DIETARY ANTIOXIDANT SUPPLEMENTATION AND DNA DAMAGE IN SMOKERS AND NONSMOKERS

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Abstract: Deficiencies of antioxidant nutrients have been implicated in the etiology of lung and other cancers. However, most intervention trials with antioxidant nutrients have not shown beneficial effects, and some have indicated that beta-carotene may be deleterious. This randomized, doubleblind, placebo-controlled study evaluated the effects of five short-term ( 4-wk) antioxidant nutrient supplement regimens [ascorbic acid (350 mg), RRR-alpha-tocopherol (250 mg), betacarotene (60 mg), selenium (80 mug as sodium selenite), ascorbic acid (350 mg) + RRR-alpha-tocopherol (250 mg)] on plasma antioxidants and mononuclear leukocyte DNA damage in male smokers (n = 9) and nonsmokers (n = 12). Plasma concentrations of ascorbic acid and tocopherol were significantly increased by supplementation, but there was no significant change in plasma beta-carotene or blood glutathione peroxidase activity after supplementation with betacarotene or selenium. DNA damage in mononuclear leukocytes, as assessed by comet assay, was not affected by any supplementation regimen. DNA damage, as assessed by 8-hydroxydeoxyguanosine in mononuclear leukocytes, was not influenced by ascorbic acid, alpha-tocopherol, or selenium supplementation in smokers or nonsmokers, but beta-carotene supplementation resulted in significant differences between smokers and nonsmokers in the level of oxidative DNA damage, with decreases in smokers and increases in smokers. This is a further indication of the differential effects of supplemental beta-carotene in smokers and nonsmokers.
Introduction

Reactive oxygen species (ROS), generated in vivo, have the potential to cause damage to DNA and other biomolecules and thus have the potential to initiate cancers and other chronic diseases ( 1). There is an array of nutrient-dependent, nonenzymatic and enzymatic mechanisms that can modulate ROS-mediated damage. Antioxidant nutrients include ascorbic acid, tocopherols, and carotenoids, which act as scavengers of ROS, and trace elements, such as copper or selenium, which, as part of antioxidant enzymes, have roles in the elimination of ROS ( 2).

A number of long-term ( 4-12yr) intervention studies have investigated the effects of dietary antioxidant supplementation on subsequent cancer onset and mortality. Promising results were obtained in a study in China, in which daily supplementation with a combination of [beta-carotene (15 mg), vitamin E (30 mg), and selenium (50 mug) resulted in reductions in total mortality and mortality from cancer ( 3). However, this population was not well nourished, and the effects may not be relevant to populations with an adequate intake of these nutrients. In trials in developed countries with smokers ( 4-6) and with a cohort of physicians ( 7), daily supplementation with beta-carotene ( 20-30mg), alone or in combination with vitamin A ( 25,000 IU) or tocopherol (50 mg), did not show beneficial effects on cancer outcomes. Indeed, there were indications that intervention with beta-carotene may increase cancer incidence, cancer mortality, and total mortality ( 4-6).

An observational epidemiological study and shorter-term supplementation studies have used markers of DNA damage, rather than cancer incidence, as the end point to investigate the significance of dietary and plasma antioxidants in smokers and nonsmokers at a molecular level. In a cross-sectional, observational study of Japanese adult male smokers and nonsmokers, lymphocyte DNA adduct levels were not significantly higher in smokers, and differences in lymphocyte DNA adduct levels were not related to differences in plasma beta-carotene or alpha-tocopherol in smokers or nonsmokers ( 8). Supplementation with ascorbic acid (250 mg) or tocopheryl acetate (100 mg) daily for two months, alone or in combination, had no effect on the urinary excretion of the DNA adduct 8-oxo-7,8-dihydro-2'-deoxyguanosine in male smokers ( 9). Supplementation with ascorbic acid (100 mg), alpha-tocopherol (280 mg), and beta-carotene (25 mg) daily for 20 weeks decreased endogenous DNA damage, as assessed by oxidation of pyrimidines, and also increased lymphocyte resistance to exogenous ex vivo DNA damage in smokers and nonsmokers ( 10). However, it was notable that, after only five weeks on this regimen, DNA damage, as assessed by oxidized pyrimidines, was significantly higher in the smokers than in the nonsmokers ( 10). Thus short-term supplementation studies with smokers and nonsmokers may provide insights into the way in which antioxidant nutrients impact on molecular damage that is potentially carcinogenic.

Cancer incidence is particularly high in Northern Ireland, a region where smoking is prevalent in a relatively high proportion of the population (11). The aim of the present study was to compare the effects of antioxidant nutrient supplements on mononuclear leukocyte DNA damage in smokers and nonsmokers, as assessed by two methods: the comet assay, which, as applied here, measures DNA single-strand breaks and alkali-labile sites, and the analysis of 8hydroxydeoxyguanosine, which is an oxidation product formed by the hydroxylation of guanine.
Materials and Methods
Subjects and Intervention Protocol

Twenty-one men, aged 26-59 years, were recruited from the academic and technical staff of the University of Ulster. All subjects were apparently healthy, were on no long-term medication, and were not taking other dietary supplements. Nine were smokers and 12 were nonsmokers. Smokers reported smoking at least one cigarette per day for at least eight years, and the nonsmokers reported never smoking or not smoking for at least one year. The trial lasted 40 weeks and used a placebo-controlled, randomized, double-blind, multiple-crossover design. Each treatment period lasted four weeks, with a four-week washout period between treatments, when the subjects received placebo. Entry to the study was staggered over four weeks. At entry, subjects were randomly allocated to placebo or one of the supplements for the first four-week period. The order of the supplements was randomized. It was found post hoc that 5 of the 9 smokers and 6 of the 12 nonsmokers had received placebo in the first four-week period. The supplements were ascorbic acid (350 mg), RRR-alpha-tocopherol (250 mg), beta-carotene (60 mg), selenium (80 mug as sodium selenite), and ascorbic acid (350 mg) + RRR-alpha-tocopherol (250 mg). Placebo and supplements were in tablet form (Larkhall Natural Health, London, UK). Blood (40 ml) was taken from subjects at entry and at the end of each four-week period into lithium heparin tubes. Plasma was separated from blood (8 ml) by centrifugation (400 g, 20 min, 4 degrees C) immediately after collection. Plasma aliquots for analysis of alpha-tocopherol, beta-carotene, and ascorbic acid were frozen in liquid nitrogen and stored at -80 degrees C. Plasma aliquots for ascorbic acid analysis were diluted 1:1 with 10% aqueous metaphosphoric acid before freezing. Aliquots of whole blood were also stored at -80 degrees C before analysis of hemoglobin and glutathione peroxidase activity. All antioxidant analyses were carried out in single batches at the end of the intervention, and storage times ranged from 1 to 10 months. Anthropometric measurements (height, weight, waist, hip, and skinfold thicknesses) were made at the start and end of the study period. Body mass index (kg/m2) and percent body fat were calculated using appropriate equations ( 12, 13). Habitual intake of macronutrients and micronutrients was assessed at the end of the study by using a modified ( 14) seven-day diet history ( 15) and a computerized food composition database (Foodbase, Royal Society of Chemistry, Cambridge, UK).
Plasma and Blood Antioxidant Analysis

Plasma alpha-tocopherol and beta-carotene were measured by high-performance liquid chromatography (HPLC) ( 16) on 100-mul aliquots, with tocopheryl acetate as internal standard. Pooled plasma samples with each set acted as internal quality controls, and external standards were used for calibration. The coefficients of variation (CVs) for alpha-tocopherol and beta-carotene analyses were 4.6% and 6.3%, respectively. Ascorbic acid in plasma was measured by a modified HPLC method ( 17) on 50-mul aliquots, with pooled plasma and standard ascorbic acid (40 muM) as controls; the CV was 7.2%. Selenium-dependent glutathione peroxidase, a functional marker for selenium status, was measured on whole blood by means of an enzymatic-spectrophotometric method (Ransel, Randox Laboratories, Crumlin, UK), and activity was calculated per gram of hemoglobin. The CV for the glutathione peroxidase assay was 9.4%. Hemoglobin was measured using 40 ml of whole blood, which were added to 20 ml of Isoton (Coulter Electronics, Luton, UK). After mixing by inversion, six drops of Zapoglobin (Coulter Electronics) were added. Hemoglobin was then measured as cyanmethemoglobin in a hemoglobinometer (Coulter Electronics).
Analysis of 8-Hydroxydeoxyguanosine in Mononuclear Leukocyte DNA

Immediately, after collection, whole blood (30 ml) was mixed with 30 ml of Dutch modified RPMI 1640 at 20 degrees C; this mixture was layered in four 15-ml aliquots onto 15-ml aliquots of lymphocyte separation medium (Nycomed, Oslo, Norway) and centrifuged (700 g, 30 min, 20 degrees C). The mononuclear leukocytes were isolated from the phase interfaces with a glass pipette, combined, washed twice [50 ml of Dutch modified RPMI and 20 ml of tris(hydroxymethyl)aminomethane (Tris)-buffered saline, pH 7.4], resuspended in 1 ml of freezing buffer (10 mM Tris, 400 mM NaCl, 2 mM disodium EDTA, pH 8.0), frozen in liquid nitrogen, and stored at -80 degrees C. At the end of the study, thawed mononuclear leukocytes were lysed in proteinase K buffer (Sigma Chemical, Poole, UK), and the cellular proteins were salted out by dehydration and precipitation. After ribonuclease treatment, DNA was precipitated in ethanol, redissolved in nitrogen-saturated buffer, and stored at -20 degrees C before hydrolysis of DNA with deoxyribonuclease I, alkaline phosphatase, and phosphodiesterases I and II. Deoxyguanosine and 8-hydroxydeoxyguanosine were determined by HPLC ( 18) with use of coulometric detection. The 8-hydroxydeoxyguanosine was calculated as moles per 105 mol of deoxyguanosine. The CV for this analysis in our laboratory is normally <5%.
Assessment of DNA Damage in Mononuclear Leukocytes (Comet Assay)

Whole fresh blood (30 mul) was added to 1 ml of RPMI 1640 (without phenol red; containing 10% fetal calf serum), mixed, left to stand for 30 minutes on ice, then underlaid with 100 mul of Histopaque 1077 (Sigma Chemical) and centrifuged (200 g, 5 min, 4 degrees C). Mononuclear leukocytes were isolated from the interface and resuspended in 1 ml of phosphate-buffered saline on ice. These cells were irradiated with X-rays at 4 degrees C to doses of 1 or 5 Gy at a dose rate of 2.65 Gy/min (300 kV Siemens Stabilipan). Unirradiated (control) mononuclear leukocytes were also prepared. Mononuclear leukocyte DNA damage was assessed using the comet assay ( 19) with minor modifications ( 20, 21). DNA damage was assessed as tail moment [(percentage of DNA in the tail x distance between head and tail centers) + 100]. Fifty cells were assessed for each treatment. The CV for this assay is about 10% ( 22).
Statistics

Data were analyzed by repeated-measures analysis of variance and independent t-tests, with a Bonferroni correction used for multiple comparisons.
Results

There was no significant difference between the smokers and nonsmokers for age, height, weight, or body mass index (Table 1) or in percent body fat or waist and hip measurements (data not presented). Although generally low, alcohol intakes were significantly and substantially higher in the smokers (Table 1). There was no significant difference between smokers and nonsmokers in the intakes of other macronutrients, fiber, or selected antioxidant micronutrients (Table 1). Although plasma concentrations of ascorbic acid, alpha-tocopherol, and beta-carotene and blood glutathione peroxidase activity were higher before supplementation in the nonsmokers than in the smokers, none of these differences was significant (Table 2). Analysis of the data for each supplementation regimen at the end of the respective subsequent placebo periods showed no significant differences, indicating that carryover effects were minimal. Analysis of the effects of supplementation on the respective plasma antioxidants showed that supplementation with ascorbic acid and alpha-tocopherol, singly or in combination, led to significant increases in the concentrations of these antioxidants in the plasma of smokers and nonsmokers (Table 2). Although supplementation with beta-carotene increased plasma beta-carotene by 26% in smokers and 9% in nonsmokers, these effects were not significant. Supplementation with selenium did not significantly affect selenium-dependent glutathione peroxidase activity (Table 2). Mononuclear leukocyte DNA damage, as assessed by the comet assay on unirradiated cells, was not influenced by any supplementation regimen in smokers or nonsmokers (Table 3). This DNA damage was increased by exogenous radiation doses (1 and 5 Gy), but the level of damage was not influenced in smokers or nonsmokers by any supplementation regimen (data not presented). Supplementation with selenium or with ascorbic acid and alpha-tocopherol, alone or in combination, had no effects on the level of oxidative DNA damage, as assessed by 8-hydroxydeoxyguanosine in mononuclear leukocyte DNA, in smokers or nonsmokers (Table 4). However, there was a significant difference between smokers and nonsmokers in response to supplementation with beta-carotene, with 8-hydroxydeoxyguanosine concentrations decreased in nonsmokers but increased in smokers (Table 4).
Discussion

Although there were significant increases in plasma ascorbic acid and alpha-tocopherol concentrations in response to supplementation with these nutrients (Table 2), these were not accompanied by any significant effects on the level of mononuclear leukocyte DNA damage, as assessed by comet assay or by 8-hydroxydeoxyguanosine (Table 3). These findings concur with recent results which showed that supplementation of male smokers with ascorbic acid (250 mg) or tocopheryl acetate (100 mg) daily for two months, either alone or in combination, had no effect on DNA damage, as assessed by urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine ( 9). Together, these results indicate that supplementation with ascorbic acid or alpha-tocopherol, alone or in combination, is ineffective at reducing oxidative DNA damage or DNA damage as assessed by strand breaks. The lack of a relationship between plasma alpha-tocopherol and DNA damage also supports a recent cross-sectional study in Japan with male smokers and nonsmokers, which found not only that lymphocyte DNA adduct levels were not significantly higher in smokers, but also that individual differences in lymphocyte DNA adduct levels were not related to differences in plasma alpha-tocopherol in smokers or nonsmokers ( 8). Furthermore, in that study, no relationship was found between lymphocyte DNA adduct levels and plasma beta-carotene levels in either group, and it was concluded that neither alpha-tocopherol nor beta-carotene is protective against smoking-induced DNA damage ( 8). However, the study was observational, and subjects were not supplemented with either micronutrient.

A protective role for supplemental beta-carotene against DNA damage was suggested in a study where smokers and nonsmokers were supplemented with ascorbic acid (100 mg), alpha-tocopherol (280 mg), and beta-carotene (25 mg) daily for 20 weeks ( 10). In that study, supplementation had no significant effects on DNA damage, as assessed by strand breaks, in smokers or nonsmokers. When DNA damage was assessed as oxidation of pyrimidines, supplemented smokers and nonsmokers showed substantial and highly significant reductions in DNA damage after 20 weeks. However, at five weeks, although the supplement had no significant effect on oxidized pyrimidines in either group, there was evidence of a differential effect, and the level of oxidized pyrimidines was significantly and substantially higher in the smokers ( 10). Differential responses in smokers and nonsmokers to short-term beta-carotene supplementation is also indicated in the present study, where four weeks of supplementation with beta-carotene (60 mg) showed that oxidative DNA damage, as assessed by mononuclear leukocyte 8-hydroxydeoxyguanosine, increased in smokers but decreased in nonsmokers. However, beta-carotene supplementation had no effect on the level of DNA damage, as assessed by strand breaks (comet assay). Taken as a whole, it appears that supplemental alphatocopherol, ascorbate, and selenium do not affect the levels of DNA damage but that supplemental beta-carotene can impact oxidative DNA damage but not DNA damage as assessed by strand breaks; furthermore there are differential responses in smokers and nonsmokers, which are dependent on the duration of supplementation.

The reasons for the different responses of smokers and nonsmokers to beta-carotene supplementation are not clear. One possible explanation is an interaction with alcohol consumption. Alcohol consumption is difficult to assess accurately, but despite the relatively modest intakes reported here, alcohol was the only nutrient to show a significant difference, with smokers having a substantially higher intake than nonsmokers. Interactions between alcohol and beta-carotene are indicated in other studies. For example, a cross-sectional study of smokers and nonsmokers showed no difference in alcohol intake between the two groups, but alcohol consumption and smoking were significantly negatively correlated with plasma beta-carotene ( 8). Furthermore, in two major antioxidant intervention trials with smokers, increases in cancer incidence were associated with higher alcohol intakes ( 23, 24). The beta-carotene supplements used here and in other studies are many times greater than normal intakes. Little is known of the biological effects of these high doses of beta-carotene, which may have prooxidant effects, or of the effects of beta-carotene metabolites, which may increase when high levels of beta-carotene are given ( 25). Toxic tissue levels of beta-carotene or its metabolites may result from the coingestion of beta-carotene and alcohol ( 26), and there may be other potentially damaging interactions between betacarotene, alcohol, and vitamin A ( 25). The mechanisms whereby long-term beta-carotene supplementation may increase cancer incidence in smokers remain unclear. However, it appears that short-term ( 4-5wk) supplementation with beta-carotene can result in increased levels of DNA damage in smokers compared with nonsmokers.
Acknowledgments and Notes

This work was supported by The Cancer Research Campaign. Address reprint requests to Dr. R. W. Welch, Northern Ireland Centre for Diet and Health, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, UK.

Submitted 6 October 1998; accepted in final form 1 April 1999.

Table 1. Age, Anthropometric Data, and Estimated Daily Intake of Energy, Macronutrients, Fiber, and Selected Antioxidant Nutrients for Smokers and Nonsmokers[a,b]

Smokers Nonsmokers
(n = 9) (n = 12)

Age, yr 40.6 +/- 2.7 43.7 +/- 2.0
Height, m 1.70 +/- 0.02 1.77 +/- 0.01
Weight, kg 71.4 +/- 2.7 80.6 +/- to 2.7
BMI, kg/m2 24.4 +/- 0.9 25.6 +/- 0.7
Total energy, kJ 9,099 +/- 540 8,805 +/- 642
Protein, g 72.1 +/- 4.4 81.3 +/- 5.5
Fat, g 80.4 +/- 6.4 76.2 +/- 6.2
Carbohydrate, g 267 +/- 16 274 +/- 21
Alcohol, g 22.5 +/- 8.6 7.9 +/- 4.3[*]
Dietary fiber,[c] g 13.4 +/- 1.6 18.8 +/- 2.7
Ascorbic acid, mg 77 +/- 14 74 +/- 8
Vitamin E, mg 71 +/- 8 100 +/- 12
beta-Carotene, mug 2,440 +/- 260 2,453 +/- 246
Retinol, mug 713 +/- 307 688 +/- 286
Retinol equivalents, mug 1,121 +/- 334 1,119 +/- 298
Selenium, mug 37 +/- 4 51 +/- 7

a: Values are means +/- SE; n, number of subjects. BMI, body mass index.

b: Statistical significance is as follows: *, significantly different from smokers, p < 0.05.

c: Nonstarch polysaccharides, Englyst method.

Table 2. Plasma Antioxidants Before and After Supplementation With Relevant Micronutrients in Smokers and Nonsmokers[a,b]

Legend for Chart:

A - Supplement
B - Plasma
C - Smokers, (n = 9), Before
D - Smokers, (n = 9), After
E - Nonsmokers, (n = 12), Before
F - Nonsmokers, (n = 12), After

A C D
B E F

Ascorbic acid (350 mg) 27.7 +/- 2.7 54.5 +/- 10.4[*]
Ascorbic acid, mumol/l 32.3 +/- 2.0 45.1 +/- 5.6[*]

alpha-Tocopherol (250mg) 23.6 +/- 1.1 30.8 +/- 3.1[*]
alpha-Tocopherol, mumol/l 25.5 +/- 0.8 28.6 +/- 2.0[*]

beta-Carotene (60 mg) 0.27 +/- 0.05 0.34 +/- 0.05
beta-Carotene, mumol/1 0.46 +/- 0.08 0.50 +/- 0.09

Selenium (80 mug) 40.2 +/- 1.4 41.3 +/- 4.9
GPx,[c] U/g Hb 41.9 +/- 1.2 38.9 +/- 3.4

Ascorbic acid (350 mg) 27.7 +/- 2.7 48.7 +/- 7.9[*]
Ascorbic acid, mumol/l 32.3 +/- 2.0 43.3 +/- 5.8[*]

+ alpha-tocopherol (250 mg) 23.6 +/- 1.1 31.6 +/- 2.5[*]
alpha-Tocopherol, mumol/l 25.5 +/- 0.8 32.9 +/- 2.5[*]

a: Values are means +/- SE at end of placebo period preceding supplementation with relevant micronutrient (Before) and at end of supplementation period with relevant micronutrient (After); n, number of subjects.

b: Statistical significance is as follows: *, significant difference between placebo and supplementation, p < 0.05.

c: Selenium-dependent glutathione peroxidase (GPx) activity per gram of hemoglobin (Hb).

Table 3. Mononuclear Leukocyte DNA Damage of Smokers and Nonsmokers Before and After Micronutrient Supplementation, as Assessed by Comet Assay on Unirradiated Samples[a,b]

Legend for Chart:

A - Supplement
B - Smokers (n = 9), Before
C - Smokers (n = 9), After
D - Nonsmokers (n = 12), Before
E - Nonsmokers (n = 12), After

A B C
D E

Ascorbic acid 1.50 +/- 0.45 3.61 +/- 2.50
3.19 +/- 1.30 3.84 +/- 1.86

alpha-Tocopherol 4.49 +/- 2.22 3.40 +/- 1.41
1.95 +/- 0.63 1.76 +/- 0.62

beta-Carotene 2.19 +/- 0.45 3.62 +/- 0.90
1.65 +/- 0.42 1.57 +/- 0.35

Selenium 2.02 +/- 0.48 2.59 +/- 1.07
2.41 +/- 0.77 2.77 +/- 1.22

Ascorbic acid + 1.50 +/- 0.49 3.40 +/- 1.41
alpha-tocopherol 2.25 +/- 0.68 3.82 +/- 1.15

a: Tail moments.

b: Values are means +/- SE at end of placebo period preceding supplementation with relevant micronutrient (Before) and at end of supplementation period with relevant micronutrient (After); n, number of subjects.

Table 4. 8-Hydroxydeoxyguanosine in Mononuclear Leukocyte DNA of Smokers and Nonsmokers Before and After Micronutrient Supplementation[a-c]

Legend for Chart:

A - Smokers (n = 9), Before
B - Smokers (n = 9), After
C - Nonsmokers (n = 12), Before
D - Nonsmokers (n = 12), After

A B C
D E

Ascorbic acid 3.70 +/- 0.41 5.91 +/- 2.39
6.86 +/- 2.42 3.09 +/- 0.80

alpha-Tocopherol 6.31 +/- 2.77 3.93 +/- 1.97
4.83 +/- 0.89 4.30 +/- 0.99

nu-Carotene 2.58 +/- 0.59 3.52 +/- 0.81[*]
5.25 +/- 1.38 2.24 +/- 0.36[*]

Selenium 6.52 +/- 2.25 12.98 +/- 4.97
5.35 +/- 1.70 6.28 +/- 2.04

Ascorbic acid + 5.79 +/- 2.15 3.99 +/- 0.95
alpha-tocopherol 11.26 +/- 6.14 4.63 +/- 1.19

a: 8-Hydroxydeoxyguanosine as moles per 102 mol deoxyguanosine.

b: Values are means +/- SE; n, number of subjects. c: Statistical significance is as follows: *, responses significantly different in smokers and nonsmokers (p < 0.05). An outlier in smokers after supplementation (46.50) was excluded before data were tested using repeated-measures analysis of variance followed by an independent t-test with a Bonferroni correction.
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By R.W. Welch; E. Turley; S.F. Sweetman; G. Kennedy; A.R. Collins; A. Dunne; M.B.E. Livingstone; P.G. McKenna; V.J. McKelvey-Martin and J.J. Strain

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