Fortification of grains has resulted in a positive public health outcome vis-a-vis reduced incidence of neural tube defects. Whether folate has a correspondingly beneficial effect on other disease outcomes is less clear. A role for dietary folate in the prevention of colorectal cancer has been established through epidemiological data. Experimental data aiming to further elucidate this relationship has been somewhat equivocal. Studies report that folate depletion increases DNA damage, mutagenesis, and chromosomal instability, all suggesting inhibited DNA repair. While these data connecting folate depletion and inhibition of DNA repair are convincing, we also present data demonstrating that genetic inhibition of DNA repair is protective in the development of preneoplastic colon lesions, both when folate is depleted and when it is not. The purpose of this paper is to (1) give an overview of the data demonstrating a DNA repair defect in response to folate depletion, and (2) critically compare and contrast the experimental designs utilized in folate/colorectal cancer research and the corresponding impact on tissue folate status and critical colorectal cancer endpoints. Our analysis suggests that there is still an important need for a comprehensive evaluation of the impact of differential dietary prescriptions on blood and tissue folate status.
Folate deficiency has been linked to a variety of pathologic conditions and cancers. Perhaps most notably, folate is required during pregnancy for normal development of the neural tube closure. Once the connection between reduced dietary folate consumption and neural tube defects (NTDs) was well established, the FDA mandated fortification of grain-based foods with folic acid. This mandate resulted in a >25% decrease in incidence of NTDs in the United States [
Folate is ingested from food, primarily from fruits and vegetables in the form of polyglutamated folate, and from folate supplements (primarily folic acid), and is ultimately metabolised into a variety of oxidized and reduced forms with varying levels of methylation, thoroughly reviewed elsewhere [
Micronuclei originate from acentric chromosomes, chromatid fragments, or whole chromosomes that fail to attach properly to the mitotic spindle during anaphase and therefore do not segregate properly during cytokinesis [
Evidence collected from a variety of laboratories over the past decades has demonstrated an accumulation of DNA damage and/or mutations when folate is deficient. The mutagenic response to ENU (ethyl nitrosourea) is greater when folate is deficient [
Folate deficiency has been shown to result in an accumulation of uracil in DNA, a BER substrate, likely through altered thymidylate synthesis and a resulting dUMP/TMP imbalance. Uracil is uniquely removed from DNA by the BER pathway in a DNA-polymerase-
Genome instability phenotypes in base excision repair mutant models.
Gene | Genotype | Phenotype | Genome instability |
---|---|---|---|
UNG |
Ung−/− | Viable |
Uracil accumulation in brain |
| |||
SMUG |
Smugtg/+ |
Viable | C to T mutagenesis |
| |||
OGG1 |
Ogg1−/− | Viable |
8-OHdG accumulation |
| |||
MYH |
Myh−/− |
Viable |
Spontaneous mutagenesis |
| |||
AAG |
Aag−/− | Viable |
Increased mutagenesis |
| |||
NTH | |||
[ |
Nth1−/− |
Viable | Increased thymine glycol in liver after X-ray irradiation |
[ |
Ogg1−/−Nth−/− mice | Viable | Gamma irradiation-induced DSB |
| |||
TDG |
Tdg−/− | Embryonic lethal | Deficient repair of mtDNA |
| |||
MBD4 |
Mbd4−/− |
Viable |
Aberrant chromatin metabolism |
| |||
FEN |
Fen1−/− |
Early embryonic lethal |
Microsatellite instability |
| |||
APE |
Ape−/− |
Embryonic lethal |
|
[ |
Apex1+/−XPC−/− | Increased UV-induced skin cancer | Increased mutagenicity |
| |||
XRCC |
Xrcc1−/− | Embryonic lethal | |
[ |
Xrcc1+/− | Increased AOM-induced ACF | SCE in embryo and cell lines |
| |||
|
|
Embryonic lethal | DSB accumulation |
[ |
|
Viable |
Increased SCE in MEFs |
| |||
LIGI |
Lig−/− | Embryonic lethal defective erythropoiesis | Oxidative stress sensitivity |
| |||
LIGII |
Lig−/− | Embryonic lethal | Elevated SCE |
Biochemistry of base excision repair in uracil removal. Uracil removal is carried out as depicted, with initiation of removal by a uracil-excising DNA glycosylase (UDG depicted). All the uracil-excising glycosylases are monofunctional and leave behind an abasic lesion with an intact DNA backbone. An endonuclease (Apex 1) incises the DNA backbone 5′ to the abasic lesion, generating a 3′hydroxyl group and a 5′deoxyribose flap. A DNA polymerase (DNA polymerase
Accordingly, we have recently shown that the inhibitory effect of folate depletion on BER is achieved in part through inhibiting transactivation of the rate-limiting activity of BER,
Glycosylase-mediated induction of BER begins a series of enzymatic reactions that induces a break in the phosphodiester backbone; a break that persists until repair is completed (Figure
Many epidemiological studies support the protective effect of folate in prevention of colorectal cancer. Most recently, a meta-analysis of 13 human studies shows a positive correlation between folate consumption and protection from colorectal cancer [
Conclusions about the potential dangers of folate on colorectal cancer development may be based, in some instances, on unequal comparisons. A primary objective of this paper is to complete a careful analysis of dietary intervention studies to evaluate the importance that differences in model systems and/or dietary interventions may have on critical colorectal cancer endpoints. In Table
Impact of experimental design on blood and tissue folate.
Animal model | Experimental diet | Abx | Wire cages | Length of feeding | In vivo folate levels | Citation |
---|---|---|---|---|---|---|
Rat studies quantifying impact of dietary intervention on blood and/or tissue folate status | ||||||
| ||||||
Sprague-Dawley rats | Amino acid defined (Dyets) |
No | Yes | 25 weeks | Folate levels (nmol/g) |
[ |
| ||||||
Sprague-Dawley rats | Amino acid defined (Dyets) |
No | Yes | 8 weeks |
|
[ |
| ||||||
Sprague-Dawley rats | AIN-76 semipurified diet |
Yes and no | Yes | 12 weeks | Whole blood folate |
[ |
| ||||||
Sprague-Dawley rats | AIN-76 semipurified diet |
Yes and no | Yes | 26 weeks | Whole blood folate |
[ |
| ||||||
Sprague-Dawley rats | AIN93 (G or M not Specified) |
No | No | 20 weeks | Hepatic folate (nmol/g) |
[ |
| ||||||
Sprague Dawley rats | AIN93 purified diet (G or M not specified) |
No | Yes | 20 weeks | Plasma folate |
[ |
| ||||||
Fischer-344 rats | AIN-93 diet |
No | Yes | 11 weeks | Plasma folate |
[ |
| ||||||
Sprague-Dawley rats | Amino acid defined (Dyets) |
Yes | No | 24 weeks | Plasma folate |
[ |
| ||||||
Male Hooded-Lister |
AIN-93G purified diet with vitamin-free casein |
No | Yes | 6 weeks | Folate value, ng/mg protein |
[ |
| ||||||
Mouse studies quantifying impact of dietary intervention on blood and/or tissue folate status | ||||||
| ||||||
C57bl/6J mice, APCMin | Amino acid defined (Dyets) |
No | No | 3 months; |
Serum folate (ng/mL): |
[ |
| ||||||
C57bl/6 mice, |
AIN-93G purified diet with vitamin-free casein |
Yes | No | 8 weeks | Serum folate |
[ |
| ||||||
C57bl/6 mice, Aag−/− | AIN-93G purified diet with vitamin-free casein |
Yes | Yes | 4 weeks | Liver folate |
[ |
| ||||||
|
||||||
C57bl/6 mice, |
AIN-93G purified diet |
No | Yes | 5 weeks (Apc+/+); 11 weeks (Apcmin/+) | Colon (fmol/ugpro) |
[ |
| ||||||
C57bl/6J mice, Shmt (+/− and −/−) | AIN-93G purified diet |
No | No | 32 weeks |
|
[ |
| ||||||
C57bl/6 mice, APC1638N | Amino acid defined (Dyets) |
No | No | 16 weeks |
|
[ |
| ||||||
C57bl/6 mice, APC1638N |
|
No | No |
|
Maternal |
[ |
| ||||||
Folate depletion studies presenting critical colorectal cancer endpoints, but without folate status information | ||||||
| ||||||
Fisher 344 rats | AIN93G |
Yes and no | No | 5 weeks | ND | [ |
| ||||||
Fisher 344 rats | NIH-31 |
No | No | 36 weeks |
ND | [ |
| ||||||
C57bl/6J mice | Casein/soy based |
No | No | 10 weeks | ND | [ |
| ||||||
BALB/cAnNCrlBR mice | Amino acid defined (Harlan Teklad) |
Yes | No | 12 to 14 months | ND | [ |
| ||||||
C57bl/6 mice Bpol+/− | AIN93G (Dyets) |
Yes | No | 12 weeks total |
ND | [ |
| ||||||
Albino rats | AIN93M |
No | No | 6 weeks total (4 weeks pre-AOM; 2 weeks post-AOM) | ND | [ |
Values in brackets
Impact of dietary intervention on blood and colon folate status.
Percent change in blood folate status by dietary intervention | |||
---|---|---|---|
2 mg/kg to 0 mg/kg |
|
Rat | 20 wk |
(with either abx or wire bottom cages) |
|
Rat | 11 wk |
|
Mouse | 8 wk | |
[ |
|
Rat | 8 wk |
|
Rat | 24 wk | |
| |||
2 mg/kg to 0 mg/kg |
|
Mouse | 12 wk |
(without abx or wire bottom cages) |
|
Mouse | 5 wk |
|
Mouse | 11 wk | |
[ |
|
Mouse | 16 wk |
| |||
2 mg/kg to 8 mg/kg | ↑58% | Rat | 20 wk |
(with either abx or wire bottom cages) | ↑62% | Rat | 8 wk |
[ |
↑140% | Rat | 24 wk |
| |||
2 mg/kg to 8 mg/kg | |||
(without abx or wire bottom cages) | ↑44% | Mouse | 12 wk |
[ |
|||
| |||
Percent change in colon folate status by dietary intervention | |||
| |||
2 mg/kg to 0 mg/kg |
|
Rat | 20 wk |
(with either abx or wire bottom cages) |
|
Rat | 8 wk |
[ |
|||
| |||
2 mg/kg to 0 mg/kg |
|
Mouse | 5 wk |
(without abx or wire bottom cages) |
|
Mouse | 16 wk |
[ |
|||
| |||
2 mg/kg to 8 mg/kg | ↑19% | Rat | 20 wk |
(with either abx or wire bottom cages) | ↑66% | Rat | 8 wk |
[ |
|||
| |||
2 mg/kg to 8 mg/kg | |||
(without abx or wire bottom cages) |
Abx: antibiotics; wk: week; *choline also depleted in this dietary intervention; **riboflavin, B6, and B12 also modified in this dietary intervention.
Impact of experimental design on critical colorectal cancer endpoints.
Animal model | Carcinogen | CRC-specific endpoints measured |
---|---|---|
Studies demonstrating beneficial effects of folate on critical colorectal cancer endpoints | ||
Rat |
DMH |
|
| ||
Rat |
5-week diet prior to DMH |
|
| ||
Rat |
DMH |
|
| ||
Rat |
3-week diet prior to DMH |
|
| ||
Mice |
None, diet only |
|
| ||
Mice |
None, diet and genotype only |
|
| ||
Mice |
None, diet and genotype only |
|
| ||
Mice |
None, diet and genotype only |
|
| ||
Mice |
DMH |
|
| ||
Rat |
AOM |
|
| ||
Rat |
AOM |
|
| ||
Rat |
AOM |
|
| ||
Mice |
None, diet and genotype only |
|
| ||
Mice |
DMH |
|
~indicates values are approximated from graphical data; N/A: not available; Shmt: serine hydroxyl methyl transferase.
Duration of dietary intervention appears to affect the impact on folate status. Very few studies have measured colonic folate status in response to dietary depletion, which is the target tissue of interest for this paper. But in two papers in which this was determined there seems to be a significant impact of increasing the length of the study on colonic folate levels. After 8 weeks of feeding, rats exhibit a 35% decrease in colon folate levels [
There also seems to be a differential sensitivity to folate depletion between mice and rats. From the limited data available, mice appear to become severely depleted (>90% reduced blood folate) after 8 weeks of feeding [
The information in Table
These considerations aside, there is a definite impact of altering blood and tissue folate status on colorectal cancer endpoints. And these differences seem to be clearly dependent on the stage of cancer development. Table
In mouse studies, data are confounded by genotype differences in models predisposed to develop gastrointestinal tumors, as well as other genetic manipulations devised to investigate the role(s) of certain pathways on colon tumorigenesis. In total we present 4 mouse studies showing a protective effect of folate on colon tumorigenesis, and 2 studies showing both detrimental and protective effects. Each study presents its own limitations preventing direct comparisons and solid conclusions. For example, in the
In two studies using a different APC model, the APC1638N mouse, riboflavin, B6, and B12 deficiencies were investigated along with folate deficiency such that the conclusions are not specific to folate. Additionally, both these studies avoided use of antibiotics and wire-bottom caging, so the impact of dietary intervention on folate status was moderate (see Table
In the only study to investigate tumorigenesis in response to folate depletion in a mouse strain other than C57bl/6, Knock et al. have shown that folate depletion increased the number of duodenal tumors in the BALB/c strain [
Two studies present data demonstrating both protective and detrimental effects of folate on critical endpoints. Song et al. [
It becomes clear that while each study presents important information regarding the impact of folate on genomic stability in the colon, that there is some problem with a lack of consistency across study designs that prevent us from arriving at definitive conclusions. As the body of literature on folate continues to grow, these gaps in knowledge will be filled. We suggest that there is still an important need for a comprehensive study investigating the impact of differential folate prescriptions on blood and tissue folate status. We have shown here that the duration of feeding, dosage of folate, and use or avoidance of antibiotics and/or wire-bottom caging all impact the severity of folate depletion. Another point to consider is the difference in total blood folate levels between rodents and humans. The normal range for serum folate in humans is 2.7–17 ng/mL, manyfold lower than the average values observed in mice and rats. The range for mouse values reported in Table
This work was supported by a grant from the Ellison Medical Foundation [DCC].