Morphological andcell kinetic effects of dietary ... · kinetic analysis. These animals received intra-peritonealvincristine (1 mgperkg)at900amonthe day of study. Individual animals

Glut, 1987, 28, 754-763

Morphological and cell kinetic effects of dietarymanipulation during colorectal carcinogenesisD J GALLOWAY, FREDA JARRETT, P BOYLE, MALLIKA INDRAN,KATHARINE CARR, R W OWEN, AND W D GEORGE

From the University Departments of Surgery, Pathology and Anatomy, Western Infirmary and University ofGlasgow, Glasgow, and Bacterial Metabolism Research Laboratory, Centre for Applied Microbiology andResearch PHLS Porton Down, Salisbury, Wilts.

SUMMARY The effect of dietary manipulation of fat and fibre on the structural and cell kineticcharacteristics of colonic mucosa was studied before and during experimental carcinogenesis in 232male Albino Swiss rats. Carcinogen treated animals were given 12 weekly injections ofazoxymethane (10 mg/kg/week). The animals were divided between four dietary groups (1) highfat, high fibre, (2) low fat, high fibre, (3) high fat, low fibre and (4) low fat, low fibre. Pathologicaland cell kinetic information together with details of certain faecal characteristics was collectedwhen the animals were killed 4, 20, and 28 weeks after starting their experimental diet. Tumourinduction was significantly influenced by diet. The highest risk of colorectal tumour developmentwas found in groups fed diet 3: high fat, low fibre (p<003). In contrast, diet 2: low fat, high fibrewas associated with the lowest risk. The proportion of histologically proven colonic tumoursoccuring in each dietary group was: diet 1 - 10-9%, diet 2 - 3-6%, diet 3 - 63.7%, diet 4 - 21-8%.Scanning electron microscopic (SEM) studies done on selected samples indicated both dietary andazoxymethane related alterations in crypt unit integrity. The most marked surface architecturalchanges were seen in carcinogen treated animals maintained on diet 3 (high fat, low fibre).Stathmokinetic analysis revealed considerable intergroup variability. Both fat and fibre producedsignificant effects, principally during the preneoplastic phase of carcinogenesis. Faster proliferativeactivity tended to be found in animals at low risk of tumour induction (diet 2), slower proliferationbeing more characteristic of animals at high risk (p<005) The findings suggest that bothtopographical and cell kinetic parameters have an important relationship with promoting andprotecting dietary factors during the development of colorectal cancer.

Colorectal cancer is at present the second mostfrequent cause of cancer related death in the WesternWorld. Despite increasing sophistication inboth diagnostic and therapeutic strategies, littleimprovement has been evident in the outcome aftermanagement of this condition. Much of the currentresearch relative to large bowel cancer is thus beingdirected towards achieving a clearer understandingof the aetiological and biological properties of thedisease.

Descriptive and analytical epidemiologicalstudies' have, by highlighting the marked inter-national incidence variation for colorectal cancer,

Address for corrcspondcncc: Mr Divivl J (Gillowaiy. Dept of Surgcry. WcstcrnInfrni.irv, (iisgzow Gil 6NTRccivced for publicitioil 3 Octohcr 1986.

consistently emphasised the importance of environ-mental factors in the aetiology of this condition. Inparticular, dietary factors appear to be important,especially dietary fat and fibre which have beenshown to have promoting and protecting influencesrespectively.Over the past decade several good animal models

for experimental colorectal cancer have beendeveloped and investigated. Wide experience hasbeen gained with a particularly reliable model whichinvolves the induction of large bowel cancer inrodents using such hydrazine carcinogens as 1, 2-dimethyl-hydrazine or azoxymethane.' Dietarymanipulation of fat and fibre during experimentalcarcinogenesis has shown respective promoting andprotecting influences attributable to these crudenutrients in the animal model.'

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Morphological and cell kinetic effects ofdietary manipulation during colorectal carcinogenesis

In the process of neoplastic transformationit is clear that functional disturbances in theproliferative characteristics of any tissue undergoingneoplastic change are required before the develop-ment of the recognisable morphological abnor-malities that constitute neoplasia. Currentunderstanding of the histogenesis of colorectalcancer owes much to morphological studies.67 Untilrecently, however, there has been relatively littleemphasis on the study of the functional changesin growth characteristics which can account forthe resulting histological pattern.' Thus kineticparameters demand scrutiny in any serious study ofthe histogenesis of colorectal cancer.

It is clear, therefore, that both dietary factors andcell kinetic characteristics bear a fundamentalrelationship to the development of colorectal cancer.The purpose of this study was to examine andcharacterise the effects of the manipulation of dietaryfat and fibre on the structural and cell kineticcharacteristics of colonic mucosa during the inductionof experimental large bowel cancer.

Methods

STUDY DESIGNThe experimental system used in this study involvedthe induction of colorectal cancer in inbred, maleAlbino Swiss rats using subcutaneous azoxymethanegiven in a dose of 10 mg/kg/week for 12 consecutiveweeks. The animals were 8-12 weeks old at the startof the experiment and they were housed in cages withstainless steel grid floors to minimise coprophagia.Two hundred and thirty two animals were studiedand divided between four dietary groups. Both theexperimental diets and water were available to theanimals ad libitum. The diets were commerciallyprepared to the crude nutrient specifications shownin Table 1. (Special Diet Services, Witham, Essex).

Table 1 Dietary composition; crude nutrient content(adjusted to 10% water content)

Diet I Diet 2 Diet 3 Diet 4High fat Lowfat High fat Lowfathighfibre highfibre lowfibre lowfibre

Moisture (%) 10 10 10 10Crude fat(%) 251 21 25-1 1-9Crude fibre (%) 266 26-9 2-1 1-9Crude protein (%) 149 148 14-9 14-7Carbohydrate (%) 16-4 38-7 43-7 67-2Ash 7-0 7-5 4-1 4-3Total (%) 100 100 100 1(0Digestive energy

13-0 8-2 17-9 13-2

20-1 14-7 2)-3 14-9

Each diet contained the same standard vitamin andmineral supplementation. The source of fat in thediets was beef tallow and the principal source of fibrewas purified cellulose. Morphological and cell kineticobservations were made at three different stages ofcarcinogenesis as follows:1 0112 categoryThe animals in this measurement category weremaintained on the respective diets for four weeksbefore being killed. No carcinogen or controlinjections were administered.2 4/12 categoryAnimals in this group were maintained on theirrespective diet for four weeks and then, in addition, acourse of control or carcinogen injections were givenand the animals killed four months from the time ofthe first injection.3 6/12 categoryIn this group the animals were treated in the sameway as the 4/12 category except that the time of deathwas six months after the first injection.

GROUP SIZEIn the 0/12 category each of the four dietary groupscontained 10 animals. In both the 4/12 and 6/12categories both control and carcinogen treatedgroups for each of the four diets comprised 12animals.

SAMPLING PROCEDUREAt the time of death an identical sampling procedurewas followed for each group of up to 12 animals.Eight of these animals were used for the stathmo-kinetic analysis. These animals received intra-peritoneal vincristine (1 mg per kg) at 9 00 am on theday of study. Individual animals within the groupwere then killed at carefully defined time intervals(30, 45, 60, 75, 90, 105, 120, and 180 minutes) overthe ensuing three hours.9 All of these animals werethen subjected to the same post mortem examination.The gastrointestinal tract was excised and openedalong its length. Standard full thickness specimenswere taken from the colon at four defined sitescorresponding to the rectum, the descending colon,the major flexure and the caecum. Two correspondingsamples were taken from each of these four areas andwere processed for histology and cell populationkinetic analysis respectively. The liver was alsoremoved from each animal and was processed forhistology as was any other abnormal feature detectedon autopsy.

HISTOLOGYThe tissue samples were fixed in buffered 10%formalin. Dehydration and blocking in paraffin waxwas carried out and four micron thick sections were

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G(allowtaY, Jarrett, BIoYle, Ini(dranl, Carr, Owen and George

cut aind then stained using a regressive haemalum andeosin staining technique before examination.Standard histological sections from three animals ineach individual subgroup - that is, 20 subgroups, andfrom each of the four anatomical sites were examinedfor evidence of crypt hyperplasia by carefully countingthe nuclei along the length of 10 axially sectionedcrypts on each slide.

SCANNING. ElECTRON MICROSCOPYParatffin embedded tissue blocks from the descendingcolon of each dietary group studied in the 4/12category were taken and reprocessed for scanningelectron microscopy by removing the paraffin waxfrom the tissue firstly by trimming and subsequentlyby melting in a pan of hot wax. The samples werethen taken through four changes of xylene and thentwo changes each of absolute alcohol and amylacetate before critical point drying with carbondioxide. They were then mounted on stubs usingsilver paint, coated with gold in a sputter coater andexamined in a JEOL T300 scanning electron micro-

scope at 20KV without any stage tilt. The recordingwas done on FP4 70mm films by exposing it for 90seconids.To facilitate the analysis of the SEM appearances a

scoring system was used. This was developed from apreviously validated system for the assessment ofvillous damage in irradiated small intestinal mucosa."The scoring system is as follows: 0: Spherical or

ovoid crypt units with round or ovoid crypt orifices;normal appearance. (Fig. 1), 2: Elongated crypt unitswith slit like orifices, 4: Large crypt units with wideorifices, 6: Crypt units showing wide orifices withswollen edges, 8: Absence of definitive crypt units(Fig. 2), 10: Total absence of epithelium.When a sample contained crypt units fitting morethan one of the descriptive categories, an average ofthe individual total score for each dietary group wascalculated.

CEI I KINETICSThe tissue samples for kinetic analysis were fixed inCarnoy's fluid for six hours, rehydrated, hydrolysed

Fig. I Normal scanning electron microscopic appearances. Crypt unit.s are spherical or ovoid with round or ovoid cryptorifices; a crvfpt 1unlit integrity .score of zero was given.

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Morphologicla(nd1 ccli kinetic effe(ts of'(lietary nanipiilatioti ldrinig colorec-dtil carciniogenesi.s

in normal hydrochloric acid at 60'C for 10 minutesbefore staining in Schiffs reagent for one hour. Themucosa was then microdissected and a squashedpreparation of whole colonic crypts was obtainedwhich was then mounted in clear epoxy resin. Meanwhole crypt metaphase counts from 10 crypts in eachsample were then plotted against time to derive thecrypt cell production rate by linear regressionanalysis.

STATISTICA1 ANAL YSISIn order to make estimates of the independent effectsof the different levels of dietary fat and fibre intakesas well as the administration of a carcinogen, the useof a multivariate analysis was required. This wasaccomplished using the computer package GLIM''(Generalised Linear Interactive Modelling). In orderto test whether the crypt cell production ratesdiffered between the dietary groups a modified linearregression analysis was used. Because each pointon any slope was calculated from a number of

observations and the standard errors of each pointvaried considerably, it was necessary to perform theregression 'weighting' each point inversely accordingto its standard error. This avoided the situationwhere one very high or low aberrant value based onfew observations could over-influence the calculatedslope.

FAECAI CHARACTFRISTICSThe weight of faeces produced by animals in the fourdietary groups within the 4/12 category was recordedduring two separate seven day periods. These periodswere located during week six and week 16, - that is,half way through the course of injections andimmediately before death.

Faeces from each animal were tested just beforedeath for the presence of occult blood using amodified guiac test. (Hlaemoccult; Eaton Labs).The identification and quantification of faecal

steroids was done as previously described.' Faecalsamples from each group were pooled and extracts

Fig. 2 Scanning electron microscopic appearances showing crypt units with wide orifices with swollen edges; the crypt ulnitintegrity score was 8.

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Galloway, Jarrett, Boyle, Indran, Carr, Owen and George

Table 2 Total body weight change as a percentage ofstairtinig weight

D)iet Group Measureenetit Category4/12 6/12

I Control + 13.9%/* +2-7%AOM Trctcd -14.5%* +1-8%

2 C>ontrol +13-3% * -25l1%*AOM Trcated + 10/% -18.4% *

3 Control + 19 2%* +29-3%*AOM Trciated -0(6% +2-7%

4 Control +20.8%* +23-1%*AOM Trcated +3-6%Yo +1 10(%*

* Denotes a statistically significant change in weight.

comprising both neutral sterols and free faecal bileacids were analysed, the individual compounds beingidentified by computerised gas liquid chromato-graphy and mass spectrometry.

Results

GENERAL CHARACTERISTICSIn total, 24 (10.34%) of the 232 animals failed tosurvive until the scheduled time of their death. Ofthese, six were in the control and 18 in the carcinogentreated groups. No pathological abnormalities weredetected among the control groups and in thecarcinogen treated animals, no deaths were clearlyattributable to gastrointestinal neoplasia or anycomplications resulting therefrom. Considerabledifficulty was encountered in achieving detailed postmortem information from these animals because ofcannibalisation. The four different dietary groups inboth the 0/12 category and the 4/12 category were notstatistically different from one another with regard toeither the weight of diet consumed or the calculatedgross energy intake appropriate to that food intake.In the 6/12 category both the weight of food taken inand the corresponding gross energy value was greaterin animals fed diet 1 compared with all the other threedietary groups (p<0001).

Table 2 shows that the pattern amongst the fourdietary groups for total body weight during thecourse of experiments was such that high fibre fedanimals tended to thrive less well than low fibre fedanimals. The only groups which consistently lostbody weight were the 6/12 groups eating diet 2 (lowfat, high fibre).

PATHOl OGYNo abnormalities were encountered in any of thecontrol animals. Reliable autopsy data are availablefor 208 (90%) animals. Among the carcinogentreated groups in the 4/12 category the only neo-plastic lesions noted were found among animals

4

Control U

0ul iL3~ E

Diet 1 Diet2 Diet3 Diet 4H igh fat Low fat High fat Low fatHigh fibre Highfibre Lowfibre Low fibre

Fig. 3 Ultrastructural appearances. A crypt unit integrityscore ofone is regarded as normal. The mean and rangeforeach group is shown where applicable.

consuming diet 3 (high fat, low fibre). Amongst these10 animals, four had large bowel neoplasia; in two,lesions in the proximal colon were easily visible andin a further two microscopic adenomatous foci weredetected on histological examination of mucosawhich appeared macroscopically normal.

Within the 6/12 category an entire range of gastro-intestinal neoplastic lesions was identified. Theinterdietary distribution of visible colonic tumourswhich were subsequently confirmed histologicallywas analysed by first comparing the median numberof tumours in each dietary group using the Kruskal-Wallis test (non-parametric analysis of variance).This revealed that the median number of tumours foreach of the dietary groups showed statisticallysignificant differences (p<0-025). Follow up Mann-Whitney U tests were carried out to compareeach diet with each of the other diets. Diet 1 hadsignificantly fewer tumours than diet 3 (p<0-02) anddiet 4 (p<0-05). Diet 2 had significantly fewertumours than diet 3 (p<0.02) and diet 4 (p<0.05).Finally, diet 3 animals had significantly more tumoursthan diet 4 counterparts.

In total, 55 gastrointestinal neoplasms wereconfirmed among all the carcinogen treated groups inthese experiments. Six (10-9%) of these lesions werefound in animals fed diet 1. Two (3-6%) of the lesionsoccurred in animals fed diet 2 (low fat, high fibre). Incontrast, 35 (63.7%) occurred in high fat, low fibrefed animals, - that is, diet 3, and the remaining 12(21-8%) were found in the low fat, low fibre fedgroup.

Careful assessment of crypt length showed noevidence of any significant differences between anycarcinogen treated groups and their correspondingcontrols. Thus in this study there is no direct evidenceof any azoxymethane induced crypt hyperplasia.

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Fig. 4 Scanning electron microscopic appearances oftiny adematousfocus involving several adjacent crypts.

TOPOGRAPHICAL CHARACTERISTICS

No major topographical changes can be attributed todiet along the colon, although slight differences insurface architecture were apparant between thedietary groups, with the highest crypt unit integrityscore applying to animals fed diet 2 (low fat, highfibre). After carcinogen treatment a much greaterdegree of deviation from the normal pattern was

seen. These changes were most severe in the animalsfed diet 3 (high fat, low fibre), see Figure 3.

Figure 4 shows an example of the scanning electronmicroscopic appearances of a lesion considered torepresent early colonic neoplasm.

CEI L POPUI ATION KINETICSTable 3 shows the values for the crypt cell productionrates (CCPR) in the animals in the 4/12 category.There was considerable variability within those dataand no clear pattern exists with respect to an effect ofany of the major variables, namely diet, anatomical

Table 3 Crypt cellproduction rate (cellslcryptlhour) 4/12 category: control and carcinogen treated groups

A naotnical siteDiet Rectumn Desceniding colon Transverew colon (aecum

Control AOM Control AOM Control AOM Control AOM

I High fatHigh fibre 7 54 7-82 12 72 12 57 12 71 9-55 8X91 9)69

2 Low fatHighfibrc 11-52 11-16 4-82 21-51 13 45 42-47 12 69 145(0

3 High fatLow fibrc 4-23 3 32 3-69 6-72 7X() 8t23 7 81 5t21

4 Low fatLow fibre 9-25 7 32 14 10 10(4( 12-6( 11 800 X 55 8X48

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Rectum

Desc.colon

Transcolon

Caecum

4/12 6/120 / 12 Control AOM Control AOM

No significant differences detected

; CCPR significantly greater indiet 1 than diet 3

Fig. 5 Summary ofstatistical analysis ofstathmokineticdata using GLIM and a weighted one way analysis ofvariance. Multiple comparisons were performed and thesigniificant interdietary differences are displayed in thegrid.

site and the use of azoxymethane. A weightedone-way analysis of variance has been carried outusing the generalised linear interactive modellingtechnique in an attempt to do multiple comparisonsbetween the different dietary groups and discern anysignificant pattern which might exist in the data.The significant differences generated are shown in

Figure 5 which shows that there was no clear orconsistent effect of any individual diet on cell kineticparameters. In the 0/12 category the only significantdietary influence in kinetic parameters occurred inthe most distal portion of the bowel. Fibre seems tohave a more pronounced individual effect than fat,the tendency being for the diets containing highlevels of fibre to be associated with more rapid cellproliferation than those containing less fibre.

In the 4/12 control category significant dietaryinfluences were again seen in the distal colorectum.Here, however, attempts to distinguish individualeffects of fat and fibre yield conflicting impressionsand the results appear inconsistent. In the 4/12carcinogen treated animals the most interesting andconsistent contrasts appear. Dietary effects can beseen at each of the anatomical sites examined. Forthe rectum the individual fat and fibre effects are suchthat in the significant differences which do appear,high fat is associated with slower CCPR and highfibre with faster CCPR. For the descending andtransverse colon exactly the same individual nutrienteffects can be seen and reinforcing this, in each

instance the low fat high fibre diet (diet 2) wasassociated with significantly faster CCPR than thehigh fat low fibre diet (diet 3). In the caecum nosignificant individual effect of fat can be identifiedbut, once again, increased dietary fibre was associatedwith faster CCPR.

In the 6/12 control animals the kinetic activityis rather more even and no significant dietarydifferences appeared. In the correspondingcarcinogen treated animals there was only oneisolated difference between diets 1 and 4.

FAECAI CHARACTERISTICSPHYSICAL PROPERTIESTable 4 shows the weight of faecal output for controland carcinogen treated groups, during both week sixand week 16 of the study, averaged and expressed asgrams of faeces per rat per 24 hours. High fibrecontaining diets (diets 1 and 2) were associated with amuch more marked bulking effect than the low fibrecontaining diets (p<0-001). Furthermore nosignificant differences were seen which could beattributed to dietary fat or the administration ofcarcinogen.

FAECAL OCCULT BLOODIn 208 animals Haemoccult tests were carried out and22 (10-57%) were positive. Sensitivity and specificityhave been calculated with respect to macroscopicallyobvious gastrointestinal lesions which subsequentlywere confirmed to be neoplastic on histologicalexamination. There were five false positive slides andone false negative. Thus the test had a sensitivity of94*4% and a specificity of 97*3% The predictiveaccuracy was 77-2% for a positive result and 99*4%for a negative result.

FAECAL BILE ACID CONCENTRATIONFigure 6 shows a summary of the total faecal free bileacid (FBA) concentration expressed as milligramsper gram dry faeces. The total FBA concentration indietary groups 1 and 2, the high fibre containing diets,

Table 4 Faecal weight (glratlday) as measured during twoseparate periodsforgroups in the 4/12 category

Diet Control groups Carcinogen treated groupsWeek 6 Week 16 Week 6 Week 16

1 High fat 12 03 12 25 11 75 9 98High fibre

2 Low fat 12-25 11-95 11-82 14(X)High fibre

3 High fat 1.39 1-88 1-35 1-82Low fibre

4 Low fat 2-01 1-40 1-9( 1-32Low fibre

T T

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Morphological and cell kinetic effects of dietary mnaniplulation during colorectal carcinogenesiLs

14

L 12a> 10

m 8-E

Ln7 6-u0

a

Q 4-

2U-

0

0/12 Category

1 High fat highfibre2 Low fat highfibre3 High fat tow fibre4 Low tat low fibre

1 2 3 4Diet

4/12 Category

LJControl J|AOM C]|

l-

I

1 2Diet

3 4

Fig. 6 Faecalfree bile acid concentration (mglg dryfaeces);0112 and 4/12 categories.

was considerably lower than the corresponding lowfibre containing diets 3 and 4 where the values werefrom 3-4-109 times greater. Neither dietary fat northe administration of azoxymethane produced a

significant or consistent effect on FBA concentration.

Discussion

Despite the many mechanisms postulated by whichdietary fat and fibre may exert promoting andprotecting influences on colorectal carcinogenesis,the exact importance of each and their inter relation-ships are still poorly understood.

TUMOUR INDUCTIONIn this study the two techniques of histology and SEMshow that both the levels of dietary fat and fibre havea bearing on the ultimate tumour risk applying tocarcinogen treated animals consuming any one ofthe four diets. As in other investigations"47 highlevels of fat enhanced carcinogenesis, and high levelsof fibre"-9 had a potent protective effect. In additionthere is good evidence of a significant interactionbetween the promoting and protecting influences offat and fibre.The deliberate variation of any single dietary

component will require the addition or displacementof other components thus altering the relativecomposition of at least two dietary variables. Therole of other crude nutrients such as protein andcarbohydrate on subsequent tumour induction isseen here to be of little importance. The proteincontent of the four study diets is closely similardespite widely differing risks for tumour develop-ment. While carbohydrate levels differed betweenthe groups they were most closely similar betweendiets 2 and 3, for which tumour induction patterns

were most dissimilar. Neither protein nor carbo-hydrates have been previously thought to influencecarcinogenesis significantly. There is no directrelationship between tumour induction and food orenergy intake in this study. It is possible, however,that the protective effect of diet 2 (low fat, high fibre)may have been accentuated by the failure of thoseparticular groups to thrive.

Current understanding of the role of dietaryfactors on intestinal carcinogenesis implicates themetabolic activity of the bacterial population of thegastrointestinal tract. The flora can be alteredby changes in dietary practices.r" Whether fibreproduces its effect by some direct physicochemicalaction or by increasing faecal bulk, thereby dilutingand possibly minimising any contact between themucosa, and as yet unidentified carcinogens, isunknown." In this study fibre was the only nutrientassociated with any significant alteration in faecalbulk.The proposed promoting mechanism for fat has

been closely linked with faecal bile acid concen-tration. It has been shown that in man, increasing thedietary intake of fat was associated with an increasein FBA concentration. In experimental animals,secondary bile acids have cocarcinogenic properties.'While in the present study a comparison of the dietswith respect to FBA concentration showed a patternwhich paralleled that of tumour induction, the majordietary determinant of FBA concentration was fibrerather than fat content.

DYNAMIC CEI I POPUI ATION KINETICSWhile the fundamental relevance of cell kineticstudies to colorectal neoplasia is not in dispute, theactual kinetic processes involved in tumour develop-ment are more controversial. Dietary manipulationin this study produced no overall clear-cut kineticeffect. Carcinogen treated groups in the preneo-plastic phase of tumour development (4/12 category)did not show some striking fat and fibre related trendsin colonic proliferative activity. The slower kineticactivity tended to occur in dietary groups at high riskfor tumour induction, the lower risk diet groupshaving more rapid cell production rates. High fibrecontaining diets were frequently associated with fastcell production, while high fat, in contrast, was oftenlinked to slower cell proliferation. There were nokinetic effects attributable to individual factors suchas azoxymethane, protein, carbohydrate, energyintake or body weight.

In view of the presence of discernable diet relatedkinetic effects at only one stage of carcinogenesis itmay be postulated that these proliferative patternsare relevant to subsequent tumour production at onecrucial part of a complex multistage process. Much

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762 Galloway, Jarrett, Boyle, Indran, Carr, Owen and George

evidence is now aivailable to support the concept thatrapid proliferative activity present at the time ofinitiation enhances carcinogenesis.4`-" This study isthe first to report proliferative differences related todietary promoting and protecting factors during thepostinitiation phase. A study"7 in which dietary fibrehas been manipulated, however, has suggestedthat the hyperproliferative effect of fibre has anenhancing effect on tumour initiation but a protectiveeffect during promotion. No hyperplasia afterazoxymethane treatment was noted in this study.Sunter2" did find such hyperplasia in a closelysimilar experimental model and it seemed to beaccompanied by a rise in cell production rate ascarcinogenesis progressed. Cooke'9 also notedprogressively increasing crypt cell production rates asneoplasia progressed, although it is of interest thatthe tumour distribution in their study correspondedto the areas where crypt cell production rate had beenslowest during the early stages of promotion.The work reported here together with that of

Tutton and Barkla3' appears to conflict with thesuggestion that an increased cell proliferation rateis a fundamental kinetic process during tumourinduction. Indeed these latter studies emphasise theimportance of a slowly proliferating cell populationto the evolution of colorectal neoplasia. Specificslowing of cell proliferation within an initiated cellpopulation may allow those cells more time withinthe mucosa to establish their selective growthadvantage. Given that tumour developmentprobably involves many stages it may be that all thesekinetic observations are not incompatible, but reflectmeasurements made at different parts of a complexprocess. The relationship between cell kinetics,tumour promotion and histogenesis requires furtherstudy to determine more accurately whether the cellkinetic differences seen in this study are crucial orepiphenomenal.

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