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Colon
Effect of maternal and postweaning folic acid supplementation on colorectal cancer risk in the offspring
  1. Karen K Y Sie1,
  2. Alan Medline2,3,
  3. Jacobine van Weel4,
  4. Kyoung-Jin Sohn5,
  5. Sang-Woon Choi6,
  6. Ruth Croxford7,
  7. Young-In Kim1,5,8,9
  1. 1Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada
  2. 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
  3. 3Department of Pathology, Humber River Regional Hospital, Toronto, Ontario, Canada
  4. 4Faculty of Medicine, University of Utrecht, The Netherlands
  5. 5Department of Medicine, University of Toronto, Toronto, Ontario, Canada
  6. 6Vitamin and Carcinogenesis Laboratory, Jean Mayer USDA Human Nutrition Research Center on Ageing at Tufts University, Boston, Massachusetts, USA
  7. 7Statistical Consultant, Toronto, Ontario, Canada
  8. 8Division of Gastroenterology, Department of Medicine, St. Michael's Hospital, Toronto, Ontario, Canada
  9. 9The Keenan Research Center, the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
  1. Correspondence to Young-In Kim, St. Michael's Hospital, 16CC-038, 30 Bond Street, Toronto, Ontario, Canada, M5B 1W8; youngin.kim{at}utoronto.ca

Abstract

Background Intrauterine and early life exposure to folic acid has significantly increased in North America owing to folic acid fortification, widespread supplemental use and periconceptional folic acid supplementation. The effect of maternal and postweaning folic acid supplementation on colorectal cancer risk in the offspring was investigated.

Methods Female rats were placed on a control or supplemental (2.5× the control) diet prior to mating and during pregnancy and lactation. At weaning, male pups from each maternal diet group were randomised to the control or supplemental diet (n=55 per each of the four maternal/pup diet groups) for 31 weeks and colorectal cancer was induced by azoxymethane at 5 weeks of age. At necropsy, colorectal cancer parameters as well as colorectal epithelial proliferation, apoptosis and global DNA methylation were determined in the offspring.

Results Maternal, but not postweaning, folic acid supplementation significantly reduced the odds of colorectal adenocarcinoma by 64% in the offspring (OR 0.36; 95% CI 0.18 to 0.71; p=0.003). Pups from the dams fed the control diet that were given postweaning folic acid supplementation had significantly higher tumour multiplicity and burden than other groups (p<0.05). Maternal and postweaning folic acid supplementation interacted in a manner that decreased rectal epithelial proliferation (p<0.05). Both maternal and postweaning folic acid supplementation significantly decreased DNA damage in the rectum (p<0.05). Maternal folic acid supplementation significantly increased (p=0.007), whereas postweaning supplementation significantly decreased (p<0.001), colorectal global DNA methylation.

Conclusions The data suggest for the first time that maternal folic acid supplementation at the level equivalent to the average postfortification total folate intake in North America and to that recommended to women at reproductive age protects against the development of colorectal cancer in the offspring. This protective effect may be mediated in part by increased global DNA methylation and decreased epithelial proliferation and DNA damage in the colorectum.

  • Folate
  • folic acid
  • maternal supplementation
  • colorectal cancer
  • animal model
  • colorectal cancer
  • dietary - colon cancer
  • folic acid
  • methylation
  • vitamins

https://doi.org/10.1136/gut.2011.238782

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    Significance of this study

    What is already known about this subject?

    • Intrauterine and early life exposure to folic acid has significantly increased in North America owing to folic acid fortification, widespread supplemental use and periconceptional folic acid supplementation.

    • The role of folic acid supplementation in colorectal cancer prevention is highly controversial as animal studies and randomised controlled trials have shown that folic acid supplementation appears to prevent the development of new colorectal cancer while it may promote the progression of existing preneoplastic lesions to colorectal cancer.

    • The embryonic stage is highly susceptible to changes in the intrauterine environment including the intrauterine nutritional milieu due to epigenetic and metabolic programming, alterations to which may influence the risk of developing cancer in adulthood.

    • Epidemiological studies have reported a protective effect of periconceptional maternal folic acid supplementation on several paediatric cancers in the offspring, although some studies have not confirmed this purported protective effect and some have even reported an increased risk. A recent animal study has shown that both maternal and postweaning folic acid supplementation significantly increased the risk of mammary tumours in the offspring. At present, no information concerning the effect of maternal folic acid supplementation on colorectal cancer risk in the offspring exists in animal models or in humans.

    What are the new findings?

    • Maternal, but not postweaning, folic acid supplementation at the level equivalent to the average postfortification total folate intake in North America and to that recommended to women at reproductive age significantly reduced the odds of colorectal cancer by 64% in the offspring (OR 0.36; 95% CI 0.18 to 0.71; p=0.003).

    • Maternal and postweaning folic acid supplementation interacted in a manner that decreased rectal epithelial proliferation in the offspring (p<0.05). Both maternal and postweaning folic acid supplementation significantly decreased DNA damage in the rectum of the offspring (p<0.05).

    • Maternal folic acid supplementation significantly increased (p=0.007), whereas postweaning supplementation significantly decreased (p<0.001), colorectal global DNA methylation in the offspring.

    How might it impact on clinical practice in the foreseeable future?

    • Given the drastically increased intrauterine and early life exposure to folic acid in North America and the incidence, mortality and morbidity of colorectal cancer globally, future studies are warranted to clarify the effect of maternal and postnatal folic acid supplementation on colorectal cancer risk in the offspring and associated mechanisms.

    • Given the organ-specific effect and tumour-promoting effect associated with supraphysiological levels of folic acid supplementation on cancer risk, future studies are required to investigate the effect of maternal folic acid supplementation on other cancer risk in the offspring as well as a dose–response relationship.

    Introduction

    Dietary intake and blood measurements of folate appear to be inversely related to colorectal cancer risk in epidemiological studies.1 2 Folic acid (the synthetic form of folate used in supplements and fortified foods) chemoprevention trials in humans using the recurrence of adenomas as the primary end point, however, have produced conflicting results. While three trials did not demonstrate a protective effect of folic acid supplementation (0.5–1 mg/day for 2–6.5 years),3–5 one of these trials reported that folic acid supplementation significantly decreased the risk of recurrent adenomas by 40% only in those with low plasma folate concentrations at baseline.5 Another trial reported that folic acid supplementation at 5 mg/day for 3 years significantly reduced the number of recurrent adenomas.6 However, the Aspirin/Folate Polyp Prevention Study7 reported that folic supplementation at 1 mg/day for 6 years significantly increased the recurrence of advanced adenomas by 67% and the incidence of multiple adenomas by twofold.

    Animal studies have suggested that folate possesses dual effects on colorectal carcinogenesis depending on the stage of cell transformation at the time of intervention and the dose of supplementation.8 9 Folate deficiency has an inhibitory, whereas folic acid supplementation has a promoting, effect on the progression of established colorectal (pre)neoplastic lesions.10–12 In contrast, folate deficiency in the normal colorectum seems to predispose it to neoplastic transformation, and modest levels of folic acid supplementation suppress, whereas supraphysiological supplemental doses enhance the development of colorectal cancer in the normal colorectum.10 11 13–15

    Several biological mechanisms exist to explain the dual effects of folate on colorectal carcinogenesis. As an essential cofactor for nucleotide biosynthesis, folate plays an important role in DNA synthesis, integrity and repair, aberrancies of which are mechanistically related to cancer development.8 In normal tissues, folic acid supplementation provides nucleotide precursors for DNA synthesis and replication, thereby ensuring DNA fidelity, maintenance of DNA integrity and optimal DNA repair; this would reduce the risk of neoplastic transformation.8 In contrast, folic acid supplementation promotes the progression of (pre)neoplastic lesions by providing nucleotide precursors to the rapidly replicating transformed cells, allowing accelerated proliferation.8 Folate also modulates DNA methylation of cytosine within the cytosine–guanine (CpG) sequences because of its role in the provision of S-adenosylmethionine, the primary methyl group donor for most biological methylation reactions.16 Neoplastic cells simultaneously harbour widespread global DNA hypomethylation and specific promoter CpG island hypermethylation.16 Global DNA hypomethylation is an early and consistent event in colorectal carcinogenesis and contributes to colorectal cancer development through several mechanisms including chromosomal instability.16 DNA methylation at promoter CpG islands silences transcription and hence inactivates the function of a wide array of tumour suppressor and cancer-related genes.16 Folic acid supplementation is able to reverse pre-existing global DNA hypomethylation and to increase the extent of global DNA methylation above the pre-existing level,16 thereby reducing the risk of neoplastic transformation. In contrast, folic acid supplementation may cause de novo methylation of CpG islands of tumour suppressor genes, with consequent gene inactivation leading to tumour development and progression.8

    Folate intake and blood levels in North America have dramatically increased over the past decade. This is due to the drastic increase in dietary folate intake from mandatory folic acid fortification17 aimed at reducing the rate of neural tube defects, and also to the consumption of supplemental folic acid by up to 30–40% of the North American population.18 Furthermore, women of childbearing age are routinely advised to take 0.4–1.0 mg of folic acid for the prevention of neural tube defects.19 These facts suggest that intrauterine exposure of the developing fetus to folate and folic acid has significantly increased in North America, for which essentially no data on cancer risk in adulthood exist. Epigenetic and metabolic programming takes place during embryogenesis and hence the embryonic stage is highly susceptible to changes in the intrauterine environment, which may influence the risk of developing colorectal cancer in adulthood.20 Given these considerations, we investigated the effect of maternal and postweaning folic acid supplementation on the risk of colorectal cancer and on epithelial proliferation, apoptosis and DNA methylation in the offspring.

    Methods

    Animals, dietary intervention and colorectal tumour induction

    Six-week-old Sprague–Dawley rats were purchased from Charles River Laboratories (St Constant, Quebec, Canada). Female rats were housed in pairs with males when breeding, and singly during pregnancy and lactation. Female rats were randomised to receive an amino acid-defined diet (Dyets, Bethlehem, Pennsylvania, USA) containing 2 (control) or 5 (supplemented) mg of folic acid/kg of diet for 3 weeks prior to mating and throughout pregnancy and lactation (figure 1). Male rats used for breeding were placed on the same diet as the female breeding mate. Litters remained with the mothers until the end of the weaning period (3 weeks of age). At weaning, 25 pups from each maternal diet group were killed for baseline plasma folate and homocysteine (an accurate inverse indicator of tissue folate status) and hepatic folate measurements (figure 1). The remaining male pups from each maternal dietary group were randomised to receive either the control or folic acid-supplemented diet (n=55 per each of the four maternal/pup diet groups) (figure 1).

    Figure 1
    Figure 1

    Study design. AOM, azoxymethane.

    Starting at 5 weeks of age, pups were given once weekly subcutaneous injections of azoxymethane (AOM) (15 mg/kg body weight; Midwest Research Institute, Kansas City, Missouri, USA) for 2 weeks for colorectal tumour induction (figure 1).12 21 At 20 weeks of age (14 weeks after the last AOM injection), 10 pups from each of the four maternal/pup diet groups were killed for folate analyses, rectal epithelial proliferation and apoptosis (figure 1). The remaining pups (n=45 per each of the four maternal/pup diet groups) were killed at 34 weeks of age (28 weeks after the last AOM injection) for folate and tumour analyses and rectal epithelial proliferation and apoptosis (figure 1).

    We wished to examine genomic DNA methylation of the normal colonic mucosa. The AOM-induced rat colon harbours ∼100 aberrant crypt foci, the purported precursor of colorectal cancer in rodents,22 by 14 weeks post-AOM injection.12 21 Thus, it is impossible to extract non-neoplastic colonic DNA from the AOM-induced colon. Therefore, we conducted a separate experiment of a similar design except that pups did not receive AOM, and pups (n=25 per each of the four maternal/pup diet groups) were killed at 14 weeks of age (figure 1).

    Amino acid-defined diets containing different levels of folic acid constitute a standard method of providing supplemental dietary folate in rodents and have been extensively used in previous studies of folate and colorectal cancer.10–14 The control diet containing 2 mg of folic acid/kg of diet is generally accepted as the basal dietary requirement for rats23 and was selected to represent the recommended dietary allowance for humans (0.4 mg/day of dietary folate equivalents).24 The supplemented diet containing 5 mg of folic acid/kg represents the likely average postfortification total folate intake of 0.8–1.0 mg/day in North American populations taking multivitamins containing 0.4 mg of folic acid,9 and the recommended dose for all women planning a pregnancy or capable of becoming pregnant (0.4–1.0 mg/day).19 The detailed composition of the diets has been published previously.11 Diets and water were provided ad libitum. Body weights were recorded weekly.

    Sample collection and analysis of colorectal tumours

    The rats were killed by CO2 inhalation followed by cervical dislocation. At necropsy, blood was collected and plasma was stored at −80°C with and without 0.5% ascorbic acid for plasma folate and homocysteine assays, respectively. The liver from each rat was excised, snap-frozen and stored at −80°C for hepatic folate analysis. The entire colorectum from each animal was immediately excised, put on a glass plate suspended on crushed ice, opened longitudinally, and flushed with phosphate-buffered saline solution to eliminate faecal debris. The rectum from each animal (the distal 2 cm of the colorectum) was cut, placed in a cassette with a foam cushion and stored in 10% neutral buffered formalin for immunohistochemical analyses of epithelial proliferation and apoptosis. The entire colon was examined in a blinded fashion for any macroscopic lesions, which were excised, fixed in formalin and processed in a standard manner for H&E staining for histological confirmation of adenomas and adenocarcinomas by a gastrointestinal pathologist (AM) blinded to the study groups. The longest diameter of each macroscopic lesion was measured. For DNA methylation analysis, the colorectal mucosa was carefully scraped with glass slides, rapidly weighed, frozen in liquid nitrogen and stored at −80°C for DNA extraction.

    Folate and homocysteine concentrations

    Plasma and hepatic folate concentrations were determined by a standard microbiological microtitre plate assay.12 Total plasma homocysteine concentrations were determined using the Axis Homocysteine EIA kit (Abbott Laboratories, Mississauga, Ontario, Canada).12

    Epithelial proliferation and apoptosis

    Rectal epithelial proliferation was determined by staining histological sections with monoclonal antibodies against Ki-67, a nuclear protein expressed in proliferating cells.12 Rectal epithelial apoptosis was determined by terminal uridine deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay using the ApopTag Peroxidase In Situ Apoptosis Detection Kit S7100 (Millipore, Billerica, Massachusetts, USA). The scoring was based on the percentage of positively stained cells in relation to the total number of cells considered, and a scoring index was used to group the range of percentages as follows: 0, <1%; 1+, 2–5%; 2+, 6–10%; 3+, 11–20%; and 4+, >21% (figure 2A–D).12

    Figure 2
    Figure 2

    Representative images of Ki-67-stained rectal epithelial cells demonstrating (A) low staining (3.3% positive stained nuclei in relation to the total number of cells considered; scoring index 1+) and (B) high staining (17.7%; 3+). Representative images of rectal epithelial apoptosis as determined by TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick end labelling) assay demonstrating (C) low staining (0%; 0) and (D) high staining (14%; 3+).

    Global DNA methylation analysis

    DNA from the colonic mucosa was extracted using a Quiagen DNeasy Blood and Tissue kit and Quiagen RNase A (Quiagen, Mississauga, Ontario, Canada). The size of DNA estimated by agarose gel electrophoresis was >20 kb in all instances. The final preparations had a ratio of A260 to A280 between 1.8 and 2.0 and were free of RNA and protein contaminations. The concentration of each DNA sample was determined as the mean of three independent spectrophotometric readings. Global DNA methylation was determined by liquid chromatography–electrospray ionisation mass spectrometry25 and is presented as the percentage of 5-methylcytosine of the overall amount of cytosine and 5-methylcytosine.

    Statistical analysis

    Statistical analyses were performed using SPSS 14.0 for Windows. Continuous variables were log-transformed due to departure from normality. Parameters measured at weaning were analysed using univariate one-way analysis of variance (ANOVA), and those measured after weaning were analysed using univariate two-way ANOVA. Tumour incidence was analysed using binary logistic regression. Body weights over time were compared using repeated measures ANOVA. Ordinal variables (tumour multiplicity and burden) were analysed using the non-parametric Kruskal–Wallis test. If a significant effect was present, pairwise comparisons using non-parametric Mann–Whitney U tests were performed to clarify the effects of maternal and pup diet. With the exception of the analyses of characteristics measured at the time of weaning, all analyses included an interaction term between maternal and pup diets in order to test whether the effect of the maternal diet depended on the pup diet, and vice versa. Significant interactions found in the two-way ANOVA were followed by the Tukey honestly significant difference (HSD) posthoc analyses to determine the nature of the interaction. Correlations between continuous dependent variables were evaluated using the Spearman rank correlation coefficient. All significance tests were two sided and were considered significant at p<0.05. Results are expressed as mean±SD.

    Results

    Body weight and growth

    Animals demonstrated no growth retardation, and no premature death occurred. The overall pattern of weight gain from weaning to 34 weeks of age was similar among the four maternal/pup diet groups. However, maternal folic acid supplementation significantly influenced the weight of pups. The mean weight of the pups from the folic acid-supplemented dams was 4% higher (95% CI 2% to 6%) than that of the pups from the dams fed the control diet (p<0.001). In contrast, postweaning folic acid supplementation did not affect the weight of pups (p=0.5). No significant interaction was found between the maternal and pup diets in influencing the body weight of pups.

    Systemic and tissue folate status

    At weaning, plasma and hepatic folate and plasma homocysteine concentrations of the pups reflected maternal folic acid supplementation (p<0.001 and p=0.003, respectively; table 1).

    View this table:
    Table 1

    Folate status of offspring at 3 (weaning; n=25/each maternal diet group), 14 (n=25/each maternal/pup diet group), 20 (n=10/each maternal/pup diet group) and 34 (n=45/each maternal/pup diet group) weeks of age

    Plasma and hepatic folate (p<0.001) and plasmas homocysteine (p<0.05) concentrations of the offspring significantly reflected postweaning folic acid supplementation at all three time points except for plasma homocysteine at 20 weeks of age (table 1). In contrast, maternal folic acid supplementation significantly increased plasma folate concentrations only at 34 weeks of age (p<0.001) and hepatic folate concentrations at 14 and 34 weeks of age (p<0.001), and decreased plasma homocysteine levels only at 14 weeks of age (p=0.02) (table 1). A significant interaction between maternal and postweaning folic acid supplementation was observed for plasma and hepatic folate at 34 weeks of age (p=0.015 and p=0.002, respectively) and for plasma homocysteine at 14 weeks of age (p=0.001).

    Effect on colorectal tumours

    From 60% to 86% of the pups developed at least one colorectal adenocarcinoma with or without co-existing adenomas (figure 3). No animals had adenomas alone. Maternal folic acid supplementation significantly reduced the odds of colorectal adenocarcinomas by 64% in the offspring (OR 0.36; 95% CI 0.18 to 0.71; p=0.003; figure 3). Postweaning folic acid supplementation, however, had no significant effect, nor was there any significant interaction between the maternal and pup diets.

    Figure 3
    Figure 3

    Effect of maternal and postweaning folic acid (FA) supplementation on the incidence of colorectal cancer in the offspring. † denotes a significant difference between the maternal diets at p<0.05.

    Tumour multiplicity and the sum of colorectal tumour diameters per rat (used as a proxy of tumour burden) depended significantly on diet (p=0.014 for the number of colorectal tumours (adenomas + adenocarcinomas) per rat, p=0.019 for the number of colorectal adenocarcinomas per rat, p=0.006 for the sum of colorectal tumour diameters, and p=0.013 for the sum of diameters of colorectal adenocarcinomas; table 2). Pairwise analyses determined that in each case, pups from the dams fed the control diet that were given postweaning folic acid supplementation had significantly higher tumour number and burden compared with the other groups (p<0.03, p<0.05, p<0.02 and p<0.02 respectively; table 2).

    View this table:
    Table 2

    Tumour multiplicity and burden at 34 weeks of age (n=45/each maternal/pup diet group)

    Effect on rectal epithelial proliferation

    At 20 weeks of age, rectal epithelial proliferation was not significantly different among the four dietary groups (table 3). However, at 34 weeks of age, both maternal and postweaning folic acid supplementation affected rectal epithelial proliferation in the offspring (p<0.05; table 3), with a significant interaction between the maternal and pup diets (p<0.001). This interaction suggests that postweaning folic acid supplementation significantly reduced rectal epithelial proliferation compared with the postweaning control diet only in pups from the folic acid-supplemented dams but not in pups from the dams fed the control diet. Maternal folic acid supplementation was associated with significantly increased rectal epithelial proliferation compared with the control maternal diet only in pups placed on the postweaning control diet but not in pups placed on the postweaning folic acid-supplemented diet.

    View this table:
    Table 3

    Rectal epithelial proliferation (Ki-67) and rectal epithelial apoptosis (TUNEL) at 20 (n=10/each maternal/pup diet group) and 34 (n=45/each maternal/pup diet group) weeks of age

    Effect on rectal epithelial apoptosis

    At 20 weeks of age, increasing the folic acid level in the maternal diet from 2 to 5 mg/kg of diet decreased rectal epithelial apoptosis in pups by a factor of 0.6 (95% CI 0.08 to 1.21; p=0.025; table 3). Similarly, increasing the folic acid level in the pup diet from 2 to 5 mg/kg of diet decreased rectal epithelial apoptosis by a factor of 1.1 (95% CI 0.58 to 1.62; p<0.001; table 3). There was no significant interaction between the maternal and pup diets. At 34 weeks of age, however, neither maternal nor postweaning folic acid supplementation had a significant effect on rectal epithelial apoptosis (table 3).

    Effect on global DNA methylation of the colon

    At weaning, the extent of global DNA methylation of the colon was significantly higher, by 3%, in the pups from the folic acid-supplemented dams than in the pups from the dams fed the control diet (p=0.007; table 4). In contrast, at 14 weeks of age, postweaning, but not maternal, folic acid supplementation significantly, by 3.3–6.4%, decreased the extent of global DNA methylation of the colon in the offspring (p<0.001; table 4). No significant interaction between the maternal and pup diets at 14 weeks of age was observed.

    View this table:
    Table 4

    Global DNA methyation of the non-neoplastic colonic mucosa in the offspring at 3 (weaning; n=25/each maternal diet group) and 14 (n=25/each maternal/pup diet group) weeks of age

    Discussion

    In the present study, maternal folic acid supplementation at the level equivalent to the average postfortification total folate intake in North America significantly reduced the odds of colorectal cancer by 64% in the offspring, whereas postweaning folic acid supplementation had no effect. No significant interaction between maternal and postweaning folic acid supplementation was observed. Interestingly, tumour multiplicity and burden were significantly higher in the pups from the dams fed the control diet that were given postweaning folic acid supplementation than those from the other groups. One possible explanation for this observation is that the pups from the dams fed the control diet might have developed a higher number of microscopic precursor lesions in the colorectum compared with those from the folic acid-supplemented dams, and postweaning folic acid supplementation might have promoted the progression of these precursors to neoplastic lesions.

    We posited that maternal and postweaning folic acid supplementation would increase global DNA methylation in the offspring, thereby reducing colorectal cancer risk. A new DNA methylation pattern is established during embryogenesis soon after implantation26 and DNA methylation of the developing fetus appears to be highly susceptible to environmental modifiers including maternal supplementation of methyl group donors.27 28 Our data demonstrate for the first time that maternal folic acid supplementation at the level equivalent to the average postfortification total folate intake in North America can significantly increase global DNA methlyation in the colon of the weanling offspring. Unexpectedly, at 14 weeks of age, postweaning folic acid supplementation significantly decreased global DNA methylation in the colon of the offspring. This seemingly paradoxical effect may be partly explained by the preferential shuttling of the flux of one-carbon units to the nucleotide synthesis pathway over the biological methylation pathway in response to folic acid supplementation. Although folic acid is an inhibitor of dihydrofolate reductase (DHFR),29 it can upregulate DHFR in certain situations.30 This upregulation may increase thymidylate synthase activity because the transcription of these genes is co-regulated by several transcription factors31 and this would increase thymidylate production at the expense of methylation reactions.32

    The observed changes in global DNA methylation in the colon of the offspring were very modest, albeit significant, and thus their functional ramifications are uncertain and a definitive mechanistic link to colorectal cancer risk in the offspring cannot be established. Increased global DNA methyation in the developing colorectum of the offspring in utero and in early postnatal life associated with maternal folic acid supplementation would protect against chromosomal instability and this might have partly contributed to the reduced colorectal cancer risk in adulthood. In contrast, decreased global DNA methylation associated with postweaning folic acid supplementation would increase chromosomal instability, leading to an increased colorectal cancer risk. However, in the present study, postweaning folic acid supplementation did not influence colorectal cancer risk in the offspring, suggesting that the observed decrease in global DNA methylation associated with postweaning folic acid supplementation did not significantly contribute to the development of colorectal cancer in the offspring. Future studies are warranted to investigate several downstream effects of global DNA methylation changes to establish their mechanistic link to the decreased colorectal carcinogenesis in the offspring associated with maternal folic acid supplementation. We did not investigate the effect of folic acid supplementation on promoter CpG island DNA methylation, which may also play a role in colorectal carcinogenesis in the offspring, because most promoters that are subject to DNA methylation are not conserved between humans and rats, and specific promoters that are presumably epigenetically regulated in rats have not been extensively characterised nor have they been implicated in rat colorectal carcinogenesis. Studies are currently underway to investigate the effect of maternal and postweaning folic acid supplementation on promoter CpG island DNA methylation of critical tumour suppressor and cancer-related genes as well as their functional ramifications in the offspring's colon using mice, which are a more appropriate animal model for studying epigenetics.

    Maternal and postweaning folic acid supplementation interacted in a manner that decreased rectal epithelial proliferation of the offspring at 34 weeks of age. This might be another mechanism by which folic acid supplementation might have contributed to the reduced colorectal cancer risk in the offspring as epithelial hyperproliferation in the colorectum increases colorectal cancer risk.33 Consistent with our observation, folic acid supplementation has been shown to inhibit proliferation of human colon cancer cells34 35 and rectal epithelium.36 Folic acid inhibits DHFR,29 thereby decreasing the intracellular folate pool and downregulating thymidylate synthase because of the co-regulation of transcription of DHFR and thymidylate synthase.31 This would result in decreased DNA synthesis.

    Both maternal and postweaning folic acid supplementation was associated with a significantly lower TUNEL score index in the rectum of the offspring at 20 weeks of age. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades that remove cells that are senescent, damaged, superfluous or potentially neoplastic.37 Our data suggest that maternal and postnatal folic acid supplementation was associated with significantly reduced DNA damage in the developing colorectum and this might have contributed to the reduced colorectal cancer risk in the offspring. Folic acid supplementation probably protected the developing colorectum from DNA damage by ensuring the provision of nucleotide precursors for optimal DNA synthesis and replication, thereby ensuring DNA fidelity, maintenance of DNA integrity and stability, and optimal DNA repair. However, at 34 weeks of age, no significant effect of folic acid supplementation on DNA damage was observed, suggesting that the effect of folic acid supplementation is primarily on the developing colorectum.

    In contrast to our study, a study using ApcMin/+ mice reported that postweaning folic acid supplementation significantly increased the number of small intestinal adenomas in the offspring.38 Interestingly, folic acid supplementation provided in utero and during lactation, and continued through the postweaning period did not increase the number of adenomas in the offspring.38 However, folate deficiency induced in utero and continued through the postweaning period significantly reduced the number of adenomas in the offspring.38 One important distinction between this and our studies is the difference in the models used. The ApcMin/+ mouse has a strong genetic proclivity towards small intestinal tumorigenesis, and (pre)neoplastic foci develop very early in life. In ApcMin/+ mice, any preventive effect of maternal folic acid supplementation on the initiation of small intestinal tumorigenesis might have been overwhelmed, whereas postweaning folic acid supplementation probably promoted the progression of the established (pre)neopalstic foci, similar to the previously observed tumour-promoting effect on the postinitiation phase.10–12 14 In the AOM rat model, colorectal cancer was induced at puberty and maternal folic acid supplementation provided a protective environment in utero and in the early postnatal period for the development of (pre)neoplastic lesions in the offspring. However, postweaning folic acid supplementation had no effect on the postinitiation phase of colorectal carcinogenesis in the offspring. We have recently reported that both maternal and postweaning folic acid supplementation at the same level as in the present study significantly increased the incidence of mammary tumours in the offspring.39 This suggests that the effect of maternal and postweaning folic acid supplementation on cancer risk in the offspring may be organ specific.

    Epidemiological studies have reported a protective effect of periconceptional maternal folic acid supplementation on several paediatric cancers in the offspring.40–43 However, some studies have not confirmed this purported protective effect44 45 and have even reported an increased risk.42 Furthermore, most of these epidemiological studies could not delineate the effect on cancer risk specific to folic acid from other vitamins in the supplements.46 At present, no information concerning the effect of maternal folic acid supplementation on colorectal cancer risk in the offspring exists in humans.

    In summary, our data suggest, for the first time, that maternal folic acid supplementation at the level equivalent to the average postfortification total folate intake in North America and to that recommended to women at reproductive age significantly protects against the development of colorectal cancer in the offspring. Although our study lacks a comprehensive mechanistic interrogation of the observed protective effect of maternal folic acid supplementation on colorectal cancer risk in the offspring, our data suggest that increased global DNA methylation associated with maternal folic acid supplementation and decreased epithelial proliferation and DNA damage associated with maternal and postweaning folic acid supplementation may be responsible for the observed decreased colorectal cancer risk in the offspring. Given the drastically increased intrauterine and early life exposure to folic acid in North America and the health burden of colorectal cancer globally, future studies are warranted to clarify the effect of maternal and postnatal folic acid supplementation on colorectal cancer risk in the offspring and associated mechanisms.

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    Footnotes

    • See Paper, p 1695

    • Presented in part at the 2008 American Association for Cancer Research Meeting, San Diego, CA, USA, April 2008 and published in abstract form in the Proceedings of the American Association for Cancer Research 2008;68:abst 2098.

    • Funding The Canadian Cancer Society (Grant # 017078; to YIK); Canadian Institutes of Heath Research (Grant # 14126; to YIK); Canadian Graduate Scholarships Master's Award from the Canadian Institutes of Health Research (to KKYS).

    • Competing interests None.

    • Ethics approval Animal experimentation was approved by the Animal Care Committee of the University of Toronto, in accordance with the guidelines of the Canadian Council on Animal Care.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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