Lynn P. Pirie

Folate Deficiency In Vitro Induces Uracil Misincorporation and DNA Hypomethylation and Inhibits DNA Excision Repair in Immortalized Normal Human Colon Epithelial Cells

Epidemiological studies have indicated that folic acid protects against a variety of cancers, particularly cancer of the colorectum. Folate is essential for efficient DNA synthesis and repair. Moreover, folate can affect cellular S-adenosylmethionine levels, which regulate DNA methylation and control gene expression. We have investigated the mechanisms through which folate affects DNA stability in immortalized normal human colonocytes (HCEC). DNA strand breakage, uracil misincorporation, and DNA repair, in response to oxidative and alkylation damage, were determined in folate-sufficient and folate-deficient colonocytes by single cell gel electrophoresis. In addition, methyl incorporation into genomic DNA was measured using the bacterial enzyme Sss1 methylase. Cultured human colonocyte DNA contained endogenous strand breaks and uracil. Folate deficiency significantly increased strand breakage and uracil misincorporation in these cells. This negative effect on DNA stability was concentration dependent at levels usually found in human plasma (1-10 ng/ml). DNA methylation was decreased in HCEC grown in the absence of folate. Conversely, hypomethylation was not concentration dependent. Folate deficiency impaired the ability of HCEC to repair oxidative and alkylation damage. These results demonstrate that folic acid modulates DNA repair, DNA strand breakage, and uracil misincorporation in immortalized human colonocytes and that folate deficiency substantially decreases DNA stability in these cells.

Sensitivity of markers of DNA stability and DNA repair activity to folate supplementation in healthy volunteers.

We have previously reported that supplementation with folic acid (1.2 mg day−1 for 12 week) elicited a significant improvement in the folate status of 61 healthy volunteers. We have examined effects of this supplement on markers of genomic stability. Little is known about the effect of folate supplementation on DNA stability in a cohort, which is not folate deficient. Preintervention, there was a significant inverse association between uracil misincorporation in lymphocyte DNA and red cell folate (P<0.05). In contrast, there were no associations between folate status and DNA strand breakage, global DNA methylation or DNA base excision repair (measured as the capacity of the lymphocyte extract to repair 8-oxoGua ex vivo). Folate supplementation elicited a significant reduction in uracil misincorporation (P<0.05), while DNA strand breakage and global DNA methylation remained unchanged. Increasing folate status significantly decreased the base excision repair capacity in those volunteers with the lowest preintervention folate status (P<0.05). Uracil misincorporation was more sensitive to changes in folate status than other measures of DNA stability and therefore could be considered a specific and functional marker of folate status, which may also be relevant to cancer risk in healthy people.

Blood Folate Status and Expression of Proteins Involved in Immune Function, Inflammation, and Coagulation: Biochemical and Proteomic Changes in the Plasma of Humans in Response to Long-Term Synthetic Folic Acid Supplementation

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

The response of human colonocytes to folate deficiency in vitro: functional and proteomic analyses.

Low folate intake is associated with colon cancer. We combined a proteomics and biochemical approach to identify proteins and pathways affected by folate deficiency in human colonocytes. Folate differentially altered activity and expression of proteins involved in proliferation [e.g., PCNA], DNA repair [e.g., XRCC5, MSH2], apoptosis [e.g., BAG family chaperone protein, DIABLO and porin], cytoskeletal organization [e.g., actin, ezrin, elfin], and expression of proteins implicated in malignant transformation [COMT, Nit2].

Folate Deficiency Alters Hepatic and Colon MGMT and OGG-1 DNA Repair Protein Expression in Rats but Has No Effect on Genome-Wide DNA Methylation

Folate deficiency is implicated in human colon cancer. The effects of feeding rats a folate-deficient diet for 24 weeks on DNA damage (8-oxo-7,8-dihydroguanine), DNA repair [O6-methylguanine-DNA methyltransferase (MGMT) and 8-oxoguanine-DNA glycosylase (OGG-1) activity], and epigenetic parameters (genome-wide cytosine methylation and indices of cellular methylation status) were investigated. Relative to control diet, the folate-deficient diet resulted in significantly reduced levels of serum (∼80%; P < 0.0001), whole blood (∼40%; P < 0.0001), and tissue folate (between 25% and 60% depending on the tissue sampled; P < 0.05); increased plasma total homocysteine (∼35%; P < 0.05); and decreased S-adenosylmethionine to S-adenosylhomocysteine concentrations (∼11%; P < 0.05). There was no significant change in the levels of 5-methyldeoxycytidine in liver or colon DNA, nor in the activity of liver DNA cytosine methyltransferase. However, there were significant increases in 8-oxo-7,8-dihydroguanine (P < 0.001) in lymphocyte DNA and in levels of the DNA repair proteins OGG-1 (∼27%; P < 0.03) and MGMT (∼25%; P < 0.003) in the liver, but not in the colon. This may reflect the ability of the liver, but not the colon, to upregulate DNA repair enzymes in response to either elevated DNA damage or an imbalance in the nucleotide precursor pool. These results show that folate deficiency can significantly modulate DNA damage and DNA repair, providing mechanisms by which it plays a role in the etiology of human cancer. We speculate that the inability of colon tissue to respond to folate deficiency occurs in humans and may increase the potential for malignant transformation. Cancer Prev Res; 3(1); 92–100

Nutritional B vitamin deficiency disrupts lipid metabolism causing accumulation of proatherogenic lipoproteins in the aorta adventitia of ApoE null mice.

SCOPE Cardiovascular disease is the major cause of death in the world. Low dietary folate, elevated homocysteine, and high circulating cholesterol are risk factors. METHODS AND RESULTS We investigated whether folate and/or B vitamin deficiency would change lipoprotein and fatty acid metabolism and lipid accumulation in the aorta adventitia of ApoE null mice. Mice (n = 10 per group) were fed a control (C; 4%) or high saturated fat (HF; 21%), and high cholesterol (0.15%) diet for 16 weeks. Folate (F-) or folate, B6 and B12 deficiency (F-B-) were imposed on these diets. Feeding a HF diet increased plasma and liver total cholesterol and HDL cholesterol (two- to threefold; p < 0.05). Total cholesterol increased (twofold; p < 0.05) in aorta adventitial lipid in response to HF. Feeding a diet depleted of folate and B vitamins (F-B-) significantly increased cholesterol accumulation in both liver and aorta adventitial lipid (approximately 50-70%; p < 0.05). Moreover, the proportions of fatty acids in hepatic and adventitial lipid was significantly changed by B vitamin depletion, measured as an increase in saturated fatty acids (approximately 15%) and a decrease (approximately 11%) in monounsaturated fatty acids (p < 0.05). CONCLUSION B vitamin deficiency perturbs lipid metabolism in ApoE null mice, causing accumulation of proatherogenic cholesterol and fatty acids in the aorta adventitia.