AJCN Cancer Health Disparities Conference
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, X.
Right arrow Articles by Yeum, K.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, X.
Right arrow Articles by Yeum, K.-J.
Agricola
Right arrow Articles by Zhao, X.
Right arrow Articles by Yeum, K.-J.
American Journal of Clinical Nutrition, Vol. 83, No. 1, 163-169, January 2006
© 2006 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Modification of lymphocyte DNA damage by carotenoid supplementation in postmenopausal women1,2,3,4

Xianfeng Zhao, Giancarlo Aldini, Elizabeth J Johnson, Helen Rasmussen, Klaus Kraemer, Herb Woolf, Nina Musaeus, Norman I Krinsky, Robert M Russell and Kyung-Jin Yeum

1 From the Jean Mayer USDA-Human Nutrition Research Center on Aging (XZ, EJJ, HR, NIK, RMR, and K-JY) and the Department of Biochemistry, School of Medicine (NIK), Tufts University, Boston, MA; the Istituto Chimico Farmaceutico Tossicologico, University of Milan, Milan, Italy (GA); BASF AG, Ludwigshafen, Germany (KK); BASF Corp, Florham Park, NJ (HW); and BASF Health and Nutrition A-S, Ballerup, Denmark (NM)

2 The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

3 Supported in part by the US Department of Agriculture, under agreement number 1950-51000-065-02A and BASF Aktiengesellschaft, Germany.

4 Address reprint requests to K-J Yeum, Jean Mayer US Department of Agriculture-Human Nutrition Research Center on Aging, Tufts University, 711 Washington St, Boston, MA 02111. E-mail: kyungjin.yeum{at}tufts.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging such as cancer and cardiovascular disease. Carotenoids could be a part of a protective strategy to minimize oxidative damage in vulnerable populations, such as the elderly.

Objective: Our aim was to determine the protective effect of carotenoids against DNA damage.

Design: A randomized, double-blind, placebo-controlled intervention study was conducted. Thirty-seven healthy, nonsmoking postmenopausal women aged 50–70 y were randomly assigned to 1 of 5 groups and were instructed to consume a daily dose of mixed carotenoids (ß-carotene, lutein, and lycopene; 4 mg each), 12 mg of a single carotenoid (ß-carotene, lutein, or lycopene), or placebo for 56 d. Plasma carotenoid concentrations were analyzed by using HPLC, and lymphocyte DNA damage was measured by using a single-cell gel electrophoresis (comet) assay.

Results: At day 57, all carotenoid-supplemented groups showed significantly lower endogenous DNA damage than at baseline (P < 0.01), whereas the placebo group did not show any significant change. Significantly less (P < 0.05) endogenous DNA damage was found as early as day 15 in the mixed carotenoid (P < 0.01) and ß-carotene (P < 0.05) groups.

Conclusions: The results indicate that carotenoid supplementation decreases DNA damage and that a combination of carotenoids (4 mg each of lutein, ß-carotene, and lycopene), an intake that can be achieved by diet, or a larger dose (12 mg) of individual carotenoids exerts protection against DNA damage.

Key Words: Mixed carotenoids • lutein • ß-carotene • lycopene • DNA damage • comet assay • elderly women


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging such as cancer and cardiovascular disease (1). Epidemiologic studies have shown that a high fruit and vegetable intake is associated with a lower risk of such chronic diseases (2-5). It is probable that carotenoids found in fruit and vegetables act as a major group of antioxidants, thereby preventing damage from harmful reactive oxygen species, which are continuously produced in the body from normal cellular functions and from exogenous sources (6). Carotenoids have also been reported to modulate the gene expression of steroid targets and inflammatory markers in animals (7) and to prevent UVA radiation–induced gene regulation in human cells (8).

Dietary supplementation with antioxidants, such as carotenoids, can be a part of a protective strategy to minimize the oxidative damage in vulnerable populations, such as the elderly. Carotenoids have in vitro antioxidant activity at physiologic oxygen tensions (9). However, this antioxidant effect of carotenoids is still controversial in vivo (10, 11). It should be pointed out that the metabolism and functions of carotenoids may differ in in vivo compared with in vitro systems. For example, antioxidant nutrients can interact with each other during gastrointestinal absorption and metabolism (12-15). Therefore, it is important to determine the biological actions of single carotenoids compared with those of mixed carotenoids in vivo.

The single-cell microgel electrophoresis assay (ie, the comet assay) was developed to detect DNA single or double strand breaks. Broken DNA fragments result in an increased migration in electrophoresis to form a diffuse DNA area, which resembles a comet tail after staining (16). Pool-Zobel et al (17, 18) reported that supplementation of the diet of healthy persons with tomato, carrot, or spinach significantly reduced the number of endogenous strand breaks in lymphocyte DNA. Given that these foods also contain various phytonutrients other than carotenoids, it is difficult to ascertain if the DNA protection was because of the carotenoids. Currently, no human studies exist on the protective role of a combination of the major dietary carotenoids, ie, lutein, ß-carotene, and lycopene, against DNA strand breaks. The present study was conducted to compare the protective effect of single carotenoid with that of mixed carotenoid supplementation for 8 wk against DNA damage in elderly women.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Forty nonsmoking postmenopausal women aged 50–70 y were enrolled in the present study. All study subjects were in good health, as determined by a medical history questionnaire, physical examination, and normal results of clinical laboratory tests. All study subjects fulfilled the following eligibility criteria: 1) no history of cardiovascular, hepatic, gastrointestinal, or renal disease; 2) no alcoholism, no smoking, and no exogenous hormone use; 3) no supplemental vitamin or carotenoid use for >6 wk before the start of the study; and 4) plasma carotenoid concentrations <200% of the National Health and Nutrition Examination Survey (NHANES) III median concentration at screening, which excluded women with extreme carotenoid diets. The study protocol was approved by the Institutional Review Board of Tufts-New England Medical Center and Tufts University Health Sciences, and written informed consent was obtained from each study subject.

Study design
A research dietitian instructed the subjects to exclude foods rich in carotenoids and to limit fruit and vegetable intake to 2 servings/d of light colored fruit and vegetables for 2 wk before starting the study and during the entire study to maximize the blood response to the low doses of carotenoid supplementation. The subjects were randomly assigned to receive one of the following treatments: 1) placebo; 2) 4 mg/d each of lutein, ß-carotene, and lycopene; 3) 12 mg lutein/d; 4) 12 mg ß-carotene/d; or 5) 12 mg lycopene/d. On sampling days 1, 15, 29, and 43, the subjects were provided with a 2-wk supply of supplements. The subjects were instructed to take the carotenoid supplements with their first meal of the day; the meal was to contain ≥10 g fat to facilitate absorption of the carotenoid supplement. The carotenoid supplements were supplied by BASF AG (Ludwigshafen, Germany) as hard gelatin capsules. ß-Carotene and lycopene were synthetic and lutein was of natural origin. To fill the capsules, the carotenoids were formulated as dry powders (beadlets) in such a way that the carotenoids were embedded in a matrix of hydrocolloid and sucrose. For mixed carotenoids, these beadlets contained a mixture of lutein, ß-carotene, and lycopene (ie, a multicore structure). Dietary compliance, which included counting any remaining pills and checking the compliance calendar, 3-d food records, and food-frequency questionnaires, was measured biweekly. The research dietitian at the Jean Mayer US Department of Agriculture-Human Nutrition Research Center on Aging at Tufts University interviewed the study subjects on each sampling day.

Fasting (12-h) blood samples were collected in evacuated containers containing EDTA at baseline and at days 15, 29, 43, and 57, and lymphocytes were isolated immediately after collecting the samples and cryopreserved until the single-cell gel electrophoresis analysis. Plasma samples were divided into aliquots and stored at –80 °C for subsequent analyses of carotenoids.

Plasma carotenoid and tocopherol analysis
all-trans-ß-Carotene (type IV), {alpha}-carotene, and lycopene standards were purchased from Sigma Chemical Co (St Louis, MO). Lutein was purchased from Kemin Industries (Des Moines, IA). All HPLC solvents were obtained from JT Baker Chemical (Phillipsburg, NJ) and were filtered through a 0.45-µm membrane filter before use.

Plasma carotenoid concentrations were measured by an HPLC system as previously described, with minor modifications (19). Plasma samples (200 µL) were extracted with 2 mL chloroform:methanol (2:1, by volume) followed by 3 mL hexane. Samples were dried under nitrogen and resuspended in 75 µL ethanol:methyl tert-butyl ether (2:1, by volume), of which 25 µL was injected onto the HPLC. The HPLC system consisted of a Waters 2695 Separation Module (Waters Instruments, Milford, MA), 2996 Photodiode Array Detector (Waters Instruments), a Waters 2475 Multi {lambda} Fluorescence Detector (Waters Instruments), a C30 carotenoid column (3 µm, 150 x 3.0 mm; YMC, Wilmington, NC), and a Waters Millenium 32 data station (Waters Instruments). The mobile phase was methanol:methyl tert-butyl ether:water (85:12:3, by volume, with 1.5% ammonium acetate in water; solvent A) and methanol:methyl tert-butyl ether:water (8:90:2, by volume, with 1% ammonium acetate in water; solvent B). The gradient procedure was reported earlier (19). The results were adjusted by an internal standard containing echinenone and retinyl acetate. The interassay CV (n = 25) was 4% and the intraassay CV was 4% (n = 9). Recovery of the internal standard averaged 97%.

Single-cell gel electrophoresis analysis (comet assay)
Lymphocyte separation
Lymphocytes were separated immediately after the blood samples were collected. Lymphocytes were isolated by density gradient sedimentation (Histopaque 1077; Sigma Diagnostics) and frozen in a mixture containing 50% fetal calf serum, 40% culture medium (RPMI 1640; Sigma Diagnostics), and 10% dimethyl sulfoxide at a freezing rate of –1 °C/min to a final temperature of –80 °C before storage in liquid nitrogen.

Cryopreserved lymphocyte recovery
Cells were recovered by submersion in a 37 °C water bath until the last trace of ice was melted. The cells were transferred to prechilled 50% RPMI 1640 medium and 50% fetal calf serum and then were centrifuged at 200 x g for 5 min at 4 °C. The cells were resuspended in cold phosphate buffered saline and checked for viability (typically ≥95% viability) and cell number (typically 1 x 105 cells/mL). For each subject, the lymphocytes at the 5 time points (days 1, 15, 29, 43, and 57) were recovered on the same day and time.

Alkaline single-cell gel electrophoresis
DNA strand breaks were measured in lymphocytes with the alkaline single-cell gel electrophoresis assay, ie, the comet assay, (20) with minor modifications. Endogenous DNA damage and hydrogen peroxide-challenged DNA damage were measured by exposing the agarose embedded with cells to either phosphate buffered saline alone or hydrogen peroxide (10 µmol/L) in phosphate buffered saline for 10 min.

Quantitation of DNA damage
DNA damage was measured by visual image analysis (21). The comet-like images were classified visually into 5 categories (0–4) by a blinded observer according to appearance, which resulted from the relative proportion of DNA in the tail, as shown in Figure 1Go. At least 100 cells were counted and categorized to avoid any selection bias. The percentage DNA in the tail [(2.5 x cells0 + 12.5 x cells1 + 30 x cells2 + 60 x cells3 + 90 x cells4)/{Sigma} cells] was calculated to express the amount of DNA damage.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 1. Visual classification of DNA damage, according to the relative proportion of DNA in the tail (scores 0–4), obtained by single-cell gel electrophoresis. Score 0 represents undamaged cells, and score 4 represents the most heavily damaged cells.

 
Statistical analysis
Values are expressed as means ± SEMs. Three subjects dropped out during the study because of personal reasons. When the randomization code was uncovered after completing the biochemical analyses, there were 6 subjects in the placebo group, 8 in the mixed carotenoid, lutein, and lycopene groups, and 7 in the ß-carotene group. A repeated-measures analysis of variance with a post hoc Dunnett's multiple comparison test was used to measure the effect of carotenoid supplementation on plasma carotenoid concentrations and to measure the endogenous DNA damage for each day with respect to day 1. The differences between the groups for each time point were measured by a one-way analysis of variance with Tukey's multiple comparison. A paired t test was used to calculate the effect of low fruit and vegetable diets for 2 wks (day –14 compared with day 1). When an equal variance test failed, a Mann-Whitney rank sum test was used. The statistical analyses were performed with SIGMASTAT (version 3.1; Systat Software Inc, Point Richmond, CA) and SPSS (version 11.5; SPSS Inc, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in plasma carotenoid concentrations after low fruit and vegetable diets and carotenoid supplementation
The low fruit and vegetable diets the subjects consumed for 2 wk before starting the intervention resulted in decreases in plasma carotenoid concentrations, as shown in Table 1Go. Two weeks on a low fruit and vegetable diet reduced the subjects' plasma carotenoid concentrations of lutein, ß-carotene, and lycopene by 62%, 64%, and 58%, respectively (day –14 compared with day 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Changes in plasma carotenoid concentrations in all the study participants after low fruit and vegetable diets for 14 d1

 
The mean (±SEM) baseline concentrations of {alpha}-tocopherol, ascorbic acid, uric acid, and characteristics of the study subjects are presented in Table 2Go. No significant difference in major antioxidant nutrients and lipid profiles were observed between the groups at baseline (day 1).


View this table:
[in this window]
[in a new window]
  		TABLE 2 Anthropometric and biochemical characteristics of the study groups at baseline (day 1)1

 
Plasma lutein concentrations were significantly increased on day 15 in the mixed carotenoids and lutein groups (P < 0.01), so that the values were 228% and 514%, respectively, of the baseline values (Figure 2Go). These concentrations were maintained throughout the study period. No significant increases in plasma lutein concentrations were observed in the placebo, ß-carotene, and lycopene groups throughout the intervention period. The concentrations of plasma ß-carotene were significantly increased in the mixed carotenoids and ß-carotene groups after 15 d of supplementation (P < 0.01) so that concentrations were 146% and 387%, respectively, of the baseline concentrations. The placebo-, lutein-, and lycopene-supplemented groups did not show any significant increases in plasma ß-carotene concentrations (Figure 3Go). Plasma lycopene concentrations were significantly increased in the lycopene group after 15 d of supplementation (P < 0.01), whereas the placebo, lutein, and ß-carotene groups had significantly lower plasma lycopene concentrations during the intervention period than at baseline (P < 0.05) (Figure 4Go). The plasma lycopene concentrations of the mixed carotenoids group did not significantly change during the intervention period.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. Mean (±SEM) plasma lutein concentrations over time by carotenoid supplementation for 56 d in postmenopausal women (aged 50–70 y). The lutein, ß-carotene, and lycopene groups were supplemented with 12 mg/d of each respective carotenoid. The mixed carotenoids group was supplemented with 4 mg/d each of lutein, ß-carotene, and lycopene. n = 6, 8, 8, 7, and 8 for placebo, mixed carotenoids, lutein, ß-carotene, and lycopene groups, respectively. No significant difference was observed between groups on day 1 (one-factor ANOVA). The time-by-treatment interaction was significant, P < 0.001. *,**Significantly different from baseline (day 1) (repeated-measures ANOVA with Dunnett's multiple comparison): *P < 0.05, **P < 0.01.

 


View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3. Mean (±SEM) plasma ß-carotene concentrations over time by carotenoid supplementation for 56 d in postmenopausal women (aged 50–70 y). The lutein, ß-carotene, and lycopene groups were supplemented with 12 mg/d of each respective carotenoid. The mixed carotenoids group was supplemented with 4 mg/d each of lutein, ß-carotene, and lycopene. n = 6, 8, 8, 7, and 8 for placebo, mixed carotenoids, lutein, ß-carotene, and lycopene groups, respectively. No significant difference was observed between the groups on day 1 (one-factor ANOVA). The time-by-treatment interaction was significant, P < 0.001. *,**Significantly different from baseline (day 1) (repeated-measures ANOVA with Dunnett's multiple comparison): *P < 0.05, **P < 0.01.

 


View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 4. Mean (±SEM) plasma lycopene concentrations over time by carotenoid supplementation for 56 d in postmenopausal women (aged 50–70 y). The lutein, ß-carotene, and lycopene groups were supplemented with 12 mg/d of each respective carotenoid. The mixed carotenoids group was supplemented with 4 mg/d each of lutein, ß-carotene, and lycopene. n = 6, 8, 8, 7, and 8 for placebo, mixed carotenoids, lutein, ß-carotene, and lycopene groups, respectively. No significant difference was observed between the groups on day 1 (one-factor ANOVA). The time-by-treatment interaction was significant, P < 0.001. *,**Significantly different from baseline (day 1) (repeated-measures ANOVA with Dunnett's multiple comparison): *P < 0.05, **P < 0.01.

 
Endogenous and hydrogen peroxide–induced DNA damage after carotenoid supplementation
The effects of carotenoid supplementation on DNA damage are shown in Table 3Go. Endogenous DNA damage at baseline was significantly higher in the ß-carotene group than in the placebo group (P < 0.05). Compared with day 1, the amount of endogenous DNA damage was significantly decreased by day 15 in the mixed carotenoid (P < 0.01) and ß-carotene (P < 0.05) groups and by day 57 in all of the carotenoid-supplemented groups (P < 0.01). The placebo group did not show any significant change in endogenous DNA damage throughout the intervention period. When DNA was challenged by hydrogen peroxide (10 µmol/L for 10 min), there were no significant time-by-treatment interaction and no changes over time or across treatment groups (Table 3Go).


View this table:
[in this window]
[in a new window]
  		TABLE 3 The effects of carotenoid supplementation on endogenous and hydrogen peroxide–induced lymphocyte DNA damage at 5 time points1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Epidemiologic evidence continues to accumulate that diets high in fruit and vegetables are associated with a reduced risk of chronic diseases, such as cardiovascular disease (5, 22-25). Carotenoids found in fruit and vegetables may have biological actions to prevent such diseases. It has been reported that supplementation with 24 mg ß-carotene/d for 12 wk, or an equal amount of a carotenoid mixture containing lutein, ß-carotene, and lycopene, ameliorates UV radiation–induced erythema in humans (26). In addition, a protective effect of a mixed carotenoid supplement (ß-carotene, {alpha}-carotene, lycopene, lutein, bixin, and mixed paprika carotenoids) against the low-density lipoprotein oxidation that is induced by fish oil has been reported (27). The subjects of the present study were supplemented with the 3 major carotenoids found in fruit and vegetables, ie, 4 mg each of lutein, ß-carotene, and lycopene. Four mg lutein can be obtained from approximately one-fourth cup of cooked spinach, 4 mg ß-carotene from about one-third of a medium carrot or one-fourth cup of pumpkin, and 4 mg of lycopene from one medium tomato or three-fourths tablespoon of tomato paste. Therefore, our mixed carotenoid dose consisted of physiologic doses of carotenoids that can be obtained from a prudent diet.

In accordance with studies conducted by others (10, 28-31), plasma lutein, ß-carotene, and lycopene concentrations were significantly and selectively increased to mean values of 0.90, 1.47, and 1.07 µmol/L, respectively, after 15 d of supplementation with 12 mg/d of lutein, ß-carotene, or lycopene. Other carotenoid concentrations were either maintained or were lower than baseline concentrations. However, note that these increased carotenoid concentrations were much higher than 90% of those obtained in NHANES III (lutein, 0.67; ß-carotene, 0.91; lycopene, 0.70 µmol/L) for the same age, sex, and ethnic group (50–70 y, non-Hispanic white women; n = 1017) as our study subjects. The plasma concentrations reached by supplementation with 12 mg individual carotenoids are generally not attained by diet alone. On the other hand, plasma carotenoid concentrations in the mixed carotenoid group, which received 4 mg each of lutein, ß-carotene, and lycopene, reached 0.40, 0.50, and 0.57 µmol/L on day 15 for lutein, ß-carotene, and lycopene, respectively. These concentrations are well within the 50th–75th percentile range of the values from NHANES III for the same age, sex, and ethnic group and are not difficult to attain with a diet that contains the recommended intakes of fruit and vegetables. Because of the low dose of the lycopene supplement compared with the usual lycopene intake of our study subjects, lycopene concentrations did not significantly increase with 4 mg lycopene supplementation in the mixed carotenoid-supplemented group.

Oxidative DNA damage is an ongoing process of aging (32-35) and is elevated in patients with disease (36). However, intervention trials that involved increased fruit and vegetable intakes have shown mixed results regarding DNA damage; ie, the studies either show decreased oxidative DNA damage (18, 28, 37-44) or no effect on DNA damage and repair (45).

It is difficult to compare DNA damage between studies because of the discordance of methodologic conditions, such as duration of alkali unwinding, electrophoresis, and visual scoring system (46). The present study applied the visual scoring system, as described by Collins et al (21). Visual scoring data accurately corresponds to the data that is measured by computer image analysis (16, 47). Another potential variable could be the use of fresh compared with stored cells. In the present study, the cryopreserved lymphocytes of each subject that were collected at 5 time points (days 1, 15, 29, 43, and 57) were analyzed at the same time for DNA damage to avoid experimental day-to-day variance. Duthie et al (48) reported that freezing lymphocytes does not increase DNA strand breaks compared with freshly isolated lymphocytes, and that fresh and frozen lymphocytes responded almost identically to hydrogen peroxide treatment.

In our study, there was detectable endogenous DNA damage at baseline (day 1) (11.0 ± 2.3% DNA in the tail), ie, after 2 wk of a low fruit and vegetable diet (<2 servings/d). It is plausible that the deprivation of fruit and vegetables could affect endogenous DNA damage. Furthermore, it has been reported that both endogenous DNA damage (49) and hydrogen peroxide–induced DNA damage (50) are positively associated with age. In addition, increased frequencies of micronuclei and chromosome aberrations with age have been reported, which suggests increased genetic instability with age (51). We also found that our older subjects showed higher endogenous DNA damage than did the younger subjects (data not shown). It is possible that the high endogenous DNA damage in our study subjects because of the low fruit and vegetable diet allowed for a significant improvement in endogenous DNA damage as early as day 15 of supplementation with either 12 mg/d of a single carotenoid or, perhaps more importantly, with a physiologic dose of mixed carotenoids.

The effects of carotenoid supplementation against hydrogen peroxide–induced DNA damage were weaker than were those shown for endogenous DNA damage. It is possible that carotenoids actively protect against endogenous DNA damage in vivo, whereas the protective effects are reduced ex vivo due to the loss of interactions with other antioxidants.

In contrast to the present study in which we found a significant decrease in endogenous DNA damage by supplementation with 12 mg of a single carotenoid, Collins et al (52) reported that there was no effect on endogenous DNA damage in men and women aged 25–45 y after a 12-wk supplementation with 15 mg/d each of {alpha}- and ß-carotene, lutein, or lycopene in a placebo-controlled parallel study design. However, note that there was an inverse correlation between total serum carotenoid concentrations and oxidized pyrimidines in their study. On the other hand, another study that used lutein, ß-carotene, or lycopene supplementation for 1 wk, in the same amounts as those in our study, showed significant increases in DNA repair in younger men and women aged 24–34 y, probably because of an antioxidant effect of carotenoids rather than a direct effect on the repair process (53). Intervention studies that used a combination of antioxidant supplements have consistently shown protective effects against DNA damage in accordance with the present study, even though different antioxidants and dosages were applied [eg, a combination of 100 mg vitamin C, 280 mg vitamin E, and 25 mg ß-carotene (54); a combination of 8 mg lycopene, 0.5 mg ß-carotene, and 11 mg vitamin C (29); and a combination of 8.2 mg ß-carotene, 3.7 mg {alpha}-carotene, and 1.75 mg {alpha}-tocopherol (10)].

In conclusion, supplementation with a single carotenoid or a combination of carotenoids resulted in beneficial effects against DNA damage in older women. In particular, a physiologic dose of mixed carotenoids was protective against endogenous DNA damage in older women. Additional studies on the effect of physiologic doses of carotenoids, in combination with other antioxidants contained in fruit and vegetables, on oxidative DNA damage are needed to support the role of a diet high in fruit and vegetables in the prevention of chronic diseases such as cardiovascular diseases and cancer.


   ACKNOWLEDGMENTS
 
We thank the volunteers who participated in this study, Hong Wang for her assistance in measuring plasma carotenoids, Paul Jacques for making the NHANES III data available, and the Metabolic Research Unit and Nutrition Evaluation Laboratory staffs at the Jean Mayer US Department of Agriculture-Human Nutrition Research Center on Aging at Tufts University. We also thank the BASF AG for providing the carotenoid supplements.

XZ helped with the comet assay, data interpretation, and manuscript preparation. GA set up the comet assay for the study with K-JY and assisted in the interpretation of the results. EJJ supervised the plasma carotenoid measurements. HR was responsible for educating and monitoring the study participants and ensuring the compliance of the study participants. KK and HW were responsible for the formulation and safety of the carotenoid supplements. NM was responsible for manufacturing the carotenoid supplements. NIK and RMR helped design the study and interpret the results. K-JY designed and conducted the interventions and supervised the project and manuscript preparation. None of the authors had any advisory board affiliations or financial interest in any organization sponsoring the research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Benzie IF. Evolution of antioxidant defence mechanisms. Eur J Nutr 2000; 39: 53–61.[Medline]
  2. Liu S, Lee IM, Ajani U, Cole SR, Buring JE, Manson JE. Intake of vegetables rich in carotenoids and risk of coronary heart disease in men: the Physicians' Health Study. Int J Epidemiol 2001; 30: 130–5.[Abstract/Free Full Text]
  3. Greenberg ER, Sporn MB. Antioxidant vitamins, cancer, and cardiovascular disease. N Engl J Med 1996; 334: 1189–90.[Free Full Text]
  4. Gaziano JM, Hennekens CH. The role of beta-carotene in the prevention of cardiovascular disease. Ann N Y Acad Sci 1993; 691: 148–55.[Medline]
  5. Hung HC, Joshipura KJ, Jiang R, et al. Fruit and vegetable intake and risk of major chronic disease. J Natl Cancer Inst 2004; 96: 1577–84.[Abstract/Free Full Text]
  6. Gate L, Paul J, Ba GN, Tew KD, Tapiero H. Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 1999; 53: 169–80.[Medline]
  7. Herzog A, Siler U, Spitzer V, et al. Lycopene reduced gene expression of steroid targets and inflammatory markers in normal rat prostate. FASEB J 2005; 19: 272–4.[Abstract/Free Full Text]
  8. Wertz K, Hunziker PB, Seifert N, et al. beta-Carotene interferes with ultraviolet light A-induced gene expression by multiple pathways. J Invest Dermatol 2005; 124: 428–34.[Medline]
  9. Zhang P, Omaye ST. Antioxidant and prooxidant roles for beta-carotene, alpha-tocopherol and ascorbic acid in human lung cells. Toxicol In Vitro 2001; 15: 13–24.[Medline]
  10. Astley SB, Elliott RM, Archer DB, Southon S. Evidence that dietary supplementation with carotenoids and carotenoid-rich foods modulates the DNA damage: repair balance in human lymphocytes. Br J Nutr 2004; 91: 63–72.[Medline]
  11. Hininger IA, Meyer-Wenger A, Moser U, et al. No significant effects of lutein, lycopene or beta-carotene supplementation on biological markers of oxidative stress and LDL oxidizability in healthy adult subjects. J Am Coll Nutr 2001; 20: 232–8.[Abstract/Free Full Text]
  12. van den Berg H, van Vliet T. Effect of simultaneous, single oral doses of ß-carotene with lutein or lycopene on the ß-carotene and retinyl ester responses in the triacylglycerol-rich lipoprotein fraction of men. Am J Clin Nutr 1998; 68: 82–9.[Abstract]
  13. Paetau I, Chen H, Goh NM, White WS. Interactions in the postprandial appearance of ß-carotene and canthaxanthin in plasma triacylglycerol-rich lipoproteins in humans. Am J Clin Nutr 1997; 66: 1133–43.[Abstract/Free Full Text]
  14. Kostic D, White WS, Olson JA. Intestinal absorption, serum clearance, and interactions between lutein and ß-carotene when administered to human adults in separate or combined oral doses. Am J Clin Nutr 1995; 62: 604–10.[Abstract/Free Full Text]
  15. White WS, Stacewicz-Sapuntzakis M, Erdman JW Jr, Bowen PE. Pharmacokinetics of beta-carotene and canthaxanthin after ingestion of individual and combined doses by human subjects. J Am Coll Nutr 1994; 13: 665–71.[Abstract]
  16. Collins A, Dusinska M, Franklin M, et al. Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen 1997; 30: 139–46.[Medline]
  17. Pool-Zobel BL, Bub A, Liegibel UM, Treptow-van Lishaut S, Rechkemmer G. Mechanisms by which vegetable consumption reduces genetic damage in humans. Cancer Epidemiol Biomarkers Prev 1998; 7: 891–9.[Abstract]
  18. Pool-Zobel BL, Bub A, Muller H, Wollowski I, Rechkemmer G. Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis 1997; 18: 1847–50.[Abstract/Free Full Text]
  19. Yeum KJ, Booth SL, Sadowski JA, et al. Human plasma carotenoid response to the ingestion of controlled diets high in fruits and vegetables. Am J Clin Nutr 1996; 64: 594–602.[Abstract/Free Full Text]
  20. Collins AR. The comet assay. Principles, applications, and limitations. Methods Mol Biol 2002; 203: 163–77.[Medline]
  21. Collins AR, Dusinska M. Oxidation of cellular DNA measured with the comet assay. Methods Mol Biol 2002; 186: 147–59.[Medline]
  22. Osganian SK, Stampfer MJ, Rimm E, Spiegelman D, Manson JE, Willett WC. Dietary carotenoids and risk of coronary artery disease in women. Am J Clin Nutr 2003; 77: 1390–9.[Abstract/Free Full Text]
  23. Mayne ST. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. J Nutr 2003; 133(suppl): 933S–40S.[Abstract/Free Full Text]
  24. Hu FB. Plant-based foods and prevention of cardiovascular disease: an overview. Am J Clin Nutr 2003; 78(suppl): 544S–51S.[Abstract/Free Full Text]
  25. Gaziano JM, Manson JE, Branch LG, Colditz GA, Willett WC, Buring JE. A prospective study of consumption of carotenoids in fruits and vegetables and decreased cardiovascular mortality in the elderly. Ann Epidemiol 1995; 5: 255–60.[Medline]
  26. Heinrich U, Gartner C, Wiebusch M, et al. Supplementation with beta-carotene or a similar amount of mixed carotenoids protects humans from UV-induced erythema. J Nutr 2003; 133: 98–101.[Abstract/Free Full Text]
  27. Kiokias S, Gordon MH. Dietary supplementation with a natural carotenoid mixture decreases oxidative stress. Eur J Clin Nutr 2003; 57: 1135–40.[Medline]
  28. Porrini M, Riso P. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J Nutr 2000; 130: 189–92.[Abstract/Free Full Text]
  29. Riso P, Visioli F, Erba D, Testolin G, Porrini M. Lycopene and vitamin C concentrations increase in plasma and lymphocytes after tomato intake. Effects on cellular antioxidant protection. Eur J Clin Nutr 2004; 58: 1350–8.
  30. Hoppe PP, Kramer K, van den Berg H, Steenge G, van Vliet T. Synthetic and tomato-based lycopene have identical bioavailability in humans. Eur J Nutr 2003; 42: 272–8.[Medline]
  31. Hughes DA, Wright AJ, Finglas PM, et al. Effects of lycopene and lutein supplementation on the expression of functionally associated surface molecules on blood monocytes from healthy male nonsmokers. J Infect Dis 2000; 182(suppl): S11–5.[Medline]
  32. Astley SB, Lindsay DG. European Research on the Functional Effects of Dietary Antioxidants (EUROFEDA). Conclusions Mol Aspects Med 2002; 23: 287–91.
  33. Sanchez P, Penarroja R, Gallegos F, Bravo JL, Rojas E, Benitez-Bribiesca L. DNA damage in peripheral lymphocytes of untreated breast cancer patients. Arch Med Res 2004; 35: 480–3.[Medline]
  34. Smith TR, Miller MS, Lohman KK, Case LD, Hu JJ. DNA damage and breast cancer risk. Carcinogenesis 2003; 24: 883–9.[Abstract/Free Full Text]
  35. Palyvoda O, Polanska J, Wygoda A, Rzeszowska-Wolny J. DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests. Acta Biochim Pol 2003; 50: 181–90.[Medline]
  36. Demirbag R, Yilmaz R, Kocyigit A. Relationship between DNA damage, total antioxidant capacity and coronary artery disease. Mutat Res 2005; 570: 197–203.[Medline]
  37. Thompson HJ, Heimendinger J, Haegele A, et al. Effect of increased vegetable and fruit consumption on markers of oxidative cellular damage. Carcinogenesis 1999; 20: 2261–6.[Abstract/Free Full Text]
  38. Riso P, Visioli F, Gardana C, et al. Effects of blood orange juice intake on antioxidant bioavailability and on different markers related to oxidative stress. J Agric Food Chem 2005; 53: 941–7.[Medline]
  39. Riso P, Pinder A, Santangelo A, Porrini M. Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage? Am J Clin Nutr 1999; 69: 712–8.[Abstract/Free Full Text]
  40. Porrini M, Riso P, Oriani G. Spinach and tomato consumption increases lymphocyte DNA resistance to oxidative stress but this is not related to cell carotenoid concentrations. Eur J Nutr 2002; 41: 95–100.[Medline]
  41. Porrini M, Riso P, Brusamolino A, Berti C, Guarnieri S, Visioli F. Daily intake of a formulated tomato drink affects carotenoid plasma and lymphocyte concentrations and improves cellular antioxidant protection. Br J Nutr 2005; 93: 93–9.[Medline]
  42. Bub A, Watzl B, Blockhaus M, et al. Fruit juice consumption modulates antioxidative status, immune status and DNA damage. J Nutr Biochem 2003; 14: 90–8.[Medline]
  43. Chen L, Stacewicz-Sapuntzakis M, Duncan C, et al. Oxidative DNA damage in prostate cancer patients consuming tomato sauce-based entrees as a whole-food intervention. J Natl Cancer Inst 2001; 93: 1872–9.[Abstract/Free Full Text]
  44. Bowen P, Chen L, Stacewicz-Sapuntzakis M, et al. Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis. Exp Biol Med (Maywood) 2002; 227: 886–93.[Abstract/Free Full Text]
  45. Moller P, Vogel U, Pedersen A, Dragsted LO, Sandstrom B, Loft S. No effect of 600 grams fruit and vegetables per day on oxidative DNA damage and repair in healthy nonsmokers. Cancer Epidemiol Biomarkers Prev 2003; 12: 1016–22.[Abstract/Free Full Text]
  46. Faust F, Kassie F, Knasmuller S, Boedecker RH, Mann M, Mersch-Sundermann V. The use of the alkaline comet assay with lymphocytes in human biomonitoring studies. Mutat Res 2004; 566: 209–29.[Medline]
  47. Garcia O, Mandina T, Lamadrid AI, et al. Sensitivity and variability of visual scoring in the comet assay. Results of an inter-laboratory scoring exercise with the use of silver staining. Mutat Res 2004; 556: 25–34.[Medline]
  48. Duthie SJ, Pirie L, Jenkinson AM, Narayanan S. Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability. Mutagenesis 2002; 17: 211–4.[Abstract/Free Full Text]
  49. Diem E, Ivancsits S, Rudiger HW. Basal levels of DNA strand breaks in human leukocytes determined by comet assay. J Toxicol Environ Health A 2002; 65: 641–8.[Medline]
  50. Piperakis SM, Visvardis EE, Sagnou M, Tassiou AM. Effects of smoking and aging on oxidative DNA damage of human lymphocytes. Carcinogenesis 1998; 19: 695–8.[Abstract/Free Full Text]
  51. Bolognesi C, Abbondandolo A, Barale R, et al. Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations, and micronuclei in human lymphocytes. Cancer Epidemiol Biomarkers Prev 1997; 6: 249–56.[Abstract]
  52. Collins AR, Olmedilla B, Southon S, Granado F, Duthie SJ. Serum carotenoids and oxidative DNA damage in human lymphocytes. Carcinogenesis 1998; 19: 2159–62.[Abstract/Free Full Text]
  53. Torbergsen AC, Collins AR. Recovery of human lymphocytes from oxidative DNA damage; the apparent enhancement of DNA repair by carotenoids is probably simply an antioxidant effect. Eur J Nutr 2000; 39: 80–5.[Medline]
  54. Duthie SJ, Ma A, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res 1996; 56: 1291–5.[Abstract/Free Full Text]
Received for publication April 13, 2005. Accepted for publication October 3, 2005.




This article has been cited by other articles:


Home page
 J. Am. Coll. Nutr.Home page
S. Devaraj, S. Mathur, A. Basu, H. H. Aung, V. T. Vasu, S. Meyers, and I. Jialal
A Dose-Response Study on the Effects of Purified Lycopene Supplementation on Biomarkers of Oxidative Stress
J. Am. Coll. Nutr., April 1, 2008; 27(2): 267 - 273.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
J. L. Watters, J. A. Satia, L. L. Kupper, J. A. Swenberg, J. C. Schroeder, and B. R. Switzer
Associations of Antioxidant Nutrients and Oxidative DNA Damage in Healthy African-American and White Adults
Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1428 - 1436.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
C. I. Gill, S. Haldar, L. A Boyd, R. Bennett, J. Whiteford, M. Butler, J. R Pearson, I. Bradbury, and I. R Rowland
Watercress supplementation in diet reduces lymphocyte DNA damage and alters blood antioxidant status in healthy adults
Am. J. Clinical Nutrition, February 1, 2007; 85(2): 504 - 510.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, X.
Right arrow Articles by Yeum, K.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, X.
Right arrow Articles by Yeum, K.-J.
Agricola
Right arrow Articles by Zhao, X.
Right arrow Articles by Yeum, K.-J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS