Epidemiological and experimental studies suggest that diminished folate status increases the risk of colorectal carcinogenesis, and that this effect may be mediated through the induction of abnormalities in DNA methylation and nucleotide synthesis. Folate’s biochemical functions within one-carbon metabolism are dependent on adequate availability of several other B-vitamins, including vitamin B2, B6 and B12. This study therefore investigated if mild depletion of vitamins B2, B6 and B12 superimposed upon the effects of mild folate depletion would impact on select molecular events in the colonic mucosa relevant to carcinogenesis: genomic DNA methylation, uracil misincorporation and gene expression. Four-month old C57BL/6J mice (n=108) were divided into 6 groups and pair-fed one of six diets for 10 wks: vitamin replete, mild folate (0 mg/kg) depletion alone, or mild folate depletion combined with mild inadequacies of vitamin B2 (0.5 mg/kg), vitamin B6 (0.5 mg/kg), and/or vitamin B12 (0 mg/kg). Genomic DNA methylation, uracil misincorporation and gene expression were measured in colonic DNA by LC/MS, GC/MS and real-time PCR, respectively. Assays of vitamin status in the blood showed that very mild, but significant, inadequacies of vitamins B2, B6 and B12 were successfully induced in the corresponding groups (p ≤ 0.05). Although genomic DNA methylation in the folate-deplete group (1.55+0.13) was not significantly reduced when compared to the folate-sufficient group (1.60±0.28), DNA hypomethylation (0.90±0.18) did occur with mild folate depletion combined with mild inadequacies of B2, B6, and B12 (P ≤ 0.05). Similarly, minor reductions in the expression of the APC and p53 genes produced by mild folate depletion alone were significantly magnified by the combined depletion of all the abovementioned B-vitamins, resulting in a 5-fold and 16-fold reduction in expression, respectively (P ≤ 0.01). Uracil incorporation was not significantly altered by vitamin depletion (P ≥ 0.05). These findings indicate that a mild depletion of folate that is of insufficient magnitude by itself to produce adverse molecular consequences in the colon may produce such effects when present in conjunction with mild inadequacies of other one-carbon nutrients, producing large decreases in the expression of relevant tumor suppressor genes even in the absence of any genetic predisposition to colon carcinogenesis. This underscores the potential impact that ‘subclinical’ B-vitamin depletions may have when they act in concert with one another.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]