Antioxidants and breast cancer risk- a population-based case-control study in Canada

Breast cancer is the most common cancer and the second leading cause of cancer-related death among Canadian women [1]. According to the most recent projection from Canadian Cancer Society, there will be 22,700 new cases of female breast cancer representing 27.9% of all cancer cases in women, and 5,400 deaths caused by breast cancer accounting for 15.1% of all cancer-related deaths in women in Canada in 2009 [1].

Recently, antioxidants such as vitamin A and its precursor (beta-carotene, lycopene, lutein, etc), vitamins C, E, and selenium, have drawn a lot of attention to the scientists and the public alike [2, 3]. They have been shown in experimental studies to neutralize or trap reactive oxygen species (also known as free radicals), thereby preventing cellular damage caused by the reaction of these species with proteins and nucleic acids [4‐9]. Inflammation, infection, extreme exercise and exposure to various environmental factors, including pollution, tobacco smoke and radiation, can increase the level of free radicals in the body [3]. Excess production of free radicals and/or deficiency of the antioxidant defence system can result in oxidative stress, causing damage to DNA and other molecules [4, 5]. Over time, such damage may become irreversible and may lead to diseases such as cancer. There is evidence showing that oxidative stress and lipid peroxidation are linked to the aetiology of breast cancer [5]. Antioxidants are often described as "mopping up" free radicals, therefore they decrease oxidative stress and oxidative DNA damage [4, 6, 7, 9]. It has also been shown that antioxidants may selectively induce apoptosis in cancer cells but spare normal cells, and inhibit cell proliferation [3, 10, 11].

Although there is considerable evidence of anticancer effect of antioxidants from cell culture and animal studies, the results from observational studies and intervention trials are inconsistent [8, 12, 13]. A recent review conducted by Michels et al summarized the findings from prospective observational studies of the role of diet on the risk of breast cancer, including antioxidants, and found that the reported results were not consistent and sometimes controversial regarding the association between breast cancer incidence and any dietary antioxidants and blood antioxidants [2]. Randomized clinical trials of the effects of antioxidants on chronic diseases including breast cancer, also reached inconsistent conclusions [8, 13, 14]. However, since antioxidant enhancement, either through increased dietary intake or by supplementation, is a preventive measure that can be easily implemented, further studies to look at this association are warranted. The objective of this study was to assess the association between antioxidants from both diet and supplement and breast cancer, using data from a large population based case-control study in Canada.

Data from 2,362 breast cancer cases (866 premenopausal cases and 1,496 postmenopausal cases) and 2,462 controls were available for analysis.

Table 1 shows the distribution of some selected characteristics of breast cancer cases and controls. For premenopausal women, compared with controls, cases were slightly older and had less live birth and longer years of menstruation. For postmenopausal women, compared with controls, cases were slightly younger and had higher education, longer smoking pack-years, higher alcohol consumption, higher BMI, less live births, and longer years of menstruation.

Table 2 shows the average intakes of antioxidants from diet and average years of antioxidant supplementation. The average intakes of beta-carotene, vitamin C, vitamin E, selenium and zinc from diet and average years of supplementation were similar for cases and controls for both premenopausal and postmenopausal women.

Table 3 presents the breast cancer risk associated with different levels of antioxidants from dietary intake including beta-carotene, alpha-carotene, lycopene, lutein and zeaxanthin, vitamin C, vitamin E, selenium and zinc. No significant association was observed between breast cancer risk and the highest quartiles of these antioxidant intakes from dietary sources compared with the lowest quartiles of intake, either for premenopausal or for postmenopausal breast cancer.

Table 4 shows the breast cancer risk associated with different categories (years) of antioxidants from supplementation. For premenopausal breast cancer, supplementation of 10 years or longer was associated with statistically significant reduction in breast cancer risk for zinc. For postmenopausal breast cancer, supplementation of 10 years or longer was associated with statistically significant reduction in breast cancer risk for multiple vitamin, beta-carotene, vitamin C, vitamin E and zinc. No significant association between antioxidants and risk of breast cancer, in either premenopausal or postmenopausal cases, was observed, for supplementation less than 10 years prior to data collection, however.

Table 5 gives the odds ratios of breast cancer associated with high intakes of beta-carotene, vitamin C, vitamin E, zinc, and selenium from diet only, from supplementation only and from both sources. High intakes of beta-carotene, vitamin C, vitamin E, Zinc, and selenium from diet only were not significantly associated with breast cancer risk compared with low intake of these antioxidants for both premenopausal and postmenopausal women. Results for high intake of these antioxidants from supplementation only were similar to table 3. High intakes from both sources were not significantly associated with breast cancer risk except for vitamin C in premenopausal women and for zinc in postmenopausal women.

Our large population based study indicated that supplementations of 10 years or longer with zinc were associated with statistically significant reductions in premenopausal breast cancer risk. For postmenopausal women, supplementations of 10 years or longer of multiple vitamins, beta-carotene, vitamin C, vitamin E and zinc were associated with statistically significant reductions for breast cancer risk. No significant difference was observed in breast cancer risk, either premenopausal or postmenopausal, across different levels of antioxidants from dietary sources, nor was there a significant effect of supplementation shorter than 10 years. With the ability to draw a national sample, our study is amongst the largest in the field. We have also been able to adjust for a number of risk factors known to be related to breast cancer such as BMI, energy intake, and physical activity. However, this study could not determine the overall effect of total dose or intake of antioxidants from both diet and supplement.

Carotenoids (precursor of vitamin A) include alpha-carotene, beta-carotene, lutein, lycopene, and beta-cryptoxanthin. Higher dietary intakes and blood concentrations of carotenoids, including beta-carotene, have been shown to reduce breast cancer risk, but the results are not entirely consistent [8, 19]. A summarized analysis of 8 case-control studies showed a 15% reduction in breast cancer risk associated with the highest category of dietary beta-carotene [20]. A meta-analysis of 11 studies published from 1982 to 1997 also suggests that intake of 7000 μg per day or more of beta-carotene was associated with 18% reduction of breast cancer risk compared with intake of 1000 μg per day or less [21]. In addition, a nested case-control study reported a 50% decrease in breast cancer risk in women with high levels of serum beta-carotene, lycopene, and total carotene compared with those with low levels of these micronutrients [22]. Higher serum beta-carotene concentration was also found to be associated with lower breast cancer risk in other cohort studies [23‐26] and case-control studies [27, 28]. However, no association was observed in some studies for serum beta-carotene [29, 30] and for dietary beta-carotene, alpha-carotene, lutein, beta-cryptoxanthin and lycopene [12, 31] and in one randomized trail on beta-carotene supplementation use [13]. The Women's Health Initiative Observational Study on postmenopausal women also suggests that the association between breast cancer risk and carotenoids differs by estrogen receptor (ER) and progesterone receptor (PR) status since this study found that dietary alpha-carotene and beta-carotene were inversely associated with the risk of ER+PR+ breast cancer only, but not with other breast cancer groups defined by ER and PR status [32]. Carotenoids have been shown to protect against DNA alterations via a decrease in oxidative and other damage to DNA in humans [3, 6, 7, 11] and protect cells in vitro against neoplastic transformation [3, 11].

Zinc is involved with metallothionine synthesis, which is believed to inhibit free radical production [33]. Zinc supplementation has also been shown to reduce oxidative stress and DNA strand breaks and early and delayed apoptosis, as well as improve immune function [33]. Two case- control studies [34, 35] demonstrated statistically significant inverse associations between Zn exposure, measured in serum and diet, respectively, and breast cancer risk. However, two nested case- control studies [36, 37] reported no association between toenail Zn or zinc in benign breast tissue and breast cancer risk. Another case-control study also found no difference in either hair or plasma level of zinc between cases and controls [38]. The use of different status biomarkers in these studies may explain partly the mixed results of different studies [39].

Although studies in cell cultures and animals showed that vitamin E and C prevent transformation of normal cells to cancer cells and selectively induce apoptosis in cancer cells, the results of epidemiologic studies have been inconsistent [19]. The review of 12 cohort studies on dietary vitamin C and vitamin E intake and the incidence of breast cancer suggested no consistent association between both vitamin and breast cancer incidence among these studies [2]. The Danish Diet Cancer and Health Cohort observed a significant inverse association for total vitamin E (including dietary and supplemental) intake among postmenopausal women (RR = 0.59, 95% CI: 0.37-0.95) [40]. An inverse association between breast cancer risk and high serum level of alpha-tocopherol (vitamin E) was also found in 3 cohort studies [24, 26, 41]. However, several cohort studies reported no significant association [22, 29, 30, 32, 42‐45]. The evidence for vitamin C is also inconsistent. A pooled analysis of 9 case-control studies found a 31% decrease in breast cancer risk associated with each 300 mg of vitamin C per day [20]. A meta-analysis of 9 case-control and cohort studies observed a summary RR of 0.80 (95% CI: 0.68-0.95) for high consumption compared with low consumption [21]. On the other hand, several cohort studies reported no significant association between vitamin C and breast cancer risk [32, 42‐45]. One randomized controlled trial found no relationship of vitamin C and E supplementation use with breast cancer risk [13].

Because of differences in study population, study design, and nutritional assessment, it is rather difficult to reconcile results from different studies. Our study did not find any association between any antioxidant from dietary sources and breast cancer risk, or an effect of supplementation shorter than 10 years on breast cancer risk, for both premenopausal and postmenopausal cases. It is difficult to measure nutrients from dietary intake by case control studies. Although the response rate was 69.7% for breast cancer cases, selection (inclusion) of cases was not related to exposure status (i.e. antioxidant intake), therefore, selection bias is unlikely to occur. Recall bias, inherent in all case-control design, is possible because cases might bias their responses to questions on diet after a few months of cancer diagnosis. The possibility that such a bias was introduced into our study was reduced by including many questions on other exposures, such as socio-demographic factors, physical activity, weight, smoking history, employment history and residential history and by not placing any particular emphasis on diet in the questionnaire. Although we used a widely validated questionnaire, measurement error of dietary intakes, for example, self-reports of long-term dietary supplements, could introduce misclassification in exposure status, but it is likely to be nondifferential. The 69-item food frequency questionnaire might fail to capture all kinds of food consumed and underestimated total dietary intake. The time window of exposure is also critical in epidemiologic studies of diseases with long incubation period such as breast cancer. To improve accuracy and precision, we used two year from diagnosis as the point of measurement in the dietary survey. Another limitation is that we could not assess the dose of antioxidants from supplementation because doses of antioxidants from different producers, years and batches may be different. This might also affect accuracy and precision of our results. Antioxidants, either from dietary intake or from supplementation, may exert their effect over a long period of time. That may be the reason why we observed the effect for supplementation of 10 years or longer only. In addition, we could not adjust for the family history of breast cancer, benign breast disease, oral contraceptive use, hormone replacement therapy, and hormone receptor status in the analyses because the information was not available for all records. Furthermore, some of the risk estimates were based on small numbers of subjects, especially for some categories of supplementation; therefore, some of the significant associations may be due to due to chance.

The recommended intakes (recommended dietary allowance) for adult women are 700 μg/day for vitamin A, 15 mg/day for vitamin E, 75 mg/day for vitamin C, 55 μg/day for selenium and 8 mg/day for zinc [46]. Canadian nutrition survey in 2004 data showed that percentages of adult women 19 years or over with a usual intake below the estimated average requirement were 35.8% for vitamin A, 16.7% for vitamin C and 14.0% for zinc [47]. Our data showed that the average dietary intake for the study population had reached the recommended dietary allowance for vitamin C, selenium and zinc, but was lower for vitamin E.

In summary, our population-based study showed that supplementation of 10 years or longer of certain antioxidants such as beta-carotene, vitamin C, vitamin E and zinc may reduce the risk of breast cancer, although this study could not determine the overall effect of total dose or intake of antioxidants from both diet and supplement. Further investigations are warranted to confirm our results. The findings of our study may shed light on the prevention and control of breast cancer.