Older age and dietary folate are determinants of genomic and p16-specific DNA methylation in mouse colon.

Older age and inadequate folate intake are strongly implicated as important risk factors for colon cancer and each is associated with altered DNA methylation. This study was designed to determine the effects of aging and dietary folate on select features of DNA methylation in the colon that are relevant to carcinogenesis. Old (18 mo; n = 34) and young (4 mo; n = 32) male C57BL/6 mice were randomly divided into 3 groups and fed diets containing 0, 4.5, or 18 mumol folate/kg (deplete, replete, and supplemented groups, respectively) for 20 wk. Genomic DNA methylation and p16 promoter methylation in the colonic mucosa were analyzed by liquid chromatography/electrospray ionization/MS and methylation-specific PCR, respectively. p16 gene expression was determined by real-time RT-PCR. Old mice had significantly lower genomic DNA methylation compared with young mice at each level of dietary folate (4.5 +/- 0.2, 4.8 +/- 0.1, and 4.9 +/- 0.1 vs. 6.0 +/- 0.1, 5.3 +/- 0.2, and 5.9 +/- 0.2%, in folate-deplete, -replete, and -supplemented groups, respectively, P < 0.05) and markedly higher p16 promoter methylation (61.0 +/- 2.7, 69.7 +/- 6.9, and 87.1 +/- 13.4 vs. 10.8 +/- 3.6, 8.4 +/- 1.8, and 4.9 +/- 1.7%, respectively, P < 0.05). In old mice, genomic and p16 promoter DNA methylation each increased in a manner that was directly related to dietary folate (P(trend) = 0.009). Age-related enhancement of p16 expression occurred in folate-replete (P = 0.001) and folate-supplemented groups (P = 0.041), but not in the folate-deplete group. In conclusion, aging decreases genomic DNA methylation and increases promoter methylation and expression of p16 in mouse colons. This effect is dependent on the level of dietary folate.

Folate supplementation increases genomic DNA methylation in the liver of elder rats.

The availability of folate is implicated as a determinant of DNA methylation, a functionally important feature of DNA. Nevertheless, when this phenomenon has been examined in the rodent model, the effect has not always been observed. Several reasons have been postulated for the inconsistency between studies: the rodent is less dependent on folate as a methyl source than man; juvenile animals, which most studies use, are more resistant to folate depletion than old animals; methods to measure genomic DNA methylation might not be sensitive enough to detect differences. We therefore examined the relationship between folate and genomic DNA methylation in an elder rat model with a newly developed method that can measure genomic DNA methylation sensitively and precisely. Thirty-nine 1-year-old rats were divided into three groups and fed a diet containing 0, 4.5 or 18 mumol folate/kg (folate-deplete, -replete and -supplemented groups, respectively). Rats were killed at 8 and 20 weeks. At both time points, mean liver folate concentrations increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend <0.001) and by 20 weeks hepatic DNA methylation also increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend=0.025). At both time points folate-supplemented rats had significantly increased levels of DNA methylation compared with folate-deplete rats (P<0.05). There was a strong correlation between hepatic folate concentration and genomic DNA methylation in the liver (r 0.48, P=0.004). In the liver of this animal model, dietary folate over a wide range of intakes modulates genomic DNA methylation.