The Action of 2-Acetylaminofluorene on Growth and LiverComposition of Rats on Low and

Adequate Dietary Sulfur*

HELMUT R. GUTMANN, DOROTHY FILBIN, AND JOHN H. PETERS

(Radioisotope Unit, Veterans Adminiatra@ion Ho.spitai, and Department of PhysiologicalChemistry, University of Minnesoki Medical School, Minneapolis, Minn.)

The observation that a variety of carcinogenicagents have a growth-inhibitory action in the rathas given rise to the theory that there is a causalrelation between the growth-inhibitory effect andthe carcinogenic activity of a compound (8). Further studies of the mechanism of the growth inhibition induced by injection of 1,@,5,6-dibenzanthracene (DBA) revealed that the inhibitionwas manifest only on an inadequate protein intake(3). Similarly, in a previous study of the growthinhibition following ingestion of @-acetylaminofluorene (AAF), a moderate growth depression wasobserved in pair-fed rats only on an 11 per centcasein diet. With the same feeding technic, a @Oper cent casein ration protected against thegrowth-retarding effect of the compound (7).These results with DBA and AAF indicate thatthese carcinogens do not inhibit growth if an adequate protein intake is provided. The growth inhibition by AAF on a low protein diet requires explanation. It may be assumed that it reflects theinterference of AAF with some phase of proteinmetabolism. This view is supported by the factthat AAF caused an early depletion of protein nitrogen in the liver cell (10) and that high levels ofcasein in the diet will reduce the number of tumorsin rats ingesting AAF (4). The possibility musttherefore be considered that the carcinogenic action of AAF is indeed the result of some earlysubtle disturbance of protein metabolism at thecellular level which does not result in a reducedgrowth rate of the rat on an adequate protein intake. The determination of the nutritional factor(s) in the @0per cent casein diet which relievesthe growth inhibition might provide a clue to thespecific mechanism of the interference of AM?

* Supported by grants from the American Cancer Society,

on recommendation of the Committee on Growth, NationalResearch Council, and the Graduate School, University ofMinnesota.

Received for publication October 19, 1955.

with protein metabolism. From a nutritionalstandpoint, the 11 per cent casein diet is madequate for satisfactory growth primarily because ofits low methionine content (16, 17). The questionarose, therefore, whether the protection againstthe growth-inhibitory action of AM? was affordedby the increased quantities of methionine in the @Oper cent casein diet or by the increase in totalnitrogen. The present experiments were designedto elucidate this problem by studying the action ofAM' on the growth of rats maintained for 100days on 11 per cent casein rations which were fortified with methionine or cystine. In another experiment, the protection which the @0per centcasein diet had provided against the carcinogenwas reinvestigated. To test this effect more rigorously, the duration of the experiment was prolonged from 8@to 119 days. In the previous experiments, a number of gross chemical changes hadbeen observed in the livers of pair-fed rats after theingestion of AM' (7). It had been inferred that thelivers of rats treated with AM? deposited appreciable amounts of glycogen, since a considerablefraction of the liver dry weight could not be accounted for by the sum of total fat and protein.The analyses of liver size and composition weretherefore repeated and extended to include estimations of liver glycogen in certain groups of rats.

MATERIALS AND METHODS

AnimaL, and dieis.—Weanling female litter-mates of theSprague-Dawley strain were used in the experiments. Femalerats are more suitable for long-term growth experiments, sincethey are more resistant to the toxic action of AAF than aremale rats (24). The animals in Experiment 1 were 20 days old,and their average weight was approximately 35 gm. at thebeginning of the experiment. The rats in Exps. 2, 8, and 4 were29 days old and had an average weight of 49 gm. at the start ofthe experiments.Exp. 1was carried out for 119days and Exps.2, 8, and 4 for 100 days. The rats were maintained and pair-fedin the manner previously described (7). Food intake and body

1 Obtained from the Holtzman Rat Company, Madison,

Win.

@40

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Gumw@ et aL—The Role of Diet and AAF on Grmvih Q@41

weight were recorded daily. The composition of the rations isgiven in Table 1. The compositions of the 11 per cent and 20per cent basal casein rations used in Exp. 1 have already beendescribed (7). In Exps. 2 and 8 the 11 per cent casein diet wassupplemented with DL-methionine (2.5 gm/kg) and L-cystine(2.0 gm/kg), respectively. These additions raised the sulfurcontent of the 11 per cent casein diet to that of the 20 per centcasein ration. In Exp. 4, the 11 per cent casein diet was supplemented with glycine (1.8 gm/kg). The amount of nitrogen inthe glycinewas the same as that furnished by the addition ofDL-methicnine (0.24 gin/kg). Glycine served as control forthe increased nitrogen intake of the rats on the 11 per centcasein diet supplemented with the sulfur amino acids. AAF,m.p. 196°-198°C., was added to the various diets as previouslydescribed (7) to give a final concentration of 0.05 per cent,which is tumor-producing in the rat (14).

Prep.ration of tissues and analytical method..—In Exp. 1liver glyongenwas estimated. The content of liver glycogenislow, in tbe rat, after 24 hours' starvation (2), and variations inthe glycogencontent might be expected as the result of differences in the rate of food intake. It had been noted that theanimals which received the 11 per cent casein diet containingAAF consumed their rations intermittently over a period of24 hours. AU other animals took the food rapidly. In this situntion, presumably all animals, except those on the 11 per centcasein ration plus AAF, would be in a fasting state if they weresacrificed 24 hours after the last feeding, and the glycogen content of their livers would consequently tend to be low. Tominimize variations in the rate of intake prior to killing, the

following feeding schedule was adopted. The rats which received the 11percent caseinration and AAFwereallowedfooduntil 8hurs before they were sacrificed, at which time the foodwas withdrawn and the intake up to that time determined.The controls and the rats on the 20 per cent casein diet (withand without AAF) received now the same amount of food ashad been consumed by the rats on the low casein diet containing AAP. This quantity of food was eaten by these rats in lessthan 2 hours. All rats were sacrificed, therefore, from 6 to 8hours following the last feeding and were probably in the postabsorptive state.

The rats in Exp. 1 were anesthetized by the intraperitonealinjection of Nembutal (5 mg/196 gm of body weight). Afterthe abdominal cavity was opened, the aorta was severed, and

samplesof liver tissue weighingabout 1 gus.wereremovedinnmediately and placed into hot 80 per cent KOH. Glycogen wasestimated on duplicate tissue samples by a slight modificationof the method of Good, Kramer, and Somogyi (5), which involves purification of the glycogen by resolution in distilled

TABLE 1

DESCRIPTION OF DIETs

CaseinAAFcontentaddedDiet

no.5(per cent)(percent)lic11.00.0lIE11.00.0520C

WE1IC+MI1E+M11C+C1IE+CIIC+GI1E+G20.0

20.011.011.011.011.011.011.00.0

0.050.00.050.00.050.00.05

Supplement

NoneNoneNoneNone

0 . 25 per cent DL-Methioninea a e a a

0 .@20per cent L-Cystine

0 . 13 per cent Glycinea a aS The compositions of the basal diets are the same as described pm

viously (7).

water and reprecipitation with 95 per cent ethanol in thepresence of saturated Na,SOi (19). Following acid hydrOlysis,glucose was determined by the photometric method of Nelson(15), with the impruved reagent of Soinogyi as the reducingagent (20). Of 26 duplicate determinations, 22 showed an aver

age deviation of less than 4 per cent and four of 4-6 per cent.The rats in experiments 2, 3, and 4 were killed by cervicaldislocation. All animals were examined for the presence oftumors. No hepatic or Other internal tumors were found. Themethods for the estimation of water, total lipids, and totalnitrogen were the same as those previously described (7).

RESULTS AND DISCUSSIONTable@ summarizes the effects of AM? on the

growth of rats maintained on 11 and @Oper centcasein diets and on 11 per cent casein diets supplemented with cystine, methionine, or glycine. As inthe previous study, no growth inhibition due to

TABLE 2

EFFECT OF 2-AAF ON GROWTH OF RATS ON 11 PER CENT, 20 PER CENT, AND

SUPPLEMENTED 11 PER CENT CASEIN DIETS

Eaperi.Av.foodProbabilityGrowthmentFeedingNo.intakeInitialwt.tFinalwt.tlevelinhibitionsSo_sDietmethodrats(gm/day)(gm.)(gin.)(P)@(per

cent)1

.11C lIEDoublepaired a75.4 5.435±1 35±1132±5107±40.0127(15)#20C

20EU a611 65.611 5.636±1 36±1147±8 146±6

0.01 16

0.01 20

0.01 16

2 11C+M Paired 8 6.6 50±2 160±511E+M a 8 6.6 50±1143±43

I1C+C a 8 6.8 48±3 159±611E+C a 8 6.8 53±2142±84

1IC+G a 8 5.5 46±2 128±4I1E+G a 8 5.5 47±2116±8S

Eap. I was carried out for 119 days; Eaps. 5, 3, and 4 for 100 days

t Averageweight ±standarderrorof the mesa.t Growth inhibition —100 (weight gain of control rats —weight gain of treated rats)/weight gain of control rats.I P is the probability for chance occurrence of the observed differencesof the means.I Per omt growth inhibition at 100 days.IIOne rat which had a slightly lower food intake than the average died during the experiment.

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Cancer Research24@

AAF was observed on the @20per cent casein diet,although in the present experiment feeding of thecarcinogen was extended from 8@to 119 days. Incontrast, AAF fed for 119 days in an 11 per centcasein ration inhibited growth to the extent of @6per cent. This supports the previous conclusionthat the inhibitory action of AAF was, in fact,counteracted by the higher protein intake. Moreover, protection was afforded for a comparativelylong time.

Addition of cystine and methionine, but not ofglycine, to the 11 per cent casein diet improved thegrowth of the control rats. This is in accord withthe current view that an 11 per cent casein diet isdeficient in sulfur, resulting in suboptimal growthof the rat (16, 17). Supplementation of the 11 percent casein diet with cystine, methionine, or glycine

terferes with the utilization of some essentialamino acid other than methionine and that theexcess of this amino acid in the @0per cent caseindiet compensates for the resulting nutritional deficiency. This suggestion takes account of the factthat an 11 per cent casein diet provides only 0.9per cent lysine, 0.1 per cent tryptophan, 0.6 percent phenylalanine, and 0.5 per cent threonine, assuming casein to consist of 100 per cent protein(1).The amountsof theseaminoacidsnecessaryfor good growth of the rat are 1 per cent, 0.@percent, 0.7 per cent, and 0.6 per cent, respectively(16). It is apparent that an 11 per cent casein ration contains only marginal amounts of theseamino acids. Another interpretation of the protective effect of the @0per cent casein diet against thegrowth-retarding action of AAF derives support

TABLE 3

EFFECT OF 2-AAF ON LIVER WEIGHTS OF RATS ON 11 PER CENT, 20 PER CENT, ANDSUPPLEMENTED 11 PER CENT CASEIN DIETS

Exp.no.5

1Diet 1ICliE20C20EWet

weightof liver(gm.)

3.90±0.144.61±0.153.55±0.086.10±0.42Dry

weightof liver

(gin.)1.16±0.051.38±0.051.06±0.031.88±0.13Water

(per cent)

70.3±0.470.1±0.470.2±0.369.2±0.2Dry

weight!100 gm body wt.

0.88±0.031.30±0.010.73±0.031.29±0.07Relative

liverenlargement

(per cent)

47.6(33.0)t

77.0(79.0)t211C+M

I1E+M4.45±0.187.08±0.361.23±0.042.11±0.1472.2±0.770.3±0.40.77±0.021.47±0.0691.0311C+C

11E+C4.84±0.207.07±0.391.30±0.052.02±0.1173.1±0.371.3±0.40.82±0.021.43±0.0575.04I1C+G

IIE+G4.27±0.135.19±0.281.13±0.031.41±0.0773.5±0.372.8±0.30.89±0.031.22±0.0437.0S 4&1Jdata are arithmetic means ± standard error of the mean

t Relative liver enlargementafter 8@days observedpreviously(7).

had no effect on the growth inhibition induced byAM?. It follows that the increased sulfur content

is presumably not the reason that the @0per centcasein diet protected against the growth-inhibitoryaction of AAF. In the light of the present experiments it appears also improbable that the growthdepression, by AM?, is due either to a specific interference of the carcinogen with the utilization ofmethionine or cystine or to sulfur deprivationwhich results from continued mercapturic acidsynthesis (@1). The question why a @0per centcasein diet can overcome the growth inhibition ofAM? remains therefore unresolved. There are re

ports in the literature which suggest that a highprotein intake as such lessens the toxicity ofpoisons such as atabrine and AM? (18, @3). Inthese instances, no specific amino acid appears tobe implicated. Relief of the growth depression observed here may likewise reflect increased resistance, resulting from a high protein intake, againsttoxic effects. However, it is possible that AAF in

from experiments which indicate that the metabolic fate of biologically active substances may bemodified by changing the dietary protein level.Thus, the inactivation of estradiol by the rat wasdecreased on low protein diets (fl). Moreover, theactivity of a number of tissue enzymes was lowered by inadequate protein intake (11, 1@). Itwould therefore seem plausible that the destruction of the carcinogen, which is likely to be carriedout by tissue enzymes, is more rapid in animalsreceiving sufficient protein in the diet.

Table S shows the effects of the various diets onthe enlargement of the liver, which enlargement istypical for rats treated with AM? (6, 7). The greatest enlargement (75—91per cent) was observed inthe groups which received the @0per cent caseindiet or the 11 per cent casein diet supplementedwith cystine or methionine. On the 11 per centcasein diet, unsupplemented or supplemented withglycine, the increase in liver size was approximately one-half (37—48per cent) of that observed

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GUTMANN et al.—The Role of Diet and AAF on Growth @43

in the other groups. Moreover, the liver enlargement which resulted from the feeding of AAF in af20 per cent casein diet appeared to be completeafter 8@days and did not progress thereafter. Onthe other hand, the liver continued to increase insize when AM? was fed in an 11 per cent caseindiet. These data suggest that the enlargement ofthe liver attributable to AM' and the rate atwhich the enlargement takes place are to some extent controlled by the dietary level of sulfur aminoacids. Tables 4 and 5 summarize the analyticaldeterminations of liver fat, protein, and glycogen

lower degree of fat deposition by AM? on the @0per cent casein ration was attributable to the excess of threonine, since this amino acid has pronounced lipotropic activity (9). Although AAFcaused accumulation of liver fat on prolonged exposure, the concentration of liver lipids never cxceeded 5 per cent of the liver wet weight. It followsthat AM? did not produce fatty livers on anydietary regime.

In agreement with previous observations (6, 7),a significant decline in the concentration of nitrogen of the livers of rats ingesting AM? in the @0per

TABLE 4

EFFECT OF 2-ACETYLAMINOFLUORENE ON LIVER COMPOsITIoN

ahVlOOgmbodyweight=(ug.oftissueortissuecomponentofAAF-treatedrats—zag.oftissueortissueenmponentofcontrolrats)/lOOgmbodyweight.

Exp.FatNitrogenGlycogenMg. fatMg. proteinMg.glycogenno.*Diet(per

cent)t(per eent)f(per cent)t100 gm. body wt.100 gm. body wt.100 gm.bodywt.I11C8.42±0.4210.1

±0.422.3±3.274±3556±19201±32liE16.9±169.3±0.3@I9.2±1.8@219±19750±19250±25@20C8.48±0.8911.7

±0.112.1±2.062±7531±2589±1620E10.5±1.3@9.2±0.124.9±2.3133±14738±31320±36211C+M11.0

±0.612.3±0.112.1*85±6591±1695#11E+M16.9±1.28.92±0.2127.3@231±28812±23403#

1IC+C7.57±0.6812.6±0.113.6@62±6643±9113#IIE+C14.1±1.49.14±0.2728.8@201±19817±33414#

S The data are arithmetic means ± standard error of the mean.

t Ona dryweightbasis.@ The differenceof control and experimental value is not considered significant (P > 0.01); in all other instances P < 0.01.

§Calculated per cent of glycogen = 100—(per cent fat + per cent protein).# Obtainedfromvaluesof total glycogencalculatedby difference.

TABLE 5

CONTRIBUTION OF FAT, PROTEIN, AND GLYCOGEN TO THE LIVER ENLARGEMENTOF RATs FED 2-ACETYLAMINOFLUORENE

‘@Dry wt.*

100 gm. body wt.Fate100 gm. body wt.FatxI00

.@drywt.i@Protein*

100 gm. body wt.@Protein

z@Glycogen5x100

@ dry wt. 100 gm. body atGlycogenx100

@ drywt.42014534.519446.24911.75607112.720737.023141.370016623.722131.630844.0Exp.no.

2

3

Diet

lIE20E

1IE+@i

139 22.8 174 28.5 3011IE+C 610 49.3

and the contribution of these compounds to theliver enlargement. The concentration of fat in thelivers of rats treated with AM? showed a significant rise, except on the @0per cent casein diet. Nochange in the concentrations of lipids had beenobserved in the previous experiment of shorterduration (7). It would appear, therefore, that prolonged intake of the carcinogen brings about deposition of liver fat on the 11 per cent, but not on the@20per cent, casein ration. Supplementation of the11 per cent casein ration with cystine and methionine did not prevent the deposition of liver fat inthe animals treated with AM?. This is somewhatunexpected in view of the established lipotropicaction of methionine and the sparing action ofcystine for methionine. It seems likely that the

cent and the supplemented 11 per cent casein dietswas observed. In these instances, the increase inliver fat and glycogen exceeded the increase in protein (Table 5). In contrast, when the increase infat and glycogen equaled the increase in protein, aswas the case on the unsupplemented 11 per centcasein ration (Table 5), the concentration of livernitrogen remained unchanged. Consequently, thedecline in the concentration of liver nitrogen is attributable to a dilution of liver protein by fat andglycogen and probably not to a diminished proteinsynthesis. The uniform increase of total protein onall diets (column 7, Table 4, and columns 5 and 6,Table .5) would, in fact, suggest that AAF accelerated protein synthesis, although, in the absence ofturnover studies, the observed net increase in total

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Cancer Research@44

protein might also be ascribed to delayed proteinbreakdown. Table 4 shows an appreciable increaseof glycogen in the livers of rats treated with AM?on the @0per cent casein ration and the supplemented 11 per cent casein diets. This confirms theprevious suggestion that AM? causes deposition ofliver glycogen (7). Accumulation of glycogen hadalso been noted previously in rats on the 11 percent casein diet, but was not found in the presentexperiment. Since these experiments were designedto control experimental variables influencing liverglycogen by determining this compound in animalswhich were presumably all in the postabsorptivestate, it was considered that these results permit abetter comparison of glycogen levels between control and experimental groups. It would, therefore,appear that ingestion of AM? does not lead to accumulation of glycogen in rats on an 11 per centcasein diet. It will be seen that the glycogen content of the livers of the control rats on the 11 percent unsupplemented diet was approximatelytwice as great as on the other rations, suggestingthat intake of a nutritionally inadequate proteininduces high levels of liver glycogen. This is consistent with the report that low protein diets favora high glycogen content of rat liver (13). Since amarked elevation of glycogen, due to AAF, occurred only on the supplemented 11 per cent caseinrations or on the @0per cent ca.sein diet, the glycogenic action of AAF seems to require adequatequantities of dietary sulfur amino acids. The dependence of increased glycogenesis, by AAF, onsufficient amounts of dietary sulfur may be explicable on the basis of the established role ofcystine and methionine as glycogen precursors.

The marked accumulation of glycogen, whichresulted from the ingestion of AAF, is also mdicated by the data of Table 5, which. show that40—50per cent of the increase in liver dry weightwas due to glycogen. These results leave no doubtthat the action of AM? involves changes in carbohydrate metabolism in addition to the previouslyreported alterations in the metabolism of proteinsand nucleic acids (6, 10). Moreover, the results ofthe determinations of liverfat, nitrogen, and glycogen, in conjunction with the data on liver enlargement, indicate that the action of AM? on theliver is modified by dietary factors.

SUMMARY

1. The carcinogen, @.-acety1aminofluorene, inhibited the growth of pair-fed rats maintained onan 11 per cent casein diet. The growth-inhibitoryaction of @,-acetylaminofluorene was eliminated byfeeding a @0per cent casein diet. Addition of DL

methionine, i,-cystine, or glycine to the ii per centcasein ration failed to prevent the growth inhibition induced by @-acetylaminofluorene.

@.The livers of the rats were analyzed for Wa

ter, fat, nitrogen, and glycogen. @-Acetylaminofluorene caused liver enlargement due to increaseddeposition of fat, protein, and, particularly, glycogen. The action of AM? in altering the chemicalcomposition of the liver is influenced by the sulfurcontent of the diet.

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1956;16:240-245. Cancer Res   Helmut R. Gutmann, Dorothy Filbin and John H. Peters  Composition of Rats on Low and Adequate Dietary SulfurThe Action of 2-Acetylaminofluorene on Growth and Liver

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