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3
,
*
Departments of Medicine and Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada;
Division of Gastroenterology, St. Michael’s Hospital, Toronto, Ontario, Canada;
**
Nutritional Immunology Laboratory and
Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA;
Divisions of Clinical Nutrition and Gastroenterology, Department of Internal Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA; and
Program in Immunology, Sackler Graduate School, Tufts University, Boston, MA
3To whom correspondence should be addressed. E-mail: youngin.kim{at}utoronto.ca
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • folate • natural killer cells • cytotoxicity • carcinogenesis • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). In several epithelial and nonepithelial tissues, folate deficiency has been observed to enhance, whereas folate supplementation suppresses, the development of cancer (1
–3
). The role of folate in carcinogenesis has been best studied for colorectal cancer (1
–3
). The majority of over 25 published epidemiologic studies indicate that dietary folate intake and blood folate levels are inversely associated with colorectal cancer risk (1
–3
). Collectively, these studies suggest an 
40% reduction in the risk of colorectal neoplasms in subjects with the highest dietary folate intake compared with those with the lowest intake (1
–3
). These studies also suggest that a modest reduction in folate status is sufficient to enhance colorectal cancer risk (1
–3
). Animal studies have also been generally supportive of a causal relationship between folate depletion and colorectal cancer risk as well as a dose-dependent protective effect of modest levels of dietary folate supplementation (4–10x) above the basal dietary requirement on the development and progression of colorectal neoplasms (4
–8
). Animal studies have also shown that the dose and timing of folate intervention are critical in providing safe and effective chemoprevention; exceptionally high supplemental folate levels (5
,9
,10
) and folate intervention after microscopic neoplastic foci are established in the colorectal mucosa (6
,7
) actually promote, rather than suppress, colorectal carcinogenesis. An accumulating body of evidence suggests that folate status may also play a modulatory role in the development of several other cancers (e.g., breast, lung, pancreas, stomach, cervix, esophagus, brain and leukemia) (1
–3
). The precise nature and magnitude of the relationship between folate status and the risk of these cancers, however, are less clearly defined compared with colorectal cancer (1
–3
). Nevertheless, the observation that the risk of neoplastic transformation in a wide range of tissues and organs may be modulated by folate status suggests that the effect of folate on carcinogenesis may be a generalized phenomenon and not limited to the colorectum. Variable cancer risk associated with folate deficiency in the aforementioned organs and tissues may be explained by different folate requirements and hence different susceptibility to folate deficiency (11
).
To date, the mechanisms by which folate deficiency enhances and supplementation suppresses carcinogenesis have not been clearly elucidated (1
–3
). Several potential mechanisms by which folate may modulate carcinogenesis at the molecular and cellular levels, including DNA damage, aberrant DNA methylation and impaired DNA repair, in specific target organs have been proposed and studied (1
–3
). The observation that the development of a growing number of cancers is linked to folate deficiency, however, suggests that a more generalized, systemic mechanism may also be operative in the folate deficiency–mediated carcinogenesis. In this regard, impaired immune surveillance and functions have been implicated in the development, progression and recurrence of several cancers (12
–15
). In particular, natural killer cells (NK)4
are non-B, non-T lymphocytes that can kill a variety of normal and virus-infected cells, cultured cell lines and tumor cells without prior sensitization and without major histocompatibility complex restriction (16
). Experimental and clinical evidence suggests that NK produce direct tumor cell destruction and may be the first line of host defense against tumorigenesis in humans (12
–15
). In vitro, NK-mediated cytotoxicity has been shown to be modulated by numerous nutritional treatments (17
–19
). These observations raise the question whether suppression or enhancement of NK-mediated cytotoxicity may be an important factor in the development/progression and prevention of certain diet related cancers, respectively. Although adverse effects of folate deficiency on immune functions, including suppressed humoral immunity, impaired cell-mediated cytotoxicity and neutrophil functions have been reported in both human and animal studies (20
,21
), the effect of folate deficiency on NK-mediated cytotoxicity has not been studied to date.
The aim of this study was to investigate the effect of dietary folate depletion on NK-mediated cytotoxicity in rats. The effects of both moderate and severe folate deficiency were studied to test the hypothesis that impaired NK-mediated cytotoxicity is a mechanism of relevance only if it is operative in moderate folate deficiency. In contrast to moderate folate deficiency, severe folate deficiency is associated with growth retardation, macrocytic anemia and lymphopenia (22
–24
), which might also mechanistically impair NK-mediated cytotoxicity. Therefore, studying both moderate and severe folate deficiency will determine whether the effect of folate deficiency on NK-mediated cytotoxicity is specific to folate deficiency or confounded by other variables such as weight loss, malnutrition and lymphopenia. Although moderate folate deficiency is more relevant to the human situation, severe folate deficiency was included in the present study because published animal studies demonstrating the relationship between folate status and cancer risk utilized both moderate and severe folate deficiency (4
–10
,25
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University.
Experiment 1 (severe deficiency).
Weaning male Sprague-Dawley rats (n = 30; 60–75 g; Charles River, Wilmington, MA) were randomly assigned to three groups. The control group (n = 10) was fed an amino acid–defined diet (Dyets Bethlehem, PA) (22
) containing 2 mg folate and 10 g succinylsulfathiazole/kg diet; 2 mg folate/kg diet is the basal dietary requirement for rats (26
). The folate-deficient and folate-supplemented groups (n = 10/group) were fed the identical diet except their diets contained 0 and 8 mg folate/kg diet, respectively. These amino acid–defined diets constitute a standard means of predictably inducing folate deficiency and repletion in rodents (5
–7
,22
–24
,27
–29
). The inclusion of succinylsulfathiazole facilitates the induction of folate deficiency (22
–24
,29
) because this nonabsorbable antibiotic eradicates intestinal microflora that are capable of de novo synthesis of folate, some of which is incorporated into tissue folate of the host (30
). Extending the 0 mg folate/kg diet beyond 5–6 wk produces a deficiency severe enough to cause marked growth retardation, illness and premature death (22
–24
). All three diets contained 50 g cellulose/kg and provided 60% of energy as carbohydrate, 23% as fat, and 17% as L-amino acids. Rats were housed individually in wire-bottomed stainless steel cages to minimize coprophagy. Body weights were recorded weekly. Rats consumed water ad libitum. The daily food consumption of each group was measured on a predetermined day of each week. Five preassigned rats from each group were killed by exsanguination under carbon dioxide anesthesia at wk 4 and 5 after the dietary treatment began. Previous animal studies utilizing severe folate deficiency of the same degree as in the present study demonstrated that animals became ill with progressive growth retardation, anemia and lymphopenia after 4–5 wk of dietary folate deficiency (22
–24
,29
). Therefore, we chose these two time points to investigate the effect of progressive severe folate deficiency as well as incremental weight loss and lymphopenia on NK-mediated cytotoxicity.
Experiment 2 (moderate deficiency).
Twenty additional weaning male Sprague-Dawley rats (60–75 g; Charles River) were randomly assigned to receive either the folate-deficient (i.e., 0 mg folate/kg diet; n = 10) or folate-supplemented (i.e., 8 mg folate/kg diet; n = 10) diet as described above except with the succinylsulfathiazole omitted. By omitting the antibiotic, less severe folate deficiency was induced in rats fed the folate-deficient diet. Rats fed this folate-deficient diet for 20–25 wk have been previously observed to develop a moderate folate deficiency without growth retardation or premature death (4
–6
,28
). In our previous studies, this moderate folate-deficient diet increased the incidence of colorectal neoplasm, whereas the 8 mg folate/kg diet (4x above the basal requirement) has consistently provided a degree of chemoprevention against colorectal cancer (4
–7
). The rats were killed 24 wk after the start of the dietary treatment.
Sample collection
Blood was collected into evacuated tubes containing EDTA and centrifuged at 800 x g for 10 min at 4°C; plasma was stored at -70°C in 5 g/L ascorbic acid for plasma folate assays. Aliquots (100 µL) of plasma were stored without ascorbate for homocysteine assays. Blood samples for blood counts were collected into tubes containing sodium EDTA and analyzed immediately (System 9000; Serono Baker Diagnostic, Allentown, PA). The liver was excised, frozen in liquid nitrogen, and stored at -70°C for subsequent analyses of folate. Spleen was excised aseptically and single cell suspensions were prepared in RPMI 1640 (Gibco BRL, Life Technologies, Gaithersburg, MD) as previously described (31
). Contaminating RBC were lysed by hemolytic Gey’s solution (17
).
Folate and homocysteine concentrations
Plasma folate concentrations were determined by a standard microbiological microtiter plate assay using Lactobacillus casei (32
). Hepatic folate concentrations were measured by the same microbiologic assay (32
), utilizing a previously described method for the determination of tissue folates (11
). Total serum homocysteine was measured by HPLC according to the fluorometric method of Vester and Rasmussen (33
).
NK-mediated cytotoxicity
A standard chromium release assay was used to assess the NK-mediated cytotoxicity of splenocytes as described previously (17
). YAC-1 target cells were labeled with sodium (51Cr)chromate (New England Nuclear, Boston, MA). Target-to-effector cell (T:E) ratios were adjusted to 1:100, 1:50, 1:25 and 1:12.5 and plated in triplicate in 96-well, V-bottom culture plates (Flow Labs, McLean, VA) containing 10,000 target cells/0.1 mL complete medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum) and corresponding ratios of effector cells in 0.1 mL complete medium. Target and effector cells were incubated for 4 h and 51Cr radioactivity in cell-free supernatant was determined. Triplicate wells of labeled target cells (10,000 cells) were incubated without spleen leukocytes in control medium to determine the spontaneous 51Cr release. Total 51Cr in target cells (10,000 cells) was determined by lysis of cells with 0.1% SDS in 0.1 mol/L NaOH. The percentage of specific 51Cr release was calculated as follows: [(t - s)/(x - s)] x 100, where t is the test 51Cr release, s is the spontaneous 51Cr release and x is the total 51Cr release. The mean percentage of specific 51Cr release was calculated for the triplicate wells and the SD between the triplicates did not vary by >5–10% of the mean.
Statistical analyses
The distribution of each variable was assessed graphically to determine whether it was normally distributed. Those variables that were not normally distributed were subjected to logarithmic transformation before a significance test was performed. In Experiment 1, differences among the three groups were determined by one-way ANOVA at each time point. Fisher’s least significance difference test was used for multiple comparisons. In Experiment 2, differences between the groups were determined by Student’s two-tailed t test. The Pearson coefficient of regression model was used to assess correlation between variables. All significance tests were two-tailed, and the significance level was set at 0.05. Results are expressed as mean ± SD of the untransformed data. Statistical analyses were performed using SYSTAT 5 for Macintosh (SYSTAT, Evanston, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weight and average daily food consumption.
All rats appeared to be healthy, and no premature deaths occurred. Consistent with previous studies (22
–24
), the folate-deficient rats showed progressive growth retardation beginning at wk 3 of the dietary intervention (Fig. 1A
). By contrast, growth curves were not significantly different between the control and folate-supplemented groups (Fig. 1
A). Daily food consumption, which was determined on a preassigned day of each week, was not different among the three groups until wk 3 when the folate-deficient group consumed on average 1 less pellet of diet (or 14% of total daily intake) compared with the control and folate-supplemented groups. The amount of food supplied to the control and folate-supplemented groups was therefore restricted to match the mean daily food consumption of the folate-deficient group starting at wk 3.
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), the hemoglobin concentrations, hematocrits and leukocyte counts in the folate-deficient group were lower than the corresponding values in the control and folate-supplemented groups at wk 4 and 5 (P < 0.005), whereas no differences were observed between the control and folate-supplemented groups at any time point (Table 1
). The mean corpuscular volume was not significantly different among the three groups at each time point. The absolute lymphocyte counts in the folate-deficient group were 47–57% (P < 0.004) and 58–65% (P < 0.002) lower than the corresponding values in the control and folate-supplemented groups at wk 4 and 5, respectively (Table 1)
. Although the absolute lymphocyte counts in the control group were 17–21% lower than the corresponding values in the folate-supplemented groups at wk 4 and 5, these differences were not significant (P = 0.07 and 0.123, respectively; Table 1
).
|
). The plasma concentrations of homocysteine, which increases during folate deficiency (34
) and is considered to be a more sensitive indicator of cellular folate depletion than blood folate levels (35
), were 12- and 22-fold higher in the folate-deficient group than in the control and folate-supplemented groups at wk 4 and 5, respectively (P < 0.001; Table 1
). By contrast, the plasma homocysteine concentrations did not differ between the control and folate-supplemented groups at wk 4 and 5 (Table 1)
.
NK-mediated cytotoxicity.
NK-mediated cytotoxicity of splenic leukocytes (expressed as percentage specific lysis) from rats fed the three diets for 4 wk did not differ at any of the 4 T:E ratios tested (Fig. 2A
). By contrast, NK-mediated cytotoxicity of splenic leukocytes from rats fed the folate-deficient diet for 5 wk was lower than the corresponding values from those fed the control and folate-supplemented diets at all 4 T:E ratios tested (P < 0.02; Fig. 2
B). NK-mediated cytotoxicity did not differ in the control and folate-supplemented groups at wk 5 (Fig. 2
B).
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Experiment 2: moderate folate deficiency
Body weight and average daily food consumption.
Consistent with previous studies (5
,6
,28
), growth curves were not significantly different between the moderately folate-deficient and folate-supplemented rats (Fig. 1
B). Daily food consumption did not differ between the two groups (data not shown).
Hematologic indices.
Consistent with previous studies (5
,6
,28
), the hemoglobin concentration, hematocrit, mean corpuscular volume, leukocyte counts and absolute lymphocyte counts were not different between the two groups (Table 2
).
|
). The plasma homocysteine concentrations were fourfold higher in the folate-deficient group than in the folate-supplemented group at wk 24 (Table 2
; P < 0.001). Plasma and liver folate and plasma homocysteine concentrations in the moderately folate-depleted rats were significantly lower and higher, respectively, than the corresponding values in the severely folate-depleted rats (Tables 1
and 2
; P < 0.001), reflecting the additional folate-depleting effect associated with the eradication of intestinal microflora (30
).
NK-mediated cytotoxicity.
NK-mediated cytotoxicity of splenic leukocytes (expressed as percent specific lysis) from rats fed the moderately folate-deficient and supplemented diets for 24 wk was not different at any of the 4 T:E ratios tested (Fig. 3
). Furthermore, no correlations were observed between plasma and liver folate and plasma homocysteine concentrations and NK-mediated cytotoxicity at wk 24.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Epidemiologic studies suggest that a mild-to-moderate degree of folate depletion, with blood folate concentrations well within the range conventionally accepted as normal, is sufficient to increase the risk of several cancers (1
–3
). Furthermore, animal studies have shown that the same degree of moderate folate depletion employed in the present study enhances colorectal carcinogenesis in chemical and genetic knockout models of colorectal cancer, if started before the establishment of neoplastic foci in the colorectal epithelium (4
–7
). Recent studies have revealed that up to 30% of ambulatory populations in the United States have a subclinical, but biochemically evident degree of folate deficiency as indicated by elevated serum homocysteine concentrations (36
). By contrast, folate deficiency as severe as that employed in the present study, causing growth retardation, macrocytic anemia, lymphopenia and profound hyperhomocysteinemia, is exceedingly uncommon among human populations in developed nations (36
,37
). These observations, as well as the fact that NK-mediated cytotoxicity was modulated only by an extremely severe degree of folate depletion in the present study, suggest that impaired NK-mediated cytotoxicity is not a likely mechanism by which folate depletion enhances carcinogenesis. Furthermore, the observations from the present study suggest that enhanced NK-mediated cytotoxicity is not a likely mechanism by which folate supplementation suppresses carcinogenesis. However, we cannot exclude the possibility that NK residing in other specific target tissue might be more sensitive to folate depletion.
Severe folate deficiency in the present study was associated with a progressive drop in hemoglobin, hematocrit, leukocyte and lymphocyte counts in rats. This observation is entirely consistent with the known biochemical function of folate in proliferating cells. Folate is an essential factor for the de novo biosynthesis of purines and thymidylate (38
). 5,10-Methylenetetrahydrofolate, an intracellular coenzymatic form of folate, is required for conversion of deoxyuridylate to thymidylate and can be oxidized to 10-formyltetrahydrofolate for de novo purine synthesis (38
). Impaired DNA synthesis resulting from folate deficiency has been observed to affect erythropoiesis, granulopoiesis and lymphopoiesis with the consequent development of anemia, neutropenia and lymphopenia in addition to megaloblastic morphologic changes (21
). The hematologic indices, however, appear to resist the effect of folate deficiency. Only severe folate deficiency of a degree that is associated with the 12- to 22-fold rise in plasma homocysteine concentrations induced significant reductions in the hematologic indices, whereas no significant change in these variables was observed with moderate folate deficiency of a degree that is associated with the fourfold rise in plasma homocysteine concentrations. One previous study also reported that rats fed a severely folate-deficient diet identical to that in the present study had significantly lower hemoglobin, hematocrit and leukocyte counts than rats fed the identical folate-supplemented diet (i.e., 8 mg folate/kg diet) (22
).
The extent to which plasma and liver folate concentrations were depleted and plasma homocysteine levels were elevated with the severely and moderately folate-deficient diets in the present study is consistent with previous observations made in rats fed similar diets for the same duration (4
–6
,22
,24
,27
–29
,39
,40
). Although the magnitude of decrease in plasma and liver folate concentrations resulting from 4 to 5 wk of severe folate deficiency appears to be modest, this was associated with a profound rise in plasma homocysteine concentrations. This indicates that the degree of severe folate deficiency achieved at wk 5 was more extreme than that at wk 4. The finding that plasma homocysteine concentrations did not differ between the control and folate-supplemented rats in Experiment 1 is consistent with observations made in humans. A strong, nonlinear, inverse association exists between plasma homocysteine and folate concentrations; in upper ranges of plasma folate concentrations, however, plasma homocysteine concentrations are less affected by plasma folate concentrations (36
,41
). Large population-based studies have demonstrated that plasma homocysteine concentrations fluctuate minimally at plasma folate concentrations above 10 nmol/L (36
,41
).
Similar to other dietary intervention studies, we wished to eliminate differences in food intake among the three groups. There was no need for pair-feeding until wk 3 of dietary intervention in the severe folate deficiency experiment. However, beginning at wk 3, the group fed the severely folate-deficient diet consumed on average 1 less pellet of diet (or 14% of total daily intake) per day than the control and folate-supplemented groups. It is unknown at present why severely folate-depleted rats consumed less food, but it may be related to anorexia and systemic ill effects of severe folate deficiency. We and others have previously documented a comparable degree of weight loss and decreased food intake associated with severe folate deficiency of the same degree employed in the present study (22
,24
). The observed growth retardation associated with severe folate deficiency, however, is not due entirely to decreased food intake because even with matched feeding, severely folate-depleted rats continued to lose weight from wk 3 to 5. Severe folate deficiency causes megaloblastic changes in the intestinal epithelium that may contribute to malabsorption (42
–44
). Therefore, the observed growth retardation in the severely folate-depleted rats was likely due to multifactorial causes including adverse physiologic effects of severe folate deficiency and decreased food intake. Despite restriction in food intake in the control and folate- supplemented groups in the severe folate deficiency experiment, the growth curves of these two groups did not show any evidence of growth retardation and continued to be linear. We do not know what the growth curves of these two groups would have looked like had we not restricted food intake, but we presume that the growth curves would have been linear at a steeper slope. This is supported by the observation that the mean weight of the control and folate-supplemented rats in the severe folate deficiency experiment was 10–12% lower than that of the rats in the moderate folate deficiency experiment at wk 5 (Fig. 1
A and B).
The percentage specific lysis observed in the moderate folate deficiency experiment (Fig. 3)
is somewhat lower than that observed in the severe folate deficiency experiment (Fig. 2
A and B). This is likely due to the day-to-day variation associated with this assay. Each NK-mediated cytotoxicity assay was performed on the day of killing the rats. Therefore, it is not appropriate to compare the absolute values of the percentage specific lysis from one experiment to another. This assay is valid only for internal comparison of the percentage specific lysis among the different dietary groups within each experiment performed on the same day.
Several lines of evidence suggest that folate deficiency may affect cell-mediated immune functions in rodents in cells other than NK (45
,46
). These include decreased number of T cells, reduced lymphocyte-mediated cytotoxicity against foreign transplanted cells and a decrease in stimulation of T lymphocytes by phytohemagglutinin (PHA; a T-cell mitogen) (45
,46
). In humans, folate deficiency has been associated with delayed-type hypersensitivity, impaired lymphocyte response to PHA stimulation, and a decrease in the capacity of PHA-stimulated peripheral lymphocytes to undergo blast transformation and to synthesize DNA (47
–49
). These defects were largely correctable by folate supplementation (47
–49
). However, one human study did not show any significant correlation between folate adequacy and several cell-mediated immunologic measures, including lymphocyte counts, delayed-type hypersensitivity and PHA response (50
).
In summary, although severe folate deficiency may adversely affect NK- mediated cytotoxicity, moderate folate depletion, which is often associated with the increased risk of several epithelial cancers (1
–3
), appears not to affect splenic NK-mediated cytotoxicity in rats. Furthermore, a modest level of folate supplementation above the basal requirement does not appear to enhance NK-mediated cytotoxicity. These data collectively suggest that NK-mediated cytotoxicity is not a likely mechanism by which folate status modulates carcinogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
4 Abbreviations used: NK, natural killer cells; PHA, phytohemagglutinin; T:E, target-to-effector.
Manuscript received 10 October 2001. Initial review completed 14 December 2001. Revision accepted 28 February 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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