Folate Deficiency in the Livers of Diethylnitrosamine Treated Rats1

[CANCER RESEARCH 36, 2775-2779, August 1976] Folate Deficiency in the Livers of Diethylnitrosamine treated Rats1

Yoon Sook Shin Buehring,2 Lionel A. Poirier, and E. L. R. Stokstad

Department of Nutritional Sciences, University of California, Berkeley, California 94720 (V. S. S. B., E. L. R. 5,1, and Carcinogen Metabolism and Toxicology Branch, National Cancer Institute, Bethesda, Maryland 20014 fL. A. P.]

SUMMARY activities of such agents as 2-acetylaminofluorene (22), N, N-dimethyl-4-aminoazobenzene (7, 19), DENA (27, 28), The effects of diethylnitrosamine on the metabolism of methylcholanthrene (24, 25), and 7,12-dimethyl folic acid and related compounds in rat liver were investi benz(a)anthracene (ii). Furthermore, the lipotropes methi gated. The administration, in the drinking water, of diethyl onine, vitamin B@9,andfolic acid are all prospective targets nitrosamine to rats for 3 weeks led to decreased hepatic . of the electrophilic activated form of 1 or more chemical levels of folate, S-adenosylmethionine, and 5-methyltetra carcinogens (12, 16, 20). The metabolic interrelations hyd rofolate :homocysteine methyltransferase . Liver methyl among the various lipotropes are very complex, and it is enetetrahydrofolate reductase levels were unaffected by ad impossible to alter the tissue levels or metabolism of one ministration of diethylnitrosamine. The polyglutamate frac without simultaneously altering the metabolism of each of tion of hepatic folates obtained from rats treated with dieth the others (3-5, 13, 42). We therefore decided to investigate ylnitrosamine for 3 weeks prior to injection with [3H]folate in detail the mechanism responsible for the folic acid defi contained less radioactivity than did the polyglutamate frac ciency observed in DENA-treated rats. tion obtained from the livers of control rats treated with [3H]folate alone. Similarly, the polyglutamate folate fraction of rat livers that were simultaneously perfused with both MATERIALS AND METHODS diethylnitrosamine and [3H]folate contained less label than the polyglutamate fraction of rat livers perfused with Animals, Diets, and Compounds. Male Sprague-Dawley [3H]folate only. Livers perfused with [2-14C]histidine and rats (Simonsen Laboratories, Gilroy, Calif.) weighing 150 to diethylnitrosamine produced more formiminoglutamate 200 g were used in all experiments. For the chronic experi and less CO2 than livers treated with [2-14C]histidine only. ment, rats were housed singly in a wire-screen-topped cage The changes noted in the hepatic folate metabolism of in a constant-temperature, light-controlled room and were diethylnitrosamine-treated rats resemble those seen in the given a commercial stock diet (Purina rat chow; Purina Co., livers of methyl-deficient rats. St. Louis, Mo.)adlibitum; experimental rats received 0.01% DENA in the drinking water for 3 weeks. In 1 experiment, 1.0% DL-methionine was added to the ground rat chow diet. INTRODUCTION Folate labeled with tritium in the 3'S', and 9 positions (specific activity, ca. 40 Ci/mmole) (Amersham/Searle Previous experiments have demonstrated that the chronic Corp., Arlington Heights, Ill.) and 5-[14C]methyl-H4PteGIu administrationofDENA3 to ratsproduces a folatedeficiency (specific activity, 50 mCi/mmole) (Amersham/Searle) were that can be reversed by the administration of high dietary purified over QAE-Sephadex (Pharmacia Fine Chemicals, levels of the methyl donors, methionine and choline (29). Piscataway, N. J.) before use (6). The [methyl-14C]AdoMet The mechanism of this effect remains unknown. The inter (specific activity, 55 mCi/mmole) (Amersham/Searle) and action between DENA, folic acid, and methionine is impor [2-14Cjhistidine (specific activity, 55 mCi/mmole) (Amer tant because of the role played by the lipotropes in modify sham/Searle) were used without further purification. In ing the activity of several chemical carcinogens. For exam some studies, folate metabolism in perfused livers was pIe, dietary methyl donors appear to diminish the carcino studied. The perfusion method used was that described by genic activity of such agents as dimethylhydrazine (29), Buehring et al. (5). Twenty @.tgofDENA were added first in aflatoxin (21), ethionine (10), and DENA (31). On the other the perfusion medium containing washed human red blood hand, vitamin B12reportedly accelerates the carcinogenic cells and then iO pCi (ca. 0.25 nmole) of [3H]folate or 5 pCi (500 @tmoles)of[2-14C]histidine were added next. After per fusion for 2 or 2.5 hr, livers were washed with ice-cold water 1 This work was supported by USPHS Grant AM-08171 from the NIH. A preliminary report of this study has been published (35). and extracted for folate, AdoMet, histidine, FIGLU, or me 2 Present address: Kinder Klinik der UniversitÃ t MUnchen, Lindwurm str. 4, thionine. 8 Munich2, W. Germany. 3The abbreviations used are: DENA, diethylnitrosamine; 5-methyl Folate Analyses. The extraction of folates from the livers H,,PteGlu, N'-methyltetrahydrofolic acid ; AdoMet , S-adenosylmethionine; of intact rats was done according to the method described FIGLU, N-formimino-L-glutamate; methylene reductase, Nâ€•Â°-methylene by Shin et al. (36). In the perfusion studies, 40 ml of perfu letrahydrofolate reductase (EC 1.1.1.68); methyltransferase, N'-methyltetra hydrofolate:homocysteine methyltransferase; H4PteGlu, tetrahydrofolic acid. sate were poured into 200 ml boiling 1.0% ascorbate solu Received November 17, 1975; accepted April 26, 1976. tion (pH 6.0); the mixture was heated in a water bath at 95Â°

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1976 American Association for Cancer Research. V. S. S. Buehring et al. and cooled in ice, and the precipitate was removed by tated KCIO4.The clear yellowish solution was then chromat centrifugation. All extracts were stored at â€”19Â°untilas ographed on Dowex 50W-X8 (Nat form). Step-wise elution sayed. Total folates were determined by microbiological was done with 0.1 M NaCI and 6 N H2S04 or 6 M HCI. The assay according to the method described by Waters and elution was carried out in both cases until the absorbance Mollin (43) and later modified by Tamura et a!. (39). Three was less than 0.03 at 256 nm. Specific radioactivities of the organisms, Lactobacillus casei (ATCC 7469), Streptococcus AdoMet peak were calculated from their UV absorption at faecalis (ATCC 8043), and Pediococcus cerevisiae (ATCC 256 nm and from radioactivity measurements. Material in 8081), were used. The assay procedures forS. faecalis and the 6 NHCI eluate and standard AdoMet migrated identically P. cerevisiae were the same as that for L. casei (39), except on thin-layerandpaper chromatography (15). that, forS. faecalis, 10 ng offolate in 5 ml of inoculum broth Methyltransferase and methylene reductase activities were and 0.1 M sodium-phosphate buffer, pH 6.7, with 0.1% measured in the livers of control and DENA-treated rats. ascorbate, instead of 0.05 M sodium-phosphate buffer, pH Livers were homogenized with 3 volumes of cold 0.05 M 6.1, were used. ForP. cerevisiae, 5 ng of Leucovorin (N5- phosphate buffer, pH 7.2, and were centrifuged at 30,000 x formyltetrahydrofolic acid) were used in the inoculum g for 30 mm. The supernatant was frozen at â€”19Â°until broth , the rest of the procedure being the same as that for L. assayed. Just before analysis, part of each sample was casei. A crude conjugase enzyme preparation was made dialyzed 2 hr against 2 liters of 0.05 M phosphate buffer, pH from hog kidney, by use of the method of Eigen and Shock 7.2, at 4Â°.The methyltransferase was assayed according to man (9). The enzyme assay was done according to the the procedure ofKutzbacheta/. (17). After2 hrof incubation, method of Bird et al. (1). DEAE-cellulose column chroma the end product, [14C]methionine, was separated from the tography with an exponential gradient of 0.01 to 0.5 M remaining substrate 5-['4C]methyl-H4PteGlu on a 0.9- x 3-cm phosphate buffer, pH 6.0 (5), was used for the separation column of Dowex i-Cl (100 to 200 mesh), and radioactivity and identiftcation of different forms of folic acid. Sephadex in the eluatewas counted (17).Methylenetetrahydrofolate G-25 column chromatography with 0.1 M phosphate buffer reductase was assayed in the reverse direction with menadi (pH 7.0) (34) was used for the separation and identification one as the artificial electron acceptor (8). of pteroylpolyglutamates. All the buffers contained 0.2 M 2- [14C]Formaldehyde formed from 5-[14C]methyl-H4PteGlu mercaptoethanol. was determined as the dimedone adduct that was extracted Amino Acid, AdoMet, and Enzyme Determinations. For with toluene (8). studies on the metabolism of histidine, after completion of Measurement of Radioactivity. Radioactivity in liver ex the perfusion, the liver was homogenized with ice-cold dis tracts, perfusate extracts, and column fractions was mea tilled water (i :3, w/v), and protein was precipitated by the sured using, scintillation liquid, 48.48 g PPO and 3.25 g addition of 10 ml 20% sulfosalicylic acid and removed by POPOP dissolved in 11.4 liters toluene for 3 to 4 hr and centrifugation. To 50 ml perfusion medium, iO ml of 20% mixed with 6.6 liters Triton X-100 overnight. sulfosalicylic were added, and precipitated protein was re moved by centrifugation. Aliquots of the supernatant were used for column chromatography and radioactivity determi RESULTS nation. Dowex 50W-X8, 200 to 400 mesh, was used for the chromatography of histidine and its metabolites (2). Five ml In Vivo. The folate contents of the livers of rats receiving of the liver or perfusate extract were applied to the column DENA for 3 weeks were determined microbiologically and (0.9 x 35 cm), and elution was carried out with 100 ml citrate compared with the corresponding values in the livers of buffer (pH 3.25), 100 ml citrate buffer (pH 4.25), and 300 ml control rats (Table 1). Ten to 33% decreases in the re citrate buffer (pH 5.28) at room temperature. The final elu sponses of the 3 organisms to hepatic folate were observed tion was made with 100 ml NaOH to remove any radioactivity when the rats were given 0.01% DENA in the drinking water, remaining on the column. Further purification of FIGLU was but these differences obtained by microbiological assays carried out by rechromatography on the same resin in the were not statistically significant. The effect of DENA on the H@form (5). For methionine determinations, the livers were distribution of folate derivatives was also studied radi homogenized in ice water aril hydrolyzed in a sealed tube in ochemically. Rats were treated with 25 pCi of [3H]folate i.p. a vacuum in 6 N HCI for 18 hr. Acid was removed by flash and were sacrificed 24 hr later. Liver extracts containing evaporation and the residues were dissolved in sodium folate derivatives were chromatographed on Sephadex G-25 citrate buffer (37). Methionine was determined on a Beck columns to separate the mono- and polyglutamate folic acid man Model 120 amino acid autoanalyzer. derivatives. The data, given in Table 1, show that [3H]folate The extraction of AdoMet was done according to the uptake by the liver was lower in the animals receiving DENA procedure of Salvatoreeta/. (33). The rat liverwas homoge in the drinking water, but the difference was of marginal nized in 1.5 M perchloric acid (1:4, w/v), to which had been statistical significance (p < 0.03). The levels of methyltrans added 200,000 dpm (1 @mole)[methyl-'4C]AdoMet, with the ferase and of methylene reductase were determined in the use of a glass tube homogenizer. The homogenate was livers of control and DENA-treated rats. Carcinogen admin centrifuged at 10,000 x g for 15 mm at 4Â°.The pH of the istration decreased the hepatic content of methyltransfer supernatant was adjusted to 6.0 with a saturated solution of ase but exerted no significant effect on hepatic levels of KHCO3 at 20Â°in the presence of a small amount of octyl methylene reductase (Table 1). Furthermore, as shown in alcohol to prevent excessive frothing. The suspension was Table 1, the chronic administration of DENA to rats led to a then centrifuged at 10,000 x g for 15 mm to remove precipi 27% decrease in the hepatic level of AdoMet. The addition

2776 CANCER RESEARCH VOL. 26

Table1 Theeffectsof chronic DENAadministration to ratson the hepatic levelsof to/atecofactors and enzymes,and of AdoMet DENA(0.01%in the drinking water)wasadministeredto maleSprague-Dawleyrats(150 to 200 g) for 3 weeks prior to sacrifice.Group

DENAFolate1-carbonmetabolite Control activityâ€• Lcasei 18.4 Â±lie (3),! 15.0 Â±1.8 (3) 0.2 S. faecalis 9.6 Â±0.4 (3) 8.6 Â±0.6 (3) >0.2 >0.2FolateP.cerevisiae 10.0 Â±2.1 (3) 6.7 Â±0.9 (3)

uptake' by liver Monoglutamates 0.34 Â±0.05 (3) 0.35 Â±0.05 (4) >0.2 Polyglutamates 4.09 Â±0.52 (3) 2.85 Â±0.25 (4) <0.07 <0.03FolateTotal' 5.59 Â±0.61 (3) 3.75 Â±0.33 (4)

enzymesâ€• Methyltransferase 0.14 Â±0.007 (9) 0.11 Â±0.003 (11) <0.005 >0.2AdoMetâ€•Methylene reductase 0.31 Â±0.02 (9) 0.35 Â±0.02 (11) 66.6 Â±4.8 (12) 48.6 Â±2.0 (16) <0.005

â€˜IStatistical significance, as determined by Student's t test. S /.Lg folate per g liver. r Mean Â± SE. â€˜INumbers in parentheses, number of livers assayed. e Twenty-four hr prior to sacrifice, each rat received an i.p. injection of 25 @Ciof [3H]folate. Values are expressed as percentage of dose injected. I The difference between the sum of mono- and polyglutamates and total radioactivity in liver represents unidentified breakdown prod ucts .

I, @moles product per mg protein per hr. S j@moles/g liver.

of 1% methionine to the diets of rats receiving DENA for 3 Table2 weeks restored the hepatic content of AdoMet to normal Effect of DENA on the metabolism of [2-14CJhistidine in the levels (68.5 Â±8.3 nmoles/g liver). perfused rat livers Perfused Livers. The interrelationships between folic ExperimentaldetailsMethods.â€•DENA given in â€œMaterialsand acid and DENA were further studied using liver perfusion in techniques of Buehring et a!. (5). Rat livers perfused with perfu- Total CO2/2hr FIGLUin liver DENA and with [2-14C]histidine formed more FIGLU and less (@moles)ChowDiet sate (@moles) CO2 than livers perfused with [2-14C]histidine alone (Table + 31 145 2). Chart 1 shows the elution patterns obtained after chro Chow + 3012930.5 matography on DEAE-cellulose of extracts prepared from 11.3Chow Â±0.7â€• 137 Â± rat livers perfused for 2 hr with 10 pCi of [3H]folate with and without DENA. There was a greater incorporation of â€” 45 96 [3H]folate into the polyglutamate fractions of untreated con Chow â€” 4110543 trol liver than of the DENA-treated liver. These perfusion 6.4â€˜, Â±2.8 100.5Â± experiments with DENA and [3H]folate were repeated with 6 more rats; the results are summarized in Table 3. The incor Mean Â± S.D. poration of labeledfolateintothe polyglutamate pool of DENA-treated livers was less than the incorporation into the ciency produced in rats by the chronic administration of polyglutamate pool of untreated livers. Similarly, the levels DENA. DENA administration in the drinking water produced of AdoMet in the DENA-treated livers were observed to be a significant drop in the polyglutamate derivatives of liver slightly less (16%) than the untreated livers but these differ folate (29), the major form of hepatic folate (12, 14, 29, 36). ences were not statistically significant (Table 3). However, In perfusion studies, the addition of DENA to the perfusate the total methionine content of DENA-treated perfused liv decreased the formation of polyglutamate derivatives of ers did not appear to be significantly different from that in folic acid from [3H]folate. The addition of DENA during perfused control livers (Table 3). perfusion also increased FIGLU levels in the liver and de creased the formation of carbon dioxide from [2- 14C]histidine. DISCUSSION Direct evidence indicates that the altered folate distribu tion in the livers of DENA-treated animals is a consequence The present results confirm and partially explain the pre of methionine deficiency which, in turn, reduces the level of vious results (29) on the methionine-reversible folate defi AdoMet. The livers of rats treated with DENA contain sign ifi

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1976 American Association for Cancer Research. Y. S. S. Buehring et al. cantly less AdoMet than the livers of untreated animals. The crease the carcinogenic activity of DENA (31) and other DENA lowering of hepatic AdoMet can be reversed by me carcinogens (10, 21, 28, 30, 31). thionine. Physiological methyl donors also appear to de The effects of DENA may be explained on the basis of the methyl trap theory (13). This postulates that an increase in the proportion of 5-methyl-H4PteGlu results in a decrease in CPM XIOâ€•3 per b the physiologically active H4PteGlu forms which are in FRACTION 4, A volved in most folate-dependent reactions (23, 40, 42). This No DENA would result in a decreased catabolism of FIGLU (3). This 4 increased proportion of 5-methyl-H4PteGIu occurs in vi tamin B12deficiency where there is a decreased activity of methyltransferase. Methionine can be converted to AdoMet C which in turn inhibitsmethylenetetrahydrofolatereductase (18), thereby reducing the formation of 5-methyl-H4PteGlu 2 and increasing the proportion of H4PteGlu (5). This is con sistent with the observed effect of methionine in decreasing FIGLU excretion in the whole animal (4), facilitating FIGLU catabolism in the perfused liver (5), and reducing the ele vated FIGLU excretion occurring in animals treated with 40 60 DENA (29). Decreased levels of polyglutamate may be the FRACTION NUMBER (2.5ml) result of decreased levels of AdoMet with a corresponding CPM XIO3 increased activity of methylenetetrahydrofolic acid reduc per tase. This would decrease the level of H4PteGlu. This, rather FRACTION B than 5-methyl-H4PteGlu, is the preferred substrate for poly glutamate synthetase (32, 38). It is also consistent with the With DENA effect of methionine in increasing the level of polyglutamate formation in the perfused liver (5). Thus the effects of DENA in decreasing polyglutamate levels may be explained by its role in reducing levels of AdoMet. Polyglutamates have a lower rate of membrane transport than folic acid monoglu e tamates, and the formation of polyglutamates represents a mechanism whereby the organism can concentrate higher levels offolic acid within the cell. The effect of DENA on the hepatic distribution of vitamin B,2 has not yet been re

20 40 60 80 ported. FRACTION NUMBER (2.5m1 ) It is tempting to speculate that the effects of DENA on Chart 1. DEAE-cellulose column chromatography of liver extracts per folate metabolism are a specific biochemical consequence fused with 10 @.tCi[3Hjfolate.A, control without DENA; B , 20 @tgDENAadded of its carcinogenic activity. As yet we have not accumulated toperfusate.Thecolumnpatternswereobtainedbythechromatographyof5 ml of liver extract equivalent to 0.5 g fresh liver. Gradient: 100 ml 0.01 M enough evidence to demonstrate this point. Possible potassium phosphate buffer, pH 6.0, with 0.2 M 2-mercaptoethanol to 0.5 M causes of the DENA-induced folate deficiency unrelated to potassium phosphate buffer pH 6.0 with 0.2 M 2-mercaptoethanol. a , elution the carcinogenic process include partial inanition, altered position of N'Â°-formyltetrahydropteroylpentaglutamate; b , 5-methyl H,PteGIu; c, H4PteGlu; d, pteroylmonoglutamate; and e, polyglutamate frac hepatic cell population, and nonspecific toxicity. tion under comparable conditions. In previousstudies,chronicDENA administrationtorats

Table 3 The intluence ofDENA on to/ate uptake, liverDistributionfo/ate distribution, and the levels methionine and AdoMet in rat perfused with 10 p@Ci(â€˜H)folateof liverDENA of [3H]folate in

in Uptakeof perfu [3H]folatebyliver(%)MethionineAdoMetPolyglutamateMonogluta Ratsate(%)fractionsliver)1â€”47.242.5 mate fractions(@.tmoles/g liver)(nmoles/ g 57.519.91112â€”54.448.3 51.71163â€”50.354.7 45.319.711850.6

3.74+59.329.5 Â±3.6â€•48.5 Â±6.1 51.5 Â±6.119.8 Â±1.34115 Â±

70.513.91175+45.839.3 60.720.0946+59.139.1 60.918.67854.7

19.6â€˜I Â±7.736.0 Â±5.6 64.0Â±5.617.5 Â±3.297 Â±

MeanÂ± S.D.

2778 CANCER RESEARCH VOL. 26

restricted both their growth rates and food consumption 17. Kutzbach, C.. Galloway, E., and Stokstad, E. L. R. Influence of Vitamin (26, 29). However, no folate deficiency, measured by ele B2 and Methionine on Levels of Folic Acid Compounds and Folate Enzymes in Rat Liver. Proc. Soc. Exptl. Biol. Med., 124: 801-805, 1969. vated FIGLU excretion following a loading dose of histidine, 18. Kutzbach, C. A., and Stokstad, E. L. A. Feedback Inhibition of Methylene could be observed in rats fasted for 3 days (L. Poirier, tetrahydrofolate Reductase in Rat Liver by S-adenosylmethionine. Biochim. Biophys. Acta, 139: 217-220, 1967. unpublished observations) or pair-fed with FIGLU-excret 19. Miller, E. C., Plescia, A. M., Miller, J. A., and Heidelberger, C. H. The ing, tumor-bearing rats (26). The histological changes pro Metabolism of Methylated Aminoazo Dyes. I. The Demethylation of 3'- Methyl-4-dimethyl-C'4-aminoazobenzene in Vivo. J. Biol. 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AUGUST 1976 2779

Yoon Sook Shin Buehring, Lionel A. Poirier and E. L. R. Stokstad

Cancer Res 1976;36:2775-2779.

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