Cardnogenesis vol.13 no. 12 pp.2471-2474, 1992

SHORT COMMUNICATION

Alterations in nucleotide pools in rats fed diets deficient in choline, methionine and/or folk acid

S.Jill James, David R.Cross and Barbara J.Miller National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA

It is well established that the metabolic pathways of methionine, choline and folate are mutually interdependent such that a deficiency in one will affect the distribution and metabolic priorities of the others (1). For example, when exogenous methionine and choline are limited in the diet, the folatedependent pathway for the endogenous resynthesis of methionine from homocysteine is up-regulated as a mechanism to maintain intracellular levels of S-adenosyl-methionine (SAM*), the major intracellular methyl donor (2). It is important to note that the increased turnover of this pathway, stimulated by decreased availability of SAM, effectively increases the intracellular requirement for folate methyl groups (3) and leads to a depletion of total folates in the liver (4,5). The indirect consequence of the irreversible diversion of folates toward the regeneration of methionine is a functional depletion of folate one-carbon groups for the de novo synthesis of purines and the pyrimidine, thymidylate (1). The central importance of folate metabolites for maintenance of both SAM-mediated mediylation reactions and •Abbreviations: SAM, S-adenosyl-methionine; MCFD, methionine-, choline-, folate-deficient diet; MCD, methionine-, choline-defrcient diet; FD, folk aciddeficient diet; TCA, trichloroacetk acid; THF, tetrahydrofolate; dNTP, deoxynucleoside triphosphate. © Oxford University Press

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The fidelity of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) percursors for the polymerases involved in replication and repair. Since folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimklines, the purpose of the present investigation was to determine whether diet-induced depletion of folates would alter intraceOular dNTP pools. Fischer 344 rats were fed one of four semi-purified diets for a period of 8 weeks: (i) supplemented control; (ii) deficient in folk acid; (iii) deficient in methionine and choline; and (iv) deficient In methionine, choline and folk add. In contrast to natural diets, semi-purified diets are nuckotide-free and consequently lack substrates for salvage pathway synthesis. This omission may place unusual stress on folate-dependent de novo nucleotide synthesis especially under conditions of dietary methyl-donor deficiency. Reversed-phase HPLC analysis of dNTP in spleen cell extracts indicated that both the thymidylate monophosphate and thymidylate triphosphate pools were decreased in spleen cells from the deficient rats consistent with a decrease in folate-dependent de novo synthesis. In addition, purine biosynthesis appeared to be negatively affect by methyl-donor deficiency as evidenced by a reduction in dGTP and dATP pools. These data indicate that deoxynucleotide pool imbalance, weO known to produce cytogenetk and mutagenk events in vitro, can also be induced in this in vivo model of diet-induced carcinogenesis.

de novo deoxynucleoside triphosphate (dNTP) synthesis is schematically shown in Figure 1. Krumdieck (1) has proposed that chronic imbalance in dNTP pools could contribute to die carcinogenic potential of methyl-donor deficiency. Since the dNTP are die immediate precursors for the DNA polymerases involved in DNA replication and repair, the fidelity of DNA syndiesis is critically dependent on the correct balance and availability of die deoxynucleotides (6,7). Environmental conditions which alter dNTP synthesis and bioavailability have been shown to disturb DNA metabolism in genetically significant ways. For example, alterations in dNTP ratios induced by folate deprivation or by anti-folate drugs in vitro, have been shown to promote certain genetic (and cancer-associated) lesions including folate-sensitive fragile site expression (8,9), DNA strand breakage (10,11), error-prone DNA repair (12,13) and mutagenesis (14,15). In humans, an increased frequency of micronuclei in erythroblasts was reported to be reversed by folate supplementation (35). More recently, lymphocyte mutation frequency was found to be positively correlated with low folate status in women with breast cancer treated widi chemotherapy (17). Increasing evidence suggests that localized folate deficiency may contribute to human carcinogenesis. In a recent study, cervical dysplasia in women taking oral contraceptives was found to be significandy reversed with folate supplementation (35). Similarly, folate (plus cobalamin) supplementation in male smokers was shown to improve bronchial squamous metaplasia (18). In patients with chronic ulcerative colitis, folate supplementation was associated with a 62% reduction in colon cancer incidence (19). Finally, pernicious anemia patients are known to have a higher risk of stomach cancer, melanoma, myeloid leukemia and cancers of the mourn and pharynx (20). The purpose of the present study was to determine whether dietary manipulation of DNA precursor synthesis by folate and methyl-donor deficiency would alter intracellular dNTP pool sizes. Methionine and choline deficiency have been recently shown to produce localized folate deficiency in the liver despite adequate folate in die diet (5,21). Therefore, in addition to folate deficiency, the effects of methionine and choline deficiency were evaluated as well as the effects of the combined deficiencies of all three methyl donors. Lymphocytes were chosen as initial indicator cells because they are well known to be highly sensitive to folate deficiency. Male Fischer 344 rats were obtained from the NCTR breeding facility, housed two/cage in a climate-controlled (24°C) room witha 12 h light/dark cycle. At6 weeks of age(110-13Ogbody wt), rats were randomly assigned to one of four experimental diets. The basal diet was purchased from Teklad Research Diets (Madison, WI) and contained 12% casein (0.3% methionine) with 10% soy oil (trace choline) and widiout folic acid in die vitamin mix. One group of rats was fed the basal diet (methionine-, choline-, folate-deficient, MCFD). A second group was fed the basal diet supplemented with 2 mg/kg folic acid (methionine-, choline-deficient, MCD). In the third group, die basal diet was supplemented with 0.3% methionine, 0.3% choline chloride but without folic acid (folic acid-deficient, FD). For the control group

SJJames, D.R.Cross and B.Miller

(C), the basal diet was supplemented with 0.3% methionine, 0.3% choline and 2 mg/kg folic acid. All four groups were allowed ad libitum access to the respective diets for a period of 8 weeks. It should be noted that the semi-purified diets and amino aciddefined diets commonly used in studies of methyl-donor deficiency do not contain sources of preformed purines and pyrimidines, in contrast to standard preparations of rodent chow containing fish meal, Brewer's yeast, etc. (e.g. Purina 5001, NIH-31). The diet provides the major source of nucleosides for salvage pathway nucleotide synthesis. The lack of nucleosides in semi-purified diets may have a significant, previously unrecognized impact on proliferating tissues by increasing the requirement for folate derivatives involved in de now nucleotide synthesis. It has been shown for example that the proliferative response of immune cells is significantly reduced in animals fed semi-purified diets lacking preformed nucleotides (22,23). The proliferative potential was restored to levels observed in animals fed chow diet by adding yeast RNA or purified bases to the diet. It should be stressed that the omission of folate in a nucleotidefree diet would be expected to place unusual stress on de novo nucleotide synthesis and nucleotide pool balance. This stress would be especially acute during initial phases of regenerative proliferation in response to cell death known to occur in both liver and lymphoid tissues with dietary methyl deficiency (24). In the present study, single cell suspensions were prepared from excised spleens by gentle massage with forceps and aspiration through a 21-gauge needle in ice-cold Hank's phosphate-buffered saline containing 0.1% glucose. Cell viability was assessed by Trypan blue exclusion and found to be > 9 5 % . An aliquot containing 108 viable cells was extracted in 0.5 ml ice-cold 0.6 M trichloroacetic acid (TCA), vortexed vigorously, and kept on ice for 20 min. The time between preparation of cell suspension and TCA addition did not exceed 10 min. After centrifugation, the acidic supernatant was transferred to a microcentrifuge tube containing 0.55 ml ice-cold freon-trioctylamine as previously described (25). The mixture was vortexed for 15 s and after centrifugation at 4°C, the lower phase was carefully removed by aspiration, leaving the aqueous solution of nucleotides. Samples were immediately lyophilized and subsequently stored at -70°C. Just before analysis, cell extracts 2472

Ftg. 2. (A) Reversed-phase HPLC profile of a standard mixture of ribonucleotides and deoxyribonucleotides. (B) Reversed-phase HPLC profile of spleen cell ribonucleotides and deoxyribonucleotides from control-fed (methionine, choline, folate-sufficient) F344 rats. The 20 /d injection volume represents the equivalent of 2.2 x 107 cells.

were resuspended in 0.2 M ammonium phosphate, pH 5.35. The 20 fil injection volume corresponded to the equivalent of 2.2 x 107 cells. The HPLC analyses were performed on a Beckman System Gold HPLC system consisting of a programmable solvent module (model 126) and wavelength detector (model 166) with an Econosphere C 18 reversed-phase column (5 micron, 300 x 4.6 mm, Alltech Associates). Absorbance was monitored at 260 nm with a flow rate of 0.8 ml/min throughout the run. Two buffers were utilized: buffer A consisted of 0.2 M (NH4)H2PO4 in 1.0 M KC1, pH 5.35; and buffer B was made of 0.2 M (NH4)H2PO4 with 1.25 M KC1 and 10% methanol, pH 5.0. Buffers were prepared fresh daily in double-distilled water, filtered (0.2 /i filter, Alltech Assoc.) and degassed by helium purging before use. Samples were isocratically eluted with 100% buffer A for the first 15 min. At t = 15 min, a 15 min linear gradient to buffer B was initiated. At t = 35 min, the system returned to 100% buffer A (over 12 s) to begin equilibrating for the next sample. A 10 min re-equilibration period was required between samples, The identity of dNTP peaks in cell extracts

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Fig. 1. Representation of the interactions between methionine, choline, folate and B12 in one-carbon metabolism emphasizing the central role of folate methyl groups for both DNA synthesis and SAM-mediated methylation reactions.

Alterations In nucleotide pools with methyl deficiency

Table I. Spleen cell nucleotide pools from control and methyl-donor deficient rats separated and quantitated by reversed-phase HPLC analysis* (pmol/106 cells) Nucleotide

dCTP dUTP dTMP dTTP dGTP dATP CTP GTP UTP ATP

FD 20.3 26.6 9.6 20.9 20.1 4.2 136.3 29.4 48.9 208.3

± ± ± ± ± ± ± ± ± ±

3.71 1.6 1.8 5.9 2.2 1.5 32.4 4.3 10.1 33.7

9.2 22.8 5.2 13.6 7.5 2.5 122.0 25.6 45.4 174.1

MCD ± ± ± ± ± ±

3.8 4.6 2.1* 6.1 4.5* 0.3* 26.0 ± 8.6 ± 10.1 ± 51.7

18.0 19.1 6.8 13.9 13.6 2.5 171.5 20.5 32.7 140.1

MCFD ± ± ± ± ± ± ± ± ±

5.5 4.5 2.4* 4.2* 3.9* 0.6* 46.2 11.3 17.1 71.1

17.3 23.2 5.7 13.3 2.0 2.3 110.7 21.4 34.5 132.1

± 4.6 6.7 1.3* ± 3.8* ± 3.5* ± 0.8* ± 52.1 ± 6.2* =fc 11.0 ± 38.2

•Values represent the mean ± SD (n = 6/group). *P < 0.05 compared to control group.

Anti-folate exposure in vitro has been associated with an

increase in dUTP pools and misincorporation of uracil in DNA in a variety of cycling cells (31,32). Uracil misincorporation has been proposed as a mechanism for genetic abnormalities associated with folate deficiency and thymidylate stress (11,33). However, in the present study, dUTP pools were not increased above control levels by dietary manipulation of folate availability. Since dNTP pool size measurement by HPLC is a static reflection of a highly dynamic and tightly regulated homeostatic process, an increase in dUTP turnover and/or misincorporation of uracil into DNA would not be detected. It is of interest to note that the dUTP pool (measured here in freshly isolated non-cycling lymphocytes) was considerably higher than that previously reported for rapidly proliferating cell lines. Resting lymphocytes have very low levels of dUTPase and uracil glycosylase which increase several-fold upon proliferative stimulation (34). The low dUTPase and uracil glycosylase activity in resting cells, coupled with an expanded dUTP pool might be expected to promote uracil misincorporation in fcJate-deficient cells. It has been hypothesized that a futile cycle of uracil misincorporation under conditions of folate deficiency contributes to inefficient DNA repair (12), DNA strand break accumulation (11) and mutation frequency (31) that may promote mechanisms of carcinogenesis (3). To summarize, we have shown that diet-induced folate and methionine/choline deficiency results in significant alterations in lymphocyte purine and pyrimidine dNTP pools. In recent preliminary studies, alterations in intracellular dNTP pool were also documented in rat hepatocytes with dietary methyl deficiency (data not shown). We hypothesize that regenerative hepatocyte proliferation in this model, under conditions of dNTP imbalance, could promote base misincorporation mutations as previously observed in vitro (14,15). Studies to explore this possibility are currently in progress. Acknowledgements The authors gratefully acknowledge the insightful comments and analysis of the manuscript by Dr Carlos Krumdieck. This study was supported by the American Cancer Society research grant no. BC-696.

References l.Krumdieck.C.L. (1983) Role of folate deficiency in carcinogenesis. In Butterworth.C.E. and Hutchenson.M.L. (eds), Nutritional Factors in the Induction and Maintenance of Malignancy. Academic Press, New York, pp. 225-245. 2. FinkelsteinJ.D. (1990) Methionine metabolism in mammals. J. Nutr. Biochem., 1, 228-237. 3. Eto.I. and Krumdieck,C.L.(1986) Rok of vitamin B12 and folate in carcinogenesis. In Poirier.L.A., Newbeme.P.M. and Pariza,M.W. (eds), Essential Nutrients in Carcinogenesis. Academic Press, New York, pp. 313—330. 4. Cook.R.J., Home.D.W. and Wagner.C. (1989) Effects of dietary methyl group deficiency on one-carbon metabolism in rats. J. Nutr., 119, 612—617.

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was verified by comparison of retention times with dNTP standards and by the increase in peak area after quantitative dNTP addition. Peak purity was confirmed by diode array detection. Chromatograms of a mixture of standards and a spleen extract from control animals are presented in Figure 2A and B respectively. Quantitative analysis of resolved peaks expressed as pmol/106 cells is presented in Table I. Decreases in the pyrimidine deoxyribonucleotides, dTMP and dTTP, were observed in all three methyl-donor deficient groups. The decrease in thymidylate derivatives in spleen cells from the deficient rats is consistent with a decrease in folate availability for de novo thymidylate synthesis. In the MCD group, the decrease in dTMP and dTTP occurred despite apparently adequate folate status as defined by normal serum folate levels (data not shown). The oneway diversion of folates towards methionine resynthesis would indirectly create a localized intracellular folate deficiency for nucleotide biosynthesis (Figure 1). In previous work, we demonstrated that de novo thymidylate synthesis was decreased in lymphocytes from methyl-deficient rats and was associated with an increase in DNA strand break accumulation and an increase in DNA repair activity (26,27). Both purine deoxynucleotide pools, dGTP and dATP, were significantly reduced in lymphocytes from the deficient rats (Table I) and may reflect a decrease in de novo purine biosynthesis. The corresponding ribonucleotide pools, GTP and ATP, were similarly reduced in the deficient groups with the most pronounced reduction in the MCFD group. The extreme sensitivity of the dGTP pool to chronic dNTP imbalance has previously been observed in a variety of cells (28-30); however, the mechanism is not clear. In the present study, it is possible that the lack of preformed nucleotides in the diet for salvage pathway synthesis interacted with folate insufficiency to severely compromise purine dNTP biosynthesis. This interpretation, however, is complicated by the observations of Home ex al. (5) who found an increase in formylated tetrahydrofolate (THF) derivatives despite a decrease in total folates in livers of methionine/choline deficient rats. The formyl group of THF provides the C-2 and C-8 carbons for de novo purine synthesis; therefore an increase in formylated THF derivatives with methyl deficiency would imply a block in purine synthesis by a mechanism other than the lack of folate. Because purine biosynthesis is energetically demanding, the decrease in ATP observed in the deficient rats could contribute to a decrease in de novo synthesis due to insufficient energy resources. The disproportionate decrease in dGTP relative to dATP, however, would imply a selective effect on dGTP biosynthesis in addition to a generalized block in purine biosynthesis.

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or folic acid.

The fidelity of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors fo...
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