Folate Deficiency and Formiminoglutamic Acid Excretion During Chronic Diethylnitrosamine Administration to Rats1

[CANCER RF.SF.ARCH 33, 383-388. February 1973] Folate Deficiency and Formiminoglutamic Acid Excretion during Chronic Diethylnitrosamine Administration to Rats1

Lionel A. Poirier2 and V. Michael Whitehead

Institut du Cancer de MontrÃ©al,HÃ´pital Notre-Dame et DÃ©partementde Biochimie, UniversitÃ©de MontrÃ©alIL. A. P.l, and Department of Haematology, and Medii! University Medical Clinic, Montreal General Hospital Â¡V.M. W./. MontrÃ©al,QuÃ©becCanada

SUMMARY restricted to certain constituents of the nucleic acids and proteins (3. 21, 23, 38) and to glycogen (11). Of the 4 Elevated levels of the histidine catabolito essential dietary compounds involved in the transfer of formiminoglutamic acid were excreted into the urine of rats 1-carbon units in vivo, 3 [methionine (19, 28, 32), folie acid that were given both 0.01% diethylnitrosamine in their (13), and vitamin B,2 (16)] can behave as classical drinking water for 1 to 5 weeks and an injection of a loading nucleophiles. Of these nucleophiles, only methionine dose of histidine. Similar histidine loading of control rats that reportedly is attacked in vivo by hepatocarcinogens (23, 24). received no carcinogen did not produce an elevation in urinary The limited quantities and the multiplicity of forms of both formiminoglutamic acid excretion. The elevation in urinary vitamin B12 and folie acid in the liver would make the direct formiminoglutamic acid excretion caused by chronic demonstration of such an interaction in vivo quite difficult. diethylnitrosamine administration was prevented by high Since each of the essential 1-carbon compounds or their dietary levels of the methyl donors, methionine, betaine, and derivatives alters the course of carcinogenesis (9, 18, 22, 24, choline; high dietary levels of folate and vitamin B12, either 31 , 33), the possible interrelationship between alone or in combination, had no significant effect on the hepatocarcinogenesis and the metabolism of 1-carbon elevated formiminoglutamic acid excretion caused by compounds is currently under investigation in these diethylnitrosamine. laboratories. The elevated formiminoglutamic acid excretion caused by This paper is concerned with the effects of the chronic the administration of diethylnitrosamine for 3 weeks was administration of diethylnitrosamine on the urinary excretion associated with a decrease in the hepatic levels of the enzymes, of the histidine catabolito FIGLU.3 Like other formiminoglutamic acid transferase and urocanase, and with a hepatocarcinogens, diethylnitrosamine appears to be decreased hepatic content of the higher conjugates of folate. metabolized to an electrophilic agent in the liver (8) and thus Whereas dietary methionine administration completely can be expected to attack nucleophilic sites in this organ. prevented this decrease in the hepatic content of the higher Studies show that urinary FIGLU excretion reflects a dietary conjugates of folate, dietary folate had no effect. Diets that or functional deficiency of the 3 essential 1-carbon contained elevated levels of methionine and choline also led to nucleophiles, methionine, folie acid, and vitamin B]2 (1,2, only a slight reversal of the decreased hepatic 14, 20, 36, 37). A block in the catabolism of FIGLU is formiminoglutamic acid transferase activity caused by generally assumed to reflect low hepatic levels of H4-folate but diethylnitrosamine. Thus the antagonistic effects on may also reflect low hepatic levels of FIGLU transferase (1). formiminoglutamic acid excretion by diethylnitrosamine and In both cases, the excess FIGLU is excreted into the urine. the methyl donors appeared to be mediated through the The tissue levels and activity of the folie acid cofactors appear hepatic content of folie acid cofactors. to be regulated by nutritional status of the animal with respect to folie acid (1), vitamin B,2 (2, 12, 37), choline (5), and methionine (2, 12, 25, 37). The hepatic levels of FIGLU INTRODUCTION transferase are controlled by dietary methionine (37), vitamin B12 (32, 37), and choline (29). Thus, elevated FIGLU Recent advances in chemical carcinogenesis have excretion can act as a sensitive indicator of a metabolic emphasized the importance of carbonium ions or electrophilic abnormality of one or more of the essential 1-carbon agents as the ultimate reactive forms for most, if not all, compounds. classes of chemical carcinogens (3, 6,8, 10,23, 38). However, the list of cellular nucleophiles implicated in the direct attack MATERIALS AND METHODS of such carbonium ions remains relatively limited, being Animals and Diets. Young, adult male Wistar rats (175 to 'Supported by grants from the National Cancer Institute and the 250 g) (Canadian Breeders, Inc., St. Constant, Quebec, Medical Research Council of Canada, and the Conseil de la Recherche MÃ©dicaledeQuÃ©bec. 'The abbreviations used are: FIGLU, jV-formimino-L-glutamic acid; 'Present address: National Cancer Institute, NIH, Bethesda, Md. H4-folate, tetrahydrofolic acid; FIGLU transferase, 20014. jV-formimino-L-j;lutamate:tetrahydrofoUc acid 5-formiminotransferase Received July 12, 1972Â¡accepted November 3, 1972. (EC 2.1.2.5).

FEBRUARY 1973 383

Canada) were used in these experiments. Upon their arrival at spcctrophotometer (27, 34). The urocanic acid, FIGLU, and this laboratory, the animals were housed (5/cage) in plastic, H4-folate used in the assays were of the highest purity wire screen-topped cages containing soft wood shavings (Bran available and were purchased from Sigma Chemical Company, de Scie, Montreal, Quebec, Canada). Food (Micro-mix St. Louis, Mo. laboratory chow, Purina Ralston of Canada, Ltd., Woodstock, Ontario, Canada) and water were available ad libitum. After 1 week, groups of rats were given either Q.Q\% RESULTS diethylnitrosamine (Eastman Organic, Rochester, New York, N. Y.), which was added to the drinking water for 1 to 5 Biological. The administration of 0.01% diethylnitrosamine weeks, or unsupplemented drinking water; the in drinking water to rats for 1 to 5 weeks led to marked diethylnitrosamine was administered in aluminum-coated symptoms of toxicity. The diethylnitrosamine-treated rats did bottles. Where appropriate, DL-methionine and folie acid not gain a significant amount of weight during the 5-week (Matheson, Coleman and Bell, East Rutherford, N. J.), choline experimental period, whereas the body weights of rats that chloride and vitamin Bi2 (General Biochemicals, Inc., Chagrin received no diethylnitrosamine increased by 68 to 101 g. The Falls, Ohio), and betaine hydrochloride (Eastman Organic high dietary levels of methionine, choline, folie acid, and Chemicals, Rochester, N. Y.) were added to the Micro-mix vitamin B! 2 appeared to have no significant effect on the diets of the diethylnitrosamine-treated and control groups, at body weights of the animals, regardless of the presence or levels of 15,000, 40, 10,000, 0.5, and 11,000 ppm. re absence of diethylnitrosamine in their drinking water. The spectively. liver weights of rats that received diethylnitrosamine for 3 Urine Collection and FIGLU Analyses. At 0. 1, 3, and 5 weeks were significantly less than the liver weights of control weeks, the rats from each group were weighed and given i.p. rats (5.5 Â±0.2 versus 8.8 Â±0.3). The addition of methionine injections of L-histidine (Eastman Organic Chemicals) (225 and choline to the diets of rats given diethylnitrosamine did jumÃ³les/100 g body weight; 225 /Â¿moles/ml 0.9% NaCl not affect the diminished liver weights caused by the solution). The histidine injections were routinely performed at carcinogen. 3:00 p.m. The urine samples were collected for 18 hr, and the Biochemical. As illustrated in Table 1, the administration of FIGLU excretion values were determined as previously 0.01% diethylnitrosamine in drinking water to rats for 1 to 5 described (35). After urine collection, the rats were returned weeks resulted in a significant increase in urinary FIGLU to the dietary and carcinogenic regimen under investigation for excretion following a loading dose of histidine. At no time was subsequent histidine injections. The experiments were there a significant change noted in the FIGLU level in the terminated at 5 weeks. urine of control rats. The addition of 1.5% methionine to the Folate Determinations. After 3 weeks of diets of rats given diethylnitrosamine prevented completely diethylnitrosamine, folate, and methionine administration, the the rise in FIGLU excretion (Tables 1 and 2). High dietary rats were sacrified by decapitation and exsanguinated. Their levels of folie acid and vitamin B]2 appeared to have no livers were quickly excised, weighed, and placed on ice. The significant effect on the diethylnitrosamine-induced rise in livers were then homogenized (Polytron homogenizer; FIGLU excretion (Table 1). A diet containing high levels of Kinematic, Lucerne, Switzerland) with 9 volumes of ice-cold both folie acid and vitamin B12 similarly exerted no effect on 0.1 M phosphate buffer, pH 7.0, containing 8.24 mM ascorbic the elevated FIGLU excretion caused by diethylnitrosamine acid. The homogenates were then placed in a water bath for 5 (Table 1). When a loading dose of histidine was injected into min at 100Â°to precipitate the proteins; then the samples were rats that received 1.1% betaine chloride in their diets cooled in ice and centrifuged at 2000 rpm for 15 min in an (equimolar to 1.0% choline chloride), as well as International Model PR-2 refrigerated centrifuge. The diethylnitrosamine in their drinking water for 3 weeks, a supernants were collected and stored at 5Â°;their folate normal urinary FIGLU level of 1.2 /umoles/100 g body weight contents were determined (before and after conjugase was observed. In the same experiment, similarly treated treatment) with Lactobacillus casei, as previously described control rats excreted 2.8 AmÃ³lesFIGLU/100 g body weight, (39). while rats treated with diethylnitrosamine alone excreted 14.3 Enzyme Assays. The hepatic levels of 3 histidine-catabolic /amÃ³les FIGLU/100 g body weight. Thus a 3rd physiological enzymes were determined in rats (9 to 12/group) that had methyl donor greatly diminished the rise in FIGLU excretion received diethylnitrosoamine, either alone or with 1.5% observed during the administration of diethylnitrosamine. methionine or 1.0% choline for 3 weeks; these levels were also Dietary supplementation with 1-carbon compounds determined in control rats. After receiving diethylnitrosamine appeared to produce other shifts in FIGLU excretion (Table for 3 weeks, the rats were given injections of histidine, and 1). High dietary levels of methionine and choline led to slight their urine was collected for FIGLU analysis, as described but statistically significant decreases in FIGLU excretion by above. At the end of the urine collection period (9:00 a.m.), rats that received no diethylnitrosamine. When diethylnitros the rats were sacrified, their livers were excised and amine was administered to rats that ingested high-methyl diets, homogenized, and the homogenates were centrifuged at urinary FIGLU levels returned to normal (Table 1). Similarly, 100,000 X g for 1 hr, as previously described (29). The rats given diets containing high levels of folate, but no supernatants were stored at -20Â° for subsequent enzyme carcinogen, excreted slightly less FIGLU than did the analysis. Histidase (L-histidine ammonia lyase, EC 4.3.1.3), corresponding control rats. The physiological significance of urocanase. and FIGLU transferase all were assayed by these observations remains obscure. When rats were given both previously described procedures on the Gilford Model 240 diethylnitrosamine and folate for 3 and 5 weeks, their urinary

384 CANCER RESEARCH VOL. 33

Table ! h IGI. U excretion during chronic diethylnttrosantine administration to rats receiving methioninc. cliolinc, folate, and vitamin B, , Uiethylnitrosamine was administered in the drinking water at a level of 0.01%; methionine elidine, folate, and vitamin B, , were administered in the diet at levels of 15,000, 10.000, 40. and 0.5 ppm, respectively. All compounds were given for I to 5 weeks.

HGLU excreted (/imoles/100 g body wt)

Without diethylnitrosamine With diethylnitrosamine

confidence confidence limits"Mean5.33.04.33.31.11.00.91.31.92.03.51.21.42.81.21.84.50.81.6Lower2.81.92.62.40.70.90.60.71.11.01.90.90.91.50.81.13.20.21.3Upper8.74.76.94.71.71.21.52.43.53.86.21.72.05.41.82.98.94.12.4Mean15.114.915.03.42.73.73.34.76.311.631.528.314.425.513.817.032.733.095%limits"Lower9.15.58.61.51.52.31.82.94.04.917.620.58.514.47.X6.024.327.7Upper24.840.426.37.85.05.95.97.610.028.056.338.924.044.327.646.344.339.6 of GroupControlMethionineCholinel-'olateVitaminfeeding013513513513513513595%

B,,1'olate

andvitamin B, ,Weeks

" I IGLU excretion values have a positive asymmetrical distribution, which causes the skewness seen in the 95% confidence limits.

Table 2 Urinary Fl(Ã¬LUand hepatic histidase. urocanase, and /â€¢'/(/'/,(/transferasein rats given 0.017f diethylnitrosamine in the drinking water and dietary melhionine and choline Â¡or3 weeks After 3 weeks on each of the dietary regimens listed, the rats were given injections of 225 jumÃ³leshistidine per 100 g body weight, and their urine was collected for 18 hr. The 1-IGLU and enzyme assays were performed as described in "Materials and Methods." Kach value represents the combined results of 3 experiments with 9 to 12 rats in each group. Urinary 1-IGLU (Â¿imoles/lOOgbody wt)

95% confidence limits Hepatic enzyme level (/jmoles/hr/g liver)

GroupControlDiethylnitrosamineDiethylnitrosamine transferase211

Â±27.0 Â±19.0 Â±132 Â±33.5 Â±21.1 Â±164 1.5';DL-methionineDiethvlnitrosamine+ Â±30.8 i22.1 i156

1.0'Vcholine + +1.5"1.61.51.6Urocanase27.4Â±1.00.91.00.91-IGLU Â±1081110 chlorideMean2.011.41.83.5Lower1.58.81.52.7Upper2.614.22.24.5Histidase29.7 0 MeanÂ±S.K.

FIGLU levels appeared to be greater than the corresponding measured. Table 2 shows that the administration of FIGLU levels noted in rats given diethylnitrosamine alone; diethylnitrosamine to rats for 3 weeks resulted in a 38% drop these differences, however, were not found to be statistically in the hepatic levels of FIGLU transferase (p <0.05). Chronic significant (p >0.05). diethylnitrosamine administration similarly resulted in a 31% Since increased FIGLU excretion may result either from decrease in the level of liver urocanase (p<0.05) (Table 2). diminished hepatic content of the enzyme FIGLU transferase The slight decrease in histidase activity observed in the livers or from hepatic deficiency of the folate cofactors, both were of diethylnitrosamine-treated rats was not statistically

FEBRUARY 1973 385

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1973 American Association for Cancer Research. Lionel A. Poirier and V. Michael Wtiitehead Table 3 deficiency that could be prevented by the simultaneous The effects of dietary folate and met/iionine on the hepatic folate administration of physiological methyl donors. Hepatic folate levels of pair-fed normal and diethylnitrosamme-treated rats distribution and urinary FIGLU excretion are reported to be Following the administration of 0.01% diethylnitrosamine in the under the metabolic control of methyl donors (2, 12, 15,25, drinking water and of 40 ppm folate and 15,000 ppm mcthionine in the diets of pair-fed rats for 3 weeks, liver folate was determined as 37). The shifts in urinary FIGLU excretion produced by described in "Materials and Methods." diethylnitrosamine and methyl donor administration appeared to reflect an altered folate metabolism rather than a changed Hepatic folate (jug/g) hepatic content of the histidine-catabolizing enzymes. Unless there was a marked difference in the activities of the wt histidine-catabolizing enzymes in vivo and in vitro, the hepatic GroupControl+ (g)7.5 conjugase9.2 conjugase27.9 levels of all of the enzymes studied appeared to be high 0.7Â°4.96.25.26.50.30.10.20.55.4i8.2 Â±20.0 enough to metabolize completely the injected histidine, even diethylnitrosamineFolie Â±10.9 i32.5 during diethylnitrosamine administration. Furthermore, the acid+ Â±7.7 Â±18.2 diethylnitrosamineMethionine+ Â±9.1 i33.4Â«29.2 block in FIGLU catabolism noted during chronic Â±4.8 diethylnitrosamine administration was associated with an diethylnitrosamineLiver 0.5Without Â±0.7Â°1.11.00.70.60.1WithÂ±1.1a2.21.31.82.72.9altered content and distribution of the hepatic folate cofactors. The methionine-reversible decrease in the "Meani S.K. of 5 to 6 rats. polyglutamate derivatives of folie acid noted in the livers of the diethylnitrosamine-treated rats also supports this proposal. significant (Table 2). The administration of high dietary levels Such a postulate appears reasonable in the light of findings of methionine and choline to rats given diethylnitrosamine for that methyl donors influence the metabolism of vitamin B12 3 weeks appeared to prevent increased FIGLU excretion and and folie acid (2, 12, 15, 25, 37) and that vitamin B12 to reverse in part the decreased hepatic enzyme levels observed deficiency leads to a secondary deficiency of folie acid (2, 37). in such animals (Tables 1 and 2). High dietary levels of the 2 Previous workers have shown that a decreased liver content of methyl donors, methionine and choline, appeared to give both folie acid and vitamin B|2 occurs during chronic partial protection aginst the decrease in hepatic FIGLU hepatocarcinogen administration (4, 7). The use of L. cosci in transferase noted with the diethylnitrosamine-treated rats the present experiments for measurement of folie acid did not (p<0.04 for diethylnitrosamine + methionine versus permit a determination of the H4-folate cofactors. However, diethylnitrosamine; p <0.05 for diethylnitrosamine + choline previous studies indicated that methionine increases the versus diethylnitrosamine). The administration of choline to availability of the H4 -folate cofactors in rat liver (25). the diethylnitrosaine-treated rats led to hepatic levels of Our findings are in general agreement with previous urocanase that were significantly above the corresponding observations that methyl donors often act as antagonists of the enzyme value in rats that had received diethylnitrosamine activity of such hepatocarcinogens as the aminoazo dyes (18, alone (/; < 0.03). The feeding of methionine to diethylnitros 22), ethionine (10), and aflatoxin (24). The antagonism amine-treated rats appeared to give some protection against between hepatocarcinogens and methyl donors is especially the decreased liver urocanase levels produced by diethylnitros interesting in view of an apparent role of methionine in amine alone, but this difference was not statistically signifi cellular differentiation (26). The decreased control by methyl cant. In no case did the dietary administration of high levels of donors of histidine catabolism could result from a diminished methyl donors completely reverse the altered enzyme levels hepatic content of the methyl derivatives essential to folate produced by diethylnitrosamine. metabolism. Such would be the result of a direct electrophilic The effects of 3 weeks of administration of attack of the activated carcinogen on methionine, for example. diethylnitrosamine on the hepatic folate content of rats are However, such an attack has been observed in vivo only with described in Table 3. In control rats, diethylnitrosamine the aromatic amines and aminoazo dyes (22, 23). The administration appeared to have no significant effect on the nitrosamines have not yet been shown to attack methionine, hepatic folate levels measured prior to conjugase treatment. although in vivo protein binding by these agents has been However, the levels of the polyglutamate derivatives of folie demonstrated (21). Carcinogen administration could also acid, which are released by conjugase treatment, were 37% indirectly influence the hepatic content or metabolism of lower in the livers of the carcinogen-treated rats than they methyl compounds. Thus, Reid et al. (30) have reported an were in the livers of the control rats. The decrease in the increased labeling of the methionine-methyl groups obtained hepatic polyglutamate levels of the diethylnitrosamine-treated from the livers of rats that were treated with rats was not affected by high dietary levels of folate but could AVV-dimethyM-aminoazobenzene and given injections of be prevented by high levels of methionine (Table 3). histidine-2-14C; also, the methylation of rRNA in the livers of rats treated with diethylnitrosamine was decreased, compared with the methylation of such RNA in normal liver (17). In DISCUSSION addition, a greatly increased synthesis of the polyamines (40) or of phospholipids in the livers of rats treated with These experiments demonstrated that chronic hepatocarcinogens could also be expected to produce a stress diethylnitrosamine administration to rats produced a folate on the labile methyl pool.

386 CANCER RESEARCH VOL. 33

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1973 American Association for Cancer Research. Folate Deficiency and Formiminoglutamic Acid Excretion during Chronic Diethylnitrosamine Administration to Rats

Lionel A. Poirier and V. Michael Whitehead

Cancer Res 1973;33:383-388.

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