Folate Deficiency Enhances the ... - Cancer Research · an association between folate deficiency and premalignant changes in several epithelial tissues, including the colon (4-7)

(CANCER RESEARCH 52, 5002-5006, September 15, 1992)

Folate Deficiency Enhances the Development of Colonie Neoplasia inDimethylhydrazine-treated Rats1

Marilia L. Cravo,2 Joel B. Mason,3 Yogeshwar Dayal, Martha Hutchinson, Donald Smith, Jacob Selhub, andIrwin H. RosenbergVitamin Bioavailability Laboratory, U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111Â¡M.L. C., J. B. M., D. S., J. S., I. H. R.], and Department of Pathology, New England Medical Center, Boston, Massachusetts 02111 [ Y. D., M. H.J

ABSTRACT

In patients with ulcerative colitis, epidemiolÃ³gica!work has suggestedan association between low folate status and an increased risk of colonieneoplasia. The aim of the present study was to determine if experimental folate deficiency increases the likelihood of developing neoplasia inrats treated with the carcinogen dimethylhydrazine. Weanling maleSprague-Dawley rats were fed with an amino acid-defined diet containing either 8 or Omg/kg folie acid. After 5 weeks of defined diet, weeklys.c. injections of dimethylhydrazine (20 mg/kg) were administered toboth groups. Serum, whole blood, liver, and colonie folate concentrationsat the time of sacrifice were significantly lower in folate-depleted animals ( /' < 0.001). There were significant differences in the incidence of

colonie neoplasia between the two groups after 20 weeks of dimethylhydrazine exposure: folate-deficient rats had a greater incidence of dys-plasia (6 of 7 versus 2 of 7 animals; P < 0.05) and carcinoma (6 of 7versus 1 of 7 animals; P < 0.01). Furthermore, a significantly greaterproportion of folate-replete rats than folate-deficient rats were free ofneoplastic lesions (5 of 7 versus 0 of 7 animals; /' < O.OS).

These results suggest that, in this animal model, folate deficiencyincreases the risk of malignancy when there is an underlying predisposition to colorectal cancer.

INTRODUCTION

Although a genetic susceptibility to colorectal cancer is wellestablished (1), environmental factors, especially nutrition, areconsidered to play an important role in most instances (2, 3). Adeficiency of the water-soluble vitamin folate is among thosenutritional factors which have been considered. The evidencewhich indicates that folate plays a role in colonie carcinogenesisis indirect at this point: some epidemiolÃ³gica! studies suggestan association between folate deficiency and premalignantchanges in several epithelial tissues, including the colon (4-7).Lashner et al. (7) have observed that among individuals withulcerative colitis, an illness which carries with it a substantialrisk of folate deficiency, folate supplementation is associatedwith a 62% reduction in the risk of developing colonie dysplasiaor cancer.

The question of whether folate deficiency plays a role incolonie carcinogenesis is an important one because folate deficiency is thought to be the most common vitamin deficiency inthe United States as well as the rest of the world (8). Further-

Received 3/9/92; accepted 7/8/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part with Federal funds from the U.S. Department of Agriculture, Agricultural Research Service Contract 53-3K06-0-1. The contents of thispublication do not necessarily reflect the views or policies of the U.S. Departmentof Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

2 M. L. C. was supported in part by a grant from IN VOTAN 1NV/FRH 12/89,Lisbon, Portugal. Present address: Servico de Gastroenterologia, Instituto PortuguÃ©sde Oncologia Francisco Gentil, Lisboa, Portugal.

3 J. B. M. was supported in part by a Future Leader in Nutrition Award from theInternational Life Science Institute. To whom requests for reprints should be addressed, at Vitamin Bioavailability Laboratory, USDA Human Nutrition ResearchCenter on Aging at Tufts University, 711 Washington Street, Boston, MA 02111.

more, folate deficiency is known to complicate several gastrointestinal illnesses in which the risk of developing intestinalmalignancies is increased (9-11).

The objective of the present study was to determine whether,and to what extent, folate deficiency enhances the likelihood ofdeveloping colonie neoplasia in rats exposed to a well-knowncolon carcinogen.

MATERIALS AND METHODS

This study was reviewed and approved by the New England MedicalCenter Animal Research Committee. Ninety-four weanling male Sprague-Dawley rats (60 g, Charles River Co., Wilmington, MA) were randomly divided into four groups. Groups 1 (n = 16) and 3 (n = 31)received a high glutamate amino acid defined diet (12) containing 8mg/kg of folie acid, whereas groups 2 (n = 16) and 4 (n = 31) receivedan identical diet except that folate was omitted (Dyets, Inc., Bethlehem,PA). Both diets contained 50 g of cellulose/kg as well as 60% of caloriesas carbohydrate, 23% as fat, and 17% as L-amino acids. No sulfonamideantibiotics were used because the marked folate deficiency associatedwith sulfonamide use is not consistent with the moderate deficiencystate we wished to examine. Furthermore, the use of antibiotics wouldhave interfered with DMH4 activation by bacterial flora (13). Rats werehoused individually in wire-bottomed stainless steel cages, diets andwater were given ad libitum, and body weights were recorded weekly.After 5 weeks of defined diet groups 3 and 4 received weekly s.c.injections of 20 mg/kg dimethylhydrazine (Sigma, St. Louis, MO) for20 weeks, while groups 1 and 2 received injections of placebo carrier.DMH was freshly dissolved in 0.05% EDTA and adjusted to pH 6.5with NaHCO3 as described (14). Three or four animals in groups 1 and2 and 5-7 animals in groups 3 and 4 were sacrificed at 5, 10, 15, 20, and22 weeks after the first injection of DMH. One rat in group 4 died atweek 5 and was not examined.

The rats were killed by cardiac puncture under methoxyflurane anesthesia. Blood was collected, stored at -20Â°C in 0.5% ascorbic acid,

and later assayed for serum and whole blood folate by microbiologieassay using Lactobacillus casei (15). One g of liver tissue was rapidlyweighed, minced, and added to 10 volumes of 100C folate extractionbuffer (15). After boiling for 10 min, the suspension was cooled to OÂ°C,

homogenized, and centrifuged. The clear supernatants were stored atâ€”¿�70Â°Cfor subsequent assay of total folates. Microbiological assay was

carried out using cryoprotected cultures of L. casei after treatment withchicken pancreas conjugase to convert all the polyglutamates to theircorresponding monoglutamate derivatives (15). The same procedurewas used to measure folate concentration in colonie mucosa! scrapings.The values obtained are expressed per gram of tissue.

The entire gastrointestinal tract was examined for the presence ofmacroscopic tumors. The colon (except for the cecum, which was usedfor mucosa! scrapings) was opened longitudinally, rinsed in saline, andcarefully examined for any macroscopic lesions. All such lesions weremeasured, mapped, and recorded. The colon was then rolled up into a"Swiss roll" (resembling a jelly roll configuration; see Ref. 16), fixed

overnight in 10% formalin, and processed for light microscopy. A singlelongitudinal section (5 /Â¿m)was cut from the midline of each Swiss roll,without regard to the presence of macroscopic lesions, and stained withhematoxylin/eosin. The Swiss roll is a means of providing a pathologist

1The abbreviation used is: DMH, dimethylhydrazine.

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FOLATE DEFICIENCY ENHANCES COLONIC NEOPLASIA IN RATS

with an intact longitudinal section of the entire length of the colon ona single microscope slide, and it has previously been used to quantitatively and qualitatively describe DMH-induced lesions in the colon ( 16).Since midline sections were cut without regard to the presence of macro- or microscopic lesions, each S-jim section in this study constituteda random and representative longitudinal section from the entire lengthof the colon of each rat, excluding the cecum. Assessment of the microscopic lesions was thereby performed in a manner that was independent of the assessment of macroscopic lesions.

Two experienced pathologists read the slides independently and in ablinded fashion. One of the two was designated as the chief pathologist;in those few instances (n = 3) where there was disagreement about thehistological interpretation, the opinion of the chief pathologist prevailed. The entire length of each colon, excluding the cecum, was carefully examined by this technique, and each focus of neoplasia wasrecorded with respect to its location (proximal versus distal) and theseverity of dysplasia (vide infra). Histological lesions in colonie mucosawere classified into three categories according to previously describedcriteria (17, 18): (a) low-grade dysplasia; (ft) high-grade dysplasia; and(c) carcinoma. The epithelium was also examined for the presence ofmegalocytosis and/or megaloblastosis (the former refers to differentiated epithelial cells possessing cytologie characteristics of folate deficiency; the latter refers to undifferentiated stem cells deep within thecrypt possessing those characteristics; the remainder of this paper usesthe more commonly used term, megaloblastosis, to encompass both celltypes). The presence of megaloblastosis was determined qualitatively aspreviously described (19, 20): epithelial cells possessing nuclei whichwere larger and more hyperchromatic than normal and whose chroma-tin pattern was more "open" than normal but whose other cytologie

characteristics, such as a columnar shape and basally located nucleus,were retained were considered to be megaloblastic. If such changes wereobserved anywhere in a Swiss roll, that colon was considered to possessevidence of megaloblastosis.

Statistical significance of the mean values was determined using thetwo-tailed Student's t test. Nonlinear regression analyses were used to

evaluate the association between two variables (MYSTAT; Systat, Inc.,Evanston, IL). Fisher's exact test was used to determine the significance

of differences in the proportion of neoplastic lesions observed in groups3 and 4.

RESULTS

Body Weight. There were no significant differences in thegrowth curves of animals fed a folate-deficient or folate-repletediet. However, DMH-injected animals (groups 3 and 4) weresignificantly lighter when compared to non-DMH-injectedanimals (groups 1 and 2) from the 13th week on (P < 0.05)(Fig. 1).

Folate Status. Table 1 shows that systemic folate deficiencywas clearly present in groups 2 and 4 as demonstrated by serum,whole blood, and liver folate concentrations. The table alsodemonstrates that folate depletion was present in the coloniemucosa itself. Progression of folate deficiency was not observedafter the fifth week of DMH or placebo injections, which is thefirst time point at which rats were sacrificed. For example,serum folate concentration, which is considered to be an accurate indicator of systemic folate status in rats by Clifford et al.(21), did not change significantly between the first time point(5 weeks) and the last time point (22 weeks) in any of the 4groups of animals. Accordingly, the average values displayed inTable 1 represent the pooled values from all of the rats in eachof the four groups.

Macroscopic Data. No neoplastic lesions were observed innon-DMH-injected animals (groups 1 and 2). Also, there wereno small intestinal or extraintestinal malignancies observed inDMH-injected animals. No mÃ©tastaseswere observed in any ofthe groups. The cecum was everted and carefully examined in

00â€¢¿�*â€¢".C.2?*3$

8001

600-

400-

200

0 10 20 30

time(week after initiation of diets)Fig. 1. Average body weights of animal groups as a function of time. Group 1.

folate-replete, no DMH; group 2, folate-deplete. no DMH; group 3, folate-replete. DMH: group 4, folate-deplete. DMH. After 13 weeks there was a significant difference in the average body weight of animals that received injections(groups 3 and 4) and animals that did not receive injections (groups 1 and 2) ofDMH. O, group 1; +, group 2; D, group 3; *, group 4.

Table 1 Folate status in the four experimental groupsGroups 1 and 3 are folate replete, whereas groups 2 and 4 are folate depleted.

Values represent pooled values from all time points and are expressed as mean +SD.

Serum folate (ng/ml)Whole blood folate (ng/ml)Liver folate (fig/g)Colonie folate (ng/g)Group

198

+ 30731 + 3176.8 + 2.0504+ 154Group

29.0+ 2.7Â°

147 + 50Â°2.2 + 0.9Â°197+ 114Â°Group

392

+ 26626+ 1576.1 +2.3558 + 212Group

415.0+ 9.8*

189 + 54*1.9 + 0.8*

171 + 129*

" P < 0.001, less than group I.* P < 0.001, less than group 3.

each rat before it was processed for biochemical analysis; nomacroscopic lesions were observed.

Macroscopically evident DMH-associated lesions appearedas either pedunculated or sessile polyps, as previously described(22, 23). In both groups the size of the lesions ranged from 2 to20 mm. At 20 weeks, a greater proportion of group 4 had polypsthan group 3, although the difference was not statistically significant (6 of 7 or 86% versus 3 of 7 or 43%; P = 0.09). Themanner in which the colons were processed in this study precluded histological analysis of macroscopic lesions. Nevertheless, previous investigators who have used this animal modelhave observed that macroscopic lesions invariably contain neoplastic foci (16, 24-28), establishing the assessment of macroscopic lesions as a reflection of neoplastic transformation.

Microscopic Data. Megaloblastosis was observed in 15%(2 of 16) of the animals in group 2, in 40% (12 of 30) of theanimals in group 4, and in none of the animals in groups 1 and3. The cancers were well-differentiated adenocarcinomas in allinstances except Â¡none case in group 4, where a signet ring-typecarcinoma was observed. Most of the highly neoplastic lesions(carcinoma and high-grade dysplasia) were located adjacent tohypertrophie lymphoid follicles.

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Fig. 2 presents the percentage of DMH-injected rats that hadeither dysplasia and/or carcinoma and gives an indication of thetime course of developing neoplastic lesions in this study. As isindicated in the figure, the earliest neoplastic lesions in DMH-injected rats were observed at 15 weeks. Although the folate-deficient animals had a higher prevalence of neoplasia at thistime point, the differences between the two groups were notsignificant. However, highly significant differences in the prevalence of neoplasia were observed by 20 weeks (P< 0.01). Table2 summarizes the histolÃ³gica! findings in DMH-injected rats at20 weeks; by this time point a significantly greater proportionof the folate-deficient animals were observed to have dysplasia(P< 0.05) and carcinoma (P< 0.01). Furthermore, the proportion of folate-replete animals that were free of any neoplasticlesions at 20 weeks was significantly greater than the proportion of neoplasia-free animals in the folate-deficient group (5 of7 versus 0 of 7, respectively; P < 0.05). Table 3 lists the individual neoplastic lesions observed in each of the DMH-injectedrats at 20 weeks; the table also specifies whether each lesion waslocated in the proximal or distal half of the colon and whethereach dysplastic lesion was high grade or low grade. There wereno statistically significant differences between groups 3 and 4with respect to the preponderance of proximal versus distallesions or with respect to the proportion of dysplastic foci whichwere high-grade lesions.

At the doses of DMH used in this study all animals would beexpected to eventually develop colon cancer. This was found tobe true; by 22 weeks all of animals in groups 3 and 4 haddeveloped dysplasia and/or carcinoma. At this time point, only5 rats in group 3 and 4 rats in group 4 were sacrificed. Therewas a trend toward a greater number of neoplastic lesions perrat in the folate-deficient group (4.8 versus 2.5 lesions/rat,group 4 versus group 3, respectively) but the relatively smallnumber of rats at this time point precluded any meaningfulstatistical analysis.

DISCUSSION

This study demonstrates that moderate folate deficiency enhances the development of DMH-related neoplasia in the rat

*iâ€”¿�Q.

I

1001

80-

p=0.005

group 4

M

2

15

group 3

20

time(weeks after initiation of DMH injections)Fig. 2. Incidence of colonie dysplasia and/or carcinoma (percentage of animals

with either of these lesions) for folate deplete (â€¢)or folate replete (O) rats. At 20weeks there were significant differences between the two groups. Groups killed at5 weeks contained S animals each; other time points contained 7 animals each.

Table 2 Incidence of neoplastic lesions in groups 3 (folate replete) and 4 (folatedepleted) at 20 weeks after the first DMH injection

Values outside of parentheses are percentages; values within parentheses arethe actual proportions.

% of ratswithdysplasiaGroups328

(2/7)486 (6/7)"%

of ratswithcarcinoma314(1/7)486

(6/7)*%

of rats free ofneoplasticlesions371

(5/7)40 (0/7)"

" P < 0.05, different from group 3.* P < 0.01, different from group 3.

Table 3 Distribution of neoplastic lesions in DMH-injected rats at 20 weeks

Dysplasia" Carcinoma"

Proximal Distal Proximal Distal

Group 3 rats (replete)

III-2III-3III-4III-5III-6III-7

Group 4 rats (depleted)IV-1IV-2IV-3H|H1"iv-4

H" IT1IV-5IV-6IV-7IL11"

1II

1" Each vertical bar indicates a distinct focus of neoplasia; dysplastic lesions are

followed by a superscript L if they were low grade and a superscript H if they werehigh grade.

colon. Several clinical studies have suggested an associationbetween folate deficiency and dysplasia in the cervical (4), bronchial (5, 6), and colonie (7) epithelium. In addition, some ofthese studies have demonstrated an apparent reversal of thepathologic lesions with supplemental folate (4,6). However, noclinical study to date has been able to establish a clear cause-and-effect relationship between folate deficiency and dysplasia.The present study provides strong evidence for such a relationship: the experimental animals, which clearly had both systemicand colonie folate deficiencies, showed an enhanced susceptibility to the effects of a colon carcinogen compared to a controlgroup consisting of folate-replete animals. It is worth emphasizing that the procarcinogenic effect of folate deficiency thatwe observed was achieved with a diet that produces a modestdegree of deficiency that is not severe enough to cause growthretardation or anemia (12). This contributes to the potentialclinical significance of these observations, since mild folate deficiency in humans is much more common than a severely deficient state.

It is also important to point out that group 2 animals did notdevelop any neoplastic changes; this confirms the concept thatfolate deficiency is not carcinogenic by itself (29). However,when superimposed on an underlying predisposition to cancer,folate deficiency seems to increase the susceptibility to neoplastic transformation. Such a relationship is underscored by gastrointestinal illnesses, such as ulcerative colitis and celiac sprue,which are associated with both an increased risk of folate deficiency and an increased susceptibility to cancer (9-11, 30, 31).

A significant problem with some of the above-mentionedclinical studies exists because of a confusing similarity betweenthe cytological characteristics of dysplastic and megaloblasticcells. In the trials involving folate supplementation (4, 6), the

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similarity between the two cell types confounds the interpreta- associated with gene activation (40, 41). Previous studies have

tion of whether dysplasia or megaloblastosis regressed withtherapy (6). In the present study, animals in groups 2 and 3enabled us to observe cases which were either solely megalo-blastic or solely dysplastic. The fact that no megaloblastosis wasobserved in group 3 and no dysplasia in group 2 suggests thatour blinded pathologists were capable of objectively distinguishing between these two cell types. Nevertheless, the prevalence of observed megaloblastosis in group 4 was greater than ingroup 2 (40% versus 15%). Since the degree of folate deficiencywas similar in the two groups, this observation raises the question of whether some of the lesions observed in group 4 animalswhich were classified as megaloblastosis were in fact dysplasiaor whether there is a more complicated functional relationshipbetween these two cell types which is not yet fully understood.

Determining whether folate deficiency was present in thecolonie mucosa was an important feature of this study, sincesome tissues appear to be more resistant to depletion thanothers (21). The cecum was chosen as a representative portionof the colon for folate analysis. Given the substantial degree offolate deficiency observed in the cecum and in the other tissueswhich were examined in the folate-deficient rats, it is unlikely

that the remainder of the colon would have been folate repletein these animals. The fact that the cecum was used to obtainmucosal tissue for folate analysis precluded histological analysis of this portion of the colon. Therefore, by necessity, ourconclusions regarding the procarcinogenic effect of folate deficiency are limited to that portion of the colon distal to thececum. It is unlikely that excluding the cecum from the histological analysis substantially skewed our observations, particularly since we observed that the geographic distributions ofneoplasia in groups 3 and 4 were not significantly different.

The mechanism by which local folate deficiency might contribute to the risk of malignant changes remains unclear. However, it is worth noting that folate is a critical element forthymidylic acid synthesis through thymidylate synthetase(29, 32). Thymidine deprivation has been shown to result inprofound alterations in nucleotide pools (33), which will resultin a reduction in the rate of DNA replication (34). In addition,reduction of the deoxythymidylate pool leads to expansion ofthe deoxyuridylate pool and abnormally high incorporation ofuracil into DNA. The repair of misincorporated uracil underconditions of folate deficiency results in the so-called catastrophic repair in which further incorporation of uracil maytake place (35). It has been postulated that this interferencewith the biosynthesis of deoxynucleotides is responsible for thecocarcinogenic effect of folate deficiency (29). In the presentstudy nucleotide pools were not measured. However, prior studies by us indicate that folate deficiency in rats on a diet identicalto that used in this study leads to an impairment of thymidylate-DNA synthesis in the colonie epithelium (36). Another possibleexplanation why folate-deficient cells apparently have a greatersusceptibility to undergo neoplastic transformation is related toDNA methylation. Rogers and Newberne (37) demonstratedthat diets deficient in methionine, choline, folate, and vitaminB12 (lipotropes) promote chemical carcinogenesis in rats. Thiscocarcinogenic effect seems to be related to a decrease in theconcentration of 5-adenosylmethionine that occurs as a consequence of dietary methyl deprivation (38). S'-adenosylmethion-

ine serves as a methyl donor for many biochemical reactions,including DNA methylation (29, 39). Although the role ofDNA methylation in gene regulation is not fully understood,many studies have shown that a decrease in methylation is

suggested that generalized DNA hypomethylation as well asspecific protooncogene hypomethylation precedes the development of malignancy in both experimental and human colonietumors (42-44). Since folate is a necessary cofactor for theregeneration of 5-adenosylmethionine, it is possible that folatedeficiency results in hypomethylation of DNA and activation ofspecific protooncogenes.

Given the high incidence of colorectal cancer in the generalpopulation and the substantial prevalence of folate deficiency incertain segments of our population (8), these observations raisethe question of whether maintenance of adequate folate statusin patients at high risk for colorectal cancer may be an important factor in minimizing the risk of developing colonie neoplasia. Further studies will be necessary to confirm our observations in experimental carcinogenesis and in order to establishwhether, and under what conditions, folate deficiency mightenhance colon carcinogenesis in humans.

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1992;52:5002-5006. Cancer Res Marilia L. Cravo, Joel B. Mason, Yogeshwar Dayal, et al. Neoplasia in Dimethylhydrazine-treated RatsFolate Deficiency Enhances the Development of Colonic

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Folate Deficiency Enhances the ... - Cancer Research · an association between folate deficiency and premalignant changes in several epithelial tissues, including the colon (4-7)