Eight cohort studies, conducted in Norway 35, Japan 36, Europe 37, the US 38394041, and Singapore 42, have examined fish or marine n-3 fatty acids intake in relation to breast cancer; only one of them, the study from Singapore, found an association 42.

Results from case-control studies of marine n-3 fatty acids (eicosapentaenoic acid and DHA) measured in adipose tissue are mixed 4344454647. Maillard and colleagues 45 found strong inverse associations for DHA, and for the marine n-3/total n-6 fatty acids ratio. In a case-control study conducted in Finland, DHA level in breast adipose tissue, along with its dietary intake, were significantly lower in breast cancer patients compared to control patients, suggesting a protective effect of DHA in the risk of breast cancer 43. Also, a case-control study conducted across five European countries found a decreased risk of breast cancer with an elevated ratio of marine n-3 fatty acids to total n-6 fatty acids in four of five centers 44. In contrast to these findings, two case-control studies conducted in the US found no consistent association between n-3 fatty acid levels in adipose tissue and breast cancer risk 4647.

The reason for the discrepancy between the US and European studies remains unresolved. One hypothesis is these discrepancies may be due to differences in ranges of fish intake across the various populations 42. Another hypothesis is that the inconsistencies may be due in part to interactions between n-3 fatty acids and antioxidant compounds in the diet, affecting their roles in breast cancer risk 454849. As pointed out above, data from experimental studies suggest that the strength of the association with marine n-3 fatty acids may be reduced in the presence of high antioxidant intake because the latter inhibit the formation of lipid peroxidation products, which have been proposed as the proximal anti-carcinogens 4850. In fact, several experimental studies showed that the suppression in cancer growth with n-3 fatty acids is enhanced by pro-oxidants 51 and eliminated by antioxidants such as vitamin E 11185152. This suggests that the anti-cancer effect of n-3 fatty acids is mediated, at least in part, by lipid peroxidation products and emphasizes the importance of the potential interactions of anti- and pro-oxidant compounds with marine n-3 fatty acids 45. Maillard and colleagues 45 suggested that the lack of an association between marine n-3 fatty acids and breast cancer risk in US studies 39464753 may be due, in part, to the interfering role of antioxidant vitamins, which are commonly taken as supplements in the US 4554.

As mentioned above, we recently published the first set of prospective results linking intake of marine n-3 fatty acids to breast cancer protection in Singapore Chinese women 42. In our study, relative to the lowest quartile of marine n-3 fatty acid intake, individuals in the higher 3 quartiles exhibited a 26% reduction in risk of breast cancer (relative risk = 0.74, 95% confidence interval (CI) = 0.58, 0.94). We also have published the first set of results in humans implicating the peroxidation products of marine n-3 fatty acids as the proximal anticarcinogens 19, a notion strongly supported by experimental evidence 18495052555657. Glutathione-associated metabolism is a major mechanism for cellular protection against agents that generate oxidative stress, acting by eliminating products of lipid peroxidation 5859. Therefore, we hypothesized that individuals possessing the low activity genotypes of GSTM1, GSTT1 and/or GSTP1 (that is, GSTM1-null, GSTT1-null and GSTP1 AB/BB genotypes, respectively) 6061 may exhibit a stronger inverse association between marine n-3 fatty acids and breast cancer than their high activity counterparts. This hypothesis was supported by study results 19.

Meta-analysis of case-control and prospective cohort studies on breast cancer have failed to demonstrate a convincing link between n-6 fatty acids and breast cancer development 626364. In contrast, diets containing n-6 fatty acids have been shown to induce breast cancer in experimental studies 65. N-6 fatty acids act as competitive inhibitors of n-3 fatty acids in fat metabolism, and it has been shown that the stimulatory or inhibitory effect of n-6 or n-3 fatty acids in experimental mammary carcinogenesis is abrogated by the addition of the other type of fatty acid 666768. Experimental studies have shown that a diet high in n-6 fatty acids decreases the concentrations of marine n-3 fatty acid-induced lipid peroxidation products in breast cancers to the lowest levels, and that the lower this concentration, the bigger the tumor volumes resulting from n-6 fatty acid administration 5255. This experimental evidence is consistent with our results 42 showing that n-6 fatty acids increase breast cancer risk only among women with a low intake of marine n-3 fatty acids; that is, when concentrations of n-3 fatty acid-induced beneficial lipid peroxidation byproducts are low. So one possible mechanism whereby n-6 fatty acids increase breast cancer growth is the decreasing of the beneficial lipid peroxidation products derived from n-3 fats.

At least 13 epidemiological studies have assessed the direct relationship between the individual dietary intake of soy products and the risk of breast cancer (reviewed by Peeters and colleagues 69). Overall, results do not show protective effects, with the exception maybe for women who consume phytoestrogens at adolescence or at very high doses 707172. Only 4 of these 13 studies were prospective, and none of them found statistically significant breast cancer reductions. The only prospective study with urinary measurements taken before breast cancer occurrence was done in a Dutch postmenopausal population and showed a non-significant breast cancer risk reduction for high excretion 73. Soy phytoestrogen levels in the Dutch study were very low, as were those in another recent study 74. In conclusion, none of the five prospective studies assessing the effects of phytoestrogens on breast cancer risk found protective effects 69. However, none of these studies took into account mechanistic pathways by which soy can operate, including the lipid peroxidation pathway, for example, analyzing soy oil, where lipid peroxidation may be higher.

Soybean and canola oils are the primary sources of alpha linolenic acid (ALA; an oxidizable n-3 fatty acid) in the diet 75. Soybeans contain lipoxygenase, which is an oxidizing enzyme 76 that catalyzes lipid peroxidation 77. It has been shown that soybean lipooxygenase increases peroxidation of membrane lipids 78 and oxidizes low-density lipoproteins 121379. A soybean oil diet fed to rabbits and rats caused an increase in lipid peroxidation compared to controls 141516; this increase is accenuated by protein insufficiency 17.

Limited in vitro data suggest that the decreased breast cancer risk associated with ALA may be related to increased lipid peroxidation products 18. The addition of ALA to breast cancer cells caused an increase in the formation of lipid peroxidation products in the cell lipids, and their content was correlated with the capacity of arresting cell growth 18. The addition of the antioxidant vitamin E to the ALA-supplemented cancer cells diminished formation of lipid hydroperoxides and restored cell growth 18. In addition, vitamin E also suppressed the inhibitory effect of ALA on tumor growth in different models of mammary carcinogenesis in rats 51. Administration of oxidative compounds to diets high in ALA led to an inhibition of tumor growth in chemically induced mammary carcinogenesis 51.

In an experimental study, a fermented soymilk product induced generation of ROS and caused apoptotic cell death in MCF-7 breast cancer cells. Growth inhibition and ROS generation induced by fermented soymilk product could be inhibited by catalase and deferoxamine, indicating that the ROS production probably was the cause of this apoptotic cell death 80. The opposite has also been reported, that is, soy having an antioxidant effect 81. The effect appears to be indirect as the antioxidant potency of isoflavones is weak and the effects appear to be due more to effects on signaling pathways that induce antioxidant enzyme systems or suppress enzymes that produce ROS.

In animal models of breast cancer, tumor growth is inhibited by brassica consumption, or ITC or indole-3-carbinol administration 82838485. In humans, the relationship between brassica consumption and breast cancer risk is uncertain. Investigations have found null associations 86878889, protective but not statistically significant associations 909192, and statistically significant protective associations 9394. The failure to consider the lipid peroxidation pathway and the implied modifying effects of oxidative stress-related genes may explain the lack of consistency among human studies. ITCs are potent inducers of lipid peroxidation 20212223. There is suggestive evidence that ITC-induced apoptosis is mediated by oxidative stress/lipid peroxidation products 2223. Depletion of the antioxidant glutathione significantly accelerated ITC-triggered apoptosis 23. ITCs are also able to induce glutathione S-transferases (GSTs) and/or other antioxidant enzymes through the stress-signaling pathway involving oxidative stress 22. Oxidizing agents enhanced ITC-induced ROS production and ITC-induced GST activities, whereas antioxidants inhibited both 22. In humans, one study has shown that individuals with genetic polymorphisms encoding lower or no activity in antioxidant enzymes (GSTM1, GSTT1) experience more breast cancer protection from ITCs than those with common alleles 95, putatively because more beneficial peroxidation agents could reach the cancer cells and cause damage (see below). However, this finding has not been confirmed in a second study 94.

Published epidemiological studies overall suggest that green tea but not black tea consumption is related to decreased risk of breast cancer 96. A recent meta-analysis 96 included three cohort and one population-based study for green tea, while five cohort and eight case-control studies were analyzed for a link between black tea and breast cancer. Overall summary odds ratio showed an approximately 20% statistically significant reduction in risk of breast cancer associated with high intake of green tea. No such protective effect was found for black tea.

Both green and black tea have demonstrated inhibitory activities against chemically induced mammary tumors in experimental animals 979899. Epigallocatechin gallate (EGCG), one of the main constituents of tea that is thought to be responsible for its anticancer properties, not only can function as an antioxidant, but it possesses the chemical property of a pro-oxidant. Previous studies on the antioxidative properties of EGCG have demonstrated that its effects include both trapping of ROS as well as inhibition of lipid peroxidation 25. However, after neutralizing the peroxyl and/or other radicals, EGCG itself could be converted to a phenoxyl radical 26. Experimental studies in HT-29 colorectal cancer cells have indicated that oxidative stress is involved in EGCG-induced cell death. The chemical property of EGCG as a potential pro-oxidant is highlighted by the blocking effects of reduced glutathione and N-acetyl-L-cysteine against EGCG-induced mitogen-activated protein kinase activation, cytochrome c release and cell death 24. In a recent study 100, Wu and colleagues noted that the protective effect of tea on breast cancer was confined to those possessing the low-activity genotype of the antioxidant catechol-O-methyl transferase (COMT), putatively because more beneficial peroxidation agents could reach the cancer cells and cause damage.

Increased mammographic breast density is strongly associated with the risk of breast cancer 101. A recent study showed that an increased intake of vitamin D and calcium was associated with decreases in mammographic breast density 102.

Evidence from both in vitro and in vivo studies has demonstrated that vitamin D compounds can inhibit the growth of breast cancer cells 103. The anticancer activity of the hormonal form of vitamin D, 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) 104, is associated with inhibition of cell cycle progression, induction of differentiation, and apoptosis. In addition, 1,25(OH)₂D₃ may exert some of its activity by cooperating with other anticancer agents. 1,25(OH)₂D₃ and its synthetic analogues increased the susceptibility of cancer cells to the cytotoxic/cytostatic action of tumor necrosis factor 105106107, interleukin 1, interleukin 6 108, doxorubicin, menadione 109, and radiation 110. A feature shared by these agents whose potency is increased by 1,25(OH)₂D₃ is their ability to bring about excessive ROS generation in their target cells 111112113. This common feature suggests the involvement of ROS in the interaction between 1,25(OH)₂D₃ and these agents. In addition, the potentiation of the cytotoxic/cytostatic action of the chemotherapy drug doxorubicin or cytokines by 1,25(OH)₂D₃ is markedly inhibited by the antioxidant N-actylcysteine 108109. Importantly, it has recently been shown that 1,25(OH)₂D₃, acting as a single agent, is also a pro-oxidant in cancer cells 29. These findings indicate that 1,25(OH)₂D₃ causes an increase in the overall cellular redox potential that could translate into modulation of redox-sensitive enzymes and transcription factors that regulate cell cycle progression, differentiation, and apoptosis 29.

The evidence that calcium or dairy products are associated with breast cancer risk is still open to debate. A recent pooled analysis of cohort studies failed to find an association 114, but some studies found some limited evidence of a protection 115116. As mentioned above, a recent study showed that an increased intake of vitamin D and calcium was associated with decreases in mammographic breast density 102. Boyapati and colleagues 116 recently reported that dietary calcium intake was negatively associated with the risk of breast cancer in both premenopausal and postmenopausal women. In the Nurses' Health Study both calcium and dairy product intake was associated with a survival benefit for women with breast cancer 117118.

After ingestion, calcium absorption can occur via an active transport process (transcellular) that requires the action of 1,25-(OH)₂D₃, or by passive diffusion (paracellular), which is a vitamin-D-independent process. The vast majority of calcium absorption (77% to 92%) relies upon the trans-cellular pathway, and thus upon the activity of 1,25-(OH)₂D₃ 119. Therefore, it is difficult to separately establish the potential effects of these two nutrients on health and disease, and it is of clear importance to evaluate the effects of these nutrients simultaneously 120.

There are several proposed mechanisms of action for calcium, including inhibition of cellular proliferation 121122, and induction of apoptotic cell death 123124 through oxidative stress 3031125126. It is, however, unclear how calcium, especially through intake, might have these hypothesized effects when blood levels are so tightly regulated. It seems that the elevation in the intracellular free calcium, which correlates with the induction of apoptosis in breast cancer cells, is brought about by vitamin D compounds 124127128129. The latter studies further support the need for investigations that consider the role of vitamin D and calcium simultaneously. The effects of these two nutrients may be strongly related to one another, and separate studies may not capture their true effects on breast neoplasia. Generally, apoptotic cell death is triggered by intracellular signaling pathways, including a rise in intracellular free calcium 30, the generation of ROS and membrane lipid peroxidation products 125126. Calcium strongly stimulates the release of ROS 130 (including superoxide anion 131132133, hydrogen peroxide and hydroxyl radicals 134), which induce membrane lipid peroxidation 130. This calcium-dependent increase in membrane lipid peroxidation triggers apoptotic cell death 3031125126. Calcium, lipid peroxidation and apoptosis are possibly interlinked through signals, as is evident from the increased activity of nuclear factor kappa-B, a critical molecule in oxidative-stress-induced apoptosis, and generation of nitric oxide 135.

Glutathione-associated metabolism is a major mechanism for cellular protection against agents that generate oxidative stress, protecting cells against cytotoxic products of lipid peroxidation 5859. GSTs are induced under conditions of oxidative stress, and alpha-, pi-, mu-, and theta-class GSTs are active in detoxification of numerous products, including reactive oxidant damage to DNA and lipids, such as organic epoxides, lipid hydroperoxides, and unsaturated aldehydes 5859. Individuals lacking these enzymes may have reduced removal of lipid peroxidation products and, thus, may experience higher cancer protection, as supported by results from our study 19. We found that women with genetic polymorphisms causing lower or no activity in detoxifying genes (GSTM1, GSTT1, GSTP1) had more protection from marine n-3 fatty acids than those with common alleles, putatively because more cytotoxic peroxidation agents could reach the cancerous and pre-cancerous cells and cause damage. Recent results on cyclin D1 (CCND1), an intracellular cell-cycle regulatory protein with checkpoint function, and breast cancer risk further support our hypothesis of an oxidative stress-induced apoptosis mechanism underlying the diet-induced breast cancer protection. CCND1 has been shown to modulate growth arrest and apoptosis following exposure to ionizing radiation oxidative DNA damage 137138. In a recent study, the protective effect of the heterozygous CCND1 GA genotype on breast cancer risk was restricted to situations of elevated oxidative stress, characterized by absence of antioxidant GSTM1 and GSTT1 enzymes 139. In addition, CCND1 also interacted with marine n-3 and n-6 fatty acids to influence breast cancer risk, with the protection being restricted to women with a high intake level of n-6 fatty acids, or a low intake level of marine n-3 fatty acids 139.

Similarly, ITCs' protective effect on breast cancer found in some studies 95, although not in others 94, was mainly confined to those possessing the low activity genotype of GSTs. There is suggestive evidence that ITC-induced apoptosis is mediated by oxidative stress/lipid peroxidation products 2223. Seow and colleagues 140 also found that individuals with genetic polymorphisms causing lower or no activity in antioxidant genes (GSTM1, GSTT1, GSTP1) had more colorectal cancer protection from ITCs than those with common alleles. These findings were attributed to the direct effects of GSTs on ITC excretion 140. However, we proposed that the oxidative products of ITCs may be responsible, at least in part, for their anti-cancer effect, and this would explain why the protection appears more pronounced among subjects with lowest antioxidant GST activity 11, in parallel with what we had described for the marine n-3 fatty acids/GST/breast cancer relationship 19. A subsequent colon cancer study, in which the effect of the CCND1 A870G polymorphism on colorectal cancer risk was found to be modified by GSTM1, GSTT1, and GSTP1 genotypes and ITC intake 141, further supports our proposed oxidative stress-based hypothesis. In that study, the presence of at least one CCND1 A-allele was associated with increased risk among low dietary ITC consumers with a high activity GST profile. In contrast, the presence of at least one A-allele was associated with a decreased risk among all remaining subjects, which led the investigators to hypothesize that subjects with low intake levels of ITCs and functional GST enzymes are left with low levels of pro-oxidative, anti-cancer acting ITCs at a cellular level 141.

The genetic polymorphisms of GSTM1 and GSTT1 have also been found to influence the risk-enhancing effect of alcohol in breast cancer. Zheng and colleagues 142 found that breast cancer risk was about 7-fold increased for postmenopausal women with the GSTT1-null genotype who consumed more than 250 kg of spirit equivalents. In our prior study 19, the GSTT1-null genotype was associated with a 30% reduced risk of breast cancer. This finding is consistent with another study that reported a decreased risk among premenopausal women lacking the GSTT1 gene 60. Most studies have found no increased risk for breast cancer with null genotypes for GSTM1 and/or GSTT1 (reviewed in 61), although some positive associations have been reported 143144145146147.

COMT is an antioxidant enzyme that catalyses the methylation of hydroxylated sites on the aromatic ring of catechol compounds, which prevents their conversion to semiquinones and quinines and, therefore, blocks the generation of ROS 148149150. In a recent study, the COMT-L low activity allele-containing genotypes (HL or LL) tended to be at decreased risk of developing breast cancer, especially the advanced stage of disease in premenopausal women and local carcinoma in postmenopausal women 151152. A tendency of decreasing risk can also be seen for both pre- and postmenopausal women in the study of Millikan and colleagues 153. Similarly, in the case-control study of Lavigne and colleagues 154, a tendency of decreasing risk was seen among premenopausal women, although the results were based on a rather small number of subjects. Thompson and colleagues 155 observed increased risk for premenopausal women carrying the COMT-L allele-containing genotypes and decreased risk for postmenopausal women with these genotypes. Among Asian-American women in Los Angeles, the protective effect of tea in breast cancer was mainly confined to those possessing the low activity genotype of COMT 100. A recent study was conducted among breast cancer families participating in the Metropolitan New York Registry, one of the six centers of the Breast Cancer Family Registry 156. The study found that COMT genotypes were not statistically significantly associated with breast cancer risk, although the study population was of modest size (160 sib-ships).

MnSOD is an antioxidant enzyme that is induced by free radical challenge, such as marine n-3 fatty acid-induced lipid peroxidation 136157158, and inhibits polyunsaturated fatty acid-induced lipid peroxidation and the subsequent killing of human breast cancer cells 159. In the mitochondrion, MnSOD catalyzes the dismutation of two superoxide radicals, producing hydrogen peroxide and oxygen. A substitution variant in the mitochondrial targeting sequence was found that changes the amino acid codon at the -9 position in the signal peptide from valine to alanine 160161162. Hiroi and colleagues 163 reported that processing efficiency of the valine-type SOD leader peptide in the presence of mitochondria was significantly lower than that of the alanine-type, which may reduce protection against superoxide radicals. This decrease in the efficiency of transport into mitochondria for the valine isoform of the protein may result in increased ROS 160163. An association between the valine allele with lung cancer risk has been reported in a recent study 164. Contrarily, an association between the alanine allele and risk of breast cancer has been found in three studies, two conducted in the US 162165 and one in Finland 166. In the study by Ambrosone and coworkers 162, premenopausal women who were homozygous for the alanine allele had a four-fold increase in breast cancer risk in comparison to those with one or two valine alleles 162. Risk was most pronounced among women who consumed below the median amount of fruit and vegetables and of ascorbic acid and α-tocopherol. In addition, in the Finnish study, MnSOD genotypes containing the alanine allele were found to be associated with a 1.5-fold (95% CI = 1.1, 2.0) increased risk of breast cancer compared with those homozygous for the valine/valine genotype 166. The association between this polymorphism and breast cancer risk was weaker in the other US study (odds ratio = 1.27; 95% CI = 0.91, 1.77) 165, and no association was found in a case-control study within the Breast Cancer Family Registry 167. However, recent results suggest that this polymorphism is also associated with breast cancer risk among Chinese in Shanghai 168.