( RESEARCH 51, 16-21. January. 199I| Effects of Deficiency and Treatment upon Content and Distribution1

Jacob Selhub,2 Elias Seyoum, Elizabeth A. Pomfret, and Steven H. Zeisel

United Stales Department of Agriculture Human Research Center on Aging, Tufts University. Boston, Massachusetts 02111 fj. S., E. S.J, and Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina 27599 [E. A. P., S. H. Z.]

ABSTRACT phospholipids including and sphingomye- lin (4-6). In rats fed a choline deficient diet, hepatic concentra We examined the effects of feeding rats a choline deficient diet, of tions of choline, betaine, and phosphatidylcholine are dimin treating rats with low doses of methotrexate (MIX. 0.1 mg/kg, daily), ished, as are concentrations of and AdoMet (7-13). and of combined choline deficiency and MIX treatment upon the content and distribution of in liver. We used a newly devised technique These decreases are due to both the impairment of normal AdoMet via the choline/betaine pathway as well for analysis of folates which utilized affinity chromatography followed as to the added requirements for "labile methyl groups" to be by high pressure liquid chromatography. Compared to control rats, hepatic folate content decreased by 31% in the choline deficient rats, by used for the synthesis of choline from phosphatidylethanola- 48% in the MTX treated rats, and by 60% in rats which were both mine (13). Home et al. (14) have shown that rats fed with a choline deficient and treated with MTX. In extracts of from control diet that was deficient in both methionine and choline ("methyl rats, folates were present predominantly as penta (35%) and hexagluta- group" deficient diet) had hepatic folate concentrations that myl (52%) derivatives. The pteridine ring structure distribution of these were one-half of those present in controls. This depletion of folates was as follows: 48% 5-methyltetrahydrofolate, 14% formylated folate was associated with changes in folate distribution which tetrahydrofolate, and 39% tetrahydrofolate. In choline deficient animals, included an increase in the relative concentrations of formylated there was a decrease in the relative concentration of pentaglutamyl folates tetrahydrofolates, a decrease in the concentrations of THF and and an increase in the relative concentration of heptaglutamyl folates. In livers from MTX treated animals, MTX-polyglutamates with 2-5 gluta no change in the proportion of 5-methyl-THF. mate residues accumulated. The consequences of MTX treatment were: that alter the susceptibility of rodents to chemical a) an elongation of the glutamate chains of the folates as the proportion carcinogens may alter the susceptibility of patients to the car- of hepta- and octaglutamyl derivatives was increased relative to penta- cinogenicity of drugs (15). Lipotrope (choline) deficiency is a and hexaglutamyl folates; b) the occurrence of unreduced folie acid; c) a powerful potentiator of chemical carcinogenesis in the liver and decrease in the relative concentration of 5-methyltetrahydrofolate and an other tissues (15). The agent, methotrexate interferes increase in the relative concentration of formylated tetrahydrofolate, and with folate , and creates biochemical perturbations glutamic acid chains of folates and novo synthesis of methionine due to alterations in folate metab those that regulate folate dependent synthesis and utilization of one olism, including diminished capacity of the liver to retain folate carbon unit. (19, 20). MTX is widely used as a chemotherapeutic agent (21- 23) and, at low doses, for the treatment of psoriasis (24), rheumatoid arthritis (25, 26), and certain liver disorders (27- INTRODUCTION 29). In chemotherapy, MTX is often administered in combi Folate and choline metabolism are interrelated, and pertur nation with other agents, many of which are carcinogens (15). bations in one pathway affect the other. The transmethylation Diminished tissue stores of choline are seen in humans fed i.v. of by 5-methyltetrahydrofolate is a most impor (30); parenteral nutrition is often used in cancer patients. In tant folate dependent reaction because the product, methionine, rats, choline deficiency and treatment with MTX significantly is the precursor of 5-adenosylmethionine (1). Methionine bio increase the incidence of procarbazine induced mammary tu synthesis via this pathway cannot meet all demands for mors (15). It is possible that lipotrope or , AdoMet' and, therefore, dietary sources of labile methyl groups caused by choline deficiency or MTX treatment, might increase (methionine and choline) are required (2). Choline, after con the carcinogenicity of chemotherapeutic agents in humans. version to betaine, can act as the methyl donor for homocysteine The present study was undertaken to determine the effects of transmethylation, a reaction catalyzed by betaine transmethy- choline deficiency and methotrexate treatment (alone and in lase (3). Choline is also the precursor for a number of membrane combination) on hepatic folate distribution. Hepatic folate dis-

Received 6/14/90; accepted 8/28/90. 3The abbreviations used are: AdoMet. 5-adenosylmethionine; THF, tetra The costs of publication of this article were defrayed in part by the payment hydrofolate; HPLC. high pressure liquid chromatography; PteGlu,_7, (Pl-7) of page charges. This article must therefore be hereby marked advertisement in pteroylglutamates, the subscripts denote the number of glutamate residues. Folie accordance with 18 U.S.C. Section 1734 solely to indicate this fact. acid or folates denote pteroylglutamates with unspecified number of glutamate 'This work was supported by Grant HD-16727 from the NIH and by the residues H2PteGlu,_, (Dl-7) and H4PteGlu,_7 (Tl-7) refer to the dihydro- and United States Department of Agriculture, Agricultural Research Service under the tetrahydrofolate derivatives, respectively. 5-Methyl-H4PteGlUi_7 (Ml-7) re Contract 53-3K06-5-10. The content of this publication does not necessarily fers to tetrahydrofolates with methyl substitution: 10-formyl-H4PteGIU| 7(Fl-7) reflect the views or policies of the United States Department of Agriculture, nor and 5-formyl-H4PteGlui-7 (Ll-7) refer to tetrahydrofolates with 10-formyl and does mention of trade names, commercial products, or organizations imply 5-formyl substitutions, respectively. Abbreviations in parentheses are the desig endorsement by the United States Government. nations for the various derivatives used to describe hepatic folate distribution 2 To whom requests for reprints should be addressed, at Labo based on Chromatographie data. Methotrexate is abbreviated in the text as MTX ratory, USDA Research Center on Aging. Tufts University, and in chromatograms as Xn. where n denotes the number of total glutamate 711 Washington St., Boston M A 02111. residues. 16

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. EFFECTS OF CHOLINE AND METHOTREXATE ON HEPATIC FOLATE

tribution was determined by using a new method which com between Tn and F„.Integrated peak areas were used for estimation of bines affinity chromatography with high pressure liquid chro- folate concentrations of the individual peaks and the sum of these matography (31, 32). One advantage of this method is that it integrated areas were used to determine total folate content of the provides information on the pteridine ring distribution as well sample. A mixture of PteGlu,_7 (1 nmol each) standards was run through the column after chromatography of 2-3 samples. These runs as changes in the glutamate chain lengths. served to determine the outer boundaries of the respective clusters (32). The monoglutamyl-MTX (Xi) and its polyglutamate cogeners (X2- MATERIALS AND METHODS X6) were identified on the basis of retention time determined with authentic standards and spectral properties. Fig. 1 shows a typical Male Sprague-Dawley rats (approximately 200 g body weight; chromatogram of MTX standards. When the affinity column was not overloaded with other folates, retention of MTX was greater than 90%. Charles River Breeding Laboratories, Wilmington, MA) were housed When data are expressed as nmol/g folate liver, MTX concentrations in suspended stainless steel wire cages in a climate controlled room (24°C),and were exposed to light from 6 a.m. to 6 p.m. daily. Animals were not included. Data were analyzed for statistical significance by using analysis of variance and appropriate critical difference tests with were fed the control diet (ICN Nutritional Biochemicals custom control Tukey's honestly significance difference method. diet: 10% casein, 10% a-soy protein, 20% lard, 56% sucrose, 4% mix W, ICN mix with choline; the diet contained final content of 0.2% choline, 0.27% cystine, and 0.35% methionine) for 1 week prior to use in order to acclimate them to a semisynthetic diet. RESULTS Rats were randomly assigned to 4 treatment groups (n = 5/group) and pair fed a control or choline deficient diet (same as the control diet All animals gained similar amounts of weight during the 2 except that we used ICN vitamin mix without choline; it contained a weeks of treatment (77-84 g). There were no significant differ final content of 0.002% choline) for 2 weeks. Five animals on each diet ences in hepatic protein concentrations (142-150 mg/g liver). were treated daily for 2 weeks with MTX (0.1 mg/kg body weight As compared with controls, rats fed the choline deficient diet administered i.p. at midday; Behring Diagnostics, La .lolla, CA). On had 3-fold more hepatic triacylglycerol and diminished hepatic day 15, the rats were anesthesized with diethylether and samples of liver concentrations of choline (<50% of control), betaine (30% of were removed, immediately weighed, and placed in 5 volumes of boiling extraction solution (10 mM 2-mercaptoethanol and 2% control), methionine (80% of control), and AdoMet (60% of ascorbate in 0.1 M [bis(2-hydroxyethyl)imino]tris(hydroxymethyl)- control). Liver S-adenosylhomocysteine concentration in methane buffer at pH 7.8). After 15 min in the boiling bath the creased by 25%. Methotrexate treatment alone was associated extraction solution was cooled on ice, the mixture was homogenized, with decreased concentrations of hepatic betaine (55% of con and the supernatant fraction was collected after centrifugation at 20,000 trol), AdoMet (75% of control), and methionine (88% of con xgfor 15 min (31). trol). Combined treatment with MTX and choline deficiency Analysis of folate was conducted by using a new method (31) which further diminished hepatic AdoMet concentrations (to 33% combines affinity chromatography with the use of immobilized control) and increased S-adenosylhomocysteine (to 150% of folate binding protein with HPLC for direct determination of folate control). A full report of these changes has been published distribution. An aliquot of the supernatant fraction (with an estimated elsewhere (13). total folate plus MTX content not exceeding 15 nmol) was mixed with a trace amount of ['Hjfolic acid (5 ßC\,40 Ci/mmol; Amersham, Fig. 2 shows ion pair HPLC of representative purified liver Arlington Heights, IL) and applied to a folate binding protein-Sepha- extracts from the four treatment groups. The two major clusters rose (affinity) column (32). The column (1 ml bed volume) was washed in Fig. 2A are typical of rat liver folates (32) and consist of sequentially with 15 ml l M phosphate buffer, pH 7.0, 15 reduced penta- and hexaglutamyl derivatives. The first peak in ml water, and then eluted with 4 x 1 ml 0.02 M trifluoroacetic acid each cluster contains 10-formyl-H4PteGlun(Fn) and unsubsti- containing 0.01 M dithioerythritol. The acidic fractions were promptly tuted H4PteGlu„(T„).Thesecond peak, which is a minor con neutralized with l M piperazine. Aliquots of these fractions were used stituent, represents 5-formyl-H4PteGlu„(L„),whereasthe last to determine 3H content; a recovery of 90% or more of applied radio peak consists of 5-methyl-H4PteGlu„(M„).Inaddition to these activity was taken as an indication that the affinity column was not two clusters, the liver extracts contained traces of heptaglutamyl overloaded. The remainder of the neutralized fractions were combined folate derivatives (T7 and M7) and of 5-methyl-H4PteGlu,(M,). and an aliquot of 0.9 ml was used for analysis of folate distribution by ion pair liquid chromatography, using diode array detection (31). Briefly, the sample was injected onto a Ci8 HPLC column (Econos- phere, 5 pm, 4.6 x 100 mm; Alltech, Deerfield, IL) which had been equilibrated with 5% tetrabutyl ammonium phosphate, 25 mM NaCl, 5 mM dithioerythritol, and 10% acetonitrile. After sample application, folates were eluted from the column by using a linear gradient (10- 8B- 65%) of acetonitrile in the same equilibration solution. In this Chro 60' matographie system, folates elute in the order of increasing number of T I me Cm 1n. ) glutamate residues. Those with the same number of residues elute as a 40 separate cluster arranged in three groups. Group 1 consists of 10- 60: 241 401 formyltetrahydrofolates (10-formyl-H4PteGlu„;F„),tetrahydrofolates 40: Wavelength (nm) (H4PteGlu„;T„),anddihydrofolates (H2PteGlu„;D„).Group2 consists 20- of 5-formyltetrahydrofolates (5-formyl-H4PteGlu„;L„),andgroup 3 of 5-methyltetrahydrofolates (5-methyl-H4PteGlu„; M„)and folates (PteGlu„;P„).Thediode array detection system permitted the monitor Fig. 1. Chromatographie separation of MTX and its polyglutamates cogeners. ing of the column effluent at 280 nm for determination of folate activity Top. chromatography of a mixture containing I nmol each of MTX and its and information on the number of glutamate residues of any eluting cogeners with total glutamate residues of 1-6 (X1-X6). Bottom, chromatography folate. A final identification of the pteridine structure of the eluting of a similar mixture which was eluted from a Sepharose-FBP (affinity) column folate was made on the basis of the UV absorption values at 350 and aimed at testing MTX retention by this column. Similar respective peak heights in the two chromatograms indicate quantitative retention of MTX and its cogeners 258 nm. Absorption values at 350 nm were used for the identification under these conditions. Right, spectral scan of MTX eluting from the column at of P„andD„.Absorption values at 258 nm were used to differentiate wavelength range of 240-400 nm. mAU, milliabsorption units. 17

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. EFFECTS OF CHOLINE AND METHOTREXATE ON HEPATIC FOLATE

Except for minor differences, which will be dealt with later, the pattern of hepatic folate distribution in choline deficient ani mals (Fig. 2B) was similar to that from the livers of control animals. I Chromatography of the purified liver extracts from the meth- otrexate treated animals (Fig. 2, C and />) yielded additional peaks (X2-X5)which represent MTX polygiutamates containing (total) 2-5 glutamate residues. These samples contained larger proportions of heptaglutamyl and octaglutamyl folates and lesser proportions of pentaglutamyl folates. In addition, in the Control Choline- MTX MTX/Choline- region of monoglutamyl folates, unmodified PteGlu,(Pi) was deficient deficient present and there was no 5-methyl-H4PteGlui(Mi). Fig. }. Effects of choline deficiency and MTX treatment on total hepatic folate Total folate in the liver was decreased by 31% in choline content in rats. Data are presented as means - SE. a denotes that the respective deficient animals (P < 0.05), by 48% in MTX treated animals value is statistically different than control (P < 0.05-0.01); ft denotes that the (P < 0.01), and by 60% in animals that were both choline difference in values between hepatic folate levels in choline deficient plus MTX treated animals and those from animals which were only choline deficient, is deficient and treated with MTX (P < 0.01) (Fig. 3). Fig. 4 statistically significant (P< O.OS). shows the distribution of MTX polygiutamates in livers of

MTX treated animals. In both the choline sufficient and the 0 75 n choline deficient groups, polyglutamation did not extend be yond the pentaglutamyl level. The degree of polyglutamation was increased in the choline deficient rats. Folate distribution (Fig. 5) was similar in the control and choline deficient rats. However, the portion of total folates present as the H4PteGlu5(T5) cogener was decreased (P< 0.05). The portion present as T7 tended to increase; however this increase was not statistically significant. Distribution based 025 upon the pteridine ring structure was similar in the livers from choline deficient and control rats (Fig. 6). MTX treatment was associated with the elongation of the glutamic acid chains of folates (Fig. 5). There was a decrease in 000 the concentration of pentaglutamyl (folate) derivatives and an MTX derivative Fig. 4. Distribution of MTX polygiutamates in livers of MTX treated animals. Difference in the distribution between the two groups was not significant except for X5 (a) which was higher in choline deficient IMI than in choline sufficient ('.j animals (P < 0.05).

increase in the relative concentration of heptaglutamyl folates. ia 40 This effect was even more pronounced in livers from the choline deficient/MTX treated rats, where pentaglutamyl folates de creased to less than 10% of the total, hexaglutamyl folates DŒE25-20-IS-100i decreased to 31% of the total, while hepta- and octaglutamyl folates increased 38 and 20% of total folates, respectively (P < 0.05) (Fig. 5). MTX treatment was associated with the doubling 1B3S^Ay>a 20 in the relative concentrations of formylated tetrahydrofolates (F„+Ln), while a relative concentration of 5-methyltetrahydro folates decreased by 26% when compared to control (P < 0.01) (Fig. 6). The relative concentration of tetrahydrofolate deriva 1 51 tives were unchanged from control. In livers from the choline deficient/MTX treated rats, the relative concentration of 5- methyltetrahydrofolates in the liver decreased by 26%, tetra hydrofolate relative concentrations decreased by 50%, while the formylated tetrahydrofolate relative concentrations tripled (P < 0.01) (Fig. 6). I

10 20 30 40 T 1me Cm 1n. ) DISCUSSION Fig. 2. Chromatographie separation of purified rat liver folates. Each chro- matogram was obtained from the purification of folates from 0.2 g liver and is a Numerous reactions use folate containing coenzymes. Regu representative of 5 liver samples from equally treated animals. A, from control rats; B, from choline deficient rats; C, from MTX treated rats; and D, from lation of folate dependent metabolism occurs, in part, at the choline deficient and MTX treated rats. The identity of folate derivatives in the level of these coenzymes. The coenzyme which mediates one various peaks is denoted as: F for 10-formyltetrahydrofolates; Mm unsubstituted tetrahydrofolates; M for 5-methyltetrahydrofolates, and X for methotrexate. reaction may inhibit a second folate dependent reaction and Numbers following these letters denote (total) number of glutamate residues. coenzyme activity/inhibitor status may depend upon the chain 18

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. EFFECTS OF CHOLINE AND METHOTREXATE ON HEPATIC FOLATE

lengths of the glutamates bound to folate (33, 34). The capacity for analyzing the distribution of the various folates within tissues is of importance, as it provides insights into the changes in tissue specific folate dependent metabolic activity in response to drugs and nutrients. This concept has not been widely used because of difficulties in resolving the

Ml PI F5 T5 M5 F6 T6 M6 F7 T7 Õ17 F8 T8 M8 many forms of folates that may exist within the same tissue. In fact, most studies on tissue folate distribution used methods which included steps to reduce the number of derivatives to a more manageable number (35, 36). The method that was de veloped in our laboratory (31, 32) and used in the present study is both simple and rapid and provides information on the I TJ content and distribution of folates with emphasis on both ends C of the folate molecule. In fact, the Chromatographie analyses of gu £ the purified liver folates from the MTX treated animals are the Ml PI F5 T5 MS F6 T6 M6 F7 T7 n7 F8 T8 M8 first of their kind. Previous studies on the effect of MTX on folate distribution in the cell relied on analysis of radioactivity distribution following the addition of radiolabeled folate and allowing time for equilibration with endogenous folate (35). We have shown that choline deficiency and MTX treatment, alone or in combination, caused both quantitative and qualita tive changes in hepatic folates. These changes are likely to represent changes in the content and distribution of folate in

Ml PI F5 T5 M5 F6 T6 M6 F7 T7 M7 F8 T8 MB hepatocytes and are not due to fatty infiltration of the liver (hepatic protein concentrations were not significantly different

30 in the various treatment groups). MTX treatment was associ ated with a) the accumulation of MTX polyglutamates; b) the appearance of unmodified PteGlu; c) elongation of the gluta mate chains; and d) a decrease in the proportion of 5-methyl- THF, an increase in the proportion of formylated THF, and no É change in the proportion of THF. Under normal conditions, PteGlu, which was the sole source Ml PI F5 T5 MS F6 T6 M6 F7 T7 M7 F8 T8- M8 of folate in the experimental diet used in this study, is reduced and methylated in the course of transport across the intestinal Fig. 5. Effect of choline deficiency and MTX treatment on hepatic folate distribution in rats. Data are presented as means ±SE of percentage of total wall (37) and/or transport into hepatocytes (38). This reduction folate. .1, percentage of hepatic folate distribution in control animals: H. percent has two important functions. It provides coenzymes for those age of hepatic folate distribution in choline deficient animals; C, percentage of hepatic folate distribution in MTX treated animals; and /). percentage of hepatic which are involved in one carbon metabolism as well folate distribution in choline deficient animals that were also treated with MTX; as the substrate (in the form of H4PteGlu) for the synthesis of a denotes values which are significantly different (P < 0.05) than the respective folate polyglutamates (39). MTX inhibits the reduction of values from control animals; b denotes values which are significantly different (P < 0.05) than the respective values from the choline deficient animals; c denotes PteGlu to H4PteGlu and, as a consequence, both the supplies values which are significantly different (P< 0.05) than the respective values from of folate coenzymes and of the H4PteGlu substrate for the the (choline supplemented) animals which were treated with MTX. conversion to polyglutamyl derivatives are impaired. The un modified PteGlu, which is poorly polyglutamated, leaks off the cell (40). MTX is also an inhibitor of the transport of PteGlu across the intestine and into hepatocytes (41, 42) and this would contribute further to the impairment of folate supply to the tissue. In fact there are indications that MTX affects the assim ilation into the body folate pool of reduced folates as well (19, 43, 44). These multiple sites of inhibition strongly suggest that in the course of MTX treatment little, if any, of the PteGlu from the diet was assimilated into the liver folate pool. This possibility is in line with the observed accumulation in the liver of large concentrations of MTX polyglutamates which are accompanied by the occurrence of unmodified PteGlu. Elongation of the glutamate chains of folates in the liver is the function of both the pteridine ring structure and the number PteGlu THF FormylTHF MethylTHF of glutamic acid residues attached (37). With tetrahydrofolates Fig. 6. Effect of choline deficiency and MTX treatment on the pteridine ring (the most effective substrates), elongation is most rapid when distribution of folates in rats. Data represent means ±SE of the pteridine ring distribution, given as percentage of total folates, irrespective of the glutamic acid the total glutamate residues number 5 or less. Beyond chain length. D, hepatic folates in control animals; D, in choline deficient animals; H4PteGlu6 additional glutamates are attached at much lower M, in MTX treated animals; and B, in choline deficient-MTX treated animals, a, b, and c denote the occurrence of significant differences (P< 0.05) among groups rates due to a sharp drop in the affinity of polyglutamate as described in the previous legend. synthase. Folate depletion which accompanies MTX treatment 19

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. EFFECTS OF CHOLINE AND METHOTREXATE ON HEPATIC FOLATE

6. Cohen, E. L.. and Wurtman. R. J. Brain acetylcholine: control by dietary may result in a slower turnover of the residual folate and hence, choline. Science (Washington DC). 191: 561-562, 1976. a greater than normal residence time to allow the synthesis of 7. Shivapurkar. N., and Poirier, L. A. Tissue levels of S-adenosylmethionine hepta- and octaglutamyl folates. Elongation of the glutamate and .C-adenosylhomocysteine in rats fed methyl-deficiency, -de fined diets for one to five weeks. Carcinogenesis (Lond.), 4: 1051-1057, chain length of folates was also reported by Cassady et al. in 1983. livers from folate deficient rats (45) and in a number of other 8. Poirier, L. A., Grantham. P. H., and Rogers, A. E. The effects of a marginally lipotrope-deficient diet on the hepatic levels of S-adenosylmethionine and on conditions including acute alcohol intake (46). In some of these the urinary metabolites of 2-acetylaminofluorene in rats. Cancer Res., 37: instances chain elongation may be explained on the basis of 744-748, 1977. increased residence time of the folate within the liver (47). In 9. Shivapurkar, N., Wilson, M. J., Hoover, K. L., Mikol, Y. B., Creasia, D., and Poirier. L. A. Hepatic DNA and liver tumor formation in other cases this relationship is not obvious. male C3H mice fed methionine- and choline-deficient diets. J. Nail. Cancer MTX and its polyglutamate derivatives are strong inhibitors Inst., 77:213-217. 1986. of aminoimidazolecarboxamide ribonucleotide transformylase 10. Barak, A. J.. Beckenhauer. H. C.. and Tuma. D. J. Use of 5-adenosylmethio- nine as an index of methionine recycling in rat liver slices. Anal. Biochem.. (33). This inhibition was thought to be responsible for the 127:312-315. 1982. inhibition of purine synthesis in MCF-7 breast cancer cells (48) 11. Freeman-Narrod. M., Narrod. S. A., and Custer, R. P. Chronic toxicity of and could explain the data from the present study which showed methotrexate in rats: partial to complete protection of the liver by choline: brief communication. J. Nati. Cancer Inst., 59: 1013-1017. 1977. an increase in the relative concentration of formylated THF in 12. Yao, Z. M., and Vance, D. E. The active synthesis of phosphatidylcholine is livers of rats treated with MTX. An alternative, but not mu required for very low lipoprotein secretion from rat hepatocytes. J. tually exclusive, interpretation is related to the observed de Biol. Chem.. 263: 2998-3004, 1988. 13. Pomfret, E. A.. da-Costa, K. A., and Zeisel, S. H. Effects of choline deficiency crease in hepatic methionine and AdoMet concentrations in and methotrexate treatment upon rat liver. J. Nutr. Biochem., /: 533-541, MTX treated rats. Undoubtedly depletion of hepatic folate 1990. 14. Home, D. E., Cook, R. J., and Wagner. C. Effect of dietary supply will impair the of methionine. How deficiency on folate metabolism in rats. J. Nutr., 119: 618-621, 1989. ever, the observation that MTX treatment is associated with a 15. Rogers, A. E.. Akhtar, R., and Zeisel. S. H. Procarbazine carcinogenicity in sharp decline in 5-methyl-THF concentrations point to the methotrexate-treated or lipotrope-deficient male rats. Carcinogenesis, in press, 1990. possibility of impairment in the synthesis of this folate. This 16. Barak, A. J., Tuma, D. J., and Beckenhauer, H. C. Methotrexate hepato- effect of MTX on 5-methyl-THF concentration in rat liver was toxicity. J. Am. Coll. Nutr., 3: 93-96, 1984. also reported earlier by Barak et al. (49) and in cell cultures by 17. Barak, A. J., and Kemmy, R. J. Methotrexate effects on hepatic betaine levels Kesavan et al. (50) and by Allegra et al. (48). Methylene-THF in choline-supplemented and choline-deficient rats. Drug-Nutri. Interact., I: 275-278. 1982. reducíase,which catalyzes the synthesis of 5-methyl-THF, was 18. Svardal, A. M., Ueland, P. M., Berge, R. K.. Aarsland, A., Aasaether, N., strongly inhibited by dihydrofolate polyglutamates (34), and it Lonning, P. E., and Refsum. H. Effect of methotrexate on homocysteine and other compounds in tissues of rats fed a normal or a defined, choline-deficient is possible that this inhibition is exerted also by MTX and its diet. Cancer Chemother. Pharmacol., 21: 313-318, 1988. polyglutamate derivatives as reported by Chabner et al. (51). 19. Bardford, P. A.. Blair, J. A., and Malghani, M. A. K. The effect of metho The above interpretation, that MTX is an antagonist of trexate on folate metabolism in the rat. Br. J. Cancer. 41: 816-820, 1980. methionine synthesis and is an inhibitor of the synthesis of 5- 20. Huang, S., and Thenen, S. W. Lymphocyte cultures and dU suppression test in folate deficient rats and cebus monkey. Fed. Proc., 46: 1158, 1987. methyl-THF is also consistent with the changes seen in hepatic 21. Berlino. J. R., Levitt, M., McCullough, J. L., and Chabner, B. New ap proaches to chemotherapy with folate antagonists: use of leucovorin "rescue" folate distribution in rats which were both choline deficient and and enzymic folate depletion. Ann. NY Acad. Sci., 186: 486-495, 1971. treated with MTX. These changes cannot be explained on the 22. Bonadonna, G., Brusamolino. E.. and Valaguasa, P. Combination chemo basis of additive effects of individual contributions of choline therapy as an adjuvant tre'ment in operable breast cancer. N. Engl. J. Med., 294:405-410. 1976. deficiency and MTX treatment. Choline deficiency, which alone 23. Hryniuk. W. M., and Berlino, J. R. Treatment of leukemia with large doses had no effect on hepatic folate distribution, in combination of methotrexate and : clinical-biochemical correlates. J. Clin. with MTX caused a net increase in the accumulation of for Invest.. ¥«.-2140-2155.1969 24. Weinstein, G. D. Methotrexate. Ann. Intern. Med., 86: 199-204, 1977. mylated THF and a sharp decrease in the concentration of 25. Willkens, R. F., and Watson, M. A. Methotrexate: a perspective of its use in THF. These changes in folate distribution are the same as those the treatment of rheumatic diseases. J. Lab. Clin. Med., 100: 314-321, 1982. observed by Home et al. in rats fed a restricted choline/methi- 26. Ward, J. R. Historical perspective on the use of mcthotrexate for the treatment of rheumatoid arthritis. J. Rheumatol.. 12: 3-6, 1985. onine diet (14). 27. Kaplan, M. M.. Knox, T. A., and Arora, S. A. Primary biliary cirrhosis The increasing use of low doses of MTX for long periods of treated with low-dose oral pulse methotrexate. Ann. Intern. Med.. 109: 429- 431, 1988. time to treat a variety of chronic illnesses raises a number of 28. Anonymous. Folate status on long-term, low dose methotrexate therapy. important questions regarding potential benefits and side effects Nutr. Rev., 47: 43-45. 1989. that this treatment causes. In humans, MTX treatment appears 29. Peterson, T. C., and Williams, C. N. Depression of peripheral mono- cyte aryl hydrocarbon hydroxylase activity in patients with liver disease: also to result in the decrease of folate body pool (43, 44). The possible involvement of macrophage factors. Hepatology (Baltimore), 7:333- present study provides basis of understanding some of the 337, 1987. complex interrelationships between choline, folate, and MTX 30. Sheard, N. F., Tayek, J. A., Bistrian. B. R., Blackburn, G. L., and Zeisel, S. H. Plasma choline concentration in human fed parentcrally. Am. J. Clin. metabolism. Nutr., «.-219-224, 1986. 31. Selhub, J. Determination of tissue folate composition by affinity chromatog- raphy followed by high-pressure ion pair liquid chromatography. Anal. REFERENCES Biochem.. 182: 84-93, 1989. 32. Selhub, J.. Darcy-Vrillon, B., and Fell, D. Affinity chromatography of 1. Krumdieck, C. L. Role of folate deficiency in carcinogenesis. In: C. E. naturally occurring folate derivatives. Anal. Biochem., 168: 247-251, 1988. Butterworth and M. L. Hutchinson (eds.). Nutritional Factors in the Induc 33. Allegra, C. J., Drake, J. C., Jolivet, J., and Chabner, B. A. Inhibition of tion and Maintenance of Malignancy, pp. 225-242. New York: Academic phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate Press. 1983. and polyglutamates. Proc. Nati. Acad. Sci. USA, 82:4881- 2. Mudd, S. H., and Poole, J. R. Labile methyl balances for normal human on 4885, 1985. various dietary regimens. Metabolism. 24: 721-735. 1975. 34. Matthews. R. G., and Baugh, C. M. Interactions of pig liver methylenetet- 3. Barak, A. J., and Tuma, D. J. Betaine. metabolic by-product or vital meth- rahydrofolate reducíasewith methylenctetrahydrofolatepolyglutamate sub ylating agent? Life Sci.. 32: 771-774, 1983. strates and with dihydropteroylpolyglutamate inhibitors. , 19: 4. Zeisel. S. H. Dietary choline: biochemistry, physiology, and pharmacology. 2040-2045, 1980. Annu. Rev. Nutr., /: 95-121, 1981. 35. Krumdieck, C. L., Tamura, T.. and Eto, S. Synthesis and analysis of the 5. Kennedy, E. P., and Weiss, S. B. The function of cytidine coenzymes in the pteroylglutamates. Vitam. Horm., 40: 45-94, 1983. biosynthesis of phospholipides. J. Biol. Chem.. 222: 193-214. 1956. 36. Selhub, J. Analysis of tissue folate composition. In: M. F. Picciano, E. L. R. 20

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. EFFECTS OF CHOLINE AND METHOTREXATE ON HEPATIC FOLATE

Stokstad, and J. Gregory (eds.), Contemporary Issues in Clinical Nutrition. 44. Morgan, J. E., Baggott, J. E., and Altz-Smith, M. Folate status of rheumatoid Folie Acid Metabolism in Health and Disease, pp. 171-194. New York: Alan arthritis patients receiving long-term, low-dose methotrexate therapy. Ar R. Liss, 1990. thritis Rheum., 30: 1348-1356, 1987. 37. Selhub, J., Brin, H., and Grossowicz, N. Uptake and reduction of radioactive 45. Cassady, I. A., Budge, M. M.. Healy, M. J., and Nixon, P. F. An inverse folate by everted sacs of rat . Eur. J. Biochem., 33: 433-438, relationship of rat liver folate polyglutamatc chain length to nutritional folate 1973. deficiency. Biochim. Biophys. Acta, 633: 258-268, 1980. 38. Whitehead, V. M., and Cooper, B. A. Absorption of unaltered folie acid from 46. Krumdieck, C. L., and Eto. I. Folates in tissues and cells. Support for a two- the gastrointestinal tract in man. Br. J. Haematol., 13: 679-686, 1967. tier hypothesis of regulation of one-carbon metabolism. In: B. A. Cooper, 39. Cook, J. D., Cichowicz, D. J., George. S.. Lawler, A., and Shane, B. and V. M. Whitehead (eds.). Chemistry and Biology of Pteridines, pp. 447- Mammalian folylpoly->-glutamate synthetase. 4. In vitro and in vivo metab 466. Berlin: Walter de Gruyter, 1988. olism of folates and analogues and regulation of folate . Bio 47. Hulmán.R. S., and Steinberg, S. E. The effects of alcohol on folate metab chemistry, 26: 530-539, 1987. olism. Annu. Rev. Med., 33: 345-354, 1982. 40. Foo, S. K., and Shane, B. Regulation of folylpoly-^-glutamate synthesis in 48. Allegra, L. J.. Fine. R. L.. Drake, J. C, and Chabner. B. A. The effect of mammalian cells. In vivo and in vitro synthesis of pteroypoly->-glutamates methotrexate on intracellular folate pools in human MCF-7 breast cancer by Chinese hamster ovary cells. J. Biol. Chem., 257: 13587-13592. 1982. cells. Evidence for direct inhibition of purinc synthesis. J. Biol. Chem., 267: 41. Strum, W. B. A pH mediated, carrier mediated transport system for the 6478-6485, 1986. folate analog, amethopterin, in rat jejunum. J. Pharmacol. Exp. Ther., 203: 49. Barak, A. J., Kemmy, R. J.. and Tuma, D. J. The effect of methotrexate on 640-645, 1977. homocysteine methylating agents in rat liver. Drug-Nutr. Interact., /: 303- 42. Hörne,D. W., Briggs, W. T., and Wagner, C. Transport of 5-methyltetra- 306, 1982 hydrofolic acid and folie acid in freshly isolated hepatocytes. J. Biol. Chem., 50. Kesavan, V., Sur, P., Doig. M. T., Scanlon, K. J.. and Priest, D. G. Effects 255:3529-3535,1978. of methotrexate on folates in Krebs ascites and L1210 murine leukemia cells. 43. Kremer, J. M., Galivan, J., Streckfuss. A., and Kamen, B. Methotrexate Cancer Lett.. 30: 55-59, 1986. metabolism analysis in blood liver of rheumatoid arthritis patients. Associa 51. Chabner, B. A., Allegra, C. J.. Curt. G. A.. Clendininn. N. J.. Baram, J., tion with hepatic folate deficiency and formation of polyglutamates. Arthritis Koizumi. S.. Drake, J. C.. and Jolivet, J. Polyglutamation of methotrexate. Rheum., 29: 832-835, 1986. Is methotrexate a prodrug? J. Clin. Invest., 76: 907-912. 1985.

21

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. Effects of Choline Deficiency and Methotrexate Treatment upon Liver Folate Content and Distribution

Jacob Selhub, Elias Seyoum, Elizabeth A. Pomfret, et al.

Cancer Res 1991;51:16-21.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/51/1/16

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/51/1/16. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research.