Gastroenterology

Gastroenterology

Volume 136, Issue 1, January 2009, Pages 227-235.e3
Gastroenterology

Basic—Alimentary Tract
Folate Deficiency Induces Genomic Uracil Misincorporation and Hypomethylation But Does Not Increase DNA Point Mutations

https://doi.org/10.1053/j.gastro.2008.10.016Get rights and content

Background and Aims

Epidemiologic studies have linked nutritional folate deficiency to an increased risk of cancer, but recent trials suggest that folate supplementation does not protect against tumor formation. Our aim was to analyze the genetic and epigenetic consequences of folate deficiency and to investigate whether impairment of the uracil base excision repair pathway can enhance its effects.

Methods

Wild-type mice and those deficient in uracil DNA glycosylase (Ung−/−) were placed on a folate-deficient diet for 8 months. We measured tumor incidence in major organs, DNA mutation rates, DNA mutation spectra, local DNA methylation, and global DNA methylation in colon epithelial cells.

Results

The experimental diet increased plasma homocysteine (60%, P < .001) and DNA uracil content (24%, P < .05) but not tumor formation. Global DNA methylation was slightly decreased in splenocytes (9.1%) and small intestinal epithelial cells (4.2%), and significantly reduced in colon epithelial cells (7.2%, P < .04). No gene-specific changes in methylation were detected at the mouse B1 element, the H19 DMR, or the Oct4 gene. By λ CII assay and sequencing analysis of 730 mutants, we found that Ung−/− mice had a higher frequency of point mutations and increased C:G to T:A transitions at non-CpG sites. However, folate deficiency had no additional effect on the DNA mutation frequency or spectrum in Ung−/− or wild-type mice.

Conclusions

Contradicting current concepts, these findings indicate that the effects of a low-folate diet on DNA methylation and point mutations are insufficient to promote tumor development, even in the presence of Ung deficiency.

Section snippets

Experimental Mice and Diet Schedule

Big Blue Mice with the CII transgene (C57BL/6J background) were purchased from Stratagene (La Jolla, CA) and crossed with Ung−/− mice (129 background) that were previously generated in our lab.25 Ung+/− intercrosses with one parent Big Blue+/− generated the 2 experimental genotypes Big Blue+/−, Ung+/+ and Big Blue+/−, Ung−/−. The genetic background of all mice was 50% C57BL/6J and 50% 129. To control for genetic background effects, we evenly distributed littermates of the same genotype between

Folate Deficiency Causes Increased Plasma Homocysteine But Does Not Increase Tumor Numbers

To verify that the experimental diet was successful in achieving folate deficiency, we analyzed plasma folate levels at 3 months and 8 months of dietary intervention (Figure 1). Severe reduction of folate levels was seen after 3 months and persisted until the end of the experiment (88% decrease, P < .001). Parallel to the observed decrease in plasma folate, we detected an increase in plasma homocysteine (60% increase at 8 months, P < .001). Folate deficiency caused a moderate but not

Discussion

The primary goal of our study was to evaluate the genetic and epigenetic consequences of folate deficiency to help clarify the role of this nutrient in tumor development. Also, we wanted to investigate whether lack of the DNA repair enzyme Ung can increase point mutations during folate deficiency. We improved on previous studies by using a long-term dietary intervention (8 months) and by focusing on tissues with a high cell turnover rate, which is important for detecting passive loss of DNA

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    The authors disclose the following: H.G.L. is supported by a postdoctoral fellowship from the Fritz-Thyssen Stiftung. This work was supported by Philip Morris International and National Institutes of Health grant NIH RO1-CA087869 to R.J., and by the US Department of Agriculture, Agricultural Research Service (agreement No. 58-1950-7-707). Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the US Department of Agriculture.

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