Overweight, obesity, or a sedentary lifestyle may account for as much as 25% of all cancers, with the risk reduction differing for various cancers (24,38). Higher levels of regular, moderate-intensity physical activity are associated with a reduced risk of several cancers, including those of the breast, colon, and endometrium (34,47). Prospective studies suggest that weight loss and maintenance are associated with a reduction in breast cancer risk (22,25,34).
DNA damage has been suggested as contributing to aging and chronic diseases, especially cancer. Genomic instability, which may be caused by DNA repair defects, is regarded as a hallmark of cancer (19). DNA repair function is critical in maintaining genome stability. Defective functioning of DNA repair enzymes underlies many of the known cancer syndromes, including Lynch syndrome (also called hereditary nonpolyposis colorectal cancer) and xeroderma pigmentosum (32). Regular physical activity can reduce oxidative stress levels, putatively because of adaptation involving an improved capacity to repair oxidative-stress–related damage incurred during exercise (training effect) (37). This is supported by trials showing reduced urinary oxidative stress after regular exercise (8).
There is increasing evidence that weight loss promotes genomic stability by increasing DNA repair capacity, particularly base excision repair (4,7). Caloric restriction can reverse the age-related decline in base excision repair capacity in rats (7). Excess caloric intake and obesity have been shown to increase generation of reactive oxygen species, which can ultimately lead to DNA damage via, for example, the creation of oxidized bases. Human studies have not yet addressed possible effects of caloric restriction on DNA repair capacity. Indirect evidence for such a link comes from studies reporting effects of calorie-restricted diets in humans on oxidative DNA damage levels (13,23): oxidative DNA damage (5-hydroxymethyl-2′-deoxyuridine) decreased after a calorie-restricted diet for a short period of 2 wk in women (13) and, likewise, caloric restriction also decreased DNA damage (8-oxo-7,8-dihydroguanine) after 1 yr in men and women (23). Rodent studies have shown that caloric restriction increases resistance to oxidative damage (14,30,48).
The alkaline comet assay is a sensitive technique for the detection of DNA damage and DNA repair, requiring only a small number of cells such as peripheral blood mononuclear cells (PBMC), and has also been shown to perform well using cryopreserved blood cells (10). As noted by Berwick and Vineis (5), the use of such tests in human populations has yielded consistent positive associations between inferred or actual DNA repair capacity and particularly breast cancer (5,27). The relevance for other cancer types is currently unknown. A caveat regarding the studies to date is that no prospective investigation of DNA repair capacity (measured by these assays) and future cancer incidence has been undertaken, largely because of the expense of cryopreserving viable lymphocytes in cohort studies.
In an ancillary study to a randomized controlled trial in postmenopausal women, we investigated the independent and combined effects of a moderate to vigorous aerobic exercise intervention and a reduced calorie weight loss diet on the level of DNA damage and DNA repair in 226 women. We hypothesized that the combination of a reduced calorie weight loss diet with moderate to vigorous aerobic exercise would result in enhanced DNA repair capacity measured in PBMC by the comet assay compared with either intervention alone and compared with no lifestyle change (control).
SUBJECTS AND METHODS
Study design and participants.
The Nutrition and Exercise for Women (NEW) study was a 12-month randomized controlled trial conducted from 2005 to 2009, which tested the effects of exercise and reduced calorie dietary weight loss on biomarkers of postmenopausal breast cancer risk (17,33). Participants were recruited from the greater Seattle area, WA, through mass mailing, media placements, and community outreach.
Inclusion criteria were as follows: 50–75 yr of age; body mass index (BMI) ≥25.0 kg·m−2 (if Asian American, ≥23.0 kg·m−2); <100 min·wk−1 of leisure-time moderate activity; postmenopausal; no postmenopausal hormone therapy for the past 3 months; no history of breast cancer, heart disease, diabetes mellitus, or other serious medical conditions; fasting glucose <126 mg·dL−1 and not taking diabetes medication; nonsmoking; alcohol intake of less than two drinks per day; ability to attend diet/exercise sessions at the intervention site; and a normal exercise tolerance test.
The trial design and recruitment have been previously reported (17). Briefly, a total of 439 eligible women were stratified according to BMI (<30 kg·m−2 or ≥30 kg·m−2) and race/ethnicity (non-Hispanic white, black, other), then randomized into one of four groups: 1) reduced calorie weight loss diet (“diet,” n = 118), 2) moderate- to vigorous-intensity aerobic exercise (“exercise,” n = 117), 3) combined reduced calorie weight loss diet and moderate- to vigorous-intensity aerobic exercise (“diet + exercise,” n = 117), or 4) no diet or exercise change (“control,” n = 87). We used permuted block randomization (ratio, 0.75/1/1/1) to assign a proportionally smaller number of women to the control group. In the present ancillary study that began after the parent trial was already underway, we enrolled a subset of 226 women (Fig. 1).
The Fred Hutchinson Cancer Research Center Institutional Review Board in Seattle, WA, approved the study; all participants signed an informed consent.
Interventions and control group.
The reduced calorie weight loss intervention was a modification of the Diabetes Prevention Program (28) and the Look AHEAD (39) lifestyle interventions and has been previously described (17). The diet had a total energy intake goal of 1200–2000 kcal·d−1 based on weight and <30% daily energy intake from fat. The weight loss goal was 10% by 6 months, with maintenance thereafter. Dietitians with training in behavior modification conducted the sessions. Participants met individually with a study dietitian at least twice and attended weekly dietitian-led group meetings (5–10 women) in months 1–6. In each of months 7–12, participants had one face-to-face individual or group contact and one contact with a dietitian via phone or email. The diet sessions of the diet + exercise group were held separately from those of the diet group, and participants were requested not to discuss diet during facility drop-in exercise training sessions.
The exercise intervention goal was 45 min of moderate- to vigorous-intensity intensity exercise 5 d·wk−1 for 12 months. Participants attended three supervised sessions per week at the facility and exercised 2 d·wk−1 at home. They gradually increased exercise training from a 15-min session at 60%–70% maximal HR to the target 70%–85% of observed maximal HR by week 7 and maintained it for the duration of the study. Facility-based exercise consisted of treadmill walking, stationary bicycling, and use of other aerobic machines, whereas a variety of home exercises were encouraged including walking/hiking, aerobics, and bicycling. Participants recorded exercise mode, duration, peak HR, and perceived exertion at each session. Activities of ≥4 METs were counted toward the prescribed target (17).
Women randomized to the control group were asked not to change their diet or exercise habits. After 12 months, they were offered four weight loss classes and 8 wk of facility exercise training.
Demographics, lifestyle behaviors, anthropometrics, and body composition were assessed at baseline and 12 months. Type, intensity, and duration of physical activity over the previous 3 months were assessed with a modified Minnesota Physical Activity Questionnaire (43). A food frequency questionnaire was employed to assess the usual dietary intake (35). Height and weight were measured with a stadiometer, and the standard scale and BMI were calculated as kilograms per meter squared. Body fat was measured by a dual-energy X-ray absorptiometry whole-body scanner (GE Lunar, Madison, WI). Exercise change from baseline was measured by average daily pedometer steps and cardiorespiratory fitness (V˙O2max in liters per minute and milliliters per kilogram per minute to allow quantification of fitness change in isolation of weight loss associated with the interventions) measured by a maximal graded treadmill test and a metabolic cart (MedGraphics, St. Paul, MN).
Blood samples, isolation, and cryopreservation of PBMC.
At baseline and 12 months, 12-h fasting blood samples (50 mL) were collected. PBMC was isolated as described (9) using density-gradient centrifugation and stored in aliquots with 3 × 106 cells at −70 °C in a freezing medium containing Roswell Park Memorial Institute-1640, fetal calf serum, and dimethyl sulfoxide.
The comet assay.
The comet assay was conducted under alkaline conditions (44), with modifications for cryopreserved PBMC as described previously (9). For this, cells were gently thawed at 37 °C and an aliquot of 4 × 103 cells was embedded in agarose onto each spot of a CometSlide™. Each measurement consisted of duplicate slides, and each slide contained two gel spots.
To induce damage, two sets of cell-embedded slides were exposed to 1.23 Gy of γ-irradiation at room temperature. Subsequently, one set of slides was immersed immediately in ice-cold lysis buffer, whereas the other set was incubated at 37 °C in 95% air/5% CO2 in complete medium for 30 min to allow repair before immersing in lysis buffer. We observed about 5- to 14-fold increases in DNA damage after γ-irradiation, which was then partially reversed during DNA repair. The mean intraassay coefficient of variation of DNA repair capacity was 10%.
After lysis, electrophoresis, neutralization, ethanol washing, and air drying, the slides were stored at room temperature until analysis. Before scoring, the slides were stained with 50 μL of SYBR Green I fluorescent dye. Automated analysis of the comet assay was performed by using a MetaCyte CometScan System on the basis of the slide scanning platform Metafer (MetaSystems, Altlussheim, FRG), which consists of a motorized microscope (AxioImager M1; Carl Zeiss, Jena, Germany) with fluorescence illumination, a motorized X/Y scanning stage (Maerzhaeuser, Wetzlar, Germany) with a CCD camera (CVM4 + CL; JAIAS, Glostrup/Copenhagen, Denmark), and a Windows compatible PC running the Metafer software. Percentage DNA in the tail, tail moment (the product of comet tail length and percentage DNA in the tail), and tail moment arm (tail moment divided by the normalized integrated tail intensity to measure the average distance of DNA migration within the tail) was randomly determined from 102 cells (scored for 2 gel spots) with a cell viability by Trypan Blue exclusion test of ≥65% as described (9). We also examined the data using both 65% and 80% as a cutoff, which did not significantly change the results. DNA repair capacity was calculated as (induced damage level − damage level after 30 min of repair)/(induced damage level) × 100% with higher percentage values reflecting a greater DNA repair capacity. Assays were performed blinded and samples were excluded with assays being repeated using a second aliquot of the PBMC if the viability was below 65% or fewer than 100 comets were scanned.
We first investigated changes in DNA repair capacity and radiation sensitivity using cell populations with different cell viabilities (≥65% vs ≥80% viability). There were no differences in results. We thus included all participants with cell viabilities ≥65% in the comparison of the intervention groups compared with control. In addition, we also examined changes stratified by degree of weight change (<5% and ≥5%) and change in V˙O2max (L·min−1) (<5%, 5%–15%, or ≥15%) within the intervention groups. We applied the generalized estimating equation approach to account for repeated measurements on the same subjects. In addition, associations between baseline DNA repair capacity and age and energy balance (BMI, weight, lean mass, and fat mass) were investigated by Pearson correlation. Statistical analyses were performed using SAS (version 9.2; SAS Institute Inc., Cary, NC).
We conducted power calculations: with sample sizes for two groups being 66 (and 44, respectively), we had 80% power (type 1 error, 0.0167) to detect a difference in DNA repair capacity of 7.0 (10.2) if the SD was 11 (16) and the intraindividual correlation between baseline and 12 months was 0.5.
A description of the women participating in the study is given in Table 1. The mean age of participants in all groups was 58.2 yr; the majority (83.6%) was non-Hispanic whites with a higher education (64.6 % had at least a college degree). The average BMI was >30 kg·m−2; aerobic fitness, 22.7–24.0 kg·mL−1·min−1; and the caloric intake, about 1900 kcal·d−1. Baseline characteristics did not differ among the intervention groups.
DNA repair capacity was assessed by three analytical variables in parallel (moment arm, percentage DNA in tail, and tail moment), which all describe the proportion of damaged cells after repair compared with the basal DNA damage. At the end of the 12-month intervention period, none of the interventions: a reduced calorie diet, a moderate- to vigorous-intensity aerobic exercise intervention, or a combination of both changed DNA repair capacity compared with control (Table 2a). DNA repair capacity did not differ across groups (∼70% moment arm, ∼62% percentage DNA in tail, and ∼85% tail moment). The same was true for the radiation sensitivity (initial DNA damage after irradiation) of the PBMC (∼1% moment arm, ∼7% percentage DNA in tail, and ∼1% tail moment; data were not further shown). Measures of the intervention efficacy, such as weight and change in V˙O2max from baseline to 12 months, have been reported for the parent trial (17); these results are summarized in Table 2a. The diet intervention resulted in a 10% decrease in body weight (P < 0.0001), and the exercise intervention resulted in an 8% improvement of V˙O2max (P = 0.0004). In addition, both variables showed statistically significant changes in the combined diet and exercise intervention (P < 0.0001 and P = 0.009, respectively; Table 2a).
When we stratified by weight loss (<5% and ≥5%), no statistically significant effects of the 12-month interventions compared with control were detectable (Table 2b). In the diet + exercise group, a statistically significant increase (P trend = 0.015) in DNA repair capacity was observed among those women with the highest gain in V˙O2max (≥15%) compared with those with the lowest V˙O2max gain (<5%) (Table 3).
At baseline, DNA repair capacity was weakly positively correlated with weight, BMI, and fat mass and weakly inversely correlated with lean mass (Fig. 2 and Table 4). No such associations with 12-month DNA repair capacity or baseline or 12-month radiation sensitivity were observed (data not shown).
This randomized controlled trial showed that an exercise program, a reduced calorie weight loss diet, or a combination of both did not significantly alter DNA repair capacity in PBMC of postmenopausal women. However, we observed a weak positive correlation between body weight, BMI, and fat mass (as well as a weak negative correlation of lean mass) with baseline DNA repair capacity. To the best of our knowledge, this is the first randomized controlled trial to examine the effects of these interventions on DNA repair capacity in postmenopausal women during a long-term (12-month) intervention period.
A prolonged single bout of exercise (e.g., 42-km marathon running) results in a significant degree of unrepaired DNA base oxidation in peripheral lymphocytes despite a concurrent increase in the urinary excretion of the repaired base, 8-hydroxy-2′-deoxyguanine (45). Regular participation in moderate-intensity training (e.g., 30,000-km cycling per year) appears to result in an adaptive response that is beneficial with respect to antioxidant activity and DNA repair function. Mechanisms include the up-regulation of DNA repair enzymes (46). Allgayer et al. (1) have described a short-term (2 wk), moderate-intensity (0.3–0.4 × maximal exercise capacity) exercise program that resulted in decreased levels of urinary 8-hydroxy-2′-deoxyguanosine. In a 12-month randomized controlled trial of moderate intensity (at 60%–75% of HR maximum) 5-d aerobic exercise per week versus the usual lifestyle control, decreased levels of the oxidative marker F2-isoprostane were observed only when accompanied by marked gains in aerobic fitness (V˙O2max) (8). In our study, DNA repair capacity increased in women with the greatest improvement in V˙O2max (≥15%) compared with those of the lowest (<5%). However, this finding was limited to women in the diet + exercise group only and may be a chance observation.
Another component of energy balance that can affect oxidative stress is obesity. The major evidence of obesity-induced oxidative stress in humans comes from cross-sectional studies reporting the effects on oxidative biomarkers, such as elevated F2-isoprostanes (6,26,42) or elevated thiobarbituric acid reactive substances (18,29) by BMI categories. Exercise training reduces F2-isoprostane concentrations among women of not only younger (2,12,40) but also older age (8). A benefit of aerobic exercise training was only apparent in young women with high baseline plasma F2-isoprostanes (2). Within longitudinal studies, dietary and exercise interventions have been used to investigate the role of weight loss on markers of oxidative stress. A combined diet and exercise intervention was reported to lower lipid peroxidation after 4% weight loss in obese persons (3). The effects of long-term (12 months) dietary weight loss intervention in postmenopausal women by diet and/or exercise on DNA repair capacity and radiation sensitivity had not previously been assessed; here we report no effect.
In contrast to other reports, we did not detect an age-dependent decrease of DNA repair capacity, which may be due to the somewhat limited age range of our study population (age 50–75 yr). Furthermore, we observed a positive correlation between DNA repair capacity and BMI, body weight, and body fat. The data from earlier studies are inconsistent (36–38). We have no explanation for our finding, which is contrary to our hypothesis. However, the correlation coefficients were of medium strength and the results should not be generalized because our study population was very homogeneous and all participants were overweight or obese (mean BMI >30 kg·m−2).
A range of methods are available for the quantification of DNA damage in cells. One of the most commonly employed methods to detect DNA damage in human studies is the comet assay (16), which has been previously used in diet intervention studies (36) and studies on physical activity (21). There are different approaches to analyze comets, including visual inspection and classification, as well as computer-supported image analysis (11). For this study, we used the latter and reported the results by three validated comet assay variables (20): moment arm, percentage DNA in tail, and tail moment. Generally, percentage DNA in tail covers the widest range of damage and is linearly related to break frequencies. The tail moment recommended is expressing both the tail length and percentage DNA in tail, but it is measured in nonstandard units and, therefore, may be difficult to interpret across studies (11). In our study, results from all three parameters were comparable. Within longitudinal studies such as ours, cryopreservation of PBMC is essential to avoid batch effects across samples collected at various time points. The method we applied produces comparable results to those obtained by the use of freshly isolated PBMC (9). There is still debate whether apoptotic or necrotic cells increase the measured comet assay variability and thus impair the results of the comet assay (20,31). We therefore included only results from PBMC with a cell viability ≥65%; however, the results did not differ when we restricted viability to those ≥80% at both time points. Cryopreserved lymphocytes are not suitable for studying DNA repair capacity after H2O2 treatment (15), with the most common genotoxic agent mimicking oxidative stress. Therefore, we used radiation as the genotoxic agent. In contrast to H2O2, which causes single- and double-strand breaks, radiation additionally gives rise to base damage, as well as DNA strand and DNA protein cross-links. However, the measurement of H2O2-induced DNA damage is not reliably measureable in cryopreserved lymphocytes, as presented here (15). Future studies might involve other measures of DNA repair pathways, e.g., gene or protein expression associated with repair enzymes in target tissue or a combination of these together with markers of oxidative stress, i.e., F2-isoprostanes.
Our study had several strengths: the large study size, with sufficient power to assess long-term changes in DNA repair capacity, a randomized controlled design with three intervention arms, high retention (91%) in the parent trial (the ancillary study was restricted to participants with two repair capacity measurements), excellent adherence to the intervention programs, and gold standard measures of aerobic fitness and body composition (17). We chose a valid and robust method to detect DNA repair capacity, with the comet assay under alkaline conditions (10), in cryopreserved PBMC of a randomized controlled trial. Measuring DNA repair in PBMC is widely used in human biomonitoring studies that provide an easy way to assess DNA repair in a human population (15). There is some evidence that PBMC are representative of the general organism; however, the level of DNA repair within individual target tissues may vary. Comparing colon cancer tissue and PBMC of the same individuals revealed that there is a positive correlation between their repair capacities despite tissue-specific differences (41).
We recognize that there are some limitations, including the assay restrictions discussed above. Our finding of no change in DNA repair capacity by any of the interventions could be due to too low radiation-induced DNA damage. With the method applied, we achieved a 5- to 14-fold increase in DNA damage by γ-radiation compared with the basal damage. These levels of induced damage are appropriate for cryopreserved lymphocytes, and the average DNA repair capacity in cryopreserved lymphocytes is lower than that of fresh samples (9). To avoid high assay batch-to-batch variability, which is of particular relevance for the comet assay, we have chosen to cryopreserve pre- and postintervention lymphocytes. These were assayed simultaneously in our study. However, compared with frozen lymphocytes, examining DNA repair using fresh lymphocytes would result in higher repair capacities. This could be a reason for a lack of effect in our study. It is possible that a higher level of DNA damage at induction before in vitro DNA repair is more appropriate to cover a broader range of DNA repair capacity. Similarly, repair capacity before the interventions was already high (in average, 70%), resulting in a possible ceiling effect. One possibility for the lack of effects is that the SD for our DNA repair measures was slightly greater (SD, 9.4–20, Table 2a) than that in our own initial work (9) and as used in the initial power calculation (SD, 11–16,). We also cannot exclude that stratification by V˙O2max as a measure of fitness may not fully capture the effects of exercise on DNA repair capacity. In the future, one would include additional measures of DNA repair capacity or markers of cellular stress adaption, such as changes of superoxide dismutase activity or gene expression ultimately directly in the target tissue (e.g., epithelial tissue or muscle). Also, our study population was a relatively homogeneous sample of postmenopausal women, primarily non-Hispanic white, and our results may thus not be generalizable to all populations or the health effect on, e.g., specific cancer types. Furthermore, only one exercise program and one dietary weight loss program were tested; therefore, we are unable to speculate on the effects of other weight loss methods or other types or intensities of exercise programs on DNA damage and DNA repair capacity changes.
In conclusion, an altered energy balance due to long-term physical activity, dietary restriction, or a combination of both did not affect DNA repair capacity in overweight and obese postmenopausal women. Further studies with other measures of DNA repair function may be needed.
This work was supported by the National Cancer Institute at the National Institutes of Health (grants U54-CA116847, R01 CA102504; 5KL2RR025015-03 to K.F.S; R25 CA94880 to A.K.) and Canadian Institutes of Health Research (fellowships to K.L.C and C.M.). None of the funding agencies were involved in the trial design or conduct. During the trial, Dr. Alfano was a faculty member at The Ohio State University and relocated to NCI after completion of her efforts on the NEW trial.
Anne McTiernan and Cornelia M. Ulrich are dual senior authors.
Trial Registration: NCT00470119, http://www.clinicaltrials.gov.
No financial disclosures were reported. Results of the present study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:© 2015 American College of Sports Medicine
DNA REPAIR; COMET ASSAY; RANDOMIZED CONTROLLED TRIAL; EXERCISE; CALORIC RESTRICTION; WOMEN