influence of dietary lipotrope and lipid content on aflatoxin...

[CANCER RESEARCH 40, 2802-2807, August 198010008-5472/80/0040-0000$02.00

Influence of Dietary Lipotrope and Lipid Content on Aflatoxin B1,N-2-Fluorenylacetamide, and 1,2-Dimethylhydrazine Carcinogenesis in Rat&

Adrianne E. Rogers,2 Gregory Lenhart, and Gayle Morrison

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT

A marginally lipotrope-deficient diet, high in fat, enhanceshepatocarcinogenesis by aflatoxin B, , diethyl- and dibutyl-nitrosamine, or N-2-fluorenylacetamide and colon carcinogenesis by 1,2-dimethylhydrazine in rats, when compared to anutritionally complete diet that differs in several componentsfrom the deficient diet. Supplementation of the deficient dietwith lipotropes and amino acids decreases diethylnitrosaminehepatocarcinogenesis, and, contrary to expectation, additionof the higher, more saturated fat content of the deficient diet tothe complete diet also decreases tumor induction. This reportpresents evidence that lipotrope or amino acid supplementationof lipotrope-deficient diets prevented their enhancement of N-2-fluorenylacetamide hepatocarcinogenesis but not of aflatoxinB1 hepatocarcinogenesis. Addition of fat to the complete diethad no eftect on tumor induction by N-2-fbuorenylacetamideand decreased tumor induction by aflatoxin B, . We concludedthat specific interactions between hepatocarcinogenesis, dietary lipotropes, and lipid vary with the carcinogen.

The effect of the marginally deficient diet on 1,2-dimethylhydrazine colon carcinogenesis appeared to be the result of itslipid content. Lipotrope or amino acid supplementation of thedeficient diet did not prevent its enhancement of 1,2-dimethybhydrazine colon carcinogenesis. Substitution of the fat content of the deficient diet into the complete diet increasedcumulative probability of death with tumor but has less effecton tumor incidence and no effect on tumor number.

INTRODUCTION

Enhancement or inhibition of chemical carcinogenesis bydietary components has been demonstrated in many animalmodels and is postulated to be partially responsible for geographic and cultural variations in human cancer incidence (2,7, 23). Of the many food constituents that influence chemicalcarcinogenesis in experimental animals, lipids and lipotropes(methionine and choline) are among the most extensively studed (2, 3, 7, 12, 20, 21). High-fat diets increase the incidenceand decrease the latent period of mammary and colon tumorsinduced by several carcinogens and may enhance carcinogenesis in skin and liver under some experimental conditions (2,10).

Lipotrope deficiency was thought initially to be hepatocarcinogenic per Se, but subsequently it was recognized that thedeficiency increases susceptibility of rats to carcinogenesis by

@ This study was supported in part by USPHS Contract NO1 CP33238 fromthe National Cancer Institute.

2Towhomrequestsforreprintsshouldbeaddressed.atEl8-615,Massachusetts Institute of Technology, Cambridge, Mass. 02139.

Received December 27, 1979; accepted May 1. 1980.

AFB, ,@which contaminated the peanut meal used to formulatethe deficient diets (5). AFB1 carcinogenesis is enhanced byseveral different, bipotrope-deficient diets. Enhancement is corrected by methionine or choline supplementation in some cases(6, 8, 19) but not others (13, 18). Correction appears to occurmore readily if diets contain vegetable oil or bard, rather thanbeef tallow, as the source of fat. Partial correction of a bipotrope-deficient diet to induce a marginal deficiency makes ratshighly susceptible to AFB1 carcinogenesis compared to ratsfed either a severely deficient or a nutritionally complete diet(13, 14). The marginally deficient diet also increases tumorincidence and cumulative probability of death with tumor inmale rats treated with DENA, dibutylnitrosamine, AAF, andDMH compared to results in rats fed the nutritionally completediet(11, 12, 15, 16).

Because the deficient and complete diets differ in severalcomponents, we performed experiments to determine whichcomponents in the deficient diet are responsible for altering theresponse of rats to chemical carcinogens. Assessment hasbeen made of the contribution of differences in dietary contentof lipotropes, amino acids, carbohydrates, vitamins, and lipidsto enhancement of carcinogenesis by the marginally deficientdiet. In an earlier study, enhancement of DENA carcinogenesisby the marginally deficient diet was reduced by lipotrope oramino acid supplementation of that diet (12). Although it hadappeared likely that the high fat content of the deficient dietcontributed to its enhancement of carcinogenesis, addition ofthe same fat to the complete diet reduced DENA carcinogenesis (12).

This article reports studies of the effects of alteration of thesame dietary components in the marginally deficient or complete diets on AFB1, AAF, and DMH carcinogenesis. The purpose of the experiments was to determine if the same components are responsible for enhanced carcinogenesis by thedifferent carcinogens in deficient rats and if enhancement ofcarcinogenesis could be prevented by dietary substitutions.

MATERIALS AND METHODS

Animals. Male GD Sprague-Dawley rats (DMH, AAF experiments) or Fischer rats (AFB1 experiments; Charles River Laboratories, Wilmington, Mass.), initially weighing 40 to 50 g,were housed individually in suspended wire-mesh cages, inrooms maintained at 70â€”74Â°F.They were given tap water andfed one of the experimental diets (Table 1) ad libitum throughout the experiment. Carcinogen treatment was begun after 3weeks of dietary treatment.

Diets. Eleven diets (Table 1) were fed in 3 experiments: Diets

3Theabbreviationsusedare:AFB, aflatoxinB; DENA,N-nitrosodiethylamine; AAF, N-2-fluorenylacetamlde; DMH, 1,2-dimethylhydrazine; AFP, a-fetoprotein.

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on June 1, 2018. © 1980 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Diet and Chemical Carcinogenesis in Rats

Table 1Composition of experimental diets

Diets were fed to rats treated with AFB1, DMH (Diets 1 to 6), or AAF (Diets 1 to 3, 7 to 11).

% of component in diCta

aAlldietsweremixedintoa 3%agarsolution;theycontained5%Rogers-Harperssaltsand2%vitaminmix.Thevitamin content of Diets 1, 3, and 7 was: vitamin A, 10 mg/kg; vitamin D2,3000 lU; vitamin E, 169 lu; menadione, 1mg/kg; niacin, 50 mg/kg; calcium pantothenate, 20 mg/kg; riboflavin, 4 mg/kg; thiamin HCI, 8 mg/kg; pyridoxineHCI,8 mg/kg;folicacid,10 mg/kg;inositol,250mg/kg;vitaminB12,0.05mg/kg.ThevitamincontentofDiet4 wasthe same except for omission of folic acid. The vitamin content of Diets 2 and 5 to 11 was: vitamin A, 10 mg/kg: vitaminD2,3000lii; vitaminE,225IU;menadione,10 mg/kg;niacin,12mg/kg;calciumpantothenate,40mg/kg;riboflavin,16 mg/kg; thiamin HCI, 16 mg/kg; pyridoxine HCI, 16 mg/kg; inositol, 250 mg/kg; biotin, 0.07 mg/kg; vitamin B2,0.05 mg/kg. The mineral content of the diets was (%): calcium, 0.592; phosphorus, 0.394; potassium, 0.493; sodium,0.493; chlorine, 0.760; magnesium, 0.049; iron, 0.0049; copper, 0.001 9; manganese, 0.001 95; zinc, 0.0004; iodine,0.00001 9; molybdenum, 0.000005; selenium, 0.0000025.

b Vitamin-free casein and alcohol-extracted peanut meal and fibrin were purchased from Teklad Test Diets, Madison,Wis.; the soy assay protein was purchased from Ralston Purina Co., St. Louis, Mo. Beef tallow was purchased fromMcKinnon and Mckenzie, Brighton, Mass.

c L-Amino acids (Grand Island Biological Co., Grand Island, N. V.) in diet(g/kg): arginine, 0.66; glutamic acid, 2.45;isoleucine, 0.64; lysine, 0.98; threonine, 0.36; tryptophan, 0.15.

d In Diets 1 , 3, and 9, the carbohydrate is mixed from 3 sources: 36% of the total from dextrose (CPC International,Englewood Cliffs, N. J.); 36% from dextrin (American Maize Products, Hammond, Ind.); and 28% from sucrose (SavageCo., Waltham, Mass.). In Diets 2 and 4 to 11, the carbohydrate is sucrose alone.

than the mixed proteins of Diet 2; they differ from each otheronly in choline and methionine content.

Carcinogens. AFB1 (Makor Chemicals, Jerusalem, Israel),dissolved in dimethybsulfoxide, was administered by gavage ina dose of 15 sg/day, 3 to 5 days/week for 7 weeks to give atotal dose of 375 jig. There were 28 to 35 rats/diet group.

DMH (Aldrich Chemical Co., Milwaukee, Wis.) was administered in 0.9% NaCI solution by gavage in a dose of 30 mg/kgbody weight once weekly for 5 weeks to give a total dose of150 mg/kg. There were 25 rats/diet group.

AAF (kindly provided by Dr. Lionel Poirier, National CancerInstitute) was fed in the diet at 0.01 25% for 16 weeks. Foodintake was measured during carcinogen treatment; dietaryconcentration of AAF was adjusted if necessary during the final6 weeks to give approximately equal total AAF intakes basedon body weight. There were 32 rats/diet group.

Necropsy Procedures. AFB1-treated rats were killed andnecropsied when moribund or losing weight consistently; surviving rats were killed 90 weeks after first AFB1 treatment.AAF-treated rats were killed and necropsied when moribund orlosing weight consistently; surviving rats were killed 64 weeksafter beginning AAF treatment. DMH-treated rats were killedand necropsied when moribund or bleeding heavily from therectum; surviving rats were killed and necropsied 50 weeksafter first DMH treatment. At necropsy, all tumors and majororgans were fixed in 10% neutral-buffered formalin, embedded,processed by routine histological methods, and stained withhematoxylin and eosin.

Statistical Procedures. Tumor incidences were compared

1 to 6 in the AFB1and DMH experiments; Diets 1 to 3 and 7 to11 in the AAF experiment. The 3 basic diets were Diets 1, 2,and 10. The other diets were modifications of the basic diets,designed to test the effect of specific nutrients or groups ofnutrients. Diet 1 is a nutritionally complete, purified diet. Diet 2is the marginally lipotrope-deficient, high-fat diet used in ourearlier studies. Diet 3 is Diet 1 with the lipid content of Diet 2(beef fat and corn oil) substituted for the Wesson oil and partof the carbohydrate of Diet 1.

Diets 4 to 6 are based on Diet 2. In Diet 4, the vitamin mix ofDiet 1 (except for folate) was substituted for the vitamin mix ofDiet 2. Both vitamin mixes are complete in most respects, butthe mix in Diet 2 does not supply the amount of niacin recommended for rats, and it contains no fobate. Folate was notincluded in Diet 4 because of its bipotropic action. In Diet 5, thelipotrope (choline, methionine, and folate) content of Diet 2was made equivalent to that of Diet 1 on a caloric basis; in Diet6, the essential amino acid content of Diet 2 was made equivalent to that of Diet 1. These 6 diets are identical to the dietsfed in a previous study of DENA carcinogenesis (12).

Diets 7 to 9 also are based on Diet 2. Diet 7 is Diet 2, fullysupplemented with both lipotropes and amino acids to make itequivalent to Diet 1, and contains the full vitamin mix from Diet1; it incorporates the changes previously studied separatelyusing Diets 4 to 6. Diet 8 is Diet 2 supplemented with methionine alone; Diet 9 is Diet 2 with the carbohydrate mixture ofDiet 1 (rather than sucrose alone).

Diets 10 and 11 are, respectively, bipotrope-supplementedand lipotrope-deficient diets that contain soy protein rather

2803AUGUST1980



II

Hepatocarcinoma incidence in AFB-treatedratsHepatocarcinomaincidence in rats fed experimental diets and givenAFB(375

@Lg)intragastrically over a 7-week period. There is a significant increaseintumorincidence, compared to rats fed Diet 1, in rats fed Diet 4 ( p < 0.001 ),Diet5

(p < 0.01), or Diet 6 (p < 0.05)(@2analysis).%

ofeffectiveEffectiveno. of rats with hep@to

Diet ratsacarcinoma1.Control

27152.Deficient 31393.Control, high fat 2974.Deficient, control vitamin mix 34825.Deficient + lipotropes 33586.Deficient + amino acids 33 48

A. E. Rogers et al.

statistically by x2 or discrete multivariate analysis (1); cumubative probability of death with tumor was calculated accordingto the method of Saffiotti et al. (17). Computer programBMDPIL, life tables, and survival functions were used for statistical evaluation of cumulative probability of death with tumor.The computer program was developed at the Health SciencesComputing Facility, UCLA, and revised in November 1978under NIH Special Research Resources Grant RR-3. It givescomparisons using both the Bresbow and Manteb-Cox procedures (9). p values derived from the comparisons are designated PB(Bresbow)and PM-C(Manteb-Cox).

RESULTS

AFB1. Diet had no significant effect on growth. Hepatocarcinoma incidence and rate of increase in cumulative probabilityof death with hepatocarcinoma were greater in rats fed Diet 2(deficient) or any of the diets based on Diet 2 (Diets 4 to 6)than in rats fed Diets 1 (control) or 3 (control, high-fat) (Table2, Chart 1). None of the supplements or substitutions to Diet 2corrected its enhancement of AFB1carcinogenesis. All rats fedDiet 2 or Diets 4 to 6 were dead by 82 weeks after AFB,treatment, at which time 20% of rats fed Diet 1 and 25% ofrats fed Diet 3 were still alive. Lipotrope (Diet 5) or amino acid(Diet 6) supplementation was associated with delays in the firstdeath with tumor, but subsequent tumor development wasequaltothatinratsfedDiet2.

Diet 3, derived by substitution of the fat from Diet 2 into Diet1 , appeared to retard tumor development, although the differences between Diet 3 and Diet 1 were not statistically significant(Chart1).Sixty-four% ofratsfedDiet1 thatdiedbefore90 weeks had hepatocarcinoma or hyperplastic hepatic nodubes;36% of rats fed Diet 3 that died before 90 weeks hadhyperplastic nodules, but none had carcinoma.

Interstitial cell tumors of the testis, which occur spontaneously in Fischer rats, were present in 7 to 26% of rats in alldietary groups; tumor incidence was not related to diet.

AAF. Growth was essentially equal in rats fed AAF in thenutritionally complete diets [Diets 1 (control), 3 (control, highfat), 7 (deficient, fully supplemented), and 10 (control, soy)],and was somewhat reduced in rats fed AAF in Diets 2 (deficient), 8 (deficient + methionine), 9 (deficient, mixed carbo

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Chart 1. Cumulative probability of death with hepatocarcinoma in AFB,treated rats. x , Diet 1 (control); 0, Diet 2 (deficient); â€¢,Diet 4 (deficient, controlvitamin mix); R, Diet 5 (deficient + lipotropes); t@,Diet 6 (deficient + aminoacids). compared to Diet 1, curves are significantly different for Diet 2 (p@â€”0.003; PM.c= 0.0002), Diet 4 (p = 0.0000 by both tests), Diet 5 (p@â€”0.0001;PM-c 0.000), and Diet 6 (p@ = 0.0008, p,.,.@â€”0.0002). Diet 3 is not shown,since there were no deaths with tumor before the terminal sacrifice. Comparisonof Diet 1 and Diet 3 gave values: p@â€”0.063; p,,,.@â€”0.049.

hydrate), and 11 (deficient, soy). The difference in averageweights between the 2 clusters of dietary groups was approximately 30 to 50 g, beginning 4 weeks after initiation of AAFfeeding. Rats fed either lipotrope-deficient diet (Diet 2 or 11)had mildly fatty livers. Fat was reduced by supplementing thediet with lipotropes and amino acids (Diet 7 or 10).

Hepatocarcinomas occurred earlier and in higher incidencein rats fed Diets 2, 9, and 11 than in rats fed the other diets(Table 3). Incidence in rats fed Diet 2 was significantly differentfrom that in rats fed Diet 1, as we reported in an earlierexperiment (11). There was complete correction of this increased susceptibility to AAF by supplementation of Diet 2 withlipotropes and amino acids (Diet 7) or with methionine alone(Diet 8). Alteration of the carbohydrate source (Diet 9) had noeffect. The difference between rats fed the deficient (Diet 11)or the supplemented (Diet 10) soy protein diet was consistentwith a lipotrope effect, but it was not statistically significant.Substitution of the fat from Diet 2 into Diet 1 (Diet 3) did notretard carcinogenesis as it did in rats given DENA or AFB1.Differences between the 2 groups of diets, i.e. , the lipotropedeficient diets (Diets 2, 9, and 11) and the diets adequate inlipotropes, were significant. The risk of developing hepatocarcinoma was increased 4.3 times by lipotrope deficiency ( p <0.05).

Full examination of cumulative probability of death with livertumor was not made since the animals were killed and necropsied at 64 weeks, at which time 41 to 84% of rats in thedifferent diet groups were still alive. Cumulative probability ofdeath with tumor up to that time is shown in Chart 2. The curvefor Diet 9 (deficient, mixed carbohydrate) coincided with thecurve for Diet 2 (deficient) and the curve for Diets 3 (control,high-fat) and 8 (deficient plus methionine) were the same asfor Diets 1 (control), 7 (deficient, fully supplemented), and 10(control, soy); they have been omitted to simplify the graph.The dietary effects are apparent in the clustering of lipotropedeficient diets and of lipotrope-adequate diets, regardless oftheir other dietary components.

Table 2

a Rats that survived until the first death with hepatocarcinoma.

b Interstitial cell tumors were present in rats fed Diet 1 (26%), Diet 2 (22%),Diet 3 (7%), Diet 4 (9%), Diet 5 (18%), and Diet 6 (24%). Two rats fed Diet 4 hadtumors in addition to hepatocarcinomas: an adenocarcinoma of the colon: andan undifferentiated brain tumor. One rat fed Diet 4 had no hepatocarcinoma butbore a s.c. sarcoma; 1 rat fed Diet 1 without hepatocarcinoma bore a small renaltubular adenoma.

CANCERRESEARCHVOL. 402804



% of effective ratswithhepatocarcinomaAv.

total AAF in

Diettake(mg/kg

body wt)Effectiveno.

of rat?Before64

wkTotal1.Control

2.Deficient469 46931 321961

559l@'3.

Control, high fat4543228567.Deficient, fully supplemented444321 9538.Deficient + methionine

9. DeficIent, mixed carbohydrate478 45631 3110 58

3910.Control, soy

11. Deficient, soy470 46132 322569

47 84b

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Chart2. CumulativeprobabilityofdeathwithhepatocarcinomainAAF-treatedrats. X , Diet 1 (control); 0, Diet 2 (deficient); â€¢,Diet 7 (deficient, fully supplemented); t@,DIet 10 (control, soy): A, Diet 11 (deficient, soy).

DMH. Growth was not significantly affected by dietary treatment. Colon tumor incidence was greater in rats fed Diets 2 to6 (defIcient, with or without supplements, or control, high-fat)than in rats fed Diet 1, but the differences were not statisticallysignificant. Tumors of the small bowel occurred in approximatebythe same incidence in all groups, and Zymbal's glandtumors were present in greatest incidence in rats fed Diet 1(Table 4). Cumulative probability of death with colon tumor waslowest at all times (except at 44 weeks) in rats fed Diet 1(control) (Chart 3). Substitution of the higher, more saturatedfat content of Diet 2 into Diet 1 (Diet 3) increased colon tumorincidence and cumulative probability of death with tumor togive results similar to results in animals fed Diet 2 (deficient) ormodifications of Diet 2. Average number of colon carcinomasper tumor-bearing animal, tumor size, and histological typewere not affected by diet.

DISCUSSION

The difference between rats fed Diet 1 (control) and thosefed Diet 2 (deficient) in response to carcinogens of severalchemical classes is consistent and has been demonstratedrepeatedly in experiments using AFB1, DENA, DMH, and AAF.It has been difficult to identify the dietary components respon


Table 3Hepatocarcinoma incidence in AAF-treated rats

Carcinogen intake and incidence of hepatocarcinoma in male Sprague-Dawley rats fed experimentaldiets containing AAF for 16 weeks.

a Number of rats alive at time of first death with hepatocarcinoma.b Risk of developing hepatocarcinoma was 4.3 times greater than in rats fed Diets 1 , 3, 7, or 8 (p <

0.05)(l).

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sible for the difference in all cases, but such identification maybe important in understanding dietary effects on carcinogene515.The simplest results in the present study have been obtamed in the experiments using AAF or DMH.

First, in examining the hepatocarcinogens, the AAF experiment demonstrated that the 2 different lipotrope-deficient diets(Diets 2 and 11) enhanced AAF hepatocarcinogenesis compared to the same diets supplemented with choline and methionine (Diet 10) or with methionine alone (Diet 8). Lombardi andShinozuka (3) report similar results in AAF-treated rats fed adiet similar to Diet 2 with or without choline. Miller and Miller(4) report that methionine blocks AAF hepatocarcinogenesis inrats fed a nutritionally complete diet. In our experiment, full

I correction of Diet 2 (Diet 7) gave no greater protection than did

70 the addition of methionine alone.

The carbohydrate content of Diet 2 (deficient) apparentlydoes not contribute to its enhancement of AAF hepatocarcinogenesis. The fat content of Diet 2, added to Diet 1 (control),did not influence tumor induction, but its presence in Diet 2contributes, of course, to the degree of lipotrope deficiencyinduced. The similarity of results in rats fed the nutritionallycomplete diets (Diets 1, 3, 8, or 10) suggests that the sourceof dietary protein does not have a major effect on AAF hepatocarcinogenesis, since Diets 1 and 3 contain casein; Diet 7,a mixture of proteins, and Diet 10, soybean protein.

In the AFB1 experiment, supplementation of Diet 2 withbipotropesor amino acids had no effect on its enhancement ofhepatocarcinogenesis. This finding contrasts with some (6, 8,19) but not all (13, 18) earlier studies. As noted in the introduction, in earlier studies we found correction by lipotropesupplementation of enhanced AFB1carcinogenesis in deficientrats when dietary fat was vegetable oil or lard, but we foundmore variable results when beef tallow was fed. A direct effectof tallow (plus 2% corn oil) on AFB1 carcinogenesis was mdicated in this experiment by the differences in tumor incidenceand cumulative probability of death with tumor between ratsfed Diet 3 (control, high-fat) and rats fed Diet 1 (control). Theeffect was a decrease rather than an increase in tumor development, and, therefore, does not explain enhancement ofcarcinogenesis in rats fed Diet 2. Diet 3 reduced DENA hepatocarcinogenesis (12), although less markedly than with AFB1,but did not influence results with AAF. The rebatedobservationthat lipotrope supplementation of Diet 2 partially corrected itsenhancement of DENA hepatocarcinogenesis suggests an in

AUGUST1980 2805



Tumor incidence in OMH-treated ratsTumor incidence in rats fed experimental diets and given DMH, 30 mg/kg intragastrically, once a wee

Differences in tumor incidence are not statistically significant.kfor 5weeks.%

of rats withcarcinomaEffective

Small intes- Intestine (toDiet no. of ratsa Colonb tine tal)Zymbal'sgland1

. Control 22 52 (1 6)C 2457332.Deficient 25 83 (2.2) 2988213.Control, high fat 24 76 (1.9) 2884164.Deficient, control vitamin mix 25 76 (2.3) 3680165.Deficient + lipotrope 25 83 (2.2) 3288166.Deficient + amino acids 25 83 (2.2) 21 880

A. E. Rogers et aI.

Table 4

a-R@sthat surviveduntilthe firstdeath withcolon tumor.b A few rats bore colon adenomas only; if they are included in colon tumor incidence, minor changes occur: Diet 2,

88%; Diet 4, 80%; Diet 5, 88%. In addition to tumors listed, there were 2 cases of hepatocarcinoma (Diets 2 and 4); 3of sarcoma. 2 renal and 1 s.c. (Diets 3, 4, and 6); 1 of renal adenocarcinoma (Diet 4); and 1 of lymphocytic leukemia(Diet 2).

C Numbers in parentheses, average number of colon carcinomas per tumor-bearing rat.

clear that interactions will have to be defined for individualcarcinogens.

Lipotrope deficiency appears to be effective at an early stageof carcinogenesis. Shinozuka et al. (21) present evidence thatthe effect is exerted during promotion. In their studies andours, appearance of hyperplastic hepatocytes is markedly accelerated in deficient animals with all carcinogens studied.Appearance of oval cells, which may be hepatocyte stem cells,and of AFP depends on the carcinogen studied and the doseused (12, 20, 21). In conjunction with the AFB1 studies described above, we measured plasma AFP in rats fed Diet 1(control) or Diet 2 (deficient).4 Rats fed Diet 2 and given nocarcinogen had a significant elevation of AFP compared to ratsfed Diet 1 [0.78 Â±0.23 (S.E.) versus 0. 12 Â±0.01 @gAFP perml plasma, respectively, at 6 weeks of age]. AFP was furtherelevated by AFB1 treatment in rats fed Diet 2 (2.53 Â±0.69versus 0.82 Â±0.13 @tg/ml).In AFB1-treated rats fed Diet 1,there was no consistent elevation of AFP. The early AFPresponse may be a useful indicator of potential dietary effectson hepatocarcinogenesis.

The results in DMH-treated rats indicate that the lipid contentof Diet 2 is the major factor in its enhancement of coloncarcinogenesis and that supplementation of Diet 2 with lipotropes, amino acids, or vitamins had no effect. These results

_____ areconsistentwithreportsfromotherlaboratoriesthatin510 creased dietary fat enhances induction of colon tumors (10,

22). The change from polyunsaturated vegetable fat to predominantly saturated animal fat may have had an influence ontumor induction also, although studies in other laboratorieshave shown that fat source is less significant than fat amountin colon tumor induction (10, 22). There may be a slightincrease in tumors in rats fed polyunsaturated fat (10).

In summary, the contribution of several dietary variables toenhancement of chemical carcinogenesis by Diet 2 appears todepend on the carcinogen and target organ studied (Table 5).Lipotrope or amino acid supplementation prevents the enhancement of hepatocarcinogenesis in some but not all cases;the result may depend on the type of fat fed. The fat content ofthe deficient diet apparently is responsible for its enhancementof DMH colon carcinogenesis. Carbohydrate source and minorvariations in vitamin content have no effect.

4 A. E. Rogers and S. Sell, unpublished observations.

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Chart 3. Cumulative probability of death with colon carcinoma in DMH-treatedrats. X , Diet 1 (control); 0, Diet 2 (deficient); 0, Diet 3 (control, high-fat); â€¢,Diet4 (deficient, control vitamin mix); , Diet 5 (deficient + lipotropes); L@,Diet 6(deficient + amino acids). Compared to Diet 1, the curves are significantlydifferent for Diet 4 (PB 0.007; PM-c 0.02), Diet 5 (ps, PM-câ€”0.0002), andDiet 6 ( Pa, [email protected]â€”0.002). For Diets 2 and 3 (versus Diet 1), the values are,respectively, PB 0. 12, PM-C 0. 14, and Pa = 0.03, p,,,@â€”0.10.

verse relationship between the protection afforded by bipotropesupplementation of Diet 2 and that afforded by addition of thefat content of Diet 2 to Diet 1. We have no explanation for thisphenomenon, but it warrants further study. Dietary lipotropesare unquestionably involved in hepatocarcinogenesis inducedby many different compounds (3, 7, 11â€”13, 20, 21), and theinteractions between these compounds and dietary lipid withrespect to carcinogenesis may be significant. It is becoming

I I t@

30 40WEEKS AFTER FIRST DOSE OF DMH

2806 CANCERRESEARCHVOL. 40



AAFDENAaDecreaseDecrease


Table 5Summary of effects on carcinogenesis of dietary variables tested

Effect on carcino9enesis by

DMH

No change

Dietary variableAFBIncreased

lipotropes in Diet 2 (choline, methi- No changebonine)Increased

essential amino acids in Diet 2 (in- No changecluding methionine)No

changeDecreaseDecreaseIncreased

fat in Diet 1 (and predominantly Decreasesaturated substituted for polyunsaturated)IncreaseNo

changeDecreaseChange

in carbohydrate source, or composi- No changetion of vitamin mixNo

changeNo changeNochangea

EffectsonDENA(12)havebeenincludedforcomparison.b Decreased AFB carcinogenesis in lipotrope-supplemented

different diets (see text).ratshas beenfound in other experiments using

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1. Bishop, Y. M. M., Fienberg, S. E., and Holland, P. W. Discrete MultivariateAnalysis. Cambridge, Mass.: M. I. T. Press, 1975.

2. Carroll,K.K.,andkhor,H.T.Dietaryfatinrelationtotumorigenesis.Prog.Blochem. Pharmacol., 10: 308-353, 1975.

3, Lombardl,B.,andShinozuka,H. Enhancementof 2-acetylaminofluoreneliver carcinogenesis in rats fed a chollne.devoid diet. Int. J. Cancer, 23:565â€”570,1979.

4. MIller, E. C.. and Miller, J. A. Approaches to the mechanisms and control ofchemical carcinogenesis. Bertner Foundation Award Lecture. In: Environment and Cancer, M. D. Anderson Hospital and Tumor Institute, pp. 5â€”39.Baftimore: willIams & Wilkins, 1972.

5. Newborns,P.M.Carcinogenicityofaflatoxin-contaminatedpeanutmeal.In:G.N.Wogan(ed.),Mycotoxinsin Foodstuffs,pp. 187-208.Cambridge,Mass.: M. I. T. Press, 1965.

6. Newberne, P. M., and Rogers, A. E. Nutrition, monocrotaline and aflatoxinB, in liver carclnogenesis. Plant Foods for Man, 1: 23â€”31. 1973.

7. Newbeme,P.M.,andRogers,A.E.Nutritionalmodulationofcarcinogenesis.In: P. N. Magee (ed.), Fundamentals In Cancer Prevention, pp. 15â€”40.BaltImore: University Park Press, 1976.

8. Newbeme, P. M., Rogers, A. E., and Wogan, G. N. Hepatorenal lesions inrats fed a low lipotrope diet and exposed to aflatoxin. J. Nutr., 94: 331 -343,1968.

9. Nixon, W. J., and Brown, M. B. (eds.). BMDP-77, BMDP Biomedical Computer Programs, P. series. Berkeley, Calif.: University of California Press,1977.

10. Reddy, B. S.. Narisawa, T., Maranpot, R., Weisburger, J. H., and Wynder,E.L.Animalmodelsforthestudyofdietaryfactorsandcancerofthelargebowel. Cancer Res., 35: 3421 -3426, 1975.

11. Rogers, A. E. Variable effects of a lipotrope-deficient high-fat diet onchemical carcinogenesis in rats. Cancer Res., 35: 2469â€”2474,1975.

12. Rogers, A. E. Reduction of N-nitrosodiethylamine carcinogenesis in rats bylipotrope or amino acid supplementation of a marginally deficient diet.

Cancer Res., 37: 194â€”199, 1977.13. Rogers, A. E., and Newberne, P. M. Aflatoxin B carcinogenesis in lipotrope

deficient rats. Cancer Res., 29: 1965â€”1972, 1969.14. Rogers, A. E., and Newberne, P. M. Nutrition and aflatoxin carcinogenesis.

Nature (Lond.), 229: 62â€”63,1971.15. Rogers, A. E., and Newberne, P. M. Dietary enhancement of intestinal

carcinogenesis by dimethylhydrazine in rats. Nature (Lond.), 246: 491â€”492,1973.

16. Rogers, A. E., Sanchez, 0. . Feinsod, F., and Newberne, P. M. Dietaryenhancement of nitrosamine carcinogenesis. Cancer Res., 34: 96â€”99.1974.

17. Saffiotti, U., Montesano, A., Sellakumar, A. R., Cefis, R., and kaufman, 0.G.Respiratorytractcarcinogenesisinhamstersinducedbydifferentnumhors of administrations of benzo(a)pyrene and ferric oxide. Cancer Res., 32:1073â€”1081,1972.

18. Salmon, W. 0., and Newberne, P. M. Occurrence of hepatomas in rats feddiets containing peanut meal as a major source of protein. Cancer Res., 23:571-575, 1963.

19. Schaefer, A. E., Copeland, 0. H.. Salmon, W. 0., and Hale, 0. M. Theinfluence of riboflavin, pyridoxine, inositol, and protein depletion-repletionupon the induction of neoplasms by choline deficiency. Cancer Res., 11:786â€”792,1951.

20. Shinozuka, H., Lombardi, B., Sell, S., and lammarino, R. M. Early histologicaland functional alterations of ethionine liver carcinogenesis in rats fed acholine-deficient diet. Cancer Res., 38: 1092â€”1098, 1978.

21. Shinozuka, H., Sells, M. A., katyal, S. L., Sell, S., and Lombardi, B. Effectsof a choline-devoid diet on the emergence of a-glutamyltranspeptidasepositive foci in the liver of carcinogen-treated rats. Cancer Res., 39: 2515â€”2521, 1979.

22. Wilson, R. B., Hutcheson, D. P., and Wideman, L. Dimethylhydrazine-induced colon tumors in rats fed diets containing beef fat or corn oil with orwithout wheat bran. Am. J. Clin. Nutr., 30: 176â€”181 . 1977.

23. Wynder, E. L. The epidemiology of large bowel cancer. Cancer Res., 35:3388-3394. 1975.

2807AUGUST1980



1980;40:2802-2807. Cancer Res Adrianne E. Rogers, Gregory Lenhart and Gayle Morrison Carcinogenesis in Rats

-2-Fluorenylacetamide, and 1,2-DimethylhydrazineN, 1Influence of Dietary Lipotrope and Lipid Content on Aflatoxin B

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