Dietary Marine n-3 Fatty Acids in Relation to Risk of Distal Colorectal Adenoma in Women

Marine n-3 fatty acids have been hypothesized to influence colorectal carcinogenesis through inhibiting cyclooxygenase 2, increasing apoptotic capacity, reducing angiogenesis, activating protein kinase C, suppressing ornithine decarboxylase, and reducing fecal bile acid and neutral sterol excretion (1-7). The results of animal and ecologic studies suggest that intake of marine n-3 fatty acids may reduce risk of colorectal cancer and adenoma (8-11). Moreover, clinical trials have found that n-3 supplementation reduced cell proliferation (12-14). However, the relationship between n-3 fatty acid intake and colorectal cancer has been inconsistent in epidemiologic studies (15-19). Two prospective studies (15, 16) and one case-control study (17) reported no significant associations between dietary marine n-3 fatty acids or the ratio of total n-3 to total n-6 fatty acids (total n-3/total n-6 ratio) in relation to risk of colorectal cancer, whereas one case-control study showed statistically significant inverse trends (18). In one case-control study on adenoma risk (19), marine n-3 fatty acid concentration in adipose tissue was nonsignificantly associated with a reduced risk of colorectal adenoma, but dietary marine n-3 fatty acids and the ratio of marine n-3 to n-6 fatty acids (n-3/n-6 ratio) were not related to lower risk.

The weak and inconsistent results for marine n-3 fatty acids could be caused in part by failure to account for the influence of other fatty acids. In particular, α-linolenic acid can be converted to long-chain n-3 fatty acids, particularly if marine n-3 fatty acid intake is low (20, 21). In addition, the conversion of α-linolenic acid to marine n-3 fatty acids is influenced by n-6 fatty acid levels because n-6 and n-3 fatty acids compete for the desaturation and chain elongation pathway (20, 21). To better understand the role of marine n-3 fatty acids and colorectal carcinogenesis, we examined the hypothesis that high intake of marine n-3 fatty acids reduces the risk of colorectal adenoma, and that this relationship is modified by intake of α-linolenic acid or total n-6 fatty acids, in the Nurses' Health Study.

The Nurses' Health Study was initiated in 1976 when 121,700 female registered nurses in the United States ages 30 to 55 years completed a mailed questionnaire about their lifestyle factors and medical history. Every 2 years, a follow-up questionnaire was sent to these women so that information could be updated and newly diagnosed major illnesses identified. A food frequency questionnaire was first given in 1980. Deaths in the cohort were ascertained by reports from family members, the postal service, and a search of the National Death Index. We estimate that over 98% of deaths were reported to us through these sources (22). The overall follow-up for this cohort was 96%. In this analysis, we included participants who returned the 1980 food frequency questionnaire and who had no diagnosis of cancer (excluding nonmelanoma skin cancer), inflammatory bowel disease, familial polyposis, or colorectal polyp before 1980. To reduce the potential for detection bias, we restricted the analysis to women who reported having undergone a colonoscopy or sigmoidoscopy between 1980 and 1998. Over 90% of the adenomas were diagnosed during endoscopic procedures for screening or for unrelated gastrointestinal conditions. Sigmoidoscopies do not examine proximal regions of the colon; thus, we analyzed only adenomas of the distal colorectum to prevent misclassification and potential detection bias.

A total of 34,451 women met all the criteria for analysis. This study and analyses were approved by the Institutional Review Boards of the Brigham and Women's Hospital and the Harvard School of Public Health.

The ascertainment of adenoma cases and controls has been described previously (23). Briefly, we asked permission from women who reported a new diagnosis of polyp on the follow-up questionnaires to obtain pertinent medical records. Study investigators, blinded to the exposure information, reviewed the medical records to record the histologic type, the anatomic location, and size of reported polyps. We considered for analysis only cases of distal colorectal adenoma confirmed by histopathologic report (including carcinoma in situ), irrespective of whether or not they also had adenomas proximal to the descending colon or had hyperplastic polyps. Eligible for analysis were 1,719 distal colorectal, 1,280 distal colon, and 505 rectal adenoma cases from 1980 to 1998. Women (n = 705) were classified as having large adenomas (≥1 cm) and 897 as small adenomas; information on size was not available for 117 cases.

The semiquantitative food frequency questionnaire used in 1980 included a survey of 61 items, including a single question assessing fish intake (24). A common unit or portion size for each food (e.g., 6-8 oz for fish) was specified, and each woman was asked how often, on average, during the previous year she had consumed that amount of the item. Nine response options were given, ranging from “almost never” to “six or more times per day”. In 1984, a second dietary questionnaire, expanded to include 116 items, was given. The dietary questionnaire included four fish and shellfish items: (a) dark meat fish (3-5 oz); (b) canned tuna (3-4 oz); (c) other fish (3-5 oz); and (d) shrimp, lobster, or scallops as main dish (3.5 oz). Similar dietary questionnaires were given in 1986, 1990, and 1994. We also asked about the types of fat used for cooking and at the table. The average daily intake of nutrients was calculated by multiplying the frequency of consumption of each item by its nutrient content and summing the nutrient contributions of all foods.

The calculation of marine n-3 fatty acids has been described in detail elsewhere (25). To calculate intake of marine n-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)], we assigned grams per servings as follows: 1.51 for dark meat fish; 0.42 for canned tuna; 0.48 for other fish; and 0.32 for shrimp, lobster, or scallops. These marine n-3 fatty acid variables were derived by weighing the mean values of n-3 fatty acids for the most common types of fish or shellfish in each category according to U.S. landing data in 1984 (U.S. Department of Commerce). To make the intake of marine n-3 fatty acids from the 1980 questionnaire as comparable as possible with the later, more detailed questionnaires, we assigned 1.16 g of marine n-3 fatty acids per portion (6-8 oz) on the 1980 questionnaire. This number was calculated as a weighed average of n-3 fatty acid composition from dark meat fish, canned tuna, and other fish by using the relative consumption of these types of fish on the 1984 dietary questionnaire. Red meat intake included beef, pork, or lamb as a main dish, beef in a sandwich, hamburger, hot dogs, bacon, and other processed meats, taking into account weight in grams of each portion size. Intakes of folate, vitamin D, and calcium included the sum of the frequency of consumption of specified portion sizes of those foods containing these nutrients and additionally intakes from multivitamins and specific supplements. In a parallel study of men, the questionnaire provided a reasonable measure of marine n-3 fatty acids when compared with adipose tissue; the energy-adjusted EPA intake was correlated with the percentage of EPA in adipose tissue (Spearman correlation coefficient = 0.49; P < 0.001; ref. 26).

The end point for this analysis was prevalence of distal colorectal adenoma at endoscopy. Controls consisted of women who did not have an adenoma of the distal colorectum at endoscopy, irrespective of whether or not they had adenomas of the proximal colon or had hyperplastic polyps. In additional analyses, we analyzed the adenomas by size because large adenomas (≥1 cm) may be more likely to reflect the influence of a tumor promoter and also separately analyzed adenomas of the distal colon and rectum. We divided women into five categories according to quintiles of dietary marine n-3 fatty acids as percentage of energy intake and n-3/n-6 ratio. In multivariable analysis, the relative risks (RR) estimated by the odds ratios were simultaneously adjusted for potential confounding variables by using multiple logistic regression. To best represent the participants' long-term dietary patterns during follow-up, we used a cumulative average method based on all available measurements of diet up to the beginning of each 2-year interval. For example, among women, dietary data from the 1980 questionnaire were used to predict colorectal adenoma diagnosed between June 1980 and June 1984, the average of the 1980 and 1984 dietary intake was used to predict outcomes between June 1984 and 1986, etc.

In the models, for covariates we included established and suspected risk factors as well as indication for endoscopy. The covariates included age at endoscopy (5-year categories), indication for endoscopy, body mass index (quintiles), pack-years smoked (nonsmokers, tertiles of pack-years for smokers), alcohol consumption (nondrinkers, 10 g/d categories), family history of colorectal cancer, history of previous endoscopy, postmenopausal hormone use (premenopausal, never, current, and past user), regular aspirin use, physical activity (quartiles), and quintiles of energy intake, energy-adjusted intakes of total fiber, methionine, vitamin D, calcium, folate, total n-6 fatty acids (linoleic acid and arachidonic acid), and red meat intake. Tests for trend were conducted by assigning the median value to each quintile and modeling this value as a continuous variable. The log likelihood ratio test was used to assess the significance of interaction terms. We also conducted analyses stratified by intakes of α-linolenic acid and total n-6 fatty acids, and aspirin use. We conducted all analyses using SAS version 8.2 (SAS Institute, Inc., Cary, NC).

Table 1 shows the age-standardized baseline characteristics according to dietary marine n-3 fatty acids grouped into quintiles. At baseline in 1980, women with high intake of marine n-3 fatty acids were older at time of endoscopy, had a higher prevalence of vigorous exercise, and a lower prevalence of previous endoscopy and aspirin use. Intakes of saturated fat and red meat decreased across quintiles, whereas intakes of calcium, vitamin D, methionine, folate, and fiber showed increasing trends. A similar pattern was observed with n-3/n-6 ratio (data not shown).

Dietary marine n-3 fatty acids were not appreciably related to risk of colorectal adenoma when adjusted only for age, or when also adjusted for dietary and nondietary risk factors [RR for the highest versus the lowest quintiles of dietary marine n-3 fatty acids = 1.04; 95% confidence interval (95% CI), 0.84-1.27; P_trend = 0.66 in multivariable analysis; Table 2]. In age-adjusted analyses, dietary marine n-3 fatty acids were significantly associated with reduced risk of large adenoma. In multivariable analyses, the association was attenuated and was not statistically significant, but the CIs did not exclude a modest inverse relation (RR for the highest versus the lowest quintiles of dietary marine n-3 fatty acids = 0.74; 95% CI, 0.54-1.01; P_trend = 0.16). However, the association of dietary marine n-3 fatty acids with risk of small adenoma tended to be positive, which was different from large adenoma. We conducted a post hoc analysis whereby we included only cases and computed the multivariable odds ratio for marine n-3 intake for large versus small adenoma. The odds ratios and 95% CIs across low to high quintile of n-3 intake were 1.0 (reference), 0.70 (0.50-0.97), 0.71 (0.50-1.01), 0.67 (0.46-0.97), 0.54 (0.36-0.83); P_trend = 0.02. Furthermore, we conducted analyses using the baseline diet (the 1980 questionnaire) but results did not differ. Similarly, after excluding the participants who had an endoscopic procedure for unrelated gastrointestinal conditions such as abdominal pain, diarrhea, and constipation, the associations that were shown with marine n-3 fatty acids or n-3/n-6 ratio and risk of distal colorectal adenoma did not change appreciably (data not shown).

We also found no appreciable associations between dietary marine n-3 fatty acids and risks of distal colon, or rectal adenoma when analyzed separately. To investigate whether there was an association with even higher intake of marine n-3 fatty acids, we categorized this variable into deciles, but no clear relations between these dietary variables and risks of adenoma were observed [RR of adenoma for the highest (>0.18% of energy intake) versus the lowest deciles (≤0.03% of energy intake) of dietary marine n-3 fatty acids = 1.14; 95% CI, 0.87-1.51; P_trend = 0.37; RR of large adenoma for the highest versus the lowest deciles of dietary marine n-3 fatty acids = 0.92; 95% CI, 0.60-1.43; P_trend = 0.23]. In addition, we found that dietary EPA or DHA had no apparent relation to risk of adenoma when analyzed separately (RR for the highest versus the lowest quintiles of dietary EPA = 1.02; 95% CI, 0.85-1.22; P_trend = 0.61; RR for the highest versus the lowest quintiles of dietary DHA = 1.08; 95% CI, 0.89-1.32; P_trend = 0.23 in multivariable analyses).

As shown in Table 3, the relations between n-3/n-6 ratio and risk of adenoma were similar to those for dietary marine n-3 fatty acids (RR for the highest versus the lowest quintiles of n-3/n-6 ratio = 1.02; 95% CI, 0.83-1.25; P_trend = 0.86 in multivariable). In addition, no significant associations were found for large or small adenoma, distal colon, or rectal adenoma.

Furthermore, we examined whether associations for dietary marine n-3 fatty acids were modified by intakes of α-linolenic acid (P_interaction for distal colorectal adenoma = 0.26; P_interaction for large adenoma = 0.95) and total n-6 fatty acids (P_interaction for distal colorectal adenoma = 0.16; P_interaction for large adenoma = 0.75). The null relations between dietary marine n-3 fatty acids and risk of distal colorectal adenoma and large distal colorectal adenoma were consistent across strata of intakes of α-linolenic acid and total n-6 fatty acids (Table 4). Similarly, dietary marine n-3 fatty acids were unrelated with risk of small adenoma, distal colon, or rectal adenoma across the strata of α-linolenic acid and total n-6 fatty acids (data not shown).

Regular aspirin use was inversely associated with risk of colorectal adenoma in this cohort (27). For the analyses evaluating whether aspirin use modified the association with dietary marine n-3 fatty acids (P_interaction for distal colorectal adenoma = 0.44; P_interaction for large adenoma = 0.67), dietary marine n-3 fatty acids had no clear relation with distal colorectal adenoma regardless of aspirin use (Table 5). No apparent relations between n-3/n-6 ratio and risks of distal colorectal adenoma were also found across these strata (data not shown).

In this large prospective study, higher intake of marine n-3 fatty acids was not associated with risk of distal colorectal adenoma. In addition, no significant associations were observed between this dietary variable and risks of small adenoma, distal colon adenoma, and rectal adenoma, although a nonsignificant, suggestive, inverse association was noted for large adenoma. Similarly, we found no significant associations between n-3/n-6 ratio and risks of distal colorectal adenoma.

We did find a suggestively decreased risk of large adenoma associated with higher dietary marine n-3 fatty acid intake (RR, 0.74; 95% CI, 0.54-1.01). Interestingly, we also noted that high n-3 intake was associated with a nonsignificant increase in risk of small adenoma. We thus conducted a post hoc analysis and found that among women with a diagnosis of adenoma, those with high dietary intake of marine n-3 fatty acids had a 46% lower odds of having a large adenoma versus a small adenoma. In combination, these results are suggestive that higher dietary intake of n-3 fatty acids may be associated with a reduction in the progression of adenomas. This interpretation should be viewed cautiously, and these findings need to be replicated. Nonetheless, given the importance of adenomas growth for cancer development, this possibility warrants further study.

The epidemiologic literature on dietary marine n-3 fatty acids and colorectal neoplasia is relatively sparse. Intake of marine n-3 fatty acids was not associated with risk of colorectal cancer in a cohort of Finnish men (16), and dietary EPA, DHA, total n-3 fatty acids, the marine n-3 to linoleic acid ratio, and total n-3/total n-6 ratio were not associated with risk of colorectal cancer in a cohort of Swedish women (15). Some inconsistent results were reported in case-control studies; dietary EPA, total n-3 fatty acids, and total n-3/total n-6 ratio were not significantly associated with risk of colon cancer in a U.S. study (17), and dietary total n-3 fatty acids were inversely related to risk of colorectal cancer, whereas the ratio of total n-6 to total n-3 fatty acids was positively related to risk of colorectal cancer in French Canadians, although dietary EPA or DHA was not statistically significantly related with risk (18). In the only case-control study to examine the relation between marine n-3 fatty acids and adenoma risk (19), the adenoma risk showed inverse trends with marine n-3 fatty acids in adipose tissue, although this association did not attain statistical significance (P_trend = 0.07); in addition, total n-3/total n-6 ratio in adipose tissue was not associated with risk. Furthermore, dietary marine n-3 fatty acids, total n-3 fatty acids, and total n-3/total n-6 ratio were not associated with risk of colorectal adenoma, consistent with the results of our study.

Regarding fish intake (the main source of marine n-3 fatty acids), three cohort studies did not find associations with risk of colorectal cancer (28-30), but in one cohort study (31), intake of fish and shellfish was inversely associated with risk of colorectal cancer. The results of case-control studies were also inconsistent; no significant associations between fish intake and risks of colorectal adenoma and cancer were observed in several studies (32-34), whereas one study showed an inverse relation to colorectal cancer (35). Recently, in Asian populations, salted fish intake was positively associated with risk of colon cancer (36), whereas raw and cooked fish intake was inversely associated with risk of colon cancer (37). Differences in these studies may be due to differences in actual intake and cooking or preservation process in the study populations, and the variables adjusted for analyses. Sasaki et al. (38) reported fish fat intake as percentage of energy intake was different between countries; 1.70% in Japan, 0.46% in Spain, 0.33% in France, 0.24% in Italy, and 0.20% in the United States and the Netherlands; thus, fatty fish intake in U.S. or European countries is much lower than that in Japan. A cohort study suggested that higher intake of N-nitrosodimethylamine, an initiator of carcinogenesis, increased risk of colorectal cancer, and that the increased risk of colorectal cancer was related to intake of smoked and salted fish, the main source of nitrosamine compounds, but not to intake of fresh unsalted fish (39). In addition, because most of these studies (28-36), except for one study (37) did not focus only on fish intake as main dietary exposure, other related dietary and nondietary variables were not well controlled; of the 10 studies, five studies adjusted for energy intake (30-32, 35, 36), only two studies adjusted for fiber (30) or vegetable and meat intake (37), and only one study adjusted for smoking (37).

Interest in marine n-3 fatty acids and colorectal cancer stems from their ability to inhibit cyclooxygenase 2 activity and arachidonic acid metabolism, thereby reducing cell proliferation or inducing apoptosis in the colonic mucosa (1, 3-7). Lower cyclooxygenase 2 expression is related to reduced risk of colorectal adenoma and cancer in humans (40-42) and to higher survival rate in patients with colorectal cancer (43). Cheng et al. (13) suggested higher intake of n-3 fatty acids elevates the apoptotic rate of normal colon mucosa in patients polypectomized for adenomas or cancers but does not influence cell proliferation. However, other clinical trials showed n-3 fatty acids supplementation with high doses (2.5 and 7.7 g/d) reduces cell proliferation in the rectal mucosa in patients with sporadic colonic adenoma (12, 14). In addition, animal studies show that marine n-3 fatty acids may be related to colon carcinogenesis by reducing cell proliferation and increasing apoptosis of mucosal crypt cells (9, 44). However, dietary marine n-3 fatty acids up to ∼1.1 g/d were not associated with risk of distal colorectal adenoma in our study, but we cannot rule out an effect at pharmacologic doses.

The effects of n-3 fatty acids may be inhibited by high intake of linoleic acid because of the competition for the desaturation and chain elongation pathway (21, 45), although Pischon et al. (46) reported that total n-6 fatty acids did not inhibit the anti-inflammatory effects of n-3 fatty acids. In a European Community Multicenter Study on Antioxidants, Myocardial infarction, and Cancer breast cancer study (47), n-3/n-6 ratio in adipose tissue showed a modest inverse associations with risk of breast cancer, suggesting that the ratio may be important for breast cancer risk. In addition, because the conversion of α-linolenic acid to long-chain n-3 fatty acids is more efficient when intake of marine n-3 fatty acids is low (21), the influence of low marine n-3 fatty acid intake on adenoma risk may differ by level of α-linolenic acid intake. However, when stratified by intakes of total n-6 fatty acids and α-linolenic acid, dietary marine n-3 fatty acids as well as n-3/n-6 ratio did not seem to reduce risk of distal colorectal adenoma. The inhibition of marine n-3 fatty acids in cancer cell growth through increased lipid peroxidation products is antagonized by antioxidants (48); however, little relation between dietary marine n-3 fatty acids and risk of distal colorectal adenoma was found across strata of β-carotene, vitamin C, and vitamin E (data not shown).

Regular aspirin use reduces the risks of colorectal adenoma and cancer in healthy men and women (27, 49), and large clinical trials (50-52) also reported inverse relations with risk of colorectal adenoma among patients with a history of colorectal adenomas or cancers. One of the possible mechanisms of aspirin's effect is through inhibition of cyclooxygenase 2 activity, which is one of the mechanisms proposed for marine n-3 fatty acids. However, we observed no clear relations between dietary marine n-3 fatty acids and n-3/n-6 ratio with risks of distal colorectal adenoma even among nonusers of aspirin.

This study included 1,719 cases of distal colorectal adenoma diagnosed during 18 years of follow-up. Diets were assessed prospectively by repeated validated questionnaires that take into account possible changes in diet over time, reduce random variation, and were not influenced by knowledge of the existence of colorectal adenoma. Dietary marine ω-3 fatty acids were correlated (r = 0.49) with those in adipose tissue in a similar study of male health professionals using a similar food frequency questionnaire (26), suggesting our food frequency questionnaire provided a reasonable measure of marine -3 fatty acids. In addition, the multiple assessments of diet over time may further decrease misclassification. Additionally, supporting our ability to detect associations with marine n-3 fatty acids is that we have observed predicted inverse associations with cardiovascular disease in this cohort (25, 53, 54). However, it is possible that some inevitable errors in dietary assessment would tend to underestimate the magnitude of associations, and if the relationship for adenoma or cancer is considerably weaker than that for cardiovascular disease, we could have missed a modest association. In addition, we cannot rule out the possibility that the dietary information collected did not correspond to the etiologically relevant time for adenoma development. Many known or suspected risk factors for colorectal adenoma and colon cancer were also controlled although the possibility of residual confounding by unknown risk factors could not be excluded. However, as higher fish consumption tended to be positively related with other healthful behaviors, we would expect residual confounding would more likely make fish intake seem protective. Another limitation is that although we analyzed only adenomas of the distal colorectum to prevent misclassification and potential detection bias, we cannot rule out the possibility that some probable misclassification may influence the associations because sigmoidoscopic examination may not cover completely the whole descending colon. Finally, only a small proportion of adenoma may progress to colorectal cancer and possibly relationships with marine n-3 intake could be stronger for a small subset of adenomas predisposed to progress to cancer. Further studies are warranted to examine the potential influences of marine n-3 fatty acids on adenoma risk of the proximal colon and on later stages of carcinogenesis.

In summary, higher intake of marine n-3 fatty acids and higher n-3/n-6 ratio, at least within the ranges as consumed in the United States, may not reduce the risk of distal colorectal adenoma in women. However, the suggestion that higher intake of marine n-3 fatty acids may reduce the progression from small adenoma to large adenoma warrants further study.

We thank the contributions of Gary Chase, Karen Corsano, Lisa Dunn, Barbara Egan, and Mary Louie for their expert help.