[CANCER RESEARCH 45, 2700-2705, June 1985]

Effects of a High-Sucrose Diet on the Development of Enzyme-altered Foci inChemical Hepatocarcinogenesis in Rats1

Tom K. Hei2 and Oscar Sudilovsky3

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT

Dietary factors can modify metabolic events involved in theinitiation, promotion, or progression of tumors. To determinewhether a high-sucrose diet has any effect on the developmentof enzyme-altered foci during the promotion step of chemicalhepatocarcinogenesis in rats, 1-day-old Sprague-Dawley rats

were given a single i.p. dose of diethylnitrosamine; controlsreceived an equivalent ¡.p.volume of 0.9% NaCI solution. At 21days of age, the rats were weaned, segregated by sex, separated in groups, and fed modified AIN76A diets containing either65% glucose or 65% sucrose, with or without 0.05% phénobarbital. At the end of a 4-week treatment period, the sucrose-fed

control rats of either sex had significantly heavier livers than didthose on the glucose diet. Enlarged livers were found also in thesucrose-fed diethylnitrosamine-treated female rats, which developed twice as many 7-glutamyltranspeptidase-positive foci per

sq cm of liver section than did those on the glucose diet. Additionof phénobarbital augmented the number of foci 3-fold in thesucrose group and 5-fold in the glucose group. Focus count persq cm was similar in animals on the two phenobarbital-supple-

mented diets. Despite the absence of statistically significant liverenlargement, results analogous to those in females were obtained in carcinogen-treated males. Differences between treat

ments, however, were smaller. In both female and male rats, theDNA-synthesizing activity of hepatocytes in enzyme-altered foci

was significantly higher than in the surrounding normal parenchyma! cells, as determined by autoradiography. These studiesindicate that a high-sucrose diet has a promoting effect during

hepatocarcinogenesis induced in the rat by diethylnitrosamineand that this effect is weaker than that of 0.05% phénobarbital.

INTRODUCTION

It has been known for quite some time that both exogenousand endogenous factors can modify the outcome of hepatocarcinogenesis in laboratory animals. Pioneer studies by Biel-

schowsky (5) and Miller ef al. (16) have indicated that diet andhormones may have a profound effect on the incidence ofhepatoma in rodents. The effects of these 2 types of modifiersare frequently intertwined since both can affect a vast array ofcellular enzymes. Most of the early studies of dietary effects onexperimental hepatocarcinogenesis, however, made no attemptto delineate whether those effects took place during the initiation

1Supported in part by Grant CA 25164 from the National Cancer Institute. Some

of the data were presented in abstract form at the 68th Annual Meeting of theFederation of American Societies for Experimental Biology, St. Louis, MO, April1984.

2 Present address: Radiological Research Laboratory, College of Physicians and

Surgeons, Columbia University, New York, NY 10032.3To whom requests for reprints should be addressed, at Institute of Pathology,

Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106.Received 10/1/85; revised 2/13/85; accepted 2/15/85.

or the promotion step, the general assumption being that thediet may exert its influence through modification of enzymeactivities responsible for the metabolism and activation of chemical carcinogens.

Among the 3 major dietary components, i.e., carbohydrate,fat, and protein, the latter 2 have been implicated more recentlyin the promotion of liver cancer (1,15, 23, 31). Little is known,however, about a similar role for carbohydrates on tumor development. Specifically, the possibility that certain carbohydrates athigh dietary levels may have a promoting effect in hepatocarcinogenesis after initiation by DEN4 has not been examined here

tofore. Studies by Bender ef al. (3) have demonstrated that ratsfed a high-sucrose diet (70%) for only 4 weeks had heavier livers

due to hyperplasia of parenchymal cells while rats fed a similarconcentration of glucose showed no comparable response. Because of its ability to cause liver enlargement, it would seemplausible that a high-sucrose diet may modulate the promoting

phase of hepatocarcinogenesis (27). Accordingly, the presentstudy was undertaken to evaluate such an effect of sucrose inrats treated previously with a small initiating dose of DEN. Thedevelopment of GGTPase-positive foci was used to quantitate

the postulated dietary effects. The sequential relationships ofthese lesions with hyperplastic nodules and hepatocellular carcinomas have been demonstrated with a number of chemicals(7, 22, 35). Since PB is a potent promoter in experimentalhepatocarcinogenesis (19, 29), it was also included in this studyto determine whether a synergistic effect with a high-sucrosediet could be demonstrated.

MATERIALS AND METHODS

Animals and Treatments. Pregnant Sprague-Dawley rats (Zivic-Miller,Allison Park, PA) were received 1 week before parturition and housedindividually in plastic cages in a 23°Cconstant-temperature room with a

12-h light, 12-h dark illumination cycle. They were given free access to

a standard pelleted laboratory diet (Purina Formulab 5008; Ralston PurinaCo., St. Louis, MO) and water. Twenty-four h after delivery, newbornrats were used as in the "neonatal rat protocol" described by Peraino ef

al. (21) and one half of the total number of offspring were each given ani.p. injection of a solution of DEN (Sigma) in 0.9% NaCI solution (saline),at a dosage of 0.15 ¿irnol/gbody weight. The other half were used ascontrol animals, and each was given an injection of an equivalent volumeof saline. The rats were weaned at 3 weeks of age, segregated withrespect to sex, and housed individually in stainless steel cages with wiremesh bottoms. Both males and females from either the carcinogen-

treated or 0.9% NaCI solution control groups were then randomized byweight into 4 subgroups which were fed ad libitum one of the 4 testdiets described below and weighed twice weekly to monitor growth. Allanimals were sacrificed 4 weeks postweaning, i.e.. at 7 weeks of age.Two h before sacrificing at 9 a.m., each animal received an i.p. injectionof [3H]dThd (specific activity, 30 ¿iCi/mmol; ICN Pharmaceuticals, Inc.,

4The abbreviations used are: DEN, diethylnitrosamine; GGTPase, f-glutamyl-

transpeptidase; PB, phénobarbital;dThd, thymidine.

CANCER RESEARCH VOL. 45 JUNE 1985

2700

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECT OF HIGH-SUCROSE DIET ON HEPATOCARCINOGENESIS

Irvine, CA) at a dosage of 50 /iCi/100 g body weight. The rats were killedby cervical dislocation, and the livers were quickly excised, washed twicein saline, blotted dry with filter paper, and weighed. Serial samples fromeach hepatic lobe were taken by a standardized procedure (12) andeither fixed in 10% buffered formaldehyde for routine hematoxylin andeosin staining or frozen in isopentane immersed in liquid nitrogen forGGTPase staining and autoradiographic studies.

Diets. The basal diet used, from which the 4 test diets were derived,was the AIN76A semipurified rat-mouse diet (American Institute of Nu

trition, recommendation oladhoc Committee, March 1980). The pelletedtest diets (United States Biochemical Corp., Cleveland, OH) with orwithout 0.05% PB (Sigma) contained 65% of either glucose or sucroseas their only carbohydrate source.

Staining Procedures. Frozen liver sections were processed for his-

tochemical demonstration of GGTPase by a modification of the procedureof Ogawa ef al. (18). Basically, tissue samples were fixed in chilledacetone for 2 h followed by rinsing in 2 changes of acetone (1 h each).Liver sections were then treated with 2 changes of cedarwood oil for 16to 24 h followed by clearing in 3 changes of benzene 20 min apart. After2 h infiltration in paraffin (Tissue Prep, M.P. 56.5; Fisher Scientific, FairLawn, NJ), they were embedded in paraffin. Tissue blocks were cut into5 Mm-thick sections and stained histochemically for GGTPase accordingto the method of Rutenburg ef al. (24) using 7-glutamyl-4-methoxy-2-

naphthylamide (Vega Biochemicals, Tucson, AZ) as substrate. The sections were then thoroughly rinsed in distilled water, mounted in bufferedglycerol, and stored at 4°Cuntil analyzed.

Quantitation of Enzyme-altered Foci. The total number of GGTPase-

positive foci from each histochemically stained slide was counted microscopically under x40 magnification. Only clearly demarcated groups of6 or more cells were regarded as foci and included in the present studies(30). All slides were coded in order to avoid bias during the countingprocedures. To measure the area of foci, slides were placed on amicroslide projector (Ernest Leitz GMBH, Wetzler, Federal Republic ofGermany) with the image projected on a screen. The actual magnificationof each section was determined with a micrometer scale; routinely, ax27 enlargement was used. The perimeters of the liver section and ofeach focus were then accurately traced out on paper and later transcribed by a Numonic Electronic Digitizer (Numonic, Lansdale, PA) intoactual area after scale adjustment. The range of representative livertissue area scanned for foci was from 2.22 to 5.93 sq cm/animal.

DMA Labeling Index. Five-^m liver sections were cut, deparaffinized,

and stained histochemically for GGTPase, as described above. Afterthorough rinsing with distilled water, autoradiography was carried out onthe same sections by the "dipping technique" using Kodak NTB photo

graphic emulsion (Eastman Kodak, Rochester, NY) as described (25).The labeling index for normal hepatocytes was determined by countinga minimum of 6000 randomly selected hepatocytes in each control animaland an equivalent number of hepatocytes surrounding the foci in carcinogen-treated animals. In rats receiving carcinogen followed by a PB-

containing diet, an average of 1500 randomly selected hepatocytes perslide were counted from the GGTPase-positive foci, while only 300 were

included in animals fed diets without PB because of the smaller numberof GGTPase-positive foci found in these animals.

Determination of Statistical Significance. Statistical analysis of datain all parameters studied was determined using the 2-tailed Student's t

test for unpaired data.

RESULTS

Body Weight and Liver Weight Distribution. There was nosignificant difference in body weight distribution at any timeamong female or male rats in either the carcinogen-treated or

the control groups fed each of the 4 dietary regimens (Tables 1and 2). Dietary modulation of the development of hepaticGGTPase-positive foci therefore cannot be attributed to a generaleffect.

Although animals were randomized by weight, the relative liverweight perl 00 g of body weight was used as a better index forcomparison. The relative liver weights were significantly higherin sucrose-fed than in glucose-fed female rats, both in the controland in the carcinogen-treated groups (Table 1). Addition of PB

caused further liver enlargement and eliminated these weightdifferences between diets. On the other hand, when the samedietary treatment was applied (i.e., glucose or sucrose), therewere statistically significant differences depending on the presence or absence of PB. Any hepatic toxicity due to the carcinogenwas not reflected by the size of the liver, since both the absoluteand relative weights of this organ in rats given a single i.p. doseof DEN were comparable to those of their respective 0.9% NaCIsolution controls.

Male rats in the 0.9% NaCI solution control group respondedto the various dietary treatments in a manner similar to that ofthe females (Table 2). Animals fed the sucrose diet had significantly heavier livers than did those fed the glucose diet. Additionof PB to the carbohydrate diets caused responses similar tothose in females. In DEN-treated males, the mean relative liver

weight of the group fed 65% sucrose, although larger, was notstatistically different from that fed the 65% glucose diet. Supplementation of these carbohydrate diets with PB resulted in meanrelative liver weight differences which were highly significantbetween the sucrose groups (P < 0.01) but not significantbetween the glucose groups.

Number and Area of GGTPase-positive Foci. Occasionalfoci were found in the 0.9% NaCI solution control group, but onlyin animals given the PB-supplemented diets. GGTPase-positive

foci, on the other hand, occurred in all animals given injectionsof DEN. Although they were randomly distributed, a midzonallocalization in the hepatic lobule was predominant (Figs. 1 and2). The response was more intense in females than in males.Similar findings have been documented by Peraino ef a/. (19, 20)and by Demi ef al. (8). DEN-treated female rats kept on the high-sucrose diet generated twice as many GGTPase-positive fociper sq cm of liver area scanned as did those on the high-glucosediet (Table 3). Addition of PB to the 2 sugar diets augmented theresponse 5-fold in the glucose and 3-fold in the sucrose diet

group. No significant difference in the number of foci was foundbetween animals fed either of the PB-supplemented diets. Therewere no significant differences in the average size of GGTPase-positive foci among any of the 4 groups.

Male animals were generally less responsive to the presenttreatment protocol than were females (Table 4). Among thosetreated with DEN, a significantly higher number of foci wasproduced in sucrose-fed than in glucose-fed groups. Inclusion of

PB seemed to further enhance the production of foci in bothglucose- and sucrose-fed rats since application of the one-tailedf test showed significant differences (P < 0.05). When theseresults were analyzed with the 2-tailed f test, however, thedifferences were not statistically significant at the P = 0.05.Thus, probably because of the small sample size, the data gaveonly a weak confirmation of the null hypothesis. Similarly, therewere no significant differences between the number of focicounted in rats fed either of the 2 PB-supplemented diets.Average sizes of the GGTPase-positive foci produced by eachof the 4 dietary treatments were not significantly different. Thesize distribution of foci was analogous to that observed infemales.

CANCER RESEARCH VOL 45 JUNE 1985

2701

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECT OF HIGH-SUCROSE DIET ON HEPATOCARCINOGENESIS

Table 1Liver weights in female rats at the termination of the study

Treatment0.9%

NaCIsolution i.p.65% glucose65% sucrose65% glucose + 0.05% PB65% sucrose + 0.05%PBDEN

i.p.65% glucose65% sucrose65% glucose + 0.05% PB65% sucrose + 0.05% PBNo.

of animals in each

group4

4455

667Liver

wt(g)8.48

±0.62a

11.20 ±3.4912.40 ±2.2715.40±2.088.60

±1.5710.10 ±1.3311.93 ±2.0612.87 ±2.28Body

wl(g)21

8.3 ±10.4'21 1.7 ±20.4'219.6 ±12.6'228.2 ±22.7'227.2

±22.7'222.0 ±17.8'

232.9 ±B.&21 7.6 ±14.7'Liver

wt (g)/100 gbodywt3.89

±0.22"' c5.23 ±1.050'"5.68±1.38c'e6.35 ±0.37*e3.76

±0.34'' a4.56 ±0.43 ' "5.13±0.899'15.91 ±0.89"' '

" Mean ±S.D.""' Differences between means with like superscripts were as follows: b' °'" P < 0.05; * not significant;

' P < 0.005; 9 P < 0.01; P < 0.005; ' not significant;' not significant.

Table 2Liver weights in male rats at the terminationof the study

Treatment0.9%

NaCIsolution i.p.65%glucose65%sucrose65%

glucose + 0.05%PB65%sucrose + 0.05%PBDEN

i.p.65%glucose65%sucrose65%

glucose + 0.05%PB65%sucrose + 0.05% PBNo.

of animalsin eachgroup55563333Liverwt(g)1

3.5 ±2.09a17.9

±3.7416.0±1.6919.8±2.6913.0

+1.6715.1±1.3916.6

+1.9722.0±1.35Body

wt(g)278.4

±36.5Õ299.5±17.S7238.0+10.0'285.9

±15.6'280.0

±18.0'281.3 ±48.9'293.9

+18.7'302.2±34.7'Liver

wt (g)/1 00 gbodywt4.86

±0.42"'°5.95±0.926'"6.72+ 0.67Cl"6.92±0.640'"4.66

±0.49''°5.43+ 0.54''"5.67±0.989''7.31±0.44"' '

a Mean ±S.D.b'! Differencesbetween means with like superscripts were as follows: b P < 0.05; c P < 0.001; dP < 0.05;

*•'•a not significant; " P < 0.01; ' not significant;' not significant.

Table 3Effect of treatmentson the number and size of GGTPase-positivefoci in femalerats

Treatment0.9%

NaCI solution¡.p.65%glucose65%sucrose65%

glucose + 0.05%PB65%sucrose + 0.05%PBDEN

i.p.65%glucose65%sucrose65%

glucose + 0.05% PB65% sucrose + 0.05% PBNo.

ofanimals44454567No.of foci/sq cm

liver000.370.0035.52±1.05a'"'c11.3±2.51£>

"29.5±6.79e' e

35.8 + 13.1* *Size

of foci (sqmm x10"2)001.651.091

.54 ±0.37''91

.72 + 0.34''"2.25±0.659' '

1.93±0.64ft''Volume

(%)000.0060.000030.085

±0.025''*0.199+0.064'''0.664±0.025*' m

0.652 ±0.026'' m

a Mean ±S.D.b~mDifferences between means with like superscripts were as follows:

"•*•'P < 0.002; "•'•9' "' '•mnot significant.•>P< 0.01; CP < 0.001;

Histological Examination. In agreement with previous observations (25), foci examined 7 weeks after the single dose of DENwere relatively small. Areas surrounding them resembled morphologically the livers of control animals on the correspondingdiets. No histological evidence of inflammation, cell necrosis, orbile duct cell proliferation was present. In contrast to animals onthe glucose diet, however, there was increased lipid content inthe hepatocytes of those on the sucrose diet (Fig. 2). Theseearly changes were mostly periportal, unlike those seen in thelivers of choline-deficient rats (in which lipid deposition occurs

first in the central zone of the hepatic lobule). PB induced hepaticfatty changes in all animals, irrespective of the type of carbohydrate diet they were on.

DMA Synthesis of GGTPase-positive Foci. The [3H]dThd

labeling indices of GGTPase-positive hepatocytes from female

rats are shown in Table 5. Male rats responded in a similarmanner (data not shown). These enzyme-positive cells had a 2-to 3-fold higher proliferative activity than did surrounding normalGGTPase-negative hepatocytes, regardless of dietary treatments. The maximum [3H]dThd mean labeling index of

GGTPase-positive hepatocytes, which was attained in animalsfed the PB-supplemented high-sucrose diet, was only 2.5%.Mean labeling indices of normal hepatocytes in any of the DEN-

treated groups were comparable to those in saline controls(Table 5, Column 4), indicating that increased labeling in theGGTPase-positive foci is not the apparent consequence of a

depression of DNA synthesis in intervening normal hepatocytes.On the other hand, hepatocytes from carcinogen-treated animals

CANCER RESEARCH VOL. 45 JUNE 1985

2702

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECT OF HIGH-SUCROSE DIET ON HEPATOCARCINOGENESIS

Table4Effect of treatmentson the number and size of GGTPase-positivefoci in male rats

Treatment0.9%

NaCIsolutioni.p.65%glucose65%sucrose65%

glucose + 0.05%PB65%sucrose + 0.05%PBDEN

i.p.65%glucose65%sucrose65%

glucose + 0.05% PB65% sucrose + 0.05% PBanimals55563333liver000.20.12.85

±0.94a'6'c6.08±2.116'"11.80±7.81c'e

10.70 ±3.42"' "mmx

IO"2)000.932.151.68±0.30''91

.93 ±0.60''"2.27±0.469' '

2.53 ±0.26"' 'Volume

(%)000.0190.0020.051

±0.001''*0.110 ±0.056'-'0.280

±0.226*' m0.261 ±0.065'' m

a Mean ±S.D.fi"m Differencesbetweenmeanswith likesuperscriptswere as follows:

significant.

i:".".>.*.'p<0.05;c'"•••'•»•'•'"not

TableófH]dThd labeling indices of GGTPase-positivefoci in female rats

% of [3H]dThdlabelingindexTreatment0.9%

NaCIsolutioni.p.65%glucose65%sucrose65%

glucose + 0.05%PB65%sucrose + 0.05%PBDEN

i.p.65%glucose65%sucrose65%

glucose + 0.05% PB65% sucrose + 0.05% PBNo.

ofanimals44454567GGTPase-positivehepatocytes1

.25 ±0.246' c-'1.91±0.19i>'"''<1.65±0.26C'"''

2.52 ±0.12d'e''Normalhepatocytes0.76

±0.14a0.68

±0.250.76±0.240.74

±0.160.54

±0.13''90.87±0.03''"0.51±0.319''

0.96 ±0.39"' '

a_Mean±S.D."^ Differences between means with like superscripts were as follows: bP <

0.005; CP < 0.05; "-'-'p < 0.001; 9'"not significant; 'P < 0.05. Significantdifferences from corresponding normal hepatocytes were as follows: ' P < 0.02;*P< 0.005.

fed the high-sucrose diet had significantly higher DMA-labeling

indices than did those fed the glucose diet. Such difference wasuniversal and was seen in both normal and GGTPase-positive

hepatocytes.

DISCUSSION

Present studies corroborate the observation that high-sucrosediets induce liver enlargement in male Sprague-Dawley and

female Wistar rats (3, 11). While Tuovinen and Bender (33) foundheavy livers in male but not in female QEC Lister rats, our datashow that Sprague-Dawley rats not treated with carcinogensdevelop sucrose diet-induced hepatomegaly of analogous magnitude in both sexes (from 116 to 134% of the relative liverweight in corresponding glucose control groups). Strain differences may account to a large extent for the dissimilar sexresponses; variations in diet composition, age of animals, andduration of treatment may have also contributed.

Although the effects of various carbohydrate diets on liverweight have been established, the underlying mechanism is notyet completely understood. Studies by Bender et al. (3) attributedliver weight increase in animals fed a high-sucrose diet to hyper-

plasia and to both hypertrophy and hyperplasia in the case ofthose fed fructose. Hyperplastic effects of dietary sucrose andfructose in rats have been reported also by Bedo and Szigeti (2).

A number of substances which induce hepatomegaly in ro

dents, such as PB, hexachlorocyclohexane, estradici, mestranol,dichlorodiphenyltrichloroethane, and butylhydroxytoluene, arealso liver tumor promoters (19, 25, 28) and, as such, enhancethe production of enzyme-altered foci (9, 25). These foci are,

mechanistically at least, associated with hepatic tumorigenesis(20) and are generally regarded as putatively preneoplastic lesions (18, 28, 35). Among several histochemical markers usedwith the "neonatal rat protocol," GGTPase showed the best

correspondence between relative frequencies of detection in fociand in tumors (20). In the experiments reported here, rats on thehigh-sucrose diet developed an increased number of GGTPase-

positive foci after initiation with a single low dose of DEN (twiceas many as in the controls), indicating that high levels of thiscarbohydrate may have a promoter effect during hepatocarcin-ogenesis. The larger response in females was probably relatedto a sex-dependent effect of the initiator, DEN (8), and was

similar to that observed when PB was given as a promoter (20,21). The average size of foci in animals fed the high-sucrose diet

alone or supplemented with PB for 4 weeks was not larger,however, than that of rats fed the glucose control diets. Althoughit may be argued that a promoter effect of sucrose should haveresulted in bigger foci, it must be noted that increase in numberof foci can be detected very early while enlargement takes longerand it is first seen only after 8 weeks of promotion with PB (27).

Promoting activity can be defined operationally by a sequentialprotocol (such as the one followed here) in which a chronicallyadministered compound promotes the development of tumors ifit is given some time after a known carcinogen but, whensupplied alone, does not cause cancer during the life span of theanimal. Consequently, the virtual absence of foci in noninitiatedanimals fed the sucrose diet also lends support to a promoterrole for high levels of this carbohydrate. This is further substantiated by the autoradiographic demonstration of increased prolif-erative activity in enzyme-altered foci with respect to the sur

rounding normal hepatocytes, as well as to hepatocytes innoninitiated livers. Similar findings have been documented previously utilizing other promoting agents (25). Although stimulationof cell replication per se may not be sufficient (29), the selectiveenhancement of proliferation in putative preneoplastic foci byany given substance is highly relevant to tumor promotion (10,28).

The low labeling indices of GGTPase-positive hepatocytes infoci of sucrose-fed rats were of the magnitude anticipated in anexperiment involving the continuous long-term administration ofa promoter (28). These levels, which were still slightly above

CANCER RESEARCH VOL. 45 JUNE 1985

2703

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECT OF HIGH-SUCROSE DIET ON HEPATOCARCINOGENESIS

those in the foci of controls, might have decreased further hadthe animals been sacrificed a few weeks later because DNAsynthesis and cell proliferation in foci appear to be susceptibleto some kind of feedback regulation (26, 28).

In spite of the hepatic hyperplasia, labeling indices not unlikethose seen in controls were observed in normal hepatocytes ofboth noninitiated and initiated rats fed 65% sucrose. This is notan unexpected finding since it has been shown, with all hepatictumor promoters tested, that uninterrupted long-term treatment

causes liver DNA synthesis to decline almost back to controlvalues (28). Under the homeostatic conditions prevailing amongnormal hepatocytes, a feedback mechanism (26) probably prevented excessive DNA synthesis in the presence of continuousmitogenic stimulation by high sucrose.

Diets containing 65% glucose, used as controls in this study,elicited practically no foci. When rats were initiated with DENand then fed the control diet, however, a small but significantnumber of foci were generated. Although a weak promotingeffect of high glucose could be postulated to explain this, thefact that dietary glucose at concentrations comparable to thoseof sucrose produces neither liver hyperplasia nor similar metabolic or functional alterations (27, 33) militates against it. Mostlikely, the dosage of DEN used for tumor initiation was higherthan necessary. This is confirmed by the identification ofGGTPase-positive foci in numbers equivalent to ours in rats

receiving the same dose of DEN at 1 day of age and kept on a30% casein diet (which does not have promoting effects) for 4weeks after weaning (30).

Addition of 0.05% PB equalized the number of foci in theglucose and sucrose groups, both of which were increased to alevel comparable to those obtained by others with a 30% caseindiet containing also 0.05% PB (20,21,30). The results in Tables3 and 4 show that the promoting effect of sucrose was completely masked by the 2 to 3 times more potent PB stimulation.Therefore, supplementation with PB was neither synergistic noradditive with the effects of the high-sucrose diet. While such an

observation makes it tempting to speculate on a common orpartly common mechanism of action, currently available evidenceis inconclusive.

In the case of high sucrose, the tumor-promoting effect may

be related to the disaccharide molecule per se, the combinationof its products of intestinal hydrolysis (glucose and fructose), orfructose alone. This carbohydrate dietary regimen can altervarious hepatic lipogenic enzymes involved in the metabolism oftriglycérides, cholesterol, and phospholipids (4, 7, 11, 13, 14,32). Whereas increased triglycéridesynthesis may account forthe fatty metamorphosis evidenced histologically in the presentstudies, either changes in cholesterol or phospholipids could beimplicated in a hypothetical promotion mechanism. Thus, increased cholesterol levels in liver (33) can affect the physicalproperties of cell membranes by reducing the cooperativity ofgel-liquid crystal transitions and by increasing the fluidity of the

acyl moiety of phospholipids (34). Alternatively, the increase inliver phospholipids observed after feeding a high-sucrose diet (6,

33) could play a role in promotion by modifying membranestructure and composition. Changes in the properties of cellmembranes by perturbation of hepatocyte membrane lipids havebeen suggested as the mechanism of promotion by PB- andcholine-deficient diets (29, 36-38). Recently, a great deal of

attention has been paid to the turnover of a special group of

membrane phospholipids, the inositol phosphatides, in connection with activation of the phospholipid-dependent enzyme protein kinase C during promotion by phorbol esters (17). It isconceivable that the resulting modifications of membrane structure or function, or both, could inhibit transmission of intercellularregulatory signals (36, 39, 40). Initiated cells could thus escapemodulation of proliferation by surrounding parenchyma and giveorigin to foci with increased mitotic indices. Future studies shouldclarify whether and how the above effects of promoters on thesedifferent membrane lipids are related.

ACKNOWLEDGMENTSWe are indebted to Dr. Carl Peraino for bringing to our attention his "neonatal

rat protocol" of hepatocarcinogenesis. We want to thank Jeanette Nagy for typing

this manuscript.

REFERENCES

1. Appleton, B. S., and Campbell, T. C. Dietary protein intervention during thepostdosing phase of aflatoxin B,-induced hepatic preneoplastic lesion development. J. Nati. Cancer Inst., 70: 547-549,1982.

2. Bedo, M., and Szigeti, A. Effects of dietary carbohydrates on metabolism ofrats. Nahrung, 77: 305-312,1967.

3. Bender, A. E., and Damji, K. B. Chemical, biological and nutritional aspects ofsucrose. In: J. Yudkin, J. Edelman, and L. Hough (eds.), Some Effects ofDietary Sucrose in Sugar, pp. 172-182. London: Butterworths, 1971.

4. Bender, A. E., Damji, K. B., Khan, M. A., Khan, I. H., McGregor, L., and Yudkin,J. Sucrose induction of hepatic hyperplasia in the rat. Nature (Lond.), 238:461-462, 1972.

5. Bielschowsky, F. Tumors of the thyroid produced by 2-acetylaminofluoreneand allyl-thiourea. Br. J. Exp. Pathol., 25: 90-95,1944.

6. Bruckdorfer, K. R., Khan, I. H, and Yudkin, J. The effect of chromium on thehypertipemic action of sucrose. Nutr. Metabol., 73: 36-43, 1971.

7. Cha, C. M., and Randall, H. T. Effects of substitution of glucose-oligosaccha-rides by sucrose in a defined formula diet on intestinal disaccharidases, hepaticlipogenic enzymes and carbohydrate metabolism in young rats. Metab. Clin.Exp., 24: 57-66,1982.

8. Demi, E., Cesterie, D., Wolff, T., and Greim, H. Age, sex and strain dependentdifferences in the induction of enzyme-altered islands in rat liver by diethylni-trosamine. J. Cancer Res. Clin. Oncol., 700: 125-134, 1981.

9. Emmelot, P., and Scherer, E. The first relevant cell stage in rat liver carcino-genesis. A quantitative approach. Biochim. Biophys. Acta, 605: 247-304,1980.

10. Farber, E., and Cameron, R. The sequential analysis of cancer development.Adv. Cancer Res., 37:125-226,1980.

11. Holt, P. R., Domínguez,A. A., and Kwartler, J. Effect of sucrose feeding uponintestinal and hepatic lipid synthesis. Am. J. Clin. Nutr., 32:1792-1798,1979.

12. Laishes, B. A., and Rolfe, P. B. Quantitative assessment of liver colonyformation and hepatocellular carcinoma incidence in rats receiving intravenousinjections of isogeneic liver cells isolated during hepatocarcinogenesis. CancerRes., 40: 4133-4143,1980.

13. MacDonald, I. Some influences of dietary carbohydrates on liver depot lipids.J. Physiol., 762: 334-344, 1962.

14. MacDonald, I., and Roberts, J. B. The incorporation of various C14 dietary

carbohydrates into serum and liver lipids. Metab. Clin. Exp., 74: 991-999,1965.

15. Madhavan, T. V., and Gopalan, C. The effect of dietary protein on carcinogen-esis of aflatoxin. Arch. Pathol., 85:133-137,1968.

16. Miller, E. C., Miller, J. A., Kline, D. E., and Rusch, H. P. Correlation of the levelof hepatic riboflavin with the appearance of liver tumors in rats fed aminoazodyes. J. Exp. Med., 88: 89-102, 1948.

17. Nishizuka, Y. The role of protein Kinase C in cell surface signal transductionand tumor promotion. Nature (Lond.), 308: 693-698,1984.

18. Ogawa, K., Solt, D. B., and Farber, E. Phenotypic diversity as an early propertyof putative pre-neoplastic hepatocytes in liver carcinogenesis. Cancer Res.,40:725-733,1980.

19. Peraino, C., Fry, R. J. M., and Staffeldt, E. Reduction and enhancement byphénobarbitalof hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res., 37: 1506-1512,1971.

20. Peraino, C., Staffeldt, G. F., Cames, B. A., Ludeman, V. A., Blomquist, J. A.,and Vesselinovitch, S. D. Characterization of histochemically detectable alteredhepatocyte foci and their relationship of hepatic tumorigenesis in rats treatedonce with diethylnitrosamine or benzo(a)pyrene within one day after birth.Cancer Res., 44: 3340-3347, 1984.

21. Peraino, C., Staffeldt, E. F., and Ludeman, V. A. Early appearance of histochemically altered hepatocyte foci and liver tumors in female rats treated withcarcinogens one day after birth. Carcinogenesis (Lond.), 2: 463-465, 1981.

22. Pitot, H. C., and Sirica, A. E. The stages of initiation and promotion in

CANCER RESEARCH VOL. 45 JUNE 1985

2704

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECT OF HIGH-SUCROSE DIET ON HEPATOCARCINOGENESIS

hepatocarcinogenesis. Biochim. Biophys. Acta, 605:191-215,1980.23. Rogers, A. E., and Newberne, P. M. Dietary effects on chemical carcinogenesis

in animal models for colon and liver tumors. Cancer Res., 35: 3427-3431,

1975.24. Rutenberg, A. M., Kim, H., Fischbein, J. W., Hanker, J. S., Wasserkrug, H. L.,

and Seligman, A. M. Histochemical and ultrastructural demonstration of -,-glutamyltranspeptidase activity. J. Histochem. Cytochem., 17:517-526,1969.

25. Schulte-Hermann, R., Ohde, G., Schuppler, J., and Timmermann-Trosiener, I.

Enhanced proliferation of putative preneoplastic cells in rat liver followingtreatment with the tumor promoters phénobarbital, hexachlorocyclohexane,steroid compounds, and nafenopin. Cancer Res., 41: 2556-2562,1981.

26. Schulte-Hermann, R., and Schmilz, E. Feedback inhibition of hepatic DMAsynthesis. Cell Tissue Kinet., 73: 371-380,1980.

27. Schulte-Hermann, R., Schuppter, J., Ohde, G., Bursch, W., and Timmermann-Trosiener, I. Phénobarbitaland other liver tumor promoters. In: C. Nicolini (ed.),Chemical Carcinogenesis, pp. 231-260. New York: Plenum Publishing Co.,

1982.28. Schulte-Hermann, R., Schuppler, J., Timmermann-Trosiener, I., Ohde, G.,

Bursch, W., and Berger, H. The role of growth of normal and preneoplasticcell populations for tumor promotion in rat liver. Environ. Health Perspect., 50:185-194, 1983.

29. Shinozuka, H., Lombardi, B., and Abanobi, E. E. A comparative study of theefficacy of four barbiturates as promoters of the development of -y-glutamyl-transpeptidase-positive foci in the liver of carcinogen treated rats. Carcinogenesis (Lond.), 3: 1017-1020, 1982.

30. Staubli, W., Bentley, P., Bien, F., Fröhlich, E„and Waechter, F. Inhibitoryeffect of nafenopin upon the development of diethylnitrosamine-induced enzyme altered foci within the rat liver. Carcinogenesis (Lond.), 5: 41-46,1984.

31. Sugai, M., Witting, L. A., Tsuchiyama, H., and Kummerow, F. A. The effect ofheated fat on the carcinogenic activity of 2-acetylaminofluorene. Cancer Res.,22:510-519,1962.

32. Szepesi, B.. and Freedland, R. A. Alterations in the activities of several rat liverenzymes at various times after feeding a high carbohydrate diet to ratspreviously adapted to a high protein regimen. J. Nutr., 94: 37-46,1968.

33. Touvinen, C. G. R., and Bender, A. E. Effect of dietary sucrose and fructoseon the metabolism and lipid fractions in liver in the rat. Nutr. MetaboL 79:1-

9,1975.34. Wallach, D. F. H. Membrane anomalies of neoplastia cells. Med. Hypothesis,

2:241-256, 1976.35. Williams, G. M. The pathogenesis of rat liver cancer caused by chemical

carcinogens. Biochim. Biophys. Acta, 605: 167-189, 1980.36. Williams, G. M. Liver carcinogenesis: the role for some chemicals of an

epigenetic mechanism of liver-tumor promotion involving modification of thecell membrane. Food Cosmet. Toxicol., 19: 577-583,1981.

37. Williams, G. M., Ohmori, T., Katayama, S., and Rice, J. M. Alteration byphénobarbital of membrane-associated enzymes including gamma glutamyltranspeptidase in mouse liver neoplasms. Carcinogenesis (Lond.), 7:813-818,1980.

38. Williams, G. M., Telang, S., and Tong, C. Inhibition of intercellular communication between liver cells by the liver tumor promoter 1,1,1 -trichloro-2,2-bis(p-chlorophenyl) ethane. Cancer Lett., 77:339-344,1981.

39. Yamasaki, H., Enomoto, T., Martel, T., Shiba, Y., and «anno, Y. Tumorpromoter-mediated reversible inhibition of cell-cell communication (electricalcoupling). Exp. Cell Res., 746: 297-308,1983.

40. Yotti, L. P., Chang, C. C., and Trosko, J. E. Elimination of metabolic cooperationin Chinese hamster cells by a tumor promoter. Science (Wash. DC), 206:1089-1091,1979.

Fig. 1. Photomicrograph of a liver section from a DEN-treated rat fed a high-sucrose diet for 4 weeks. A GGTPase-

stained focus (arrowheads) is identified in the midzonal regionof the hepatic lobule/between a portal space with positivelystained bile duct (B) and a longitudinally sectioned centralvein (C). GGTPase stain, x150.

Fig. 2. Same section as in Fig. 1 after counterstaining todemonstrate histological details. Notice the fatty changes inthe peripheral region of the hepatic lobules. Focus is demarcated by arrowheads. B, bite duct; C, central vein. Hamshematoxylin, X130.

,*Ca.* T^'t

CANCER RESEARCH VOL. 45 JUNE 1985

2705

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1985;45:2700-2705. Cancer Res   Tom K. Hei and Oscar Sudilovsky  Enzyme-altered Foci in Chemical Hepatocarcinogenesis in RatsEffects of a High-Sucrose Diet on the Development of

  Updated version

  http://cancerres.aacrjournals.org/content/45/6/2700

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/45/6/2700To request permission to re-use all or part of this article, use this link

on July 6, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from