[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 Università 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, N―°-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 95° AUGUST 1976 2775 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 —19°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 —19°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 4°.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).