NUTRITION AND CANCER, 56(1), 11 21 Copyright 2006, Lawrence Erlbaum Associates, Inc. Colorectal Cancer Protective Effects and the Dietary Micronutrients Folate, Methionine, Vitamins B6, B12, C, E, Selenium, and Lycopene Gabriel Kune and Lyndsey Watson Abstract: The data reported here were obtained from the case-control arm of a large, comprehensive, populationbased investigation of colorectal cancer incidence, etiology, and survival, the Melbourne Colorectal Cancer Study, conducted in Melbourne, Australia. This part of the case-control study was designed to identify dietary factors associated with colorectal cancer risk in 715 incident cases compared with 727 age/sex frequency matched randomly chosen community controls, in which a quantitative assessment of all foods eaten was made. New data are presented on the potential of two groups of micronutrients as protective agents, namely, those involved in DNA methylation, synthesis, and repair (folate, methionine, and vitamins B6 and B12) and those with antioxidant properties (selenium, vitamins E and C, and lycopene). The adjusted odds ratios showed that for folate there was significant protection for rectal cancer in second and third quintiles of consumption but not for colon cancer, and this was similar for methionine consumption. Vitamin B6 consumption was significantly protective for both colon and rectal cancer at the higher quintiles, and this was similar for vitamin B12. Dietary selenium was significantly protective at middle quintiles of consumption at both cancer sites. Dietary vitamins E and C were statistically significantly protective for both colon and rectal cancer at all levels of consumption, and for both vitamins there was a dose-response effect of increasing protection, particularly so for colon cancer. Lycopene was not associated with colorectal cancer risk. A combined model included vitamins E, C, and B12 and selenium as micronutrients protective for colorectal cancer and folate, which, however, showed an increased risk at the highest level of consumption. These data support the proposition that a diet containing the dietary micronutrients involved in DNA methylation (folate, methionine, and vitamins B6 and B12) and some of those with antioxidant properties (selenium and vitamins E and C) may have a role to play in lowering colorectal cancer risk and also that such protection can be achieved by dietary means alone. Introduction Colorectal cancer remains a major global health problem, and it can be estimated that during 2006 there will be at least one million new cases of colorectal cancer diagnosed globally and that mortality from this cancer in 2005 will be approximately 500,000 (1 3). Diet has been the most studied etiological association of colorectal cancer, with an attributable risk of approximately 50% in high-risk countries for colorectal cancer, such as the United States, the United Kingdom, and Australia (4). Dietary preventive strategies are likely to be of importance in lowering colorectal cancer incidence and mortality. This article presents new data on the potential of several dietary micronutrients, namely, folate, methionine, vitamin B6, vitamin B12, vitamins C and E, selenium, and lycopene, to act as colorectal cancer chemopreventive agents. The Melbourne Colorectal Cancer Study The data presented here are from the Melbourne Colorectal Cancer Study, a large, comprehensive, population-based investigation of colorectal cancer incidence, etiology and survival, in a single data set, making this a unique study design (5,6). The data for this publication are derived from the etiological arm of the study. This part of the Melbourne study examined all of the then-hypothesized risks and protective factors for colorectal cancer, such as a detailed examination of previous diet, family history of colorectal cancer, smoking, alcohol consumption, previous colorectal polyps, reproductive and hormonal factors, chronic illnesses, operations and medication use, bowel habit, laxative use, and several other factors. The etiological arm was a case-control study comprising 715 histologically proven incident cases of colorectal cancer diagnosed within a 12-mo period during 1980 and G. Kune is affiliated with the Faculty of Medicine, Dentistry and Health Sciences of the University of Melbourne, Australia. L. Watson is affiliated with Mother and Child Health Research, La Trobe University, Victoria, Australia.
1981, frequency matched by age and sex with 727 randomly chosen community controls, all from metropolitan Melbourne, Australia, which at the time of the study had a population of 2.8 million. The results derived from all three arms of the Melbourne Colorectal Cancer Study have been extensively published in peer-reviewed journals (7 22). The dietary and alcohol findings were published in an issue of Nutrition and Cancer devoted entirely to the Melbourne study (19 22). The main dietary findings of relevance for this investigation were a statistically significant protective effect present for a high consumption of vegetables, a high intake of vitamin C containing foods, and a high consumption of fish (20). Since the publication of these data, new hypotheses have emerged regarding dietary factors in colorectal cancer etiology, especially regarding the possible protective effects of the micronutrients folate, methionine, vitamin B6, vitamin B12, selenium, vitamin E, and lycopene. These micronutrients were not available in food composition tables at the time of the original analyses; hence, this investigation. The micronutrients examined belong to one of two generic groups, nutrients involved in one-carbon metabolism or methylation processes (folate, methionine, and vitamins B6 and B12) or those with antioxidant properties (selenium, vitamins E and C, and lycopene). Methods Dietary Methodology The dietary data were obtained through personal interviews by university-qualified nutritionists who also held a science degree and who were specially trained to administer the questionnaire. The questionnaire involved a quantitative estimation of the usual diet of all foods eaten during the study period, and this amounted to 587 food items. Extensive measures were taken to assess the reliability and validity of the study design and the diet history method, and assessments were made regarding the presence of the major potential biases inherent in case-control studies, such as selection bias, recall bias, between-interviewer variation, reproducibility, within-interview variation over time, and interviewer bias regarding the dietary causes of colorectal cancer (20,22). It was concluded that the nutritional data were reliable and valid and that, with some minor reservations, no substantial bias was present in the dietary data (20,22). Moreover, indirect validation of the nutritional data in the study by comparison with other studies, by national per capita consumption and by a comparison of energy intake with estimated energy requirements based on height, weight, and activity levels, suggested that the dietary data were not an overestimate of intake and that cases and controls were both overestimating and underestimating their dietary intakes to a similar degree (22). Using food tables, the quantity of each food item consumed was determined in each subject and also translated into food groups and nutrients (20). The primary dietary data were recovered for the present study, and the United States Department of Agriculture (USDA) food tables were used to estimate the consumption of the several micronutrients under investigation (23). When food retention factors were available, they were included. Therefore, folate; vitamins B12, B6, and C; and lycopene were adjusted by retention factors. Statistical Analysis Quintiles of consumption of micronutrients for cases and controls combined were determined, and study subjects were categorized into appropriate quintiles. Logistic regression analysis to estimate the association between the level of consumption and case-control status was undertaken using Stata statistical software (Stata Corp., College Station, TX) (24). Subgroup analyses were undertaken for colon and rectal cancer separately comparing with controls. All models were adjusted for the design constraints, namely, age group and sex (termed unadjusted in the tables). Moreover, adjustment was also made for alcohol consumption (yes/no), body mass index (quintiles), energy intake (quintiles), family history of colorectal cancer (yes/no), oral contraceptive pill use (yes/no), cigarette pack-years (none, 1 19, 20 39, or 40), aspirin use (yes/no), and nonaspirin nonsteroidal antiinflammatory drug use (yes/no), and this was termed as adjusted in the tables. Effects are presented as odds ratios (ORs), 95% confidence intervals (95% CIs), with symbols for levels of significance. Finally, a data-driven model combining methylating and antioxidant micronutrients was developed by forward stepwise regression. Variables were included if they contributed more than 9.50 [P(χ 2 4) = 0.95] in quintiles or 3.84 [P(χ 2 1)= 0.95] if linear to the χ 2 goodness-of-fit statistic. At the completion of this process all eliminated variables were added individually to see that they remained nonsignificant, and all included variables were excluded individually to see that their effect remained significant. Results Quintiles for daily consumption of the micronutrients examined and the distribution for controls and cases and the distribution by cancer subgroup into colon and rectum are shown in Table 1. Folate The adjusted intake of folate in quintile 3 compared with quintile 1 approached a statistically significant protective effect for colorectal cancer; however, no other statistically significant effects were noted (Table 2). 12 Nutrition and Cancer 2006
Table 1. Upper Levels of Intake per Quintile of Micronutrients and Number and Percentage Distribution by Controls and Cases for the Melbourne Colorectal Cancer Study Cancer Cases Controls Colorectal Colon Rectal Quintile Upper Level of Intake/Day of Quintile N % N % N % N % Total 727 715 392 323 Folate µg/day 1 246 133 18 156 22 79 20 77 24 2 297 152 21 136 19 81 21 55 17 3 347 159 22 130 18 84 21 46 14 4 419 158 22 130 18 70 18 60 19 5 1,367 125 17 163 23 78 20 85 26 Methionine g/day 1 1.3 144 20 173 24 95 24 78 24 2 1.6 182 25 157 22 92 23 65 20 3 1.9 157 22 129 18 79 20 50 15 4 2.3 136 19 120 17 63 16 57 18 5 8.9 108 15 136 19 63 16 73 23 Vitamin B6 mg/day 1 1.7 128 18 167 23 94 24 73 23 2 2.1 151 21 139 19 81 21 58 18 3 2.6 142 20 142 20 87 22 55 17 4 3.4 156 21 129 18 66 17 63 20 5 402.6 150 21 138 19 64 16 74 23 Vitamin B12 µg/day 1 4.1 133 18 158 22 94 24 64 20 2 5.7 125 17 163 23 85 22 78 24 3 7.6 145 20 142 20 81 21 61 19 4 11.1 152 21 136 19 74 19 62 19 5 1,007.9 172 24 116 16 58 15 58 18 Selenium µg/day 1 80 123 17 166 23 94 24 72 22 2 99 147 20 144 20 87 22 57 18 3 118 157 22 131 18 74 19 57 18 4 145 164 23 122 17 69 18 53 16 5 530 136 19 152 21 68 17 84 26 Vitamin E mg/day 1 5.1 121 17 191 27 110 28 81 25 2 7.1 138 19 133 19 75 19 58 18 3 9.3 155 21 128 18 78 20 50 15 4 12.7 156 21 133 19 68 17 65 20 5 1,006.0 157 22 130 18 61 16 69 21 Vitamin C mg/day 1 69.4 117 16 172 24 95 24 77 24 2 103.6 144 20 144 20 80 20 64 20 3 141.2 144 20 145 20 74 19 71 22 4 197.8 154 21 134 19 75 19 59 18 5 1,863.0 168 23 120 17 68 17 52 16 Lycopene µg/day 1 280 142 20 147 21 77 20 70 22 2 706 138 19 150 21 88 22 62 19 3 1,336 144 20 145 20 80 20 65 20 4 2,731 149 20 139 19 74 19 65 20 5 44,690 154 21 134 19 73 19 61 19 Vol. 56, No. 1 13
14 Nutrition and Cancer 2006 Table 2. Odds Ratios and 95% Confidence Intervals and Levels of Significance of Colorectal, Colon, and Rectal Cancers for Micronutrients in the Melbourne Colorectal Cancer Study a Colorectal Cancer Colon Cancer Rectal Cancer Unadjusted b Adjusted c Unadjusted b Adjusted c Unadjusted b Adjusted c Quintile OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) Folate 2 0.76 (0.54 1.06) 0.76 (0.53 1.08) 0.91 (0.61 1.34) 0.89 (0.59 1.35) 0.62** (0.40 0.93) 0.60** (0.39 0.93) 3 0.70** (0.50 0.98) 0.71* (0.50 1.02) 0.90 (0.60 1.33) 0.91 (0.60 1.38) 0.50*** (0.32 0.77) 0.50*** (0.31 0.79) 4 0.72* (0.51 1.01) 0.75 (0.51 1.10) 0.81 (0.53 1.22) 0.85 (0.54 1.34) 0.63** (0.41 0.96) 0.63* (0.39 1.02) 5 1.15 (0.81 1.63) 1.24 (0.81 1.89) 1.17 (0.76 1.79) 1.31 (0.79 2.16) 1.11 (0.72 1.70) 1.10 (0.65 1.85) Methionine 2 0.72** (0.52 0.98) 0.75* (0.53 1.05) 0.79 (0.54 1.13) 0.81 (0.55 1.21) 0.63** (0.42 0.95) 0.67* (0.43 1.03) 3 0.68** (0.48 0.95) 0.73 (0.50 1.06) 0.78 (0.52 1.15) 0.83 (0.54 1.30) 0.56*** (0.36 0.87) 0.61** (0.38 0.99) 4 0.74 (0.51 1.05) 0.84 (0.55 1.27) 0.76 (0.49 1.16) 0.83 (0.51 1.38) 0.71 (0.45 1.12) 0.83 (0.49 1.41) 5 1.07 (0.73 1.55) 1.22 (0.75 1.97) 1.01 (0.64 1.58) 1.20 (0.67 2.13) 1.11 (0.70 1.75) 1.23 (0.68 2.21) Vitamin B6 2 0.68** (0.48 0.95) 0.65** (0.45 0.93) 0.73 (0.49 1.07) 0.68 * (0.45 1.03) 0.63** (0.40 0.96) 0.60** (0.38 0.96) 3 0.74* (0.52 1.04) 0.69* (0.48 1.01) 0.84 (0.56 1.24) 0.78 (0.50 1.20) 0.62** (0.39 0.96) 0.60** (0.37 0.96) 4 0.60*** (0.42 0.84) 0.54*** (0.36 0.80) 0.57*** (0.37 0.86) 0.51*** (0.32 0.81) 0.63** (0.40 0.97) 0.57** (0.34 0.93) 5 0.66** (0.46 0.94) 0.52*** (0.34 0.80) 0.60** (0.38 0.91) 0.47*** (0.28 0.77) 0.73 (0.46 1.14) 0.57** (0.34 0.96) Vitamin B12 2 1.08 (0.76 1.50) 1.08 (0.76 1.53) 0.97 (0.65 1.42) 0.96 (0.64 1.43) 1.23 (0.80 1.88) 1.24 (0.81 1.92) 3 0.80 (0.56 1.11) 0.78 (0.54 1.11) 0.80 (0.54 1.18) 0.76 (0.50 1.15) 0.80 (0.51 1.23) 0.78 (0.50 1.24) 4 0.72* (0.51 1.01) 0.71* (0.49 1.01) 0.69* (0.46 1.02) 0.68* (0.45 1.04) 0.77 (0.49 1.18) 0.73 (0.50 1.24) 5 0.54 (0.37 0.76) 0.49 (0.34 0.71) 0.49 (0.32 0.74) 0.45 (0.29 0.71) 0.60** (0.38 0.94) 0.53*** (0.33 0.86)
Vol. 56, No. 1 15 Selenium 2 0.70** (0.50 0.98) 0.68** (0.47 0.97) 0.76 (0.51 1.11) 0.71 (0.47 1.08) 0.63** (0.41 0.97) 0.61** (0.39 0.97) 3 0.59*** (0.42 0.83) 0.54*** (0.37 0.79) 0.61** (0.40 0.90) 0.53*** (0.34 0.82) 0.57*** (0.36 0.88) 0.53** (0.33 0.86) 4 0.51 (0.35 0.73) 0.47 (0.31 0.71) 0.55*** (0.35 0.83) 0.48*** (0.29 0.79) 0.48*** (0.30 0.75) 0.44*** (0.26 0.76) 5 0.78 (0.54 1.12) 0.66 * (0.42 1.04) 0.68* (0.43 1.05) 0.57* (0.33 0.97) 0.89 (0.56 1.39) 0.76 (0.43 1.33) Vitamin E 2 0.60*** (0.42 0.83) 0.53 (0.37 0.75) 0.61*** (0.41 0.89) 0.54*** (0.36 0.81) 0.59*** (0.38 0.89) 0.51*** (0.33 0.80) 3 0.51 (0.36 0.71) 0.44 (0.31 0.62) 0.56*** (0.38 0.81) 0.47 (0.31 0.70) 0.45 (0.29 0.69) 0.39 (0.25 0.61) 4 0.53 (0.38 0.74) 0.47 (0.33 0.67) 0.48 (0.32 0.71) 0.4 (0.27 0.62) 0.60** (0.39 0.91) 0.55*** (0.35 0.84) 5 0.51 (0.36 0.71) 0.41 (0.29 0.59) 0.43 (0.28 0.64) 0.34 (0.22 0.52) 0.61** (0.40 0.92) 0.51*** (0.33 0.79) Vitamin C 2 0.65*** (0.46 0.91) 0.66** (0.47 0.93) 0.66** (0.45 0.98) 0.65** (0.44 0.98) 0.67* (0.44 1.01) 0.67* (0.43 1.03) 3 0.61*** (0.43 0.86) 0.61*** (0.43 0.86) 0.61*** (0.41 0.90) 0.53*** (0.35 0.80) 0.76 (0.50 1.15) 0.70 (0.46 1.08) 4 0.53 (0.37 0.75) 0.55*** (0.39 0.79) 0.58*** (0.39 0.85) 0.54*** (0.36 0.81) 0.58*** (0.37 0.88) 0.56*** (0.36 0.87) 5 0.39 (0.27 0.56) 0.39 (0.27 0.57) 0.49 (0.32 0.72) 0.39 (0.25 0.59) 0.47 (0.30 0.72) 0.40 (0.25 0.63) Lycopene 2 1.05 (0.75 1.45) 1.03 (0.73 1.44) 1.19 (0.80 1.75) 1.16 (0.77 1.72) 0.89 (0.58 1.35) 0.88 (0.58 1.35) 3 0.97 (0.69 1.34) 1.00 (0.71 1.40) 1.00 (0.67 1.48) 1.05 (0.70 1.56) 0.93 (0.61 1.41) 0.95 (0.62 1.45) 4 0.89 (0.64 1.24) 0.89 (0.63 1.26) 0.92 (0.61 1.36) 0.92 (0.61 1.39) 0.86 (0.57 1.30) 0.85 (0.56 1.30) 5 0.83 (0.59 1.15) 0.86 (0.61 1.21) 0.88 (0.59 1.30) 0.92 (0.61 1.39) 0.78 (0.51 1.17) 0.81 (0.53 1.24) a: Abbreviations are as follows: OR, odds ratio; CI, confidence interval. Levels of significance: (marginal) *P < 0.10; **P 0.05; ***P 0.01; P 0.001. b: Adjusted for age and sex. c: Adjusted for age and sex, alcohol (yes/no), body mass index (quintiles), energy intake (quintiles), family history of colorectal cancer (yes/no), oral contraceptive pill use (yes/no), cigarette pack-years (none, 1 19, 20 39, or 40), aspirin use (yes/no), and nonaspirin nonsteroidal antiinflammatory drug use (yes/no).
When the quintile ORs were estimated for colon and rectal cancer separately, a statistically significant protective effect was present for quintiles 2 and 3 and more marginally significant for quintile 4 for rectal cancer subjects; however, no significant effects were noted for colon cancer (Table 2). Methionine The adjusted findings for methionine mirrored those for folate, with a protective effect approaching statistical significance for colorectal cancer in quintile 2 and a significant protective effect present in quintile 3 for rectal cancer and approaching statistical significance in quintile 2; however, there were no significant effects for colon cancer (Table 2). Vitamin B6 Consumers of vitamin B6 containing foods were protected at all levels of consumption above quintile 1 for colorectal cancer in the adjusted model (Table 2). The effect was similar for colon and rectal cancer but statistically significant at lower levels of intake for rectal cancer compared with colon cancer (Table 2). Vitamin B12 For colorectal cancer in the adjusted model, there was a dose-response of protection with increasing levels of consumption of vitamin B12 containing foods, and this became almost statistically significant in quintile 4 and highly significant in the quintile 5 (Table 2). The vitamin B12 effect was similar when cases were divided into colon and rectum but was stronger for colon cancer (Table 2). Selenium Statistically significant protective effects were noted in the adjusted data for quintiles 2, 3, and 4 and approaching significance for quintile 5 for colorectal cancer, and these effects were similar for colon and rectal cancer (Table 2). Vitamin E Statistically highly significant protective effects were noted for all quintiles of vitamin E consumption for colorectal cancer, colon cancer, and rectal cancer (Table 2). Vitamin C In the adjusted model, statistically significant protective effects were noted for all levels of consumption of vitamin C containing foods for colorectal cancer and colon cancer and for quintiles 4 and 5 for rectal cancer (Table 2). A clear dose-response protective effect was noted for increasing consumption of vitamin C containing foods for colorectal cancer and colon cancer and, with the exception of quintile 3, for rectal cancer also (Table 2). Lycopene In all quintiles of consumption for colorectal cancer, colon cancer, and rectal cancer, the ORs were close to 1, and no statistically significant effects were recorded (Table 2). Combined Model The model developed using forward stepwise regression accounting for mutually competing factors is shown in Table 3, ordered according to their contribution to the model. Vitamin E (in quintiles) offered significant protection at all levels of consumption above quintile 1, vitamin C (linear) and vitamin B12 (linear) offered increasing protection with increasing consumption, and selenium was protective at quintile 4 only. On the other hand, folate (in quintiles) showed a significantly elevated risk at quintile 5. The model was consistent for both colon and rectal cancer. Methionine was correlated with folate and vitamin B6 with B12; hence, neither remained in the model. Discussion This article focuses on the possible role of several dietary micronutrients as chemoprotective agents in colorectal cancer based on data derived from the dietary part of the case-control arm of the Melbourne Colorectal Cancer Study. A strength of the present data is that extensive precautions were taken and checks made to assess major biases inherent in case-control studies, in particular, selection bias, recall bias, interviewer variation and bias, and dietary underreporting and overreporting, and no substantial biases were detected (20,22,25). Of particular relevance is that both cases and controls underreported and overreported their diet intakes to a similar degree, so this factor is unlikely to have been responsible for significant case-control differences (22,25). Up to the present, few studies have reported quantitative dietary micronutrient intake data as opposed to qualitative or so-called semiquantitative data in relation to colorectal cancer risk. Most case-control studies and all cohort studies up to date have reported only on food-frequency questionnaires. It is appreciated that, in cohort studies of diet and cancer, because of the large number of subjects needed, cost constraints limit the food-frequency questionnaire to relatively few items. For example, the Nurses Health Study initially relied on an examination of only 61 food items, which included only 6 fruits and 11 vegetables (26). Therefore, a further strength of the present data is that they involve a wholly quantitative estimation of all foods eaten, a total of 587 food items (20,25). Many previous micronutrient data in relation to colorectal cancer risk involve nutritional supplements rather than dietary components. In these Melbourne data, 10 respondents were identified as taking multivitamin supplements, which at that time contained vitamins B12 and B6 but did not contain 16 Nutrition and Cancer 2006
Table 3. Odds Ratios and 95% Confidence Intervals and Levels of Significance for the Combined Model of Colorectal, Colon, and Rectal Cancers and Micronutrients for the Melbourne Colorectal Cancer Study a Colorectal Cancer Colon Cancer Rectal Cancer Adjusted b Adjusted b Adjusted b Quintile OR (95% CI) OR (95% CI) OR (95% CI) Vitamin E 1 1.00 1.00 1.00 2 0.56*** (0.39 0.80) 0.58*** (0.39 0.88) 0.54*** (0.35 0.85) 3 0.50 (0.35 0.72) 0.53*** (0.35 0.81) 0.45 (0.28 0.71) 4 0.55*** (0.38 0.78) 0.48 (0.31 0.73) 0.64** (0.41 1.00) 5 0.48 (0.33 0.69) 0.39 (0.25 0.61) 0.59** (0.37 0.93) Vitamin C Linear c 0.82 (0.75 0.89) 0.80 (0.72 0.89) 0.83 (0.74 0.93) Vitamin B12 Linear d 0.87*** (0.79 0.95) 0.87** (0.78 0.98) 0.86** (0.76 0.97) Selenium 1 1.00 1.00 1.00 2 0.78 (0.53 1.14) 0.79 (0.51 1.22) 0.74 (0.46 1.21) 3 0.63** (0.42 0.96) 0.58** (0.36 0.95) 0.68 (0.40 1.15) 4 0.58** (0.36 0.93) 0.56** (0.32 0.97) 0.59* (0.32 1.09) 5 0.85 (0.50 1.44) 0.70 (0.38 1.31) 1.03 (0.53 2.00) Folate 1 1.00 1.00 1.00 2 1.03 (0.71 1.50) 1.25 (0.81 1.93) 0.80 (0.50 1.27) 3 1.14 (0.77 1.69) 1.53* (0.96 2.43) 0.75 (0.45 1.24) 4 1.35 (0.88 2.07) 1.65* (0.99 2.76) 1.04 (0.61 1.77) 5 2.27 (1.39 3.69) 2.67 (1.50 4.76) 1.81** (1.00 3.27) a: Abbreviations are as follows: OR, odds ratio; CI, confidence interval. Nutrients appear in the decreasing order of variance contribution to the model. Levels of significance: (marginal) *P < 0.10; **P 0.05; ***P 0.01; P 0.001. b: Adjusted for age and sex, alcohol (yes/no), body mass index (quintiles), energy intake (quintiles), family history (yes/no), oral contraceptive pill use (yes/no), cigarette pack-years (none, 1 19, 20 39, or 40), aspirin use (yes/no), and nonaspirin nonsteroidal antiinflammatory drug use (yes/no). c: The linear effect for colorectal cancer and vitamin C model gives ORs for quintiles (q) as (OR) q 1, that is, ORs of 0.82, 0.67, 0.55, and 0.45, respectively. d: The linear effect for colorectal cancer and vitamin B12 model gives ORs for quintiles (q) as (OR) q 1, that is, ORs of 0.87, 0.76, 0.66, and 0.57, respectively. folate, methionine, or selenium. Their exclusion did not influence the magnitude of the findings (data not shown). At that time also, folate fortification of foods had not yet been introduced in Australia. It is recognized that the micronutrients presented here may be closely correlated in foods with others, such as vitamin B6 foods correlated with B12 foods, and possibly also with other compounds not yet identified but which may be associated with colorectal cancer risk. This hazard cannot be completely eliminated and in the final analysis needs to be assessed in concert with other studies, with animal experimental data and studies that demonstrate biologically plausible mechanisms of action of the nutrient under investigation. Vitamin C and beta-carotene containing foods have been previously reported in the Melbourne study showing that a high intake of vitamin C containing foods was an independent protective factor, whereas beta-carotene containing foods had no association with colorectal cancer risk (20). Micronutrients Involved in DNA Methylation and Synthesis Folate: Data gleaned from recent reviews suggest from most, but not all, studies that in humans there is an inverse relationship between folate intake (both dietary and supplemental) and colorectal cancer and adenoma risk, that similar effects are present in animal models, and that there are plausible biological mechanisms that explain this folate protection for colorectal tumors (27 29). The present data add partial support to the conclusions from the accumulated evidence quoted previously. However, folate protection was not dose dependent in the present data but rather occurred in the middle three quintiles of consumption, producing a U-shaped curve, and was statistically significant for rectal cancer only, and in the same direction as rectal cancer, but not statistically significant for colon cancer (Table 2). Although the highest quintile of folate consumption in this study showed only a very slight and statistically nonsignificant risk elevation when it alone was modeled, this reached statistical significance in the combined model (Table 3). These appear to be the first human data showing a statistically highly significant risk of colorectal cancer for high dietary folate intakes of between 419 and 1,367 µg/day. Therefore, the data can be considered consistent with the recent cautionary suggestions, derived from minimal human data and from genetically predisposed rodent models of colorectal cancer, that under certain circumstances a very high folate intake may in fact promote rather than suppress colorectal Vol. 56, No. 1 17
carcinogenesis, especially in the presence of already existing premalignant or malignant colorectal epithelial cells (29 31). We do not have a mechanistic explanation for these folate findings of risk in the combined model nor can we explain why these findings are confined to folate and not to the other micronutrients involved in one-carbon metabolism. Regarding the possible mechanism of this folate risk, the observation that some cancer chemotherapeutic agents are antifolate drugs may be relevant (30,31). These findings related to dosage and timing of folate consumption and colorectal cancer risk need further elucidation because this may become a public health issue given the widespread folate fortification of foods in several countries including the United States and Australia. Methionine: Although the collected data are more limited than for folate, they do suggest that dietary methionine is protective for colorectal cancer (27,28). As for dietary folate, the Melbourne methionine data presented here add only partial support to the previous conclusions. Interestingly, the protective effects are statistically significant only for rectal cancer in quintile 3, and marginally in quintile 2, mirroring the folate findings (Table 2). It is difficult to draw firm conclusions regarding methionine because it is mainly found in high-protein foods, such as red meat, poultry, fish, and dairy products, and a high consumption of some of these foods, such as red meat, are likely to be a risk for colorectal cancer through mechanisms other than those suggested for folate/methionine protection (27,28,32). Methionine did not have an independent effect when adjusted for folate. Vitamin B6: The limited previous data available suggest an inverse relationship between vitamin B6 intake and risk of both colorectal cancer and colorectal adenoma as well as in rodent models of chemically induced colon tumor formation (27,33,34). The study by Harnack and co-workers that first reported on all four micronutrients involved in DNA methylation found a significant protection for high folate and high vitamin B6 intakes taken together (33). These data are supported by the present study in which consumers of vitamin B6 containing foods were protected for colorectal cancer in all quintiles of consumption, and this protection was similar for colon and rectal cancer (Table 2). It has been suggested that, apart from participating in one-carbon metabolism, vitamin B6 is additionally involved in suppression of cell proliferation and antiangiogenesis (34,35). Vitamin B6 did not have an independent effect when adjusted for vitamin B12. Vitamin B12: There has been one previous report of reduced colon cancer risk of a high combined intake of folate and vitamin B12 (33). The present data appear to be the first report of an independent protective effect of vitamin B12 containing foods for colorectal cancer. In this study there was a dose-response effect with increasing consumption of vitamin B12 foods, highly statistically significant in the highest quintile of consumption, and the protection remained in the highest quintile of consumption for both colon and rectal cancer (Table 2). Although these data appear to be valid in view of the dose-response effect and uniform protection for both colon and rectal cancer, confirmation is required from future studies. As with vitamin B6, the possible mechanisms of this vitamin B12 protective effect are unclear; however, DNA repair and synthesis together with other methylating micronutrients are likely to be involved (33 36). In a randomized double-blind, placebo-controlled dietary intervention using moderately high doses of both folate (700 µg/day) and vitamin B12 (7 µg/day), plasma homocysteine status was inversely correlated with these nutritional supplements (36). To our knowledge, there is no evidence in humans that dietary exposure to micronutrients involved in one-carbon metabolism gives rise to high plasma homocysteine levels. Micronutrients With Antioxidant Properties Selenium: Selenium is an essential trace element for humans, largely obtained from plant food, such as cereals and vegetables (such as onion, garlic, and asparagus), and from fish and poultry. Some of the plant sources, such as garlic and onion, are apparently able to concentrate selenium even in selenium-deficient soils (37). The selenium content of the earth shows large geographical variations; therefore, dietary selenium consumption will also be variable (37,38). The soil of the east coast of Australia, which includes Melbourne, and therefore the region from where most of the food eaten by the subjects of this study would have originated, is regarded as a selenium-deficient area (39). In the data presented here, selenium intake was assessed using food-quantity values from the USDA because no equivalent data are available for Australian foods (23). The selenium content of plants, in particular, cereal grains, is strongly influenced by the quantity of biologically available selenium in the soil in which the plants grow, that is, by geographical origin. Values in the USDA tables are U.S. national averages and should be used with caution when levels of selenium in foods grown in Australia are of interest. Although absolute estimates of intake may be inaccurate, estimates based on relative effects within the study population (for example, ORs) are likely to be reasonable; nevertheless, our data need to be interpreted cautiously. Current collected data show an inverse relationship between selenium intake or plasma selenium levels and colorectal tumor risk in most though not all human and animal studies (27,39 41). Although there are a number of chemopreventive trials in progress, up to now the most important study is that of Clark and co-workers in the United States reported in 1996 (42). This group undertook a randomized placebo-controlled trial of over 1,300 subjects with previously resected nonmelanotic basal and/or squamous cell skin cancers using 200 µg of selenium daily, administered as 0.5 g selenium-enriched brewer s yeast, with the primary 18 Nutrition and Cancer 2006
endpoint as recurrent skin cancers. In fact, selenium did not reduce skin cancer incidence after 4- to 5-yr observation, but an unexpected finding was a significant reduction of lung, prostate, and colorectal cancer incidence, the OR for colorectal cancer being 0.61; 95% CI = 0.17 0.90; P = 0.03 (42). These trends were confirmed after 10 yr, with colorectal cancer incidence reduced by 53% (43). The Melbourne study subjects relied on a diet that was not selenium enriched in a part of the world in which the soil is regarded as being to some extent selenium deficient. Also, at the time of the study, nutritional supplements did not contain selenium. In these data a statistically significant protective effect was present for both colon and rectal cancer, particularly in quintiles 3 and 4 of consumption, this being in the range of approximately 100 120 µg of selenium per day (Table 2). Interestingly, in the highest quintile of consumption, there was less protection and it was not statistically significant. The U-shaped dose-response curve for risk found for selenium in this study appears to be mirroring currently held views that extremely low levels of selenium consumption are a risk, moderate levels are protective, and very high intakes of selenium can be toxic to humans (39). The U-shaped risk curve could also be a chance finding. Regarding selenium dosage, there is some evidence that the range of 100 200 µg of selenium per day inhibits genetic damage and carcinogenesis in humans, that 400 µg of selenium per day is the upper safety limit, and that it would be unwise to consume more than 600 µg of selenium per day for fear of toxicity (39). The recommended daily allowance by the Food and Agriculture Organization and World Health Organization is 26 µg/day for women and 34 µg/day for men, which experts regard as too low to prevent genetic damage. With the cautionary proviso that USDA selenium levels were used for Australian dietary data, the statistically significant protective range in our study of 100 120 µg selenium per day fits in well with an anticarcinogenesis effect. It is of interest that this selenium intake could be achieved without selenium fortification in an area of Australia where the soil is regarded as moderately selenium deficient. One of the hazards of selenium-containing supplements is inadvertent overdosage by overenthusiastic consumers, resulting in toxicity, something that is very unlikely to occur when selenium is derived entirely from the diet. Vitamin E: The protective effect of vitamin E containing foods was striking and consistent, and statistically highly significant for both colon and rectal cancer, at all levels above quintile 1 of consumption (Table 2). This protective effect was in keeping with some, but not all, other studies; however, in several series a quantitative estimate was not made (27,44). Of interest is that, in most colorectal adenoma studies, vitamin E had a protective effect, suggesting that it operates early in the tumorigenesis process (27). Vitamin C: The adjusted data for vitamin C containing foods showed a statistically significant protective effect for all quintiles of consumption for colorectal cancer, colon cancer, and in quintiles 4 and 5 for rectal cancer, and there was also a dose-response effect for the cancer sites, with just a marginal deviation in quintile 3 for rectal cancer (Table 2). These findings were in keeping with many, but not all, other studies (27,44). Again, it is noted that, in many of the reported series, a quantitative estimate was not made, a critical factor given the relatively high vitamin C content of approximately 70 mg/day in quintile 2 that produced protection in the Melbourne study (Table 1). As with vitamin E, most adenoma studies of the past yielded a protective effect for vitamin C also (27). Lycopene: A null result was obtained for all levels of dietary lycopene consumption for both colon and rectal cancer, and this absence of effect occurred for both unadjusted and adjusted models (Table 2). The collected evidence for colorectal cancer protection by carotenoids, including lycopene and beta-carotene, is not strong, so the null result in this study was not surprising (27,44 47). Combined Model The model shown in Table 3 allowing for mutual adjustments for all micronutrients showed that both generic groups (methylation processes and antioxidants) are independent effects for colorectal cancer. Vitamins E, C, and B12 maintain protective effects with increasing intake, whereas the relationships for folate and selenium show a more complex picture, and especially so for folate, with the possibility of increased risk with high intakes, consistent with other, though preliminary, findings discussed earlier. Biological Mechanisms Involved Although the Melbourne study did not investigate biological mechanisms of colorectal carcinogenesis, it is striking that the micronutrients shown to have significant protective effects fell into one of two groups: those associated with one-carbon metabolism and DNA methylation, synthesis, and repair (folate, methionine, and vitamins B6 and B12) or those with antioxidant properties (vitamins C and E and selenium). Recent reviews concur that for, normal so-called one-carbon metabolism and for normal DNA methylation, synthesis, and repair to take place, adequate intake of folate, methionine, and vitamins B6 and B12 is required and also that normal one-carbon metabolism is protective, inter alia, for colorectal cancer (28,31,33,35,48). Therefore, it is of interest that all the four major players in these important metabolic pathways were shown to have protective effects in the Melbourne study and that these effects could be expressed quantitatively. Moreover, these protective effects could be satisfied by diet alone without recourse to supplements. The strongest effects were noted for vitamin B12 and B6 containing foods, less strong effects were noted for folate, and Vol. 56, No. 1 19
least strong effects were noted for methionine-containing foods. A review of the few human studies that examined the other B group vitamins in relation to colorectal cancer risk, namely, vitamins B1 and B2 and niacin (nicotinic acid), shows inconsistent results ranging from risk to null results to protective effects (27). In a rat model, huge doses of niacin (equivalent to approximately 35 g of niacin per day for an average-sized human adult) resulted in elevated homocysteine levels (49). This rat study cannot be extrapolated to humans. At present these B vitamin data do not enhance the understanding of the dietary aspects of colorectal cancer risk. There is also experimental and human evidence, as indicated by recent reviews, that oxidative stress or oxidative damage to DNA caused by environmental factors, such as smoking, alcohol, and ionizing radiation, is an important mechanism of carcinogenesis in general (50 53). In the Melbourne study, three of the five dietary micronutrients examined, vitamins C and E and selenium containing foods (but not lycopene and beta-carotene), had significant protective effects, adding weight to the contention that oxidative damage to DNA is another likely mechanism of colorectal carcinogenesis. Protection was afforded by diet alone without the use of supplements. Much further research and new data derived from controlled studies are needed; however, the data presented here support our proposition that dietary micronutrients involved in DNA methylation, synthesis, and repair (folate, methionine, and vitamins B6 and B12) and some dietary micronutrients with antioxidant properties (vitamins C and E and selenium) may have a place in lowering colorectal cancer risk and also that this can be achieved by dietary means alone. Acknowledgments and Notes We thank Dr. Maxwell Watson, who assisted with matching the food items derived from the data in the Melbourne Colorectal Cancer Study with those in the USDA database, and Dr. Keith Wise, who converted food item intake into micronutrient intake for the respondents in the Melbourne study. Address correspondence to G. Kune, 41 Power Street, Toorak, Victoria, 3142, Australia. E-mail: gkune@unimelb.edu.au. Submitted 21 February 2006; accepted in final form 18 July 2006. References 1. Wingo PA, Ries LA, Rosenberg HM, Miller DS, and Edwards BK: Cancer incidence and mortality 1973 1995. A report card for the US. Cancer 82, 1197 1207, 1998. 2. Landis SH, Murray T, Bolden S, and Wingo PA: Cancer statistics. CA Cancer J Clin 49, 8 31, 1999. 3. Parkins DM, Pisani P, and Ferley J: Global cancer statistics. CA Cancer J Clin 49, 33 64, 1999. 4. Kune GA, Bannerman S, and Watson LF: Attributable risk for diet, alcohol and family history in the Melbourne Colorectal Cancer Study. Nutr Cancer 18, 231 235, 1992. 5. Kune GA and Kune S: The Melbourne Colorectal Cancer Study. A Description of the Investigation. Melbourne: University of Melbourne, 1986. 6. Kune G and Kune S: New design to examine colorectal cause and survival. Dig Surg 4, 156 159, 1987. 7. Kune S, Kune GA, and Watson LF: The Melbourne Colorectal Cancer Study. Incidence findings by age, sex, site, migrants and religion. Int J Epidemiol 15, 483 493, 1986. 8. Kune GA, Kune S, and Watson LF: History of colorectal polypectomy and risk of subsequent colorectal cancer. Data from the Melbourne Colorectal Cancer Study. Br J Surg 74, 1064 1065, 1987. 9. Kune GA, Kune S, and Watson LF: Colorectal cancer risk, chronic illnesses, operations and medications. Case control results from the Melbourne Colorectal Cancer Study. Cancer Res 48, 4399 4404, 1988. 10. Kune GA, Kune S, Field B, and Watson LF: The role of chronic constipation, diarrhea, and laxative use in the etiology of large-bowel cancer. Data from the Melbourne Colorectal Cancer Study. Dis Colon Rectum 31, 507 512, 1988. 11. Kune GA, Kune S, and Watson LF: Large bowel cancer after cholecystectomy. Am J Surg 156, 359 362, 1988. 12. Kune GA, Kune S, and Watson LF: The role of heredity in the etiology of bowel cancer: data from the Melbourne Colorectal Cancer Study. World J Surg 13, 124 131, 1989. 13. Kune GA, Kune S, and Watson LF: Children, age at first birth and colorectal cancer risk. Data from the Melbourne Colorectal Cancer Study. Am J Epidemiol 129, 533 542, 1989. 14. Kune GA, Kune S, and Watson LF: Oral contraceptive use does not protect against large bowel cancer. Contraception 41, 19 24, 1990. 15. Kune GA, Kune S, Field B, White R, Brough W, et al.: Survival in patients with large bowel cancer. A population based investigation from the Melbourne Colorectal Cancer Study. Dis Colon Rectum 33, 938 946, 1990. 16. Kune S, Kune GA, Watson LF, and Rahe RH: Recent life change and large bowel cancer. Data from the Melbourne Colorectal Cancer Study. J Clin Epidemiol 44, 57 68, 1991. 17. Kune GA, Kune S, Watson LF, and Bahnson CB: Personality as a risk factor in large bowel cancer: data from the Melbourne Colorectal Cancer Study. Psych Med 21, 29 41, 1991. 18. Kune GA, Kune S, Vitetta L, and Watson LF: Smoking and colorectal cancer risk: data from the Melbourne Colorectal Cancer Study and brief review of the literature. Int J Cancer 50, 1 4, 1991. 19. Kune GA and Kune S: The nutritional causes of colorectal cancer. An introduction to the Melbourne study. Nutr Cancer 9, 1 4, 1987. 20. Kune S, Kune GA, and Watson LF: Case-control study of dietary etiological factors: the Melbourne Colorectal Cancer Study. Nutr Cancer 9, 21 42, 1987. 21. Kune S, Kung GA, and Watson LF: Case-control study of alcoholic beverages as etiological factors: the Melbourne Colorectal Cancer Study. Nutr Cancer 9, 43 56, 1987. 22. Kune S, Kune GA, and Watson LF: Observations on the reliability and validity of the design and diet history method in the Melbourne Colorectal Cancer Study. Nutr Cancer 9, 5 20, 1987. 23. U.S. Department of Agriculture: USDA National Nutrient Database for Standard Reference, Release 17, Nutrient Lists. Beltsville, MD: U.S. Department of Agriculture, Agricultural Research Service, USDA Nutrient Data Laboratory, 2004. 24. StataStatisticalSoftware, release8.0. CollegeStation, TX: Stata, 2003. 25. Kune S: An Epidemiological Study of Colorectal Cancer. Ann Arbor, MI: University Microfilms International, 1986. 26. Michels KB, Giovannucci E, Joshipura KJ, Rosner BA, Stampfer MJ, et al.: Prospective study of fruit and vegetable consumption and incidence of colon and rectal cancers. JNCI 92, 1740 1752, 2000. 27. Kune GA: Causes and Control of Colorectal Cancer: A Model for Cancer Prevention. Boston: Kluwer, 1996. 28. Giovannucci E: Epidemiologic studies of folate and colorectal neoplasia: a review. J Nutr 132, S2350 S2355, 2003. 29. Kim Y-I: Role of folate in colon cancer development and progression. J Nutr 133, S3731 S3739, 2003. 20 Nutrition and Cancer 2006
30. Kim Y-I: Will mandatory folic acid fortification prevent or promote cancer? Am J Clin Nutr 80, 1123 1128, 2004. 31. Ulrich CM and Potter JD: Folate supplementation: too much of a good thing? Cancer Epidemiol Biomarkers Prev 15, 189 193, 2006. 32. Sandhu MS, White IR, and McPherson K: Systematic review of the prospective cohort studies on meat consumption and colorectal cancer risk: a meta-analytic approach. Cancer Epidemiol Biomarkers Prev 10, 439 446, 2001. 33. Harnack L, Jacobs DR Jr, Nicodemus K, Lazovich D, Anderson K, et al.: Relationship of folate, vitamin B6, vitamin B12 and methionine intake to incidence of colorectal cancer. Nutr Cancer 43, 152 158, 2002. 34. Matsubara K, Komatsu S, Oka T, and Kato N: Vitamin B6-mediated suppression of colon tumorigenesis, cell proliferation and angiogenesis (review). J Nutr Biochem 14, 246 250, 2003. 35. Selhub J: Folate, vitamin B12 and vitamin B6 metabolism. Nutr Health Aging 6, 39 42, 2002. 36. Fenech M, Aitken C, and Rinaldi J: Folate, vitamin B12, homocysteine status and DNA damage in young Australian adults. Carcinogenesis 19, 1163 1171, 1998. 37. Rayman MP: The importance of selenium to human health. Lancet 356, 233 241, 2000. 38. Combs GF: Selenium in global food systems. Br J Nutr 85, 517 547, 2001. 39. Whanger PD: Selenium and its relationship to cancer: an update. Br J Nutr 91, 11 28, 2004. 40. Langman M and Boyle P: Chemoprevention of colorectal cancer (review). Gut 43, 578 585, 1998. 41. Courtney EDJ, Melville DM, and Leicester RJ: Review article: chemoprevention of colorectal cancer. Aliment Pharmacol Ther 19, 1 24, 2004. 42. Clark LC, Combs GF, and Turnbull BW: The nutritional prevention of cancer with selenium 1983 1993: a randomized clinical trial. JAMA 276, 1957 1963, 1996. 43. Duffield-Lillico AJ, Reid ME, and Turnbull BW: Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the nutritional prevention of cancer risk. Epidemiol Biomarkers Prev 11, 630 639, 2002. 44. Murtaugh MA, Ma K-N, Benson J, Curtin K, Caan B, et al.: Antioxidants, carotenoids, and risk of rectal cancer. Am J Epidemiol 159, 32 41, 2004. 45. Francheschi S: Tomatoes and risk of digestive-tract cancers. Int J Cancer 59, 181 184, 1994. 46. Jain CK, Agarwal S, and Rao AV: The effects of dietary lycopene on bioavailability, tissue distribution, in vivo antioxidant properties and colonic preneoplasia in rats. Nutr Res 19, 1383 1391, 1999. 47. Slattery ML, Benson J, Curtin K, Ma K, Schaeffer D, et al.: Carotenoids and colon cancer. Am J Clin Nutr 71, 575 582, 2000. 48. Bailey LB and Gregory JF: Folate metabolism and requirements. J Nutr 129, 779 782, 1999. 49. Basu TK, Makhani N, and Sedgwick G: Niacin (nicotinic acid) in non-physiological doses causes hyperhomocysteinaemia in Sprague-Dawley rats. Br J Nutr 87, 115 119, 2002. 50. Valko M, Izakovic M, Mazur M, Rhodes CJ, and Telser J: Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 266, 37 56, 2004. 51. Evans MD, Dizdaroglu M, and Cooke MS: Oxidative DNA damage and disease: induction, repair and significance. Mutat Res 567, 1 61, 2004. 52. Cajas P, Casado E, Belda-Iniesta C, DeCastro J, Espinosa E, et al.: Implications of oxidative stress and cell membrane lipid peroxidation in human cancer (Spain). Cancer Causes Control 15, 707 719, 2004. 53. Klaunig JE and Kamendulis LM: The role of oxidative stress in carcinogenesis. Annu Rev Pharmcol Toxicol 44, 239 267, 2004. Vol. 56, No. 1 21