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Artilce

Biochemical and Molecular Aberrations in the Rat Colon Due to Folate Depletion Are Age-Specific

Sang-Woon Choi^*,3, Simonetta Friso^,#, Gregory G. Dolnikowski^**, Pamela J. Bagley, Antoinette N. Edmondson, Donald E. Smith and Joel B. Mason^*,

^* Vitamins and Carcinogenesis Laboratory, Vitamin Metabolism Laboratory, ^** Mass Spectrometry Laboratory and Comparative Biology Unit, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA; Divisions of Clinical Nutrition and Gastroenterology, Tufts University School of Medicine, Boston, MA; School of Pharmacy, University at Buffalo, Buffalo, NY; ^# Department of Clinical and Experimental Medicine, Policlinico G.B. Rossi, University of Verona, Verona, Italy

³To whom correspondence should be addressed. E-mail: sang.choi{at}tufts.edu.

ABSTRACT

Elder adulthood and diminished folate status are each associated with an enhanced risk of colorectal carcinogenesis. We therefore examined whether these two factors are mechanistically related. Weanling male Sprague-Dawley rats (n = 44) and 1-y-old rats (n = 44) were each divided into three groups and fed diets containing 0, 4.5 or 18 µmol folic acid/kg (deplete, replete and supplemented groups, respectively). Rats were killed at 0, 8 and 20 wk. The folate concentrations, the distribution of the different coenzymatic forms of folate, uracil incorporation into DNA and genomic DNA methylation were measured in the colonic mucosa. Folate-deplete and folate-replete elder rats had 30–45% lower colonic folate concentrations than young rats. Furthermore, 5-methyltetrahydrofolate was uniformly depleted in colons of the elder, folate-deplete rats, whereas this depletion occurred in only a minority of the younger rats. By the end of the experiment, the folate-deplete and folate-replete elder rats had 50% more uracil incorporated into their colonic DNA than the corresponding young groups (P < 0.05). In elder rats, this uracil misincorporation was incremental across the three diet groups (P-test for trend < 0.05), whereas no excess uracil incorporation was observed in young rats. Neither age nor dietary folate affected genomic DNA methylation in the colon. In conclusion, the colon of elder rats is more susceptible to biochemical and molecular consequences of folate depletion than that of young rats. However, folate supplementation is as effective at sustaining adequate colonic folate status in elder rats as it is in the young.

KEY WORDS: • folate • colon cancer • aging • rats • uracil misincorporation

A growing body of evidence from epidemiologic, animal and human studies suggests that diminished folate status leads to an increased risk of colorectal cancer (1 ). Animal studies, as well as a few preliminary clinical trials, suggest that supplementation of folate may convey a protective effect against development of this cancer (2 –6 ). Two of the leading putative mechanisms to explain the procarcinogenic effect of folate depletion are inappropriate uracil incorporation into DNA and aberrations in DNA methylation (7 ), both of which are phenomena whose normal homeostasis is dependent on adequate availability of one-carbon groups donated by folate coenzymes (1 ,8 –10 ).

Age is perhaps the single strongest determinant of colorectal cancer risk, with the risk doubling each decade after the age of 40 y (11 ). Although age is not a predominant risk factor for diminished folate status, several studies suggest that the elderly may be more susceptible under some circumstances (12 ,13 ) due to dietary preferences or an impaired ability to assimilate folate from dietary sources. For example, Russell et al. (14 ) showed that atrophic gastritis, an eminently age-associated condition, impairs folate absorption. However, whether age alone is a risk factor for diminished systemic folate status, it is apparent that some tissues, such as the colon, are more susceptible to folate depletion than others (15 ). Therefore, demonstrating an age-related susceptibility to folate depletion may be obscured if an examination of the relevant tissue is not undertaken.

Folate-derived one-carbon groups are essential for the de novo synthesis of thymidylate, as well as the purines. Because deoxyribonucleotides are the substrates for the polymerases involved in DNA replication and repair, the fidelity of DNA synthesis is critically dependent on the correct balance and availability of deoxynucleotides (7 ). Disruptions in intracellular nucleotide pools, such as occurs with folate deficiency (16 ) or pharmacologic inhibition of folate metabolism (17 ), lead to inappropriate base substitution, and this loss of replicative fidelity greatly enhances the risk of neoplastic transformation (18 ). In mammalian cells, the de novo synthesis of thymidylate from deoxyuridylate is a rate-limiting step for DNA synthesis, and inhibition of folate metabolism results in inappropriate uracil incorporation into DNA (19 ). Recent observations have demonstrated that folate depletion in humans results in excess uracil incorporation into DNA as well (10 ,20 ).

Folate is also essential for the synthesis of S-adenosylmethionine (SAdoMet), the universal donor for biological methylation reactions. SAdoMet donates its labile methyl group, obtained from 5-methyltetrahydrofolate, to >80 methylation reactions, including those reactions whereby specific sites within DNA become methylated. Although the alternative betaine pathway for methionine synthesis may partially compensate, dietary folate depletion alone is a sufficient perturbing force to impair biological methylation (21 ). Although the cellular pool of SAdoMet may diminish with folate depletion (22 ,23 ), the more consistent consequence of depletion (and the one that appears to play a larger role in impairing biological methylation) is the rise in S-adenosylhomocysteine (SAdoHcy), a potent inhibitor of methylation reactions (24 ,25 ). Undermethylation due to folate depletion is pertinent to carcinogenesis because a decreased level of genomic methylation is a nearly universal finding in neoplasms, i.e., this has been observed in cancers of the colon, stomach, uterine cervix, prostate, thyroid and breast (26 ). Genomic hypomethylation has been observed in several animal models of carcinogenesis as well, including cancers of the intestine (27 ,28 ). This decrease in genomic methylation appears early in carcinogenesis and precedes the better-described mutation and deletion events that occur later in the evolution of cancer. Increasing evidence implicates aberrations in methylation as playing a mechanistic role in carcinogenesis (29 ).

We therefore sought to determine the relationships between age and folate status in the target tissue of interest, the colonic mucosa. We used a well-described rodent model of chronic, moderate folate depletion (2 ,22 ,30 ,31 ), a considerably more clinically relevant diet than the short-term, severe deficiency model (4 ,32 ).

Juvenile and elder rats were fed a folate-deplete, folate-replete or folate-supplemented diet for 20 wk. Total folate concentration, distribution of folate coenzymatic forms and folate related-metabolites such as SAdoMet and SAdoHcy were measured in the colonic mucosa. We also measured two molecular end points in the colon, uracil incorporation into DNA and genomic DNA methylation, which are each thought to be instrumental in folate-related colorectal carcinogenesis, as described above.

MATERIALS AND METHODS

Animals.

This study was approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University. Young (weanling, n = 44) and middle-aged (12 mo old, n = 44) male Sprague-Dawley rats (Zivic-Miller, Zelienople, PA) were assigned randomly to one of three identical amino acid–defined diets except that they contained three different folate levels: 1) 0 µmol folate/kg (folate-deplete), 2) 4.5 µmol/kg (basal requirement of folate, i.e., replete), or 3) 18 µmol/kg (folate-supplemented state) (Dyets, Bethlehem, PA). These amino acid–defined diets constitute a standard means of predictably inducing folate deficiency and repletion in rodents (33 ,34 ).

Rats were housed individually in wire-bottomed stainless steel cages to minimize coprophagy. Body weights were recorded biweekly. Rats drank water ad libitum. The amount of food supplied to each diet group was matched to the mean daily food consumption of the group within that age category with the least food consumption. Five young and five elder rats were killed before beginning the study diets to establish baseline values. At 8 wk after initiation of the diets, five rats from each group were killed. At 20 wk, eight rats from each group were killed. Rats were anesthetized in a carbon dioxide breathing chamber, and the abdomen was then opened. Plasma and colonic mucosa samples were immediately obtained from the rats, which were then killed by cross-cutting the aorta. Complete blood counts and mean corpuscular volume were analyzed immediately (System 9000, Serono Baker Diagnostic, Allentown, PA). Plasma samples were stored at -70°C in 25 mmol/L ascorbic acid for subsequent folate assays. The colorectum was excised and opened longitudinally on an ice-cold glass plate. After being washed with ice-cold saline, the mucosa was scraped off gently with glass microscope slides, as previously described (35 ). The preparation contained not only epithelial cells but submucosal cells as well. Aliquots of mucosal scraping were flash-frozen in liquid nitrogen and subsequently stored at -70°C.

Measurement of total folate concentration and folate form distribution.

Plasma and dietary folate concentration were determined by a conventional microbiological microtiter plate assay using Lactobacillus casei (36 ). Total folate concentration of colon was determined by the same method after treatment with chicken pancreas conjugase (36 ).

Folate distribution analyses of the colonic mucosa were determined by a previously validated affinity/HPLC method using an ESA Four Channel Coularray Detector (ESA, Chemsford, MA), which gives a unique, characteristic electrochemical response for the different pteridine ring structures of folate (37 ). Quantitation and identification of individual folates were done by comparison with external folate standards of known concentrations.

SAdoMet, SAdoHcy and homocysteine concentrations.

Colonic SAdoMet and SAdoHcy concentrations were measured by HPLC with UV detection using the method described by Fell et al. (38 ) with slight modifications as recently reported (21 ). Total plasma homocysteine (tHcy) was determined by HPLC using the fluorometric method of Vester and Rasmussen (39 ).

Uracil misincorporation.

The amount of uracil in DNA was measured by a previously described gas chromatography/mass spectrometry method (10 ,19 ). Uracil was removed from 15 µg DNA by incubating with 4 U uracil DNA glycosylase for 3 h at 37°C. After the glycosylase digestion, 4,5-¹³C₂ uracil (200 pg; Cambridge Isotope Laboratories, Cambridge, MA) was added as an internal standard and the samples were dried in a speed vacuum concentrator. The residue was resuspended in a mixture of 50 µL acetonitrile, 10 µL triethylamine and 1 µL 3,5-bis(trifluoromethyl)benzyl bromide and mechanically shaken for 25 min at 37°C to derivatize the uracil, followed by the addition of 50 µL water (HPLC grade) to improve the extractability. N1,N3-(3,5-bis[trifluoromethyl]benzyl)uracil, a derivatized form, was extracted into 100 µL isooctane. The sample (5 µL) was injected onto a Series 6890 gas chromatograph equipped with a 7683 automated liquid sampler (Agilent Technology, Palo Alto, CA) in the splitless mode. Adequate separation of ura-diBTFMBz from interfering compounds was achieved on a capillary column (HP-5MS, Hewlett Packard, Billerica, MA). A 5973N mass selective detector (Agilent Technology) using negative chemical ionization/mass spectrometry was used for analysis, with elected ion monitoring at m/z 337 and 339.

DNA methylation.

Liquid chromatography/electrospray ionization mass spectrometry (Hewlett Packard/Bruker) was used to analyze genomic DNA methylation. We previously described and validated a detailed method (40 ,41 ).

Briefly, 1 µg of DNA was hydrolyzed by sequential digestion with three enzymes, nuclease P1 (Roche Molecular Biochemicals, Mannheim, Germany), venom phosphodiesterase I (Sigma, St. Louis, MO) and alkaline phosphatase (Sigma) (42 ). The hydrolyzed DNA solution was delivered directly onto the analytical column (Supelco, Bellefonte, PA) in isocratic mode. This allowed the separation of the four DNA bases as well as the identification of 5-methylcytosine. Electrospray ionization mass spectrometry was performed in positive ion mode. Identification of cytosine and 5-methylcytosine was obtained by mass spectrometric analysis of chromatographic peaks. The isotopomers ¹⁵N₃ 2'-deoxycytidine and methyl-D3, ring-6-D1 5-methyl-2'-deoxycytidine (Cambridge Isotope Laboratories) were used as internal standards. DNA methylation status was defined as the percentage of 5-methylcytosine of the overall amount of cytosine and methylcytosine.

Statistical analysis.

Data were analyzed by two-way ANOVA for the age and dietary folate at each time point. When the F-test was significant, means that differed were identified by a two-tailed Student’s t test. Logarithmic transformation was performed on all skewed variables to normalize their distributions and statistical comparison was based on log-transformed data. The P-trend was analyzed by regression. The level of significance was set at P < 0.05 for all analyses. Statistical analysis was performed using Systat 7.0.1 for Windows (SPSS, Chicago, IL). Values in the text are means ± SEM.

RESULTS

The body weight of the young rats increased progressively during the entire experiment and there were no differences among the diet groups. However, elder rats fed folate-deplete diets lost significantly more weight than did folate-replete and -supplemented rats, starting at 10 wk. All rats survived the 20-wk experiment except two elderly rats from the folate-deplete group who died at 8 and 19 wk.

Young rats fed the folate-deplete diet developed significantly lower blood hemoglobin concentrations compared with the folate-replete and -supplemented groups after 20 wk (Table 1 ), whereas elder rats fed a folate-deplete diet developed a significantly lower mean hemoglobin level than rats fed the folate-supplemented diet by 8 wk. By 20 wk, one of the elder folate-deplete rats had developed a very low hemoglobin of 63 g/L, resulting in a wide variance in values, thereby obscuring our ability to detect a significant difference between the mean hemoglobin concentrations in the elder deplete group compared with the replete and supplemented groups. Mean corpuscular volume was significantly higher in both folate-deplete groups by 20 wk compared with replete and supplemented rats.

By 20 wk, folate depletion in the elder rats produced a 99% decline in the proportion of total colonic folate in the form of methylated folates (P < 0.001) and a commensurate increase in the proportion of folates containing formyl and methylene substitutions (the last-mentioned two forms coelute in the same peak) (P = 0.018) (Table 3 ). Young folate-deplete rats also had a lower proportion of methylated folates (P < 0.01) and a greater proportion of formylated folates (P < 0.05) compared with young folate-replete and -supplemented groups. However, in contrast to the elder rats, the decrease of methylated folates with depletion was quite heterogenous among the young rats. Only 2 of 5 rats experienced a drop, leading to a mean with a large variance (see Table 3 ). In both young and elder rats, the effect on methylated folates was a stepwise decrease, and the effect of formylated folates was a stepwise increase across the three diet groups (P-test for trends, 0.011–0.001).

Elder rats had a significantly higher mean tHcy concentration than young rats at baseline (Table 2) . With folate depletion, both young and elder rats had a 9- to 10-fold rise in tHcy levels over 20 wk, although the absolute rise in tHcy levels was much greater in the elder rats. Interestingly, folate supplementation in the elder rats resulted in significantly lower plasma tHcy concentrations compared with the young rats. Thus, the elder rats’ tHcy level, which is an inverse index of folate status, was greater than that in the young rats at baseline, but more responsive to folate supplementation.

By 20 wk, the folate-deplete and folate-replete elder rats had a 50–60% increase in the uracil incorporated into colonic DNA compared with the corresponding young groups (P < 0.05). In elder rats, the effect was incremental across the three diet groups (folate-deplete group, 45.8 ± 6.2 fmol/µg DNA; folate-replete group, 39.7 ± 2.2 fmol/µg DNA;, folate-supplemented group, 35.0 ± 2.7 fmol/µg DNA; P-test for trend < 0.05; Fig. 1 ). Young rats fed the three diets did not differ in uracil misincorporation. Although a very similar pattern of uracil incorporation in the various groups was observed at 8 wk (data not shown), only at 20 wk were the differences significant. At supplemental levels of dietary folate (18 µmol/kg diet), the elder and young rats had comparable levels of uracil in their DNA.

View larger version (34K): [in this window] [in a new window]   FIGURE 1 Uracil incorporation into colonic DNA according to dietary folate status at 20 wk in young and elder rats. Values are means ± SEM, n = 8. *Different from young rats consuming the same diet, P < 0.05. In elder rats, the effect was incremental across the diet groups. (P-trend < 0.05).

DISCUSSION

Among the factors that affect the risk of colon cancer, age is regarded as one of the most important. The risk for colorectal cancers begins to increase slightly at age 40 y, increases more sharply at age 50 y, doubling with each decade, and reaching a maximum at age 75 y (43 ). Because folate status is also thought to be a determinant of colorectal cancer risk in humans (44 ,45 ) and in rodent models (2 ,46 –48 ), we were interested in determining the effect of aging on the response to different dietary folate levels by measuring folate-related metabolites and molecular markers in the colon.

This study provides strong evidence that biochemical indicators of folate depletion are considerably more prominent in the colon of elder animals than in the juvenile colon. Age-related differences in systemic folate status were less evident. Very importantly, these biochemical alterations in the colon of the elder rats were accompanied by a molecular aberration in DNA, uracil misincorporation. These two observations are consistent because folate depletion diminishes cofactor availability for the reaction in which thymidine is synthesized from uridine. It thereby disturbs the intracellular uridylate:thymidylate ratio and has been shown previously to produce excess uracil incorporation into white blood cell DNA (10 ,19 ).

Elder age had a prominent effect on total colonic folate concentrations. A more modest effect on plasma levels of folate was observed, and was evident only in those consuming the diet containing the least amount of folate. In all instances, elder rats fed the deplete and replete diets had a colonic folate concentration that was about one half to two thirds of the concentration in the corresponding young rats. Even when fed a diet that was considered to fully meet their metabolic needs (49 ), elder rats did not have as high a colonic folate concentration as young rats. This was not a subtle effect, i.e., by the end of the study, elder rats fed the "replete" diet had a 36% lower level of folate in the colon. This observation indicates that the colon of elder rats is more susceptible to folate depletion. On the other hand, a level of dietary folate that is four times the level presently considered to be basal requirement is equally effective in elder rats in sustaining colonic folate concentrations. To achieve the concentrations of folate observed in the colon of young rats, elder rats require more dietary folate than is provided by a "replete" diet.

In 1990, Holt suggested that the state of hyperproliferation that occurs in the aging intestinal tract might act in concert with genetic and environmental factors in the induction or promotion of gastrointestinal carcinogenesis (50 ). On the other hand, folate supplementation has been observed to decrease colorectal mucosal proliferation in both animal and human studies (51 ,52 ). From these observations, we hypothesize that adequate dietary folate may minimize aging-related colorectal mucosa hyperproliferation, an early step in colorectal carcinogenesis.

The heightened susceptibility to a selective depletion of methylfolate that we observed in the colon of elder rats is reminiscent of the pattern observed with the common 677CT polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene. In 1997, Ma et al. (53 ) reported that men with the homozygous mutation have half the risk of colorectal cancer as the homozygous wild-type or heterozygous genotypes. Among men with adequate folate levels, a 60% decrease in risk was observed. However, protection associated with the mutation was largely absent in men with low systemic folate status. Indeed, subsequent studies of this nature indicated that homozygosity for the MTHFR polymorphism in combination with low folate status may actually increase the risk of colorectal cancer (54 –56 ). Under low, but not high folate conditions, availability of 5-methyltetrahydrofolate for biological methylation in the homozygous MTHFR mutant is so compromised (40 ,57 ) that it presumably becomes the critical determinant of whether the cell is directed down the pathway towards neoplasia (7 ). Although in this study folate depletion produced a disproportionately large decrease in methylfolates in colons of both young and elder rats, the effect was more uniform and of larger magnitude in the latter. Therefore, folate depletion in the elder colon may mimic the situation in folate-depleted individuals with the homozygous C677T MTHFR mutation. The shift away from methylated folates that accompanied folate depletion in this study may represent a compensatory mechanism to supply adequate cofactor for nucleotide synthesis; if so, it is a mechanism that is apparently sufficient for compensation in young, but not elder rats.

The excess uracil incorporation into DNA in folate-deplete and -replete elder rats, a phenomenon not observed in the corresponding young rats, has important implications for carcinogenesis. Thus, the provision of dietary folate at less than supplemental levels in elder rats leads to abnormal base substitution in colonic DNA which is thought to make mutations (16 ,58 ) and DNA strand breaks more likely (10 ,59 ). Repair of uracil residues that are closely spaced on the opposite strands of plasmid DNA results in double-strand breaks, deletions (60 ), chromosomal breaks (61 ,62 ), micronucleus formation (63 ) and loss of heterozygosity (64 ). In turn, double-strand DNA breaks increase the chances of translocations, rearrangements, deletions and duplications that can activate protooncogenes or inactivate tumor suppressor genes (65 ). The effect of age was quite marked, i.e., elder rats that were either deplete or replete had 60% more uracil in their DNA than the corresponding young rats. Only with folate supplementation were uracil levels in the colon of elder rats as low as the values in the young rats.

Although the age-specific consequences of folate depletion were most obvious in the colon, there were some systemic indications of heightened susceptibility to depletion in the elder rats. Weight change was the initial phenomenon we observed in this regard. Paradoxically, actively growing young rats, which are traditionally perceived as having high folate requirements per gram of body weight, did not show any effect of moderate folate deficiency on their growth and looked healthy at the end of the 20-wk folate-deplete diet period, whereas elder rats fed a folate-deplete diet lost significantly more weight than folate-replete and -supplemented rats. Furthermore, two elder, folate-deplete rats died before the end of study and one additional elder rat fed the deplete diet developed severe anemia at the end of the study. Elder rats, therefore, were more susceptible to the chronic folate-deficient diet than young rats in these respects. This is supported by the small, but significantly lower plasma folate concentration in the elder rats at the end of the study as well as the higher homocysteine levels. tHcy concentration is regarded as an excellent marker of folate status (66 ). In this study, elder rats had a significantly higher tHcy concentration than the young rats at baseline as well as after depletion. This is entirely consistent with human studies (67 ,68 ). By itself, the higher homocysteine level does not prove that elder rats have lower folate status because the age-related elevation in tHcy appears to be due to age-specific differences in homocysteine metabolism as well (67 ). More important is the fact that the folate-supplemented group of elder rats had significantly lower tHcy concentrations during the entire experiment compared with the young rats. This observation suggests that folate supplementation, at four times the basal requirement, is as effective at eliminating evidence of intracellular inadequacy of folate in elder rats as it is in the young.

Both plasma and colonic folate concentrations reflected the level of dietary folate in all instances (Table 2) . These observations agree with earlier studies demonstrating that this animal model was an excellent one for predictably producing varying levels of folate status (2 ,4 ,22 ,30 ,32 ). Plasma folate concentration did not differ between the age groups in those fed the folate-replete and -supplemented diets. However, as mentioned above, the elder rats fed a folate-deplete diet developed a significantly lower plasma folate concentration than the juvenile rats. This finding is in partial agreement with a previous observation by Horne et al. (68 ), in which aged rats developed diminished serum levels of the vitamin after consuming both deplete and replete diets. Nevertheless, the essential feature of both the study of Horne et al. and our study is that elder rats have difficulty maintaining adequate folate status.

Over the wide range of folate status that was examined in this study, folate availability is a determinant of colonic SAdoHcy concentrations. Stepwise decreases in dietary folate led to stepwise increases in colonic SAdoHcy in both the young and elder rats. Although some of our earlier studies did not find colonic SAdoHcy to be responsive to dietary folate depletion (2 ,22 ), such responsiveness might become apparent only with the extended period of folate depletion that was used in this experiment. This is consistent with other studies that have observed that the magnitude of change in SAdoHcy concentrations as a result of alterations in folate status are considerably larger than those in SAdoMet because the concentration of the latter seems to be more tightly controlled (69 ). Although SAdoHcy is a direct inhibitor of methylation reactions (25 ), it did not appear to affect genomic DNA methylation status in the rat colon in this experiment. This does not exclude the possibility that the observed increases in SAdoHcy might have produced gene-specific hypomethylation of DNA, as was observed previously in a folate-deficient rat study in the absence of genomic hypomethylation (23 ).

In conclusion, these data indicate that the colonic mucosa of elder rats is more susceptible to folate depletion, and its consequences, than juvenile rats. Unlike young rats, depletion results in a predictable shift in the forms of folate present in the colon, as well as increased uracil incorporation into DNA. However, folate supplementation at four times the basal requirement is just as effective at eliminating evidence of inadequate folate status in elder rats as it is in the young. Although not proven by this study, this causal relationship between age and folate status in the colon suggests one means by which age may modulate colorectal cancer risk. These data also suggest that this animal model holds excellent potential for further studies that examine the interaction of folate status and age on colorectal carcinogenesis. Last, the observations in this study indicate that the age of a rodent has a substantial effect on colonic physiology and therefore may be an important factor to consider when such animals are used as a model for colorectal carcinogeneis. Molecular features of the human colonic mucosa related to DNA methylation have also been observed to change with aging (11 ), and the age-related alterations in one-carbon metabolism in the aging colon observed in this study may provide insight into the reasons for this changing environment.

FOOTNOTES

¹ Presented in poster form at Digestive Disease Week, May 2000, San Diego, CA [Choi, S. W., Bagley, P. J., Gee, A., Tsui, K. & Mason, J. B. (2000) The colorectal mucosa of old rats is more susceptible to folate depletion than young rats: implications for folate-related colorectal carcinogenesis. Gastroenterology 118: A70 (abs.)].

² This material is based upon work supported by the U.S. Department of Agriculture, under agreement No. 581950–9-001. Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Dept of Agriculture. This project was supported in part by the Cancer Research Foundation of America (S.W.C.), National Cancer Institute grant (RO3 CA96004–01, S.W.C.) and National Institutes of Health Grant (RO1 CA59005–05S1, J.B.M.)

⁴ Abbreviations used: MTHFR; methylenetetrahydrofolate reductase, SAdoHcy; S-adensylhomocysteine, SAdoMet; S-adenosylmethionine, tHcy; total plasma homocysteine.

Manuscript received 20 September 2002. Initial review completed 22 October 2002. Revision accepted 24 December 2002.

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