ORIGINAL CONTRIBUTION A Prospective Study of Folate Intake and the Risk of Breast Cancer Shumin Zhang, MD, ScD David J. Hunter, MBBS, ScD Susan E. Hankinson, ScD Edward L. Giovannucci, MD, ScD Bernard A. Rosner, PhD Graham A. Colditz, MD, DrPH Frank E. Speizer, MD Walter C. Willett, MD, DrPH LOW FOLATE INTAKE HAS INcreased tumor occurrence in animal models 1 and has been associated with higher risk of colon cancer in epidemiologic studies, particularly in the presence of alcohol consumption. 2-4 Deficient folate status can reduce the availability of S- adenosylmethionine for DNA methylation 1,5 and may thereby influence gene expression. 1 Diminished folate status may also result in abnormal DNA synthesis due to misincorporation of uracil into DNA leading to chromosome breaks and disruption of DNA repair. 1,6 Alcohol is a known folate antagonist 7 and thus could plausibly increase the requirement for folate intake. We hypothesized that higher folate intake might reduce risk of breast cancer, particularly among women with greater alcohol consumption, which itself moderately increases breast cancer risk. 8-11 Because no prospective data are available, we examined these hypotheses in the Nurses Health Study, a large cohort study among US women. METHODS Study Cohort In 1976, 121 700 female registered nurses aged 30 to 55 years living in 11 states completed a mailed questionnaire about Context Folate is involved in DNA synthesis and methylation and may reduce breast cancer risk, particularly among women with greater alcohol consumption. Objectives To assess the association between folate intake and risk of breast cancer and whether higher folate intake may reduce excess risk among women who consume alcohol. Design Prospective cohort study performed in 1980, with 16 years of follow-up. Setting and Participants A total of 88 818 women who completed the dietary questionnaire section of the Nurses Health Study in 1980. Main Outcome Measure Incidence of invasive breast cancer by levels of folate and alcohol intake. Results A total of 3483 cases of breast cancer were documented. Total folate intake was not associated with overall risk of breast cancer. However, among women who consumed at least 15 g/d of alcohol, the risk of breast cancer was highest among those with low folate intake. For total folate intake of at least 600 µg/d compared with 150 to 299 µg/d, the multivariate relative risk (RR) was 0.55 (95% confidence interval [CI], 0.39-0.76; P for trend =.001). This association was only slightly attenuated after additional adjustment for intake of beta carotene, lutein/zeaxanthin, preformed vitamin A, and total vitamins C and E. The risk of breast cancer associated with alcohol intake was strongest among women with total folate intake of less than 300 µg/d (for alcohol intake 15 g/d vs 15 g/d, multivariate RR, 1.32; 95% CI, 1.15-1.50). For women who consumed at least 300 µg/d of total folate, the multivariate RR for intake of at least 15 g/d of alcohol vs less than 15 g/d was 1.05 (95% CI, 0.92-1.20). Current use of multivitamin supplements, the major source of folate, was associated with lower breast cancer risk among women who consumed at least 15 g/d of alcohol (for current users of supplements vs never users, RR, 0.74; 95% CI, 0.59-0.93). Conclusions Our findings suggest that the excess risk of breast cancer associated with alcohol consumption may be reduced by adequate folate intake. JAMA. 1999;281:1632-1637 www.jama.com their medical history and lifestyle. Biennially, a mailed questionnaire has been sent to update information on potential risk factors and to ascertain newly diagnosed diseases. In 1980, a 61-item food frequency questionnaire was included to obtain dietary information. In 1984, the dietary questionnaire was expanded to 126 items. Similar questionnaires were used to update diet in 1986 and 1990. Through May 31, 1996, the follow-up rate was 95% complete as percentage of potential person-years. After up to 4 mailings, 98 462 women returned the 1980 dietary questionnaire. Women were excluded from analyses if they did not complete the 1980 dietary questionnaire, had total energy intake less than 2100 kj/d (500 kcal/d) or more than 14 700 kj/d (3500 Author Affiliations: Departments of Epidemiology (Drs Zhang, Hunter, Hankinson, Colditz, and Willett) and Nutrition (Drs Zhang, Giovannucci, and Willett), Harvard Center for Cancer Prevention (Drs Hunter and Colditz), Harvard School of Public Health, and the Channing Laboratory (Drs Hunter, Hankinson, Giovannucci, Rosner, Colditz, Speizer, and Willett), Department of Medicine, Harvard Medical School and Brigham and Women s Hospital, Boston, Mass. Corresponding Author and Reprints: Shumin Zhang, MD, ScD, Nurses Health Study, Channing Laboratory, 181 Longwood Ave, Boston, MA 02115 (email: Shumin.Zhang@channing.harvard.edu). 1632 JAMA, May 5, 1999 Vol 281, No. 17 1999 American Medical Association. All rights reserved.
kcal/d), left 10 or more items blank, or had a previous diagnosis of cancer (other than nonmelanoma skin cancer); 88 818 participants remained. Among the women included, 80% also completed the 1984 dietary questionnaire, and 76% completed the 1986 and 1990 dietary questionnaires (TABLE 1). Semiquantitative Food Frequency Questionnaires The validity and reliability of the food frequency questionnaires used in the Nurses Health Study have been described elsewhere. 12-14 For each food in the questionnaires, a commonly used unit or portion size (eg, 1 orange) was specified, and women were asked how often on average over the previous year they had consumed that amount of each food. There were 9 responses, ranging from never to 6 or more times per day. Nutrient intake was computed by multiplying the frequency response by the nutrient content of the specified portion sizes. Total alcohol intake was the sum of the alcohol content contributed from beer, wine, and liquor, assuming 12.8 g of ethanol for 360 ml (12 oz) of beer, 11.0 g for 120 ml (4 oz) of wine, and 14.0 g for a shot of liquor (45 ml [1.5 oz], 80 proof as a standard). We also asked questions on duration, brand, and type of multivitamin supplement use, which were updated biennially. A comprehensive database on the folate content of the multivitamin preparations was developed. In a sample of 173 participants, the correlation coefficient between alcohol intake as assessed by the 1980 dietary questionnaire and by four 1-week food diaries was 0.90. 8 In a sample of 188 participants, the correlation coefficients between folate intake calculated from the 1980 dietary questionnaire and erythrocyte folate concentrations in 1987 were 0.55 for total folate (including supplements) and 0.38 for folate from foods only. 2 In this sample, the mean (±SEM) erythrocyte folate concentrations for increasing quintiles of total folate intake were 682 ± 34, 773 ± 23, 804 ± 25, 804 ± 25, and 920 ± 48 nmol/l. 2 All were within the normal range ( 340 nmol/l). Ascertainment of Breast Cancer Cases Incident cases of invasive breast cancer were identified by self-report on each biennial questionnaire from 1982 to 1996. Deaths in the cohort were identified by reports from family members, the postal service, and a search of the National Death Index 15 ; we estimated that 98% of all deaths were identified. Women who reported breast cancer (or their next of kin if they had died) were asked for permission to obtain hospital records and pathology reports. We excluded noninvasive breast cancer cases in this analysis, but we included 217 invasive breast cancer cases for which no medical records could be obtained because the accuracy of self-reporting was extremely high among those for whom we were able to obtain medical records ( 99%). Physicians without knowledge of di- Table 1. Age-Standardized Baseline Characteristics by Total Folate Intake in 1980 in the Nurses Health Study* 150 (n = 7072) 150-299 (n = 42 785) Total Folate Intake in 1980, µg/d 300-449 (n = 16 089) 450-599 (n = 8126) 600 (n = 14 746) Characteristics Age, y 45 46 47 46 47 Total folate, µg/d 124 223 357 527 814 Dietary folate, µg/d 126 223 329 290 326 Multivitamin supplement use, % 7 11 28 79 95 Alcohol consumption 15 g/d, % 13 12 11 14 11 Parity (births) 2.9 3.0 2.9 2.9 2.8 Age at first birth, y 25 25 25 25 25 Age at menarche, y 12.6 12.5 12.5 12.6 12.5 Postmenopausal, % 33 33 33 32 33 Mother or sister with breast cancer, % 6 6 6 6 6 History of benign breast disease, % 23 24 25 26 25 Height, cm 163 164 164 164 164 Weight change since age 18 y, kg 8.5 8.6 8.0 7.9 7.5 Body mass index, kg/m 2 24.4 24.6 24.4 24.1 24.1 Intake Total energy, kj/d (kcal/d) 6170 (1469) 6577 (1566) 6791 (1617) 7409 (1764) 6094 (1451) Beta carotene, µg/d 1969 3703 6054 5131 5530 Lutein/zeaxanthin, µg/d 1714 3992 7473 6209 6648 Preformed vitamin A (including supplements), IU/d 2212 3099 4385 6972 11 095 Total vitamin C (including supplements), mg/d 137 217 312 391 586 Total vitamin E (including supplements), IU/d 33 39 56 92 171 *All factors except age are directly standardized. Values presented are means unless indicated otherwise. Nutrients are adjusted for total energy intake. 1999 American Medical Association. All rights reserved. JAMA, May 5, 1999 Vol 281, No. 17 1633
etary information of each study participant reviewed the records. During 16 years of follow-up, 3483 breast cancer cases were documented (827 premenopausal cases, 2345 postmenopausal cases, and 311 cases with uncertain menopausal status). Statistical Analysis Person-years for each participant were calculated from the date of returning the 1980 questionnaire to the date of diagnosis of breast cancer, death, or May 31, 1996, whichever came first. Nutrient intakes were adjusted for total energy by the residual method. 16 We also classified women according to their duration of multivitamin use. Because the US Food and Drug Administration regulations limited the maximum folate content in supplements to 100 µg before 1973, we considered 1973 as the starting point for calculation of multivitamin use relevant to folate intake. 17 We calculated incidence rate by dividing the number of cases by personyears of follow-up in each category. The relative risk (RR) was calculated as the rate in a specific category of total folate divided by that in the reference category (150-299 µg/d), with adjustment for 5-year age categories by the Mantel- Haenszel method. 18 In multivariate analysis using pooled logistic regression with 2-year time increments, 19,20 we simultaneously adjusted for 5-year age categories and other covariates listed in the footnotes to Table 2. We did not adjust for use of oral contraceptives, 21 lactation, 22 or smoking status, as they were not associated with breast cancer risk in multivariate analyses. The incidence of breast cancer was related to the cumulative average of dietary intake from all available dietary questionnaires, using methods for repeated measures described elsewhere. 23 Nondietary covariates were updated biennially. Analyses were further stratified by menopausal status, alcohol intake ( 15 g/d, 15 g/d, about 1 drink), and methionine intake (quintiles). For all RRs, we calculated 95% confidence intervals (CIs). All P values were 2-tailed. The test for trend was conducted using the median value of the cumulative average nutrient intake for each quintile analyzed as a continuous variable. Log likelihood ratio tests were used to compare models with or without interaction terms between total folate intake and alcohol consumption. RESULTS In the 1980 baseline population, the first 3 categories of total folate intake mainly represented folate from food sources (cold breakfast cereal, orange juice, and leafy vegetables were the major sources); Table 2. Relative Risk (RR) and 95% Confidence Interval (CI) of Breast Cancer According to the Categories for Cumulative Average Intake of Total Folate and Alcohol Intake in a Cohort of 88 818 Women From 1980 to 1996 Total Folate Intake, µg/d 150 150-299 300-449 450-599 600 P for Trend Overall Cases, No. 112 1411 953 535 472 Person-years 50 452 552 975 356 862 183 488 184 204 Age-adjusted RR 1.03 1.00 (Referent) 0.94 1.04 0.94.50 Multivariate RR (95% CI) Model 1* 1.09 (0.89-1.32) 1.00 (Referent) 0.92 (0.85-1.00) 0.99 (0.90-1.10) 0.91 (0.82-1.01).11 Model 2 1.07 (0.88-1.31) 1.00 (Referent) 0.93 (0.85-1.02) 1.00 (0.91-1.11) 0.93 (0.83-1.03).26 Cases, No. 93 1161 819 451 429 Person-years 44 052 480 217 313 378 159 562 163 066 Age-adjusted RR 1.03 1.00 (Referent) 0.97 1.06 1.01.55 Multivariate RR (95% CI) Model 1* 1.10 (0.89-1.36) 1.00 (Referent) 0.94 (0.86-1.03) 1.00 (0.90-1.12) 0.98 (0.88-1.10).74 Model 2 1.10 (0.89-1.37) 1.00 (Referent) 0.95 (0.86-1.04) 1.01 (0.90-1.13) 0.99 (0.89-1.12).96 Cases, No. 19 250 134 84 43 Person-years 6400 72 758 43 484 23 926 21 138 Age-adjusted RR 1.02 1.00 (Referent) 0.82 0.96 0.57.003 Multivariate RR (95% CI) Model 1* 0.98 (0.61-1.57) 1.00 (Referent) 0.82 (0.66-1.02) 0.95 (0.74-1.22) 0.55 (0.39-0.76).001 Model 2 0.89 (0.55-1.45) 1.00 (Referent) 0.85 (0.68-1.06) 0.98 (0.76-1.26) 0.56 (0.41-0.79).006 *The model included indicator variables for age (5-year categories), length of follow-up (8 periods), total energy intake (quintiles), parity (0,1 or2,3or4,or 5births), age at first birth ( 24, 25-29, or 30 years), age at menarche ( 12, 13, or 14 years), history of breast cancer in mother or a sister (yes or no), history of benign breast disease (yes or no), alcohol intake (0, 0.1-4.9, 5-14.9, or 15 g/d), body mass index at age 18 years ( 20, 20 to 22, 22 to 24, 24 to 27, or 27 kg/m 2 ), weight change from age 18 years (loss 2.0, loss or gain of 2.0, gain 2.1-5.0, gain 5.1-10.0, gain 10.1-20.0, gain 20.1-25.0, or gain 25.0 kg), height in cm, age at menopause (premenopausal, uncertain menopausal, 45, 45-49, 50-54, or 55 years), and postmenopausal hormone use (premenopausal, uncertain menopausal, never, past 5 years, past 5 years, current 5 years, or current 5 years). Additional controlling for quintiles of beta carotene, including supplements. Alcohol intake was excluded. 1634 JAMA, May 5, 1999 Vol 281, No. 17 1999 American Medical Association. All rights reserved.
79% of women in the fourth category and 95% of women in the fifth category were multivitamin supplement users (Table 1). Women who consumed more folate were more likely to have greater intakes of beta carotene; lutein/zeaxanthin; preformed vitamin A, including supplements; and total vitamins C and E, including supplements. We did not observe any important differences in established breast cancer risk factors at baseline across the 5 categories of total folate intake. Total folate intake was not associated with risk of breast cancer among the whole cohort (TABLE 2) or among premenopausal women (multivariate RRs for increasing categories of total folate = 1.23, 1.00 [referent], 0.99, 1.15, and 0.99 [95% CI, 0.79-1.23]; P for trend =.98). Total folate intake was weakly inversely associated with risk of postmenopausal breast cancer; the comparable multivariate RRs were 1.10, 1.00 (referent), 0.93, 0.94, and 0.86 (95% CI, 0.76-0.98, P for trend =.02). Folate intake derived from foods alone was not significantly related to overall risk of breast cancer (multivariate RRs = 1.10, 1.00 [referent], 0.96, 1.10, and 1.22 [95% CI, 0.81-1.83]; P for trend =.82). Higher intake of alcohol increased breast cancer risk; the multivariate RRs were 1.07 (95% CI, 0.98-1.17) for women consuming 0.1 to 4.9 g/d, 1.11 (95% CI, 1.00-1.22) for women consuming 5 to 14.9 g/d, and 1.24 (95% CI, 1.11-1.39) for women consuming at least 15 g/d compared with nondrinkers. Because a benefit of folate may be particularly important among women regularly consuming alcohol, we further examined the association between total folate intake and breast cancer risk by level of alcohol consumption (Table 2). Among women consuming at least 15 g/d of alcohol, for total folate intake of at least 600 µg/d compared with 150 to 299 µg/d, the multivariate RR was 0.55 (95% CI, 0.39-0.76, P for trend =.001) (Table 2). The RR was essentially the same after further adjustment for beta carotene, including supplements (RR = 0.56; 95% CI, 0.41-0.79) (Table 2), and did not appreciably change after controlling one at a time for lutein/zeaxanthin (RR = 0.57; 95% CI, 0.41-0.79); preformed vitamin A, including supplements (RR = 0.62; 95% Figure. Multivariate Relative Risk of Breast Cancer by Total Folate Intake and Alcohol Consumption Relative Risk 1.5 1.0 0.5 0 1.09 1.40 1.00 1.32 0.95 1.05 1.01 1.21 0.98 <150 150-299 300-449 450-599 600 Total Folate, µg/d 0.73 <15 g/d 15 g/d Alcohol Consumption The reference group for all comparisons was women who consumed 150 to 299 µg/d of total folate and less than 15 g/d of alcohol. The relative risks have been adjusted for variables listed in the footnotes to Table 2 for model 1. Table 3. Relative Risk (RR) and 95% Confidence Interval (CI) of Breast Cancer According to the Categories for Cumulative Average Intake of Total Folate and Dietary Methionine Intake in a Cohort of 88 818 Women From 1980 to 1996 Folate Intake, µg/d P for Methionine 150 150-299 300-449 450-599 600 Trend Quintile 1 ( 1.50 g/d) Cases, No. 49 319 136 82 61 Multivariate RR (95% CI)* 1.26 (0.93-1.72) 1.00 0.81 (0.66-0.99) 0.95 (0.74-1.21) 0.79 (0.60-1.04).03 Quintile 2 (1.51-1.69 g/d) Cases, No. 21 350 192 89 74 Multivariate RR (95% CI)* 0.85 (0.54-1.32) 1.00 0.86 (0.72-1.03) 0.81 (0.64-1.03) 0.74 (0.57-0.95).01 Quintile 3 (1.70-1.85 g/d) Cases, No. 14 307 193 109 97 Multivariate RR (95% CI)* 0.76 (0.44-1.31) 1.00 0.87 (0.72-1.04) 0.96 (0.77-1.20) 0.95 (0.76-1.20).88 Quintile 4 (1.86-2.07 g/d) Cases, No. 17 217 191 132 101 Multivariate RR (95% CI)* 1.54 (0.93-2.55) 1.00 1.03 (0.85-1.26) 1.29 (1.03-1.61) 1.11 (0.87-1.41).23 Quintile 5 ( 2.08 g/d) Cases, No. 11 218 241 123 139 Multivariate RR (95% CI)* 0.96 (0.52-1.77) 1.00 1.03 (0.85-1.24) 0.96 (0.77-1.21) 0.96 (0.78-1.19).63 *The model included the same indicator variables as in Table 2. 1999 American Medical Association. All rights reserved. JAMA, May 5, 1999 Vol 281, No. 17 1635
Table 4. Relative Risk (RR) and 95% Confidence Interval (CI) of Breast Cancer According to Duration of Multivitamin Supplement Use and Alcohol Consumption From 1980 to 1996 Use of Multivitamin Supplements Cases, No. Person-Years Multivariate-Adjusted RR (95% CI)* Never users 1097 457 700 1.00 (Referent) Past users 549 187 834 0.99 (0.89-1.10) Current users, y 961 358 394 0.97 (0.89-1.06) 4 122 64 264 0.91 (0.75-1.10) 5-9 140 54 140 1.02 (0.86-1.22) 10-14 290 114 269 0.94 (0.82-1.07) 15 409 125 721 1.01 (0.90-1.14) Never users 199 59 315 1.00 (Referent) Past users 64 22 365 0.77 (0.57-1.03) Current users, y 122 45 988 0.74 (0.59-0.93) 4 18 8347 0.69 (0.42-1.13) 5-9 20 6941 0.84 (0.53-1.33) 10-14 33 14 530 0.67 (0.46-0.97) 15 51 16 170 0.79 (0.57-1.08) *The model included the same indicator variables as in Table 2. CI, 0.41-0.92); total vitamin C, including supplements (RR = 0.56; 95% CI, 0.39-0.80); and total vitamin E, including supplements (RR = 0.50; 95% CI, 0.34-0.72). The RR was only slightly attenuated after inclusion of all these nutrients simultaneously in the model (RR = 0.62; 95% CI, 0.40-0.94). In the latter model, neither beta carotene, including supplements (RR = 0.90; 95% CI, 0.60-1.35, for top vs bottom quintile); lutein/zeaxanthin (RR = 0.92; 95% CI, 0.61-1.39, for top vs bottom quintile); preformed vitamin A, including supplements (RR = 0.73; 95% CI, 0.51-1.06, for top vs bottom quintile); total vitamin C, including supplements (RR = 0.95; 95% CI, 0.68-1.32, for top vs bottom quintile); nor total vitamin E, including supplements (RR = 1.10; 95% CI, 0.79-1.54, for top vs bottom quintile) were significantly associated with breast cancer risk. Thus, these other nutrients are unlikely to explain the association we observed with total folate. The inverse association between total folate intake of at least 600 µg/d and breast cancer risk among women consuming at least 15 g/d of alcohol was present among both premenopausal (RR = 0.65; 95% CI, 0.33-1.28) and postmenopausal (RR = 0.49; 95% CI, 0.33-0.74) women. We observed no association between total folate intake and breast cancer risk among women who consumed less than 15 g/d of alcohol. The test for interaction between total folate and alcohol consumption and breast cancer risk was statistically significant (P =.02). When total folate and alcohol intakes were examined in combination among the whole cohort, higher total folate intake appeared to mitigate the excess risk of breast cancer because of higher consumption of alcohol (FIGURE). The risk of breast cancer associated with alcohol intake was strongest among women with total folate intake less than 300 µg/d (for alcohol 15 g/d vs 15 g/d, multivariate RR = 1.32; 95% CI, 1.15-1.50). For women consuming at least 300 µg/d of total folate, the multivariate RR for at least 15 g/d of alcohol vs less than 15 g/d was 1.05 (95% CI, 0.92-1.20). Because intake of methionine, mainly from meats, may influence the availability of methyl groups and thus affect the requirement for folate intake, 1,5 we also evaluated the association between total folate intake and breast cancer risk by methionine intake (TABLE 3). We observed a 21% to 26% lower risk of breast cancer with intake of total folate of at least 600 µg/d compared with intake of 150 to 299 µg/d among women in the lowest 2 quintiles of methionine intake. Among women who consumed at least 15 g/d of alcohol and were in the lowest 2 quintiles of methionine intake, the multivariate RRs for increasing categories of total folate were 1.01, 1.00 (referent), 0.85, 0.93, and 0.45 (95% CI, 0.27-0.74). Because multivitamin supplements were the major source of folate intake, especially among women with intake of at least 450 µg/d of total folate, we examined multivitamin use in relation to risk of breast cancer according to alcohol consumption (TABLE 4). Among women who consumed at least 15 g/d of alcohol, the multivariate RRs were 0.77 (95% CI, 0.57-1.03) for past multivitamin users and 0.74 (95% CI, 0.59-0.93) for current multivitamin users compared with never users. These associations existed among both premenopausal (for current users, RR = 0.60; 95% CI, 0.37-0.98) and postmenopausal (for current users, RR = 0.76; 95% CI, 0.58-1.01) women. We observed no association with multivitamin use among women who consumed less than 15 g/d of alcohol. To evaluate possible distortion of the findings by differences in mammography rates among women with higher intake of total folate, we compared the cumulative average intake of total folate of women who had mammography during 1993 or 1994 with the intake of those who had not. The mean of cumulative average total folate intake (385 µg/d) of women who had mammography was only slightly higher than that of women who had not (369 µg/d). COMMENT In this large prospective cohort, total folate intake was not associated with overall risk of breast cancer. However, higher total folate intake or multivitamin use was associated with a lower risk of breast cancer among women who regularly consumed alcohol. Epidemiological data relating folate intake to breast cancer risk are limited. In 1 case-control study with 297 cases and 311 controls, premenopausal women consuming at least 460 µg/d of dietary 1636 JAMA, May 5, 1999 Vol 281, No. 17 1999 American Medical Association. All rights reserved.
folate had a statistically significant 50% lower risk compared with women who consumed 304 µg/d or less. 24 In another case-control study with 439 breast cancer cases and 494 controls, a statistically nonsignificant 30% lower risk was found among postmenopausal women consuming at least 451 µg/d of folate compared with women who consumed 289 µg/d or less. 25 A direct association between moderate alcohol consumption and breast cancer occurrence has been consistently observed in epidemiological studies across study designs, including in this cohort. 8-11 In a pooled analysis from 6 prospective cohorts, 11 an increment of 10 g/d of alcohol was associated with a 9% increase in breast cancer risk. Our data suggest that alcohol consumption modified the association of folate intake with breast cancer risk, similar to the interaction between folate and alcohol observed for colon cancer and coronary heart disease. 2,3,26 Alcohol interferes with several aspects of normal folate transport and metabolism including dietary intake, intestinal absorption, transport to tissues, storage, and release by liver, thereby disrupting folate supply to tissues. 7 The prospective design and high follow-up rates in this study reduce the concern that methodological biases explain these findings. Although the folate intake calculated from our dietary questionnaire reasonably reflects longterm intake of study subjects, 2 some misclassification still exists, which would tend to weaken any true associations. Residual confounding by nondietary factors is unlikely to explain our findings because controlling for recognized risk factors of breast cancer had minimal effect on the RRs. Our findings for folate were also independent of beta carotene and vitamins A, C, and E. Although we cannot completely exclude the possibility that other constituents of multivitamins contribute to lower breast cancer among regular alcohol consumers, our findings strongly suggest that folate is the primary factor. Folate intake above the amounts consumed by many persons appears necessary for maximal reduction of neural tube defects, 27 cardiovascular disease, 26,28,29 and colorectal cancer 1-4 as well as breast cancer in this study. In recognition of these relationships, the Recommended Dietary Allowance (RDA) for folate was recently increased from 180 µg/d 30 to 400 µg/d. The basis for the previous RDA was to protect against development of clinical folate deficiency. 30 The Food and Drug Administration now requires the fortification of grain products with folic acid, which is estimated to increase average folate intake by about 100 µg/d. 31 Our findings suggest that this may have an unplanned positive effect on breast cancer incidence. In summary, our findings suggest that the excess risk of breast cancer associated with alcohol consumption may be reduced by ensuring adequate folate intake. Because of the potential practical importance of these findings, the relation of folate intake to risk of breast cancer among women who consume alcohol needs to be examined in other studies. Funding/Support: This study was supported by research grant CA 40356 from the National Institutes of Health, Bethesda, Md. Acknowledgment: We are indebted to the participants of the Nurses Health Study for their continuing outstanding dedication and commitment to the study. We thank Meir Stampfer, MD, for his invaluable comments on the manuscript. We are also indebted to Karen Corsano and Laura Sampson, RD, for computer assistance, and Gerry Groff and Stephanie Smith-Warner, PhD, for their assistance with preparing the manuscript. REFERENCES 1. Mason JB, Levesque T. Folate: effects on carcinogenesis and the potential for cancer chemoprevention. Oncology. 1996;10:1727-1736, 1742-1743. 2. Giovannucci E, Stampfer MJ, Colditz GA, et al. Folate, methionine, and alcohol intake and risk of colorectal adenoma. J Natl Cancer Inst. 1993;85:875-884. 3. Giovannucci E, Rimm EB, Ascherio A, et al. Alcohol, low-methionine-low-folate diets, and risk of colon cancer in men. J Natl Cancer Inst. 1995;87:265-273. 4. Freudenheim JL, Graham S, Marshall JR, et al. Folate intake and carcinogenesis of the colon and rectum. Int J Epidemiol. 1991;20:368-374. 5. Cooper AJL. Biochemistry of sulfur-containing amino acids. Annu Rev Biochem. 1983;52:187-222. 6. Blount BC, Mack MM, Wehr CM, et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage. Proc Natl Acad Sci USA.1997;94:3290-3295. 7. Hillman RS, Steinberg SE. The effects of alcohol on folate metabolism. Annu Rev Med. 1982;33:345-354. 8. Willett WC, Stampfer MJ, Colditz GA, et al. Moderate alcohol consumption and the risk of breast cancer. N Engl J Med. 1987;316:1174-1180. 9. Schatzkin A, Longnecker MP. Alcohol and breast cancer. Cancer. 1994;74(suppl 3):1101-1110. 10. Swanson CA, Coates RJ, Malone KE, et al. Alcohol consumption and breast cancer risk among women under age 45 years. Epidemiology. 1997;8:231-237. 11. Smith-Warner SA, Spiegelman D, Yaun S-S, et al. Alcohol and breast cancer in women: a pooled analysis of cohort studies. JAMA. 1998;279:535-540. 12. Willett WC, Sampson L, Stampfer MJ, et al. Reproducibility and validity of a semiquantitative food frequency questionnaire. Am J Epidemiol. 1985;122: 51-65. 13. Willett WC, Sampson L, Browne ML, et al. The use of a self-administered questionnaire to assess diet four years in the past. Am J Epidemiol. 1988;127:188-199. 14. Salvini S, Hunter DJ, Sampson L, et al. Foodbased validation of a dietary questionnaire. Int J Epidemiol. 1989;18:858-867. 15. Stampfer MJ, Willett WC, Speizer FE, et al. Test of the National Death Index. Am J Epidemiol. 1984; 119:837-839. 16. Willett W, Stampfer MJ. Total energy intake: implications for epidemiologic analyses. Am J Epidemiol. 1986;124:17-27. 17. US Food and Drug Administration. Statement of general policy or interpretation: subchapter B-food and food products, part 121-food additives. Federal Register. August 2, 1973;38:20725-20726. 18. Rothman KJ. Modern Epidemiology. Boston, Mass: Little Brown & Co Inc; 1986. 19. Cupples LA, D Agostino RB, Anderson K, Kannel WB. Comparison of baseline and repeated measure covariate techniques in the Framingham Heart Study. Stat Med. 1988;7:205-222. 20. D Agostino RB, Lee ML, Belanger AJ, et al. Relation of pooled logistic regression to time dependent Cox regression analysis: the Framingham Heart Study. Stat Med. 1990;9:1501-1515. 21. Hankinson SE, Colditz GA, Manson JE, et al. A prospective study of oral contraceptive use and risk of breast cancer (Nurses Health Study, United States). Cancer Causes Control. 1997;8:65-72. 22. Michels KB, Willett WC, Rosner BA, et al. Prospective assessment of breastfeeding and breast cancer incidence among 89 887 women. Lancet. 1996; 347:431-436. 23. Hu FB, Stampfer MJ, Rimm E, et al. Dietary fat and coronary heart disease. Am J Epidemiol. 1999; 149:531-540. 24. Freudenheim JL, Marshall JR, Vena JE, et al. Premenopausal breast cancer risk and intake of vegetables, fruits, and related nutrients. J Natl Cancer Inst. 1996;88:340-348. 25. Graham S, Hellmann R, Marshall J, et al. Nutritional epidemiology of postmenopausal breast cancer in western New York. Am J Epidemiol. 1991;134:552-566. 26. Rimm EB, Willett WC, Hu FB, et al. Folate and vitamin B 6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA. 1998;279:359-364. 27. Rayburn WF, Stanley JR, Garrett E. Periconceptional folate intake and neural tube defects. J Am Coll Nutr. 1996;15:121-125. 28. Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. JAMA. 1995;274:1049-1057. 29. Selhub J, Jacques PF, Bostom AG, et al. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med. 1995; 332:286-291. 30. National Research Council, Subcommittee on the Tenth Edition of the RDAs, Food and Nutrition Board, Commission on Life Sciences. Folate. In: Recommended Dietary Allowances. 10th ed. Washington, DC: National Academy Press; 1989:150-158. 31. Oakley GP Jr. Eat right and take a multivitamin. N Engl J Med. 1998;338:1060-1061. 1999 American Medical Association. 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