alkalis and detergents.

Microgels were stained using 50 l SYBR green (Trevigen, Gaithersburg, MD) and analyzed visually at 250 magnification using an Olympus BX51 reverse fluorescence microscope (Kinetic Imaging, Merseyside, UK) at 460 nm. On each microgel, 100 randomly selected, nonoverlapping cells were visually assigned a score on a standardized arbitrary scale of 0-4 (i.e., 0 = no DNA damage; 4 = extensive DNA damage) based on comet tail length migration and relative proportion of DNA in the comet tail [see (17)]. A total damage score for each microgel 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.

The assay was carried out at 4C to minimize activity of DNA repair enzymes, and all incubation steps were carried out in the dark to reduce additional light-induced DNA damage. All samples were analyzed under blind conditions.

Apoptosis assessment in PBMC

Levels of spontaneous and induced apoptosis were measured in PBMC using a commercially available Annexin V-fluorescein assay kit (R & D Systems, Abingdon, UK). Apoptosis was induced using 2-deoxy-D- ribose (Sigma-Aldrich), which exerts oxidative stress upon the cell by depleting cellular glutathione levels. Briefly, PBMC were resuspended in RPMI-1640 (Invitrogen, Paisley, UK) containing penicillin/streptomycin (Invitrogen) and supplemented with 2% autologous serum at a concentration of 1 10^sup 6^ cells/ml. Cells were seeded at a density of 1 10^sup 5^ cells per well into 96- well flat-bottomed microplates (Corning Costar, High Wycombe, UK). Experimental wells contained 10 mM 2-deoxy-D-ribose and control wells contained media only. Plates were incubated at 37C in a 95% humidified, 5% CO2 atmosphere for 72 h.

After incubation cells were transferred to 12 75 mm polystyrene Falcon tubes and washed in PBS by centrifugation at 400 g for 7 min. PBMC were then incubated at room temperature for 15 min with the Annexin V reagent. At the end of the incubation period 400 l of prechilled binding solution was added.

Samples were analyzed immediately using a FACSCalibur E2500 flow cytometer (Becton Dickinson, Oxford, UK) and CELLQuest V3.2 analysis software (Becton Dickinson). The flow cytometer was calibrated routinely using FACSComp V4.1 software (Becton Dickinson) and Calibrite beads (Becton Dickinson). Data acquisition was stopped after 1 min on HI flow rate. Relative percentages of apoptotic cells were recorded. Treatments were run in duplicate.

Statistical analysis

Statistical analyses were carried out using STATGRAPHICS Plus for Windows 4.1 (Manugistics, Rockville, MD). The data were analyzed using a one-way ANOVA followed by a Student-Newman-Keuls test where appropriate. Differences among groups were accepted as significant when the P value was <0.05.

RESULTS

FRAP values, which signify extracellular protection against free radicals, were consistent with age, suggesting the maintenance of plasma antioxidant levels (Fig. 1). In contrast, spontaneous (p = 0.019) and hydrogen peroxide-induced (p = 0.025) DNA damage significantly increased with age (Fig. 2) together, paradoxically, with significantly reduced spontaneous (p = 0.036) and 2-deoxy-D- ribose-induced (p = 0.006) apoptosis (Fig. 3).

FIGURE 1 Age-related differences in the antioxidant potential of serum in Labrador Retriever dogs. FRAP values (+ SEM). No significant differences were identified, n = 17.

DISCUSSION

The aims of this study were based upon the predictions of the free-radical theory of aging, which states that age-related deterioration in physiological fitness is caused by the damaging effects of ROS and nitrogen intermediates upon cells.

To demonstrate the effect of free radicals on the aging canine immune system, levels of DNA damage and apoptosis were measured after periods of exposure to hydrogen peroxide and 2-deoxy-D- ribose, respectively. The total antioxidant potential of plasma was measured to estimate relative levels of antioxidant protection in the different age groups.

The results from this study can be incorporated into the free radical theory of aging to predict which processes are substantially affected by age (Fig. 4). As the total antioxidant potential of plasma did not change significantly with age, it can be proposed that a reduction in extracellular antioxidant defense is not responsible for the accumulation of DNA damage in cells from older dogs. It might, therefore, be suggested that there is either an increased production of free radicals in the elderly or that intracellular defense and repair mechanisms are compromised. When PBMC were put under oxidative pressure, levels of DNA damage were seen to increase whereas levels of apoptosis were decreased, suggesting an apparent paradox. However, it is likely that relatively low levels of oxidative pressure, such as those inflicted here, may cause damage to cellular DNA, which, in elderly subjects, may trigger cells to enter a state of replicative senescence rather than undergoing apoptosis (18).

Cells in a state of replicative senescence have a reduced capability to enter into either apoptosis or proliferation. These cells are typically chronically expanded clones (19) and likely contribute to the filling of immunological space as proposed by Franceschi et al. (20), who suggested that a lifetime’s exposure to antigenic stress causes extensive clonal expansion of memory lymphocytes, thus reducing the pool of naive lymphocytes available to respond to nonrecall antigens. Indeed, Khan et al. (19) showed that in elderly humans CD8 lymphocytes displayed extensive oligoclonality toward cytomegalovirus (CMV), with up to a quarter of the entire CD8 lymphocyte population being CMV-specific memory cells. With fewer naive lymphocytes available to react to infection by a novel antigen in the aged, it is perhaps not surprising that mortality levels due to infection with bacteria (e.g., pneumonia) and viruses (e.g., influenza) with high rates of mutation are so many times greater in elderly than in younger human subjects (3).

FIGURE 2 Age-related changes to levels of DNA damage to peripheral blood mononuclear cells from Labrador Retriever dogs (17 per age group) in presence (treated) or absence (untreated) of 50 M hydrogen peroxide in PBS. Values are accumulative comet scores from 100 analyzed cells (+ SEM) where a value of 400 would denote maximum damage to all cells. Asterisks denote significantly different results (p < 0.05) as determined by one-way ANOVA followed by a student-Newman-Keuls test.

FIGURE 3 Age-related changes to levels of apoptosis in peripheral blood mononuclear cells from Labrador Retriever dogs (17 per age group) after 72 h in the presence (treated) or absence (untreated) of 10 mM 2-deoxy-D-ribose. Values are expressed as percentage apoptotic cells (+ SEM) as measured using an Annexin V antibody. Asterisks denote significantly different results (p < 0.05) as determined by one-way ANOVA followed by a Student-Newman-Keuls test.

FIGURE 4 Suggested effects of exposure of PBMC to free radicals in aging Labrador Retriever dogs as identified by the results reported here. (1) Levels of extracellular antioxidant defense appear to be intact (levels of intracellular defense were not measured); (2) increased spontaneous DNA damage with age and greater potential for induced DNA damage; and (3) reduced progression into apoptosis with age, which suggests that more cells may be entering into a state of replicative senescence. This, in turn, would lead to a filling of immunological space as proposed by Franceschi et al. (20)

The data from this study supports the theory that free-radical damage to immune cells may be a major factor contributing to a reduction in immune efficiency in older dogs. Furthermore, these data support previous findings by this research group in which it was found that aspects of immune competence were significantly reduced with age in Labrador Retriever dogs (8,9).

1 Presented as part of the WALTHAM International Science Symposium: Nature, Nurture, and the case for Nutrition held in Bangkok, Thailand, October 28-31, 2003. This symposium and the publication of the symposium proceedings were sponsored by the WALTHAM Centre for Pet Nutrition, a division of Mars, Inc. Symposium proceedings were published as a supplement to The Journal of Nutrition. Guest editors for this supplement were D’Ann Finley, James G. Morris, and Quinton R. Rogers, University of California, Davis.

2 This research was jointly funded by the WALTHAM Centre for Pet Nutrition and the University of Nottingham, United Kingdom.

4 Abbreviations used: CMV, cytomegalovirus; FRAP, ferric reducing antioxidant power; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species.

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Daniel G. Blount,*[dagger] Paul R. Heaton,*3 and David I. Pritchard[dagger]

* WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, Leicestershire LE14 4RT, UK and [dagger] Immune Modulation Research Group, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK

3 To whom correspondence should be addressed. E-mail: paul.heaton@eu. effem.com.

Copyright American Institute of Nutrition Aug 2004