Vol. 9, 647–652, July 2000 Cancer Epidemiology, Biomarkers & Prevention 647 The Effects of Vitamin C and Vitamin E on Oxidative DNA Damage: Results from a Randomized Controlled Trial1 Han-Yao Huang,2 Kathy J. Helzlsouer, and 8-OHdG in nonsmoking adults. However, several aspects Lawrence J. Appel of a healthy lifestyle were associated with lower oxidative Department of Epidemiology, School of Hygiene and Public Health [H-Y. H., DNA damage. K. J. H.], and Department of Medicine, School of Medicine [L. J. A.], Johns Hopkins University, Baltimore, Maryland 21205-2223 Introduction Oxidative damage to DNA by ROS3 and RNS, such as hydro- Abstract gen peroxide, hydroxyl radicals, singlet oxygen, and peroxyni- Oxidative DNA damage may be important in mutagenic, trite, may be important in mutagenic, carcinogenic, and aging carcinogenic, and aging processes. Although it is plausible processes (1–3). Sources of ROS and RNS are multiple, of that antioxidant vitamins may reduce oxidative DNA which some are inevitable, such as normal aerobic metabolism damage, evidence from human studies has been sparse and phagocytosis. During mitochondrial respiration, oxygen and inconsistent. We determined the short-term effects of may undergo single electron transfer and generate superoxide vitamin C (500 mg/day) and vitamin E (400 IU anion radicals, which are normally converted to hydrogen per- d-␣-tocopheryl acetate/day) supplements on oxidative oxides by superoxide dismutase. Hydrogen peroxides can be DNA damage in a double-masked, placebo-controlled, transported across nuclear membrane and, in the presence of 2؋2 factorial trial in 184 nonsmoking adults. Mean metal ions, produce hydroxyl radicals that cause oxidative duration of supplementation was 2 months. Oxidative modification of DNA. In addition, ROS and RNS may initiate DNA damage was measured by 24-h urinary excretion of and propagate lipid peroxidation, of which the major end prod- -8-hydroxy-2؅-deoxyguanosine (8-OHdG). At baseline, ucts, malondialdehyde and 4-hydroxynonenal, possess geno .(urinary 8-OHdG (mean ؎ SE; ng/mg creatinine) was toxic and mutagenic properties (4 -associated with race (15.6 ؎ 0.8 in African Americans Oxidative damage to DNA may result in base modifica .prior tion, sugar damage, strand break, and DNA-protein cross-links ,(0.001 ؍ versus 20.3 ؎ 1.2 in Caucasians, P antioxidant supplement use (18.6 ؎ 0.8 in users versus Of these, modification of guanine by hydroxyl radicals at the -and regular exercise C-8 site, frequently estimated as 8-OHdG, is the most com ,(0.007 ؍ in non-users, P 1.5 ؎ 13.8 in exercisers versus 16.6 ؎ 0.9 in monly studied lesion (5). Urinary excretion of 8-OHdG, the 1.1 ؎ 19.2) Fruit and vegetable intake repair product from oxidative DNA modification by excision .(0.04 ؍ non-exercisers, P and serum ascorbic acid were inversely associated with enzymes, is an in vivo measure of overall oxidative DNA ,and 0.016, respectively). damage (6). In in vitro studies, exposure of DNA to oxidants 0.02 ؍ urinary 8-OHdG (P-trend The benefits of fruit and vegetable intake became evident such as singlet oxygen, UV light, radiation, and Fenton reac- with the consumption being at least three servings/day. tion, results in production of 8-OHdG (7–11). Also, high oxi- At the end of supplementation, change from baseline in dative stress, such as smoking or extreme exercise, is associated -urinary 8-OHdG (mean ؎ SE; ng/mg creatinine) was with high 8-OHdG production (12–14). Although direct evi dence that links 8-OHdG with cancer risk is lacking, increased 1.0 ؎ 0.5 ,(0.59 ؍ P) 1.1 ؎ 0.6 ,(0.61 ؍ ؊0.6 ؎ 1.4 (P in the placebo, 8-OHdG has been found in cancerous tissues (15). Urinary (0.27 ؍ and 1.6 ؎ 1.4 (P ,(0.61 ؍ P) vitamin C alone, vitamin E alone, and combined vitamins 8-OHdG was higher in small cell lung carcinoma patients C and E groups, respectively. In overall and subgroup compared with normal controls and increased in non-small cell analyses, there was no significant main effect or lung carcinoma patients during the course of radiotherapy (16). interaction effect of the supplements on urinary 8-OHdG. Evidence from observational epidemiological studies sug- In conclusion, supplementation of diet with vitamin C gests that antioxidant vitamins may reduce the risk of devel- (500 mg/day) and vitamin E (400 IU d-␣-tocopheryl oping cancer (17). Vitamin C and vitamin E are considered the acetate/day) had no significant main effect or interaction most important water-soluble and lipid-soluble micronutrient effect on oxidative DNA damage as measured by urinary antioxidants, respectively. In vitro studies suggest that vitamin C and vitamin E may protect against oxidative damage to DNA as measured by 8-OHdG (9, 18, 19), and that vitamin C may regenerate vitamin E by reducing tocopherol radicals (20, 21). Received 11/2/99; revised 4/15/00; accepted 4/23/00. However, in an animal feeding study, combinations of three The costs of publication of this article were defrayed in part by the payment of dietary levels of vitamin C and vitamin E resulted in no pro- page charges. This article must therefore be hereby marked advertisement in tective effect on oxidative damage in the liver DNA of guinea accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant RR00722 from the National Center for Research Resources of the NIH. 2 To whom requests for reprints should be addressed, at Department of Epide- miology, Johns Hopkins School of Hygiene and Public Health, 615 North Wolfe 3 The abbreviations used are: ROS, reactive oxygen species; RNS, reactive Street, E-6006, Baltimore, MD 21205-2223. Phone: (410) 502-9094; Fax: nitrogen species; 8-OHdG, 8-hydroxy-2Ј-deoxyguanosine; CV, coefficient of (410) 614-2632. variation. Downloaded from cebp.aacrjournals.org on September 24, 2021. © 2000 American Association for Cancer Research. 648 Effects of Vitamin C and Vitamin E on Oxidative DNA Damage pigs (22). In human studies (one cross-sectional study and four placebo and vitamin E/placebo) each day and to not change clinical trials), the effects of these antioxidant vitamins on their diets or take other vitamin supplements for 2 months. 8-OHdG have been inconsistent (23–27). Adherence with pill-taking was assessed by the average of pill We determined the effects of vitamin C and vitamin E, counts (observed/expected number of pills consumed ϫ 100%) alone and combined, on oxidative DNA damage as measured at each follow-up visit, changes in serum levels of ascorbic acid by urinary excretion of 8-OHdG in a double-masked, placebo- and ␣-tocopherol from baseline, and self-reports (30). controlled, 2ϫ2 factorial trial. Outcome Measures. The outcome measure in this study was change in urinary excretion of creatinine-adjusted 8-OHdG. Materials and Methods Change was the difference between measurements obtained at baseline and the end of pill-taking. Reproducibility (intra-assay Study Population. The study population consisted of 184 CV) of the laboratory measurements was assessed in 40 pairs of community-dwelling nonsmokers recruited between February duplicate samples that were randomly inserted into the array of 1996 and June 1997 in Baltimore, Maryland. Eligibility criteria specimen tubes in a pairwise fashion. were willingness to provide written informed consent and to Laboratory Assays. Urinary 8-OHdG was measured by an take study pills, but no other vitamin supplements, for 2 ϫ months. Exclusion criteria were current tobacco use, regular ELISA (16). Urine samples were centrifuged at 300 g for 10 Ն min to remove any particulate material. To each well of the exposure to passive tobacco smoke for 1 h/day, and consump- ␮ ␮ tion of Ն14 alcohol beverages/week. Individuals who took ELISA kit, a 50- l urine sample and 50 l of reconstituted vitamin supplements were eligible after a 2-month period of primary antibody were added. The plate was covered, incubated abstinence. at 37°C, and mixed continuously for 1 h. The antibodies bound to the 8-OHdG in the sample were washed with 0.05% Tween Conduct of Trial. Institutional review boards of the Johns 20/phosphoric acid buffer. An enzyme-labeled secondary anti- Hopkins Medical Institutions approved the protocol of this trial. body was added to the plate, which was then incubated at 37°C All participants provided written informed consent. and mixed continuously for 1 h, and the unbound enzyme- An in-person screening visit was conducted to ascertain labeled secondary antibody was washed away. The amount of eligibility and to obtain baseline data, including demographic antibody bound to the plate was determined by the color de- characteristics, weight, height, body mass index (weight/ 2 2 veloped from the addition of a chromatic substrate (o- height , kg/m ), medical history, medication use, prior supple- phenylenediamine) and read at 492 nm. Quantification of the ment use, regular exercise (regularly engage in any activity 8-OHdG was achieved by comparing the optical densities of the long enough to work up a sweat at least once a week), alcohol chromogenic signal of each sample to that of an internal stand- consumption, and habitual diet assessed by the Health Habits ard of known 8-OHdG concentrations. The intra-assay CV of and History Questionnaire (National Cancer Institute version this assay was 8.8%. HHHQ FULL.JAN92; Ref. 28). The dietary questionnaire Serum ascorbic acid levels were measured based on the listed three serving sizes (small, medium, and large) and stated reduction of Fe(III) to Fe(II) by ascorbic acid, followed by medium portions for food items, including fruits and vegeta- chromogenic chelation of Fe(II) with ferrozine (31). Intra-assay bles.