This study examined the effects of dietary folate depletion/repletion on gene expression-and select other molecular events-in the human colon, as well as changes occurring after one month of folate repletion; and 2) the effects of two months of folic acid supplementation in folate-replete subjects. Subjects who by history were at modestly increased risk of colon cancer were studied since the underlying concept was to define which cancer-relevant pathways are altered by varying levels of folate intake in individuals whose colons are susceptible to neoplastic transformation.
Although folate depletion is difficult to achieve in the present era of mandatory folic acid fortification, the first study successfully induced a significant degree of depletion, as evidenced by substantial declines in serum and colonic folate, and an increase in serum homocysteine (). In fact, homocysteine rises when intracellular folate concentrations can no longer adequately support biological re-methylation (22
), so the degree of depletion achieved in the study is indicative of a true intracellular depletion of the vitamin. Nevertheless, the degree of depletion at the end of the study was rather modest: since after depletion the mean serum folate concentration (=5.8 ng/mL) did not fall below the threshold that is conventionally considered to define the lower limit of normality (i.e: 5 ng/mL). This is clinically relevant since enhancement of colorectal cancer risk due to folate inadequacy often occurs in segments of the population whose low folate status is at the lower end of the normal range rather than in the zone of frank deficiency (23
). Furthermore repletion of folate also was successful since indicators of folate status, both systemic and colonic, returned nearly to baseline status. RBC folate status did not similarly increase to a significant degree presumably since the half-life of red cell folate parallels the half-life of those cells. These data agree with previous studies which have demonstrated that serum folate is a more accurate proxy measure of human colonic folate status than RBC folate (14
). In the second study, in which folic acid supplementation was examined, serum and colonic folate concentrations rose by 50–60%, confirming successful augmentation of both systemic and colon-specific folate status.
A seminal observation in the supplementation protocol is increased expression of genes involved in immune and pro-inflammatory processes. Moreover, the gene expression data from the depletion/repletion protocol are highly complementary to those of the supplementation study: all three phases (i.e.: depletion, repletion, and supplementation) indicate that higher folate status increases expression of pro-inflammatory and immune pathways (). In particular, chemokine and cytokine genes including those of the complement and coagulation systems and immune recognition system were downregulated by folate depletion and this effect was reversed with folate repletion and supplementation (Supplement S1–3
). Although repleting folate-deplete subjects with folic acid supplementation is physiologically distinct from supplementing folate-replete subjects, the observed effects on the expression of pro-inflammatory and immune-related genes were very similar, suggesting that the effect occurs incrementally over the entire range of folate status in these two protocols. Along these lines, a recent human study showed that administration of 1.2 mg folic acid for 12 weeks resulted in an increase of serum proteins involved in regulation and activation of immune function and complement cascade (24
). Interestingly, these results might in part explain the mechanistic basis for recent observations emerging from the human Aspirin/Folate Polyp Prevention trial, which suggested that folic acid administration antagonizes the suppressive effects of aspirin on circulating inflammatory markers (10
). Furthermore, in preclinical studies, supraphysiologic levels of dietary folic acid also led to enhanced inflammation in a liver tissue injury model (25
Our data also show that neuronal receptor pathways were also linked to folate status. Expression of genes such as GABA receptors or other G-protein coupled receptors such as taste transduction receptors were increased during folate depletion and reciprocally decreased during folate repletion. The potential association between these genes and colon carcinogenesis is unclear but the GABA may be over-expressed in colon cancer and GABA receptor signaling was linked to metastatic behavior of colon cancer cells in experimental models (26
One-carbon metabolism is complex, making it difficult to accurately predict the net metabolic effects of the observed changes in gene expression. However, some of the observed changes warrant speculation: thymidylate synthase expression was suppressed by supplementation (confirmed by qRT-PCR, ), which could diminish thymidylate availability for DNA synthesis and thereby enhance uracil incorporation into DNA, a potentially mutagenic event. Thus, our observations are consistent with a report in which subjects receiving 5 mg of folic acid per day developed increased levels of uracil in their colonic mucosa (27
Our results suggest that altered expression in 1-carbon enzymes may represent a protective mechanism against an oversupply of folate co-factor. By down-regulating DHFR, folic acid would be less capable of entering into the 1-carbon metabolic network (). Moreover, the terminal enzymes for two critical functions of 1-carbon metabolism--thymidylate synthesis and biological methylation of DNA--were both downregulated, an effect which might limit the synthesis of end-products in the face of excess co-factor. This result is consistent with those of Basten et al, who observed that supplemental folic acid administered to healthy volunteers in a similar dose and duration as in our study diminished DNA excision repair (28
). They postulated that the surfeit of nucleotides provided for by excess folic acid might downregulate pathways related to DNA repair, a concept that was borne out by our observation that supplementation led to a downregulation of the pathways integral to both purine and thymidine synthesis. Feasibly these changes also may alter cell cycle dynamics since we observed significant down-regulation of genes that are involved in regulation of cell cycle and DNA replication including cyclins, cyclin-dependent kinases and PCNA (Supplement S2
Both genomic DNA methylation and promoter methylation were examined as well. No significant changes in genomic DNA methylation were observed in folate depletion-repletion study or in the parallel supplementation study. The observation that the colon is relatively resistant to changes in genomic methylation due to altered folate status has also been noted in animal studies (29
), as well as some (30
), but not all (31
) clinical trials.
Probably more relevant to mechanistic issues than genomic methylation were the observations pertaining to loci-specific methylation. We found no significant changes that occurred in promoter methylation in response to folate depletion, or in the subsequent phase of repletion. Although modification of promoter methylation has been observed with folate depletion in some cell culture studies (32
), the severity of deficiency in such pre-clinical models is rather profound and it is entirely feasible that the magnitude of depletion that is encountered in clinical situations is simply not robust enough to alter promoter methylation. Additional caveat is that small shifts in methylation (<5%) may not have been reliably detected by the technology used in this study. A case-control study has explored this issue previously and the results suggested that low folate intake may be related to hypermethylation of the p16
), but this is the first time that any study has directly tested whether folate depletion in the human alters promoter methylation in the colon. The fact that many substantial shifts in gene expression were observed in the depletion/repletion protocol in the absence of changes in gene methylation suggest that the alterations in expression, as well as the changes in DNA strand breaks, were mediated by processes other than changes promoter methylation. This is consistent with a recent review of the topic, which proposed that other molecular anomalies that arise in the setting of folate depletion, such as impaired DNA synthesis and repair, are the primary drivers of enhanced carcinogenesis (34
). Our observations regarding gene-specific methylation are nevertheless limited to the folate depletion-repletion protocol, by the number of subjects with a complete set of samples, and by the marked stringency of the multiple comparisons test we imposed on our data, so we cannot exclude the possibility that some modest changes in promoter methylation occurred that went undetected or that supplementation of folate-replete subjects might induce changes.
We also assessed DNA strand breaks in two exonic loci within the colonic p53 gene. Whereas supplementation produced no apparent changes (data not shown), an increase in exon 6 breaks was observed at week 12 of the depletion protocol; strand breaks in exon 8 followed a nearly identical course but the changes in the latter never achieved statistical significance. This is consistent with an earlier study in rodents, where instability of the so-called ‘hypermutable region’ of the p53 gene was noted to increase progressively with increasing severity of folate depletion (19
). Interestingly, this was one molecular marker that did not reverse at all with repletion; indeed, numerically it continued to increase during the repletion phase, suggesting that the effect is either irreversible or that it takes longer than 4 weeks to return to its former state. The ramifications of single- and double-stranded DNA breaks in the exons of the p53 gene are not at all well understood. On the one hand, they may be a marker of instability of that gene and indicate an increased risk of mutations, which is consistent with the very high rate of mutations found at these loci (35
). On the other hand, base excision DNA repair transiently creates strand breaks (36
), and therefore higher levels of strand breaks may instead indicate exceptionally robust and effective DNA repair activity. Consistent with the last statement was the observed significant increase in gene expression of 2 genes in DNA ligase IV complex at 8 weeks of the study (Supplement S1
, genes XRCC4 and LIG4), although changes in DNA repair pathways overall fell short of statistical significance.
In conclusion, these two studies demonstrate that mild dietary folate depletion over eight weeks decreases the expression of genes involved in pro-inflammatory and immune-related pathways, and that repletion of deplete individuals or supplementation of replete individuals produces changes in expression of these pathways that are reciprocal to those observed with depletion. All of these altered profiles of gene expression occurred in the absence of significant changes in promoter methylation, thereby implicating other mechanisms that would mediate the changes in expression. Over the entire course of the depletion/repletion protocol there was a progressive increase in p53 DNA strand breaks within the hypermutable region of the gene, although the increase did not achieve significance until after the repletion phase, thereby obscuring the phase of the study that was actually responsible for the increase. These data suggest that folate status modulates mucosal inflammation, and are therefore consistent with recent observations in two clinical trials (10
). Although the functional ramifications of what we have observed remain to be fully defined, our observations make it quite clear that alterations in folate availability significantly impact on the molecular milieu of the human colonic mucosa in ways that may modulate the risk of carcinogenesis.